SSP-SAP References

SSP-SAP (Cat. #IT-11)

see also: SP-SAP (Cat. #IT-07)discontinued

101 entries found for : it-11

PARVing the way to cap translation for seizure control

Gross C (2021) PARVing the way to cap translation for seizure control. Epilepsy Curr 21(5):360-362. doi: 10.1177/15357597211027010

Summary: Loss of GABAergic interneurons leads to spontaneous recurrent seizures that persist over months if the amount and spatial spread of initial inhibitory neuron loss is sufficient.

Usage: Intrahippocampal injections of SSP-SAP (0.4 ng/10 nL) were performed using a 0.5-μL Neuros Syringe lowered into four hippocampal sites along both the transverse and longitudinal hippocampal axes bilaterally.

See: Chun E et al. Targeted hippocampal GABA neuron ablation by Stable Substance P-saporin causes hippocampal sclerosis and chronic epilepsy in rats. Epilepsia 60(5):e52-e57, 2019.

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Possible contribution of cerebellar disinhibition in epilepsy

Ming X, Prasad N, Thulasi V, Elkins K, Shivamurthy VKN (2021) Possible contribution of cerebellar disinhibition in epilepsy. Epilepsy Behav 118:107944. doi: 10.1016/j.yebeh.2021.107944

Summary: The authors hypothesize that loss of inhibition from the cerebellum can lead to cortical activation and seizures. An animal study showed microinjection of SSP-SAP produced a selective ablation of hippocampal inhibitory interneurons in vivo and a highly focal disinhibition. These results also demonstrate that the ‘‘epileptic” pathophysiology produced by experimental status epilepticus or head trauma can be replicated by focal interneuron loss, without involving principal cell loss and other interpretive confounds inherent in the use of global neurologic injury models.

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Featured Article: Targeted hippocampal GABA neuron ablation produces hippocampal sclerosis, epilepsy, and dissociable effects on the Morris water maze and object-place paired association tasks

Bumanglag AV, Truckenbrod LM, Chun E, Hernandez AR, Federico QP, Maurer AP, Sloviter RS, Burke SN (2019) Featured Article: Targeted hippocampal GABA neuron ablation produces hippocampal sclerosis, epilepsy, and dissociable effects on the Morris water maze and object-place paired association tasks. Targeting Trends 20

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Read the featured article in Targeting Trends.

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Central circuit mechanisms of itch.

Chen XJ, Sun YG (2020) Central circuit mechanisms of itch. Nat Commun 11:3052. doi: 10.1038/s41467-020-16859-5

Summary: The authors summarize the progress in elucidating the neural circuit mechanism of itch at spinal and supraspinal levels.

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A role for neurokinin 1 receptor expressing neurons in the paratrigeminal nucleus in bradykinin-evoked cough in guinea-pigs.

Driessen AK, McGovern AE, Behrens R, Moe AAK, Farrell MJ, Mazzone SB (2020) A role for neurokinin 1 receptor expressing neurons in the paratrigeminal nucleus in bradykinin-evoked cough in guinea-pigs. J Physiol 598(11):2257-2275. doi: 10.1113/JP279644

Objective: This study aimed to assess the involvement of paratrigeminal neurokinin 1 receptor neurons in the regulation of cough, breathing and airway defensive responses.

Summary: These findings warrant further investigations into targeting the jugular ganglia and paratrigeminal nucleus as a therapy for treating cough in disease.

Usage: Targeted toxin lesions across three sites of the paratrigeminal nucleus (200nl per injection site).

Related Products: SSP-SAP (Cat. #IT-11)

Episodic stimulation of central chemoreceptor neurons elicits disordered breathing and autonomic dysfunction in volume overload heart failure.

Díaz HS, Andrade DC, Toledo C, Pereyra KV, Schwarz KG, Díaz-Jara E, Lucero C, Arce-Álvarez A, Schultz HD, Silva JN, Takakura AC, Moreira TS, Marcus NJ, Del Rio R (2020) Episodic stimulation of central chemoreceptor neurons elicits disordered breathing and autonomic dysfunction in volume overload heart failure. Am J Physiol Lung Cell Mol Physiol 318(1):L27-L40. doi: 10.1152/ajplung.00007.2019

Objective: To determine the role of the central chemoreflex in the development of respiratory and autonomic dysfunction in volume overload heart failure (HF).

Summary: Episodic hypercapnic stimulation triggers ventilatory plasticity and elicits cardiorespiratory abnormalities in HF that are largely dependent on retrotrapezoid nucleus (RTN) chemoreceptor neurons.

Usage: Bilateral injections of SSP-SAP, 0.6 ng/30 nL, into the RTN were administered to destroy chemoreceptor neurons.

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Contribution of the retrotrapezoid nucleus and carotid bodies to hypercapnia- and hypoxia-induced arousal from sleep.

Souza GMPR, Stornetta RL, Stornetta DS, Abbott SBG, Guyenet PG (2019) Contribution of the retrotrapezoid nucleus and carotid bodies to hypercapnia- and hypoxia-induced arousal from sleep. J Neurosci 39(49):9725-9737. doi: 10.1523/JNEUROSCI.1268-19.2019

Objective: To examine the contribution of two lower brainstem nuclei that could be implicated in CO2 and hypoxia-induced arousal.

Summary: RTN, a brainstem nucleus that mediates the effect of brain acidification on breathing, also contributes to arousal elicited by CO2 but not hypoxia.

Usage: To ablate the retrotrapezoid nucleus (RTN), SSP-SAP was administered (2.4 ng) through bilateral injections via a dorsal craniotomy.

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Episodic stimulation of central chemoreflex elicits long-term breathing disorders and autonomic imbalance in heart failure rats.

Díaz HS, Andrade DC, Toledo C, Lucero C, Arce-Álvarez A, Del Rio R (2019) Episodic stimulation of central chemoreflex elicits long-term breathing disorders and autonomic imbalance in heart failure rats. Eur Respir J 54(suppl 63):OA4936. doi: 10.1183/13993003.congress-2019.OA4936

Objective: To determine the role of CC in the development of cardiorespiratory dysfunction in a HF model.

Summary: Rats were exposed to pisodic hypercapnic stimulation (EHS) The effects of EHS in rats with heart failure were attenuated by SSP-SAP treatment.

Usage: Selective destruction of chemoreceptor neurons within the retrotapezoid nucleus (RTN) was performed via SSP-SAP injections (0.6 ng/30 nL).

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Nociceptors expressing TRPV1 and trigeminal nucleus neurons expressing NK1 mediate orthodontic pain

Wang S, Kim M, Ong K, Pae E-K, Chung M-K (2019) Nociceptors expressing TRPV1 and trigeminal nucleus neurons expressing NK1 mediate orthodontic pain. Neuroscience 2019 Abstracts 052.10. Society for Neuroscience, Chicago, IL.

Summary: Orthodontic force produces mechanical irritation and inflammation in periodontium, which inevitably accompanies pain. Despite its high prevalence, treatment of orthodontic pain is not effective. Determining detailed neural mechanisms involving peripheral and central nervous system should be critical to improve the management of orthodontic pain. Periodontal ligament is projected by peptidergic nociceptors, which is enriched with transient receptor potential vanilloid 1 (TRPV1), a receptor for capsaicin. Trigeminal subnucleus caudalis (Vc), is critical for relaying orofacial nociceptive signal into brain. A group of second- order neurons in the superficial dorsal horn of Vc express neurokinin 1 receptor (NK1), a receptor for substance P, and receive inputs from peptidergic nociceptors. However, the contribution of these nociceptive neurons to orthodontic pain has not been determined. Orthodontic force of 10g produced reliable tooth movement in mice. Orthodontic pain was evaluated by measuring mouse grimace scale (MGS) and bite force (BF), which could represent spontaneous pain and chewing-evoked pain, respectively. Orthodontic force increased MGS and decreased BF, which peaked at 1d and returned near to sham level at 7d. Using targeted chemical ablation of specific subsets of neurons, we determined the contribution of TRPV1+ nociceptors and NK1+ Vc neurons to orthodontic pain behaviors in mice. Ablation of TRPV1+ nociceptors by injecting resiniferatoxin into trigeminal ganglia significantly attenuated orthodontic force assessed by MGS and BF. Chemical ablation of NK1+ Vc neurons by injecting saporin conjugated with substance P into Vc also significantly reduced the extent of changes in MGS and BF by orthodontic force. These results suggest that TRPV1+ trigeminal nociceptors and NK1+ Vc neurons constitute a major neural pathway for transmission of orthodontic pain, which is a fundamental neural mechanism of orthodontic pain transmission. The new mouse model of orthodontic pain will be useful for mechanistic study to develop novel approaches for painless orthodontics.

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Targeted hippocampal GABA neuron ablation produces hippocampal sclerosis, epilepsy, and dissociable effects on the Morris water maze and object-place paired association tasks

Truckenbrod LM, Bumanglag AV, Chun E, Hernandez A, Federico QP, Maurer AP, Sloviter RS, Burke SN (2019) Targeted hippocampal GABA neuron ablation produces hippocampal sclerosis, epilepsy, and dissociable effects on the Morris water maze and object-place paired association tasks. Neuroscience 2019 Abstracts 158.03. Society for Neuroscience, Chicago, IL.

Summary: An epileptogenic role for hippocampal GABAergic dysfunction has recently been reported (Chun et al. 2019). Specifically, selective ablation of hippocampal GABA neurons by Stable Substance P-saporin (SSP-saporin) conjugate caused dorsal hippocampal sclerosis and chronic epilepsy, without involving convulsive status epilepticus or widespread brain injury. The current study assessed cognitive function in chronically epileptic SSP-saporin-treated rats and their vehicle-injected controls ~8 months following injection. First, rats completed the Morris Water Maze test of spatial learning and memory (Morris et al., 1982). Animals then underwent testing with the object-place paired association (OPPA) task, which requires the hippocampus as well as functional connectivity between the hippocampus and cortical areas (Jo and Lee, 2010; Hernandez et al., 2017), and then a simple object discrimination task. Interestingly, both controls and rats with dorsal hippocampal sclerosis and chronic epilepsy were able to learn the location of the hidden platform in the Morris Water Maze task and could also acquire a simple pair-wise object discrimination. However, epileptic rats with dorsal hippocampal sclerosis were significantly impaired on the OPPA task, which requires animals to integrate spatial location memory with a correct object choice and is a more sensitive measure of cognitive dysfunction (Hernandez et al., 2015). These data indicate that, similar to humans with medial temporal lobe epilepsy, selective hippocampal sclerosis and epilepsy in this model do not result in global cognitive decline. Rather, cognitive functions that require functional connectivity between the hippocampus and cortical areas are selectively affected.

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ATS Poster of the Year Winner. Read the featured article in Targeting Trends.

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The retrotrapezoid nucleus: Central chemoreceptor and regulator of breathing automaticity.

Guyenet PG, Stornetta RL, Souza GMPR, Abbott SBG, Shi Y, Bayliss DA. (2019) The retrotrapezoid nucleus: Central chemoreceptor and regulator of breathing automaticity. Trends Neurosci 42(11):807-824. doi: 10.1016/j.tins.2019.09.002

Summary: This review describes the neurons of the retrotrapezoid nucleus (RTN), their transcriptome, developmental lineage, and anatomical projections. The authors also review their contribution to CO2 homeostasis and to the regulation of breathing automaticity during sleep and wake.

Usage: Local injection of SSP-SAP to kill RTN neurons.

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Spinal cord projection neurons: A superficial, and also deep, analysis.

Wercberger R, Basbaum AI (2019) Spinal cord projection neurons: A superficial, and also deep, analysis. Curr Opin Physiol 11:109-115. doi: 10.1016/j.cophys.2019.10.002

Summary: Modern approaches to map complex neural circuits require knowledge of the molecular language that defines cell type specificity. However, with few exceptions, NK1R remains the marker consistently used to define projection neurons and even to interrogate their contribution to pain and itch (Mantyh et al.) The first of two studies demonstrating that SP-SAP-mediated ablation of dorsal horn NK1R-expressing neurons reduces injury-induced hyperalgesia. (Carstens et al.) In this paper SP-SAP-mediated ablation of dorsal horn NK1R-expressing neurons reduced pruritogen-evoked scratching.

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Role of nociceptor toll-like receptor 4 (TLR4) in opioid-induced hyperalgesia and hyperalgesic priming.

Araldi D, Bogen O, Green PG, Levine JD (2019) Role of nociceptor toll-like receptor 4 (TLR4) in opioid-induced hyperalgesia and hyperalgesic priming. J Neurosci 39(33):6414-6424. doi: 10.1523/JNEUROSCI.0966-19.2019

Objective: To evaluate the hypothesis that hyperalgesia and priming induced by opioids are mediated by similar nociceptor mechanisms.

Summary: Treatment with isolectin B4 (IB4)-saporin or SSP-saporin (which deplete IB4! and peptidergic nociceptors, respectively), or their combination, prevented systemic LDM-induced hyperalgesia, but not priming. HDM-induced priming, but not analgesia, was markedly attenuated in both saporin-treated groups.

Usage: IB4-SAP was diluted in saline and a dose of 3.2 μg in a volume of 20 μl administered intrathecally. SSP-SAP was diluted in saline and a dose of 100 ng in a volume of 20 μl was administered intrathecally.

Related Products: IB4-SAP (Cat. #IT-10), SSP-SAP (Cat. #IT-11)

Spinal neuropeptide Y1 receptor-expressing neurons form an essential excitatory pathway for mechanical itch.

Acton D, Ren X, DiCostanzo S, Dalet A, Bourane S, Bertocchi I, Eva C, Goulding M (2019) Spinal neuropeptide Y1 receptor-expressing neurons form an essential excitatory pathway for mechanical itch. Cell Reports 28(3):625-639.e6 . doi: 10.1016/j.celrep.2019.06.033

Objective: To determine the central pathway for mechanical itch.

Summary: NPY-Y1 signaling regulates the transmission of innocuous tactile information by establishing biologically relevant thresholds for touch discrimination and mechanical itch reflexes. Neither the evoked nor spontaneous scratching seen following activation of Y1Cre neurons was affected by ablation of the GRPR+ neurons. NK1R+ neuron ablation failed to modulate mechanically evoked itch.

Usage: P28 mice were given a single intrathecal (i.t.) injection of either Bombesin-SAP (400 ng in 5 mL 0.9% sterile saline) to ablate GRPR+ cells or SSP-SAP to ablate NK1r+ neurons (100 ng in 5 mL 0.9% sterile saline). Littermate controls received Blank-SAP (equal mass in 5 mL 0.9% sterile saline).

Related Products: Bombesin-SAP (Cat. #IT-40), SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

Targeted hippocampal GABA neuron ablation by stable substance P-saporin causes hippocampal sclerosis and chronic epilepsy in rats.

Chun E, Bumanglag AV, Burke SN, Sloviter RS (2019) Targeted hippocampal GABA neuron ablation by stable substance P-saporin causes hippocampal sclerosis and chronic epilepsy in rats. Epilepsia 60(5):e52-e57. doi: 10.1111/epi.14723

Objective: Hippocampal GABA neurons were targeted for selective elimination to determine whether a focal hippocampal GABAergic defect in an otherwise normal brain can initiate cryptogenic temporal lobe epilepsy with hippocampal sclerosis.

Summary: Hippocampal GABAergic dysfunction is epileptogenic and can produce the defining features of cryptogenic temporal lobe epilepsy.

Usage: Intrahippocampal injections of SSP-SAP (0.4 ng/10 nL) were performed using a 0.5-μL Neuros Syringe lowered into four hippocampal sites along both the transverse and longitudinal hippocampal axes bilaterally.

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Read the featured article in Targeting Trends.

Contribution of retrotrapezoid nucleus and carotid bodies to asphyxia‐induced arousal in rats.

Souza G, Stornetta R, Stornetta D, Abbott S, Guyenet P (2019) Contribution of retrotrapezoid nucleus and carotid bodies to asphyxia‐induced arousal in rats. FASEB J 33(1):733.6. Experimental Biology 2019 Meeting Abstracts doi: 10.1096/fasebj.2019.33.1_supplement.733.6

Objective: To determine whether the retrotrapezoid nucleus (RTN) is implicated in CO2-induced arousal.

Summary: The authors confirm the importance of the CBs to hypoxia-induced arousal and demonstrate that arousal to hypercapnia is selectively reduced after RTN lesion.

Usage: RTN was nearly completely destroyed with microinjections of SSP-SAP (2.4 ng).

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Interaction between the retrotrapezoid nucleus and the parafacial respiratory group to regulate active expiration and sympathetic activity in rats

Zoccal DB, Silva JN, Barnett WH, Lemes EV, Falquetto B, Colombari E, Molkov YI, Moreira TS, Takakura AC (2018) Interaction between the retrotrapezoid nucleus and the parafacial respiratory group to regulate active expiration and sympathetic activity in rats. Am J Physiol Lung Cell Mol Physiol 315(5):L891-L909. doi: 10.1152/ajplung.00011.2018

Objective: To investigate the microcircuitry responsible for the distribution of the excitatory signals to the the parafacial respiratory group (pFRG) and the the respiratory central pattern generator (rCPG) in conditions of high CO2.

Summary: The activation of the pFRG late-E neurons during hypercapnia require glutamatergic inputs from the RTN neurons that intrinsically detect changes in CO2/pH.

Usage: Bilateral injections of SSP-SAP (0.6 ng in 100 nL of saline per side).

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Substance P and neurotensin in the limbic system: Their roles in reinforcement and memory consolidation.

Lénárd L, László K, Kertes E, Ollmann T, Péczely L, Kovács A, Kállai V, Zagorácz O, Gálosi R, Karádi Z (2018) Substance P and neurotensin in the limbic system: Their roles in reinforcement and memory consolidation. Neurosci Biobehav Rev 85:1-20. doi: 10.1016/j.neubiorev.2017.09.003

Summary: A specific neurotoxin, SSP-SAP can cause selective ablation of NK1 receptor expressing cells. This lesion in the BLA of rats resulted in anxiogenic effect, while in the CeA had no effect in EPM test.

See: Truitt WA et al. Anxiety-like behavior is modulated by a discrete subpopulation of interneurons in the basolateral amygdala. Neuroscience 160:284-294, 2009.

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Breathing regulation and blood gas homeostasis after near complete lesions of the retrotrapezoid nucleus in adult rats.

Souza GMPR, Kanbar R, Stornetta DS, Abbott SBG, Stornetta RL, Guyenet PG (2018) Breathing regulation and blood gas homeostasis after near complete lesions of the retrotrapezoid nucleus in adult rats. J Physiol 596(13):2521-2545. doi: 10.1113/JP275866

Objective: To test how important the retrotrapezoid nucleus (RTN) is to PCO2 homeostasis and breathing during sleep or wake.

Summary: Near complete RTN destruction in rats virtually eliminates the CRC but HVR persists and sighing and the state-dependence of breathing are unchanged. Under normoxia, RTN lesions cause no change in VE but alveolar ventilation is reduced by at least 21%, probably because of increased physiological dead volume. RTN lesions do not cause sleep apnea during SWS, even under hyperoxia.

Usage: A total of 6 microinjections (120 nl/injection; 3 rostrocaudally aligned injections per side) were made 100-200 μm below the lower edge of the facial motor nucleus 2 mm lateral to the midline. Experimental rats received either 0.6 ng, 1.2 ng, or 2.4 ng of SSP-SAP per injection.

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Toxins in Neurobiology: New tools from old molecules.

Vetter I (2018) Toxins in Neurobiology: New tools from old molecules. Neurosci Lett 679:1-3. doi: 10.1016/j.neulet.2018.05.008

Summary: The selective ablation of neurokinin-1 receptor-expressing neurons by SP-SAP revealed a key role for the preBötzinger complex in the generation of respiratory rhythm. Toxin-mediated neuronal ablation may also find therapeutic applications, such as the treatment of severe refractory pain in terminally ill patients by intrathecal SP-SAP which causes selective loss of neurokinin-1 receptor-expressing neurons in the spinal cord dorsal horn.

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Role of GPCR (mu-opioid)-receptor tyrosine kinase (epidermal growth factor) crosstalk in opioid-induced hyperalgesic priming (type II).

Araldi D, Ferrari LF, Levine JD (2018) Role of GPCR (mu-opioid)-receptor tyrosine kinase (epidermal growth factor) crosstalk in opioid-induced hyperalgesic priming (type II). Pain 159(5):864-875. doi: 10.1097/j.pain.0000000000001155

Objective: To determine the the mechanisms mediating the induction of opioid-induced hyperalgesia and the prolongation of prostaglandinE2-induced hyperalgesia in type II hyperalgesic priming.

Summary: Understanding the mechanisms responsible for the induction of type II hyperalgesic priming, a form of neuroplasticity in the peripheral terminal of the primary afferent nociceptor, may provide useful information for the design of drugs with improved therapeutic profiles to treat neuroplasticity induced by chronic use of opioids.

Usage: SSP-SAP was prepared in saline (5 ng/mL), and 20 mL was injected intrathecally into rats, 14 days before nociceptive tests.

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Modulation of chronic post-thoracotomy pain by NK-1 neurons in the rostral ventromedial medulla is not paralleled by changes spinal MAPKinase activation.

Strichartz G, Khasabov S, Barr T, Wang J, Simone D (2018) Modulation of chronic post-thoracotomy pain by NK-1 neurons in the rostral ventromedial medulla is not paralleled by changes spinal MAPKinase activation. J Pain 19:S14. doi: 10.1016/j.jpain.2017.12.065

Objective: To evaluate SSP-SAP in treatment of tactile hypersensitivity for Chronic Post-Thoractotomy Pain (CPTP).

Summary: SSP-SAP 3 weeks before thoracotomy and rib retraction (TRR) was able to completely prevent CPTP, assayed by tactile hypersensitivity.

Usage: Ablation of Neurokinin-1 receptor (NK-1R)- expressing neurons in the rat rostral ventromedial medulla (RVM), by micro-injection of the specific neurotoxin SSP-SAP.  No effect with treatment of control, Blank-SAP (IT-21).

Related Products: SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

The transient intermediate plexiform layer, a plexiform layer-like structure temporarily existing in the inner nuclear layer in developing rat retina

Park HW, Kim H-L, Park YS, Kim I-B (2018) The transient intermediate plexiform layer, a plexiform layer-like structure temporarily existing in the inner nuclear layer in developing rat retina. Exp Neurobiol 27:28-33. doi: 10.5607/en.2018.27.1.28 Related Products: SSP-SAP (Cat. #IT-11)

CD44 signaling mediates high molecular weight hyaluronan-induced antihyperalgesia.

Ferrari LF, Khomula EV, Araldi D, Levine JD (2018) CD44 signaling mediates high molecular weight hyaluronan-induced antihyperalgesia. J Neurosci 38(2):308-321. doi: 10.1523/JNEUROSCI.2695-17.2017

Objective: To study the role of the cognate hyaluronan receptor, CD44, signaling in anti-hyperalgesia induced by high molecular weight hyaluronan (HMWH).

Summary: These results demonstrate the central role of CD44 signaling in HMWH-induced anti-hyperalgesia, and establish it as a therapeutic target against inflammatory and neuropathic pain.

Usage: Both IB4-SAP and SSP-SAP were diluted in saline to doses previously shown to deplete nonpeptidergic (3.2 mcg/rat for IB4-SAP) and peptidergic (100 ng/rat for SSP-SAP) fibers. The toxins were administered intrathecally, in a volume of 20 mcl, 14 d before intradermal injection of LMWH on the dorsum of the hindpaw. Treatment with either conjugate, or a combination of the two, did not significantly affect mechanical nociceptive threshold.

Related Products: IB4-SAP (Cat. #IT-10), SSP-SAP (Cat. #IT-11)

Local glutamatergic transmission in the RTN/pFRG is critical for active expiration and sympathetic overactivity during hypercapnia

Barnett WH, Molkov YI, Lemes E, Falqueto B, Colombari E, Takakura AT, Moreira TS, Zoccal DB (2017) Local glutamatergic transmission in the RTN/pFRG is critical for active expiration and sympathetic overactivity during hypercapnia. Neuroscience 2017 Abstracts 233.1 / FF22. Society for Neuroscience, Washington, DC.

Summary: The retrotrapezoid nucleus (RTN) contains chemosensitive cells that distribute CO -dependent excitatory drive to the brainstem respiratory network. This drive facilitates the function of the respiratory central pattern generator (CPG), modulates sympathetic activity and determines the emergence of active expiration during hypercapnia via activation of the late expiratory (late-E) oscillator in the parafacial respiratory group (pFRG). However, the microcircuitry responsible for distribution of the chemoreflex signal to the pFRG and the respiratory CPG is not well understood. Previously, we developed a computational model of the brainstem respiratory network, which was subsequently extended to include the central and peripheral chemoreflexes as well as presympathetic circuits. We present here experiments performed on the decerebrated, arterially-perfused in situ rat, aimed to test a key assumption of this model that chemosensitive and late-E neurons in the RTN/pFRG are two distinct populations, and the latter receives local glutamatergic input from the former. The model predicts: (1) suppression of RTN chemosensitive neurons will diminish the changes to the respiratory pattern and the emergence of active expiration associated with hypercapnia; (2) the disruption of local glutamatergic neurotransmission in the RTN will specifically suppress active expiration and the appearance of late-E discharges in the sympathetic motor output. To test prediction (1) we lesioned NK1 -positive chemosensitive neurons of the RTN with microinjections of substance P-saporin (SSP-SAP) conjugate. This suppressed the emergence of late-E activity in abdominal (AbN) and sympathetic nerves, and attenuated the increase in phrenic burst amplitude during hypercapnia. However, SSP-SAP and control animals exhibited late-E AbN activity in response to peripheral chemoreflex activation. Prediction (2) was tested with bilateral microinjections of kynurenic acid (Kyn, 100 mM) in the RTN/pFRG, which suppressed the emergence of late-E AbN activity but not the change in phrenic nerve amplitude during hypercapnia. Our results support the notion that RTN chemosensitive neurons are critical for inspiratory and expiratory reflex responses to hypercapnia. Our findings indicate that activation of late-E neurons in the pFRG during hypercapnia requires glutamatergic inputs from a separate neuronal population in the RTN that intrinsically detects changes in CO . During peripheral chemoreflex stimulation, pFRG late-E neurons are activated via excitatory pathways bypassing the RTN central chemoreceptors. We recapitulate these results in our computational model.

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Chemosensitive Phox2b-expressing neurons are crucial for hypercapnic ventilatory response in the nucleus tractus solitarius.

Fu C, Xue J, Wang R, Chen J, Ma L, Liu Y, Wang X, Guo F, Zhang Y, Zhang X, Wang S (2017) Chemosensitive Phox2b-expressing neurons are crucial for hypercapnic ventilatory response in the nucleus tractus solitarius. J Physiol 595:4973-4989.. doi: 10.1113/JP274437

Objective: To investigate whether paired-like homeobox 2b  (Phox2b)-expressing NTS neurons are recruited in hypercapnic ventilatory response (HCVR)  and whether these neurons exhibit intrinsic chemosensitivity.

Summary: Respiratory deficits caused by injection of SSP-SAP into the NTS are attributable to proportional lesions of CO2/H+-sensitive Phox2b-expressing neurons.

Usage: Two protocols were applied for SSP-SAP injections. In immunostaining experiments, to determine how many Phox2b-containing cells were destroyed, a total volume of 100 nl of PBS containing 6 ng of SSP-SAP (3 ng in 50 nl; 2 injections) was injected into one side of the NTS and the contralateral NTS was used as a control (no injection).  For in vivo experiments, to determine whether loss of Phox2b cells led to impaired HCVR, bilateral injections with a total volume of 200 nl of PBS containing 6 ng (1.5 ng in 50 nl per injection; 2 injections per side) or 12 ng (3 ng in 50 nl per injection; two injections per side) of toxin into NTS.  Breathing was studied in conscious, freely moving mice treated with SSP-SAP and Blank-SAP.

Related Products: SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

Current and future issues in the development of spinal agents for the management of pain.

Yaksh T, Fisher C, Hockman T, Wiese A (2017) Current and future issues in the development of spinal agents for the management of pain. Curr Neuropharmacol 15:232-259.. doi: 10.2174/1570159x14666160307145542

Summary: Although conscious pain experience is driven by signals mediated supraspinally, the more high intensity pain generated by strong stimuli, tissue injury, and nerve injury is encoded at the spinal dorsal horn level. The control of pain signals at the spinal dorsal horn level is a tempting target for targeted pain therapy. This review discusses the potential targets for pain therapeutics in the spinal dorsal horn, and some of the spinal agents used to modulate pain transmission through that location. The use of SSP-SAP (Cat. #IT-11) is mentioned as a neurokinin-1 targeted molecule that can block some pain transmission.

Related Products: SSP-SAP (Cat. #IT-11)

Gi-protein-coupled 5-HT1B/D receptor agonist sumatriptan induces type I hyperalgesic priming.

Araldi D, Ferrari L, Levine J (2016) Gi-protein-coupled 5-HT1B/D receptor agonist sumatriptan induces type I hyperalgesic priming. Pain 157:1773-1782. doi: 10.1097/j.pain.0000000000000581

Summary: The present study explored the possibility that, like MOR and A1-adenosine receptor agonists, triptans would also induce type II hyperalgesic priming. In addition, they explored the 5-HT receptor subtypes at which triptans act (5-HT1B, 5-HT1D and 5-HT7) to induce priming. They report that while sumatriptan, a prototypical 5-HT1B/D receptor agonist induces hyperalgesic priming, this priming meets the criteria for type I rather than type II priming. Isolectin B4 (IB4)-saporin (Cat. #IT-10), was diluted in saline, and a dose of 3.2 μg, in a volume of 20 μL was administered intrathecally to rats. The neurotoxin [Sar9,Met(O2) 11]-substance P-saporin (SSP-Saporin, Cat. #IT-11) was diluted in saline, and a dose of 100 ng, in a volume of 20 μL was administered intrathecally. In a model of pain chronification, sumatriptan induces both mechanical hyperalgesia at the site of injection and type I hyperalgesic priming, in nociceptors innervating the cutaneous injection site.

Related Products: IB4-SAP (Cat. #IT-10), SSP-SAP (Cat. #IT-11)

Functional characterization of a mouse model for central post-stroke pain.

Gritsch S, Bali K, Kuner R, Vardeh D (2016) Functional characterization of a mouse model for central post-stroke pain. Mol Pain 12:1744806916629049. doi: 10.1177/1744806916629049

Summary: While clinical evidence has pointed toward central pain pathway dysfunction in central post-stroke pain (CPSP), the underlying mechanisms have not been defined. In this work the authors created a mouse model of CPSP through lesions of the thalamic ventral posterolateral nucleus. In order to examine the role of neurokinin-1 receptor-expressing (NK1R) neurons in lamina I/III of the spinal cord in the development and maintenance of CPSP the authors administered 1 μmol intrathecal injections of SSP-SAP (Cat. #IT-11). Saporin (Cat. #PR-01) was used as a control. While the NK1R+ neurons in the spinal cord were not involved in establishing CPSP, the data indicate that sensory changes in the mice are comparable to those observed in human patients with CPSP.

Related Products: SSP-SAP (Cat. #IT-11), Saporin (Cat. #PR-01)

Pain from intra-articular NGF or joint injury in the rat requires contributions from peptidergic joint afferents.

Kras J, Weisshaar C, Pall P, Winkelstein B (2015) Pain from intra-articular NGF or joint injury in the rat requires contributions from peptidergic joint afferents. Neurosci Lett 604:193-198. doi: 10.1016/j.neulet.2015.07.043

Summary: Both peptidergic and non-peptidergic neurons innervate the facet joint, which is the source of pain in a majority of neck trauma. In this work the authors examined these subpopulations of neurons to determine the contribution of each in facet joint pain. 100 ng of SSP-SAP (Cat. #IT-11) was injected into bilateral C6/C7 facet joints of rats. Alternatively, rats received 5 μg of rIB4-SAP (Cat. #IT-10) via the same method. Saporin (Cat. #PR-01) was used as control. SSP-SAP, but not rIB4-SAP was able to prevent NGF-induced mechanical and thermal hypersensitivity. SSP-SAP administration also prevented behavioral hypersensitivity and NGF upregulation in the dorsal root ganglion after facet joint distraction. The data indicate that interference with peptidergic signaling within the facet joint may be a treatment for pain originating in that location.

Related Products: SSP-SAP (Cat. #IT-11), IB4-SAP (Cat. #IT-10), Saporin (Cat. #PR-01)

A central role for spinal dorsal horn neurons that express neurokinin-1 receptors in chronic itch.

Akiyama T, Nguyen T, Curtis E, Nishida K, Devireddy J, Delahanty J, Carstens M, Carstens E (2015) A central role for spinal dorsal horn neurons that express neurokinin-1 receptors in chronic itch. Pain 156:1240-1246. doi: 10.1097/j.pain.0000000000000172

Summary: Chronic itch is caused by increased sensitivity of itch-signaling pathways. It can be generated by normally itchy stimuli (hyperknesis) and by normally non-itchy light touch (alloknesis). The authors used an ovalbumin-induced atopic dermatitis model to study chronic itch in mice. The mice received 400-ng intrathecal injections of Bombesin-SAP (Cat. #IT-40), SSP-SAP (Cat. #IT-11), or the control Blank-SAP (Cat. #IT-21). While Bombesin-SAP significantly attenuated hyperknesis, it had no effect on spontaneous scratching or alloknesis. SSP-SAP reduced all behavioral signs of chronic itch.

Related Products: Bombesin-SAP (Cat. #IT-40), SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

NK1-receptor-expressing paraventricular nucleus neurones modulate daily variation in heart rate and stress-induced changes in heart rate variability.

Feetham C, Barrett-Jolley R (2014) NK1-receptor-expressing paraventricular nucleus neurones modulate daily variation in heart rate and stress-induced changes in heart rate variability. Physiol Rep 2:e12207. doi: 10.14814/phy2.12207

Summary: Neurons in the paraventricular nucleus (PVN) project to the medulla and spinal cord, regulating heart rate and blood pressure. Although the activity of these neurons becomes elevated during heart failure, their role in overall cardiovascular control is unclear. The authors lesioned the PVN of rats with 2 ng injections of SSP-SAP (Cat. #IT-11). Heart rate variability during the experiment was measured using a high/low frequency ratio in response to psychological stress. The variability response of lesioned rats was lower than that of controls, and a shift in daily heart rate variation was seen as well. The authors conclude that neurokinin-1 expressing neurons in the PVN couple the cardiovascular system to the daily heart rate as well as the sympathetic response to psychological stress.

Related Products: SSP-SAP (Cat. #IT-11)

Expression of different neurokinin-1 receptor (NK1R) isoforms in glioblastoma multiforme: potential implications for targeted therapy.

Cordier D, Gerber A, Kluba C, Bauman A, Hutter G, Mindt TL, Mariani L (2014) Expression of different neurokinin-1 receptor (NK1R) isoforms in glioblastoma multiforme: potential implications for targeted therapy. Cancer Biother Radiopharm 29(5):221-226. doi: 10.1089/cbr.2013.1588

Summary: The neurokinin-1 receptor (NK1r) has been found to be consistently over-expressed in gliomas, making it a potential target for therapeutic strategies. However, treatments with therapies utilizing substance P (SP), the ligand for the NK1r, have at best yielded uneven results. In this work the authors investigated factors that may predict the response to therapies directed at NK1r gliomas. SSP-SAP (Cat. #IT-11) was used at a concentration of 1 nM in cytotoxicity assays on several different glioma cell lines. Using this and other data it was shown that only the cell line with the most full-length NK1r RNA transcripts displayed high levels of binding, internalization, and cell killing necessary for NK1r to be a therapeutic target using SP.

Related Products: SSP-SAP (Cat. #IT-11)

Ablating spinal NK1-bearing neurons eliminates the development of pain and reduces spinal neuronal hyperexcitability and inflammation from mechanical joint injury in the rat.

Weisshaar CL, Winkelstein BA (2014) Ablating spinal NK1-bearing neurons eliminates the development of pain and reduces spinal neuronal hyperexcitability and inflammation from mechanical joint injury in the rat. J Pain 15(4):378-386. doi: 10.1016/j.jpain.2013.12.003

Summary: A high percentage of chronic neck pain involves the facet joint. Although the facet joint is innvervated by peptide-responsive nociceptive afferents, the role of these cells in the development and modulation of nociceptive signaling remains unclear. Using a previously developed rat model of facet joint injury, the authors examined the role of neurokinin-1 receptor-expressing spinal cells in this pathway. Rats received 100 ng SSP-SAP (Cat. #IT-11) via lumbar puncture. Blank-SAP (Cat. #IT-21) was used as a control. The results demonstrate that spinal NK1r-expressing cells are essential for nociception and inflammation due to a mechanical joint injury.

Related Products: SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

Phox2b-expressing retrotrapezoid neurons and the integration of central and peripheral chemosensory control of breathing in conscious rats.

Takakura AC, Barna BF, Cruz JC, Colombari E, Moreira TS (2014) Phox2b-expressing retrotrapezoid neurons and the integration of central and peripheral chemosensory control of breathing in conscious rats. Exp Physiol 99(3):571-585. doi: 10.1113/expphysiol.2013.076752

Summary: Previous work has shown that lesions to the retrotrapezoid nucleus (RTN) have at least a modest effect on breathing, but it is unclear whether those lesions affected the entire nucleus or were incomplete. The authors used bilateral lesions of the RTN with 0.3 to 1.2 ng total of SSP-SAP (Cat. #IT-11) to eliminate neurokinin-1 receptor-expressing neurons; these are also Phox2b+TH- neurons. The results indicate that loss of Phox2b(+)TH(-) neurons may cause deficits seen after RTN lesion, and help define the ways in which these cells are involved in controlling central and peripheral chemoreflexes.

Related Products: SSP-SAP (Cat. #IT-11)

Loss of neurons in rostral ventromedial medulla that express neurokinin-1 receptors decreases the development of hyperalgesia.

Khasabov SG, Simone DA (2013) Loss of neurons in rostral ventromedial medulla that express neurokinin-1 receptors decreases the development of hyperalgesia. Neuroscience 250C:151-165. doi: 10.1016/j.neuroscience.2013.06.057

Summary: Previous data has indicated that neurokinin-1 receptors are located on ON cells in the rostral ventromedial medulla (RVM). ON cells are considered pronociceptive because noxious stimulation is stimulatory. In this work the authors eliminated ON cells using 0.3-μl injections of 1 μM SSP-SAP (Cat. #IT-11) into the left and right side of the RVM. Blank-SAP (Cat. #IT-21) was used as a control. SSP-SAP treatment did not change mechanical or heat withdrawal responses, or change morphine-induced analgesia. A significant reduction in the duration of nocifensive behaviors induced by various hyperalgesic stimulators indicated that these neurons are involved in pain facilitation rather than modulation.

Related Products: SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

Evaluating the role of neuronal nitric oxide synthase-containing striatal interneurons in methamphetamine-induced dopamine neurotoxicity.

Fricks-Gleason AN, Keefe KA (2013) Evaluating the role of neuronal nitric oxide synthase-containing striatal interneurons in methamphetamine-induced dopamine neurotoxicity. Neurotox Res 24(2):288-297. doi: 10.1007/s12640-013-9391-6

Summary: Using the fact that neuronal nitric oxide synthase (nNOS)-containing neurons in the striatum express the substance P receptor, the authors injected four locations in the striatum with 3 ng each of SSP-SAP (Cat. #IT-11). Blank-SAP (Cat. #IT-21) was used as a control. Although there was a significant loss of nNOS-containing neurons, the lesions did not attenuate NO production.

Related Products: SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

Sudden death following selective neuronal lesions in the rat nucleus tractus solitarii.

Talman WT, Lin LH (2013) Sudden death following selective neuronal lesions in the rat nucleus tractus solitarii. Auton Neurosci 175(1-2):9-16. doi: 10.1016/j.autneu.2012.11.008

Summary: The nucleus tracts solitarii (NTS) is the terminus of cardiovascular reflex nerves. Early work in this area sought to identify transmitters involved in the control of this region. The authors used SSP-SAP (Cat. #IT-11) and anti-DBH-SAP (Cat. #IT-03) to examine the role of NK-1r-expressing neurons and catecholaminergic neurons in baroreflex control in the NTS. Either 3 ng of SSP-SAP or 42 ng of anti-DBH-SAP was injected into the NTS of rats and baroreflex function was compared 7 days later. Although each toxin had a specific effect in terms of the types of cells eliminated, both toxins initiated a disturbance of the central baroreflex control that led to the death of some animals.

Related Products: SSP-SAP (Cat. #IT-11), Anti-DBH-SAP (Cat. #IT-03)

Evidence that focal hippocampal interneuron loss disrupts theta- and gamma- band activity.

Rossi CA, Lehmkuhle MJ, Dudek FE (2012) Evidence that focal hippocampal interneuron loss disrupts theta- and gamma- band activity. Neuroscience 2012 Abstracts 918.06. Society for Neuroscience, New Orleans, LA.

Summary: Hippocampal theta (6-12 Hz) and gamma (40-100 Hz) activity are oscillatory local field potentials (LFPs) that are thought to play a critical role in the encoding and storage of new information. GABA-ergic interneurons are hypothetically involved in the generation and pacing of these oscillatory patterns of activity. The current study aimed to directly test the hypothesis that interneurons are responsible for local gamma and theta generation in the dorsal CA1 region of the hippocampus in mice. Selective focal interneuron lesions were made by intrahippocampal injection of the targeted neurotoxin SSP-Saporin into dorsal CA1 in the hippocampus of GAD67-GFP transgenic mice. Chronic recording electrodes were also implanted in the lesion area. LFPs were monitored continuously, along with video recordings of the subjects, for a period of several weeks. LFP recordings were analyzed over 24-hour periods for the occurrence of theta- and gamma-band activity. Analysis of LFP data revealed attenuation of both local theta and gamma activity in SSP-saporin-injected animals compared to controls. These results suggest a direct role of GABA-ergic interneurons in the generation of local rhythmic activity in these two frequency bands, and, by extension, an important role in learning and memory processes.

Related Products: SSP-SAP (Cat. #IT-11)

Selective damage to glia in the nucleus tractus solitarii attenuates cardiovascular reflexes.

Talman WT, Jones S, Nitschke Dragon D, Lin L-H (2012) Selective damage to glia in the nucleus tractus solitarii attenuates cardiovascular reflexes. Neuroscience 2012 Abstracts 524.05. Society for Neuroscience, New Orleans, LA.

Summary: Lesions of the nucleus tractus solitarii (NTS) are known to attenuate or abolish cardiovascular reflex responses. We have previously reported that lesions produced by saporin (SAP) conjugates and focused on neurons that express the neurokinin-1 (NK1) receptor or on other neurons that express both tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH), also attenuate baroreflex function in rats. We found that lesions of both types of neurons also led to loss of glia that stained with glial fibrillary acidic protein (GFAP). Further, we found that injection of SAP alone into the NTS led to loss of GFAP staining while leaving neurons in the region unaffected. Because both of the lesions directed at neurons were made by a toxic conjugate containing SAP, we sought to determine if SAP alone produced changes in cardiovascular reflex function. We found that injection of SAP (3 ng in 100 nl) into the NTS led to loss of the glial marker GFAP as well as connexin 43 (Cx43) immunofluorescent labeling in the NTS but did not affect the neuronal markers NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), neuronal nitric oxide synthase (nNOS), TH, DBH, vesicular glutamate transporters (VGluTs), choline acetyl transferase (ChAT), NK1, and protein gene product 9.5 (PGP 9.5). In animals treated with bilateral injections of SAP into the NTS, reflex responses were decreased during testing of the baroreflex, the chemoreflex, or the von Bezold Jarisch reflex. Comparable decreases in baroreflex responses were seen in animals treated with SAP alone when compared with other animals treated with SAP conjugates that targeted and concentrated damage to TH/DBH neurons or NK1 neurons in NTS. In contrast, when TH/DBH neurons were targeted by the toxin 6-hydroxydopamine (6-OHDA) lability of arterial pressure did not occur as it did in the other SAP and SAP conjugate studies and reflex responses to the activation of the baroreflex, the chemoreflex, and the von Bezold Jarisch reflex did not differ from control. Furthermore, injections containing SAP or a SAP conjugate, but not those containing 6-OHDA, led to lability of arterial pressure as well as cardiac arrhythmias and cardiac myocytolysis. Our studies cannot exclude a physiological effect of SAP on neurons nor can it exclude an indirect effect of glial damage on NTS neurons. However, the similarity of responses when glia seem to have been targeted alone in contrast to those responses when select neuronal types seem to have been targeted suggests that each of the cardiovascular reflexes relies on intact glia in the NTS for full reflex expression.

Related Products: Anti-DBH-SAP (Cat. #IT-03), SSP-SAP (Cat. #IT-11)

Poster: Evaluating the role of neuronal nitric oxide synthase-containing striatal interneurons in methamphetamine-induced dopamine neurotoxicity.

Fricks-Gleason AN, Keefe KA (2012) Poster: Evaluating the role of neuronal nitric oxide synthase-containing striatal interneurons in methamphetamine-induced dopamine neurotoxicity. Neuroscience 2012 Abstracts 360.06. Society for Neuroscience, New Orleans, LA.

Summary: It is well established that exposure to multiple high doses of methamphetamine (METH) produces damage to central monoamine systems. A number of factors, including the production of nitric oxide (NO), have been implicated in this neurotoxicity. While it is relatively clear that NO contributes to METH-induced neurotoxicity to the dopamine (DA) nerve terminal, the source of this NO has not been clearly delineated. There is considerable evidence suggesting that the generation of NO arises a consequence of the activation of neuronal nitric oxide synthase (nNOS). In striatum, nNOS is located post-synaptic to the DA nerve terminal in a subpopulation of striatal interneurons. Thus, we have hypothesized that DA-mediated activation of the nNOS-containing striatal interneurons is necessary for METH-induced neurotoxicity. These interneurons, along with the cholinergic neurons of striatum, selectively express the neurokinin-1 (NK-1) receptor, which is activated by the neuropeptide Substance P. Consequently, toxins targeted to NK-1 receptor-containing neurons can be used to lesion this population of striatal interneurons. One such toxin, a conjugate of Substance P to the ribosome inactivating protein saporin (SSP-SAP), has been shown to be effective in selectively destroying neurons expressing the NK-1 receptor in striatum. Therefore, using targeted deletion of the nNOS-containing interneurons via SSP-SAP, we examined the extent to which impairing post-synaptic production of NO attenuates METH-induced neurotoxicity. The SSP-SAP lesions resulted in a significant and selective loss of nNOS-containing interneurons throughout the striatum, although it was not possible to completely eliminate all of the neurons. Surprisingly, however, this marked deletion of nNOS-containing interneurons did not confer resistance to METH-induced DA neurotoxicity, even in areas completely devoid of nNOS-positive cell bodies and histochemical detection of NOS activity with NADPH diaphorase histochemical staining. Furthermore, these lesions did not attenuate NO production, as assessed via nitrotyrosine immunohistochemistry, even in areas devoid of nNOS. Taken together, these data suggest that nNOS-containing interneurons either are not necessary for METH-induced DA neurotoxicity, leaving open the potential contribution of other sources of NO, such as endothelial NOS (eNOS), or produce NO/RNS that can diffuse extensively through striatal tissue and thereby still mediate the neurotoxicity.

Related Products: SSP-SAP (Cat. #IT-11)

A nociceptive signaling role for neuromedin B.

Mishra SK, Holzman S, Hoon MA (2012) A nociceptive signaling role for neuromedin B. J Neurosci 32(25):8686-8695. doi: 10.1523/JNEUROSCI.1533-12.2012

Summary: Previous work suggests that neuromedin B (NMB) is involved in nociception. Direct injection of the peptide causes nociceptive sensitization, while NMB antagonists attenuate sensitization in reponse to nerve stimulation with mustard oil. Specific subsets of dorsal horn interneurons were eliminated by administering either 10 μg of the custom conjugate neuromedin B-SAP, 0.13 μg of SSP-SAP (Cat. #IT-11), or 1.3 μg of bombesin-SAP (Cat. #IT-40). Blank-SAP (Cat. #IT-21) was used as a control. The data indicate that NMB may be involved in the perception of thermal sensation, but not mechanical or pruritic sensation.

Related Products: NMB-SAP (Cat. #IT-70), SSP-SAP (Cat. #IT-11), Bombesin-SAP (Cat. #IT-40), Blank-SAP (Cat. #IT-21)

C1 neurons excite locus coeruleus and A5 noradrenergic neurons along with sympathetic outflow in rats.

Abbott SB, Kanbar R, Bochorishvili G, Coates MB, Stornetta RL, Guyenet PG (2012) C1 neurons excite locus coeruleus and A5 noradrenergic neurons along with sympathetic outflow in rats. J Physiol 590(12):2897-2915. doi: 10.1113/jphysiol.2012.232157

Summary: C1 neurons are known to activate sympathetic tone and stimulate the hypothalamic-pituitary-adrenal axis. C1 activation is also reported to inhibit locus coeruleus (LC) neurons. Rats received 0.6 ng of SSP-SAP (Cat. #IT-11) injected under the caudal edge of the facial motor nucleus to destroy the retrotrapezoid nucleus, increasing the proportion of C1 ChR2-expressing neurons. Stimulation of C1 neurons resulted in activation of noradrenergic neurons that are involved in hypoxia and hypotension.

Related Products: SSP-SAP (Cat. #IT-11)

Collateral damage and compensatory changes after injection of a toxin targeting neurons with the neurokinin-1 receptor in the nucleus tractus solitarii of rat.

Lin LH, Nitschke Dragon D, Talman WT (2012) Collateral damage and compensatory changes after injection of a toxin targeting neurons with the neurokinin-1 receptor in the nucleus tractus solitarii of rat. J Chem Neuroanat 43(2):141-148. doi: 10.1016/j.jchemneu.2012.02.001

Summary: Loss of substance P receptor (SPR)-expressing neurons in the nucleus tractus solitarii (NTS) results in reduced baroreflex strength and unstable arterial pressure. The neuronal population expressing the SPR also expresses several other types of receptors – such as N-methyl-D-aspartate (NMDA), and aalpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic (AMPA) receptors. The authors administered 9 ng of SSP-SAP (Cat. #IT-11) into the NTS and looked for differences in the expression of NMDA and AMPA receptors, as well as several different transporters and neuronal markers. he data suggest that the pathological effects of SSP-SAP lesions result from disruption of other neurotransmission systems than those using the SPR.

Related Products: SSP-SAP (Cat. #IT-11)

Carrageenan induced phosphorylation of Akt is dependent on neurokinin-1 expressing neurons in the superficial dorsal horn.

Choi JI, Koehrn FJ, Sorkin LS (2012) Carrageenan induced phosphorylation of Akt is dependent on neurokinin-1 expressing neurons in the superficial dorsal horn. Mol Pain 8(1):4. doi: 10.1186/1744-8069-8-4

Summary: In this work the authors administered 100 ng SSP-SAP (Cat. #IT-11) into the intrathecal space of rats (saporin, Cat. #PR-01, was used as a control). Lesioned animals displayed decreased carrageenan-induced mechanical allodynia, and carrageenan-induced phosphorylation of Akt was blocked throughout the spinal cord gray matter. Anti-NK-1 (Cat. #AB-N33AP) was used for immunohistochemistry.

Related Products: SSP-SAP (Cat. #IT-11), Saporin (Cat. #PR-01), NK-1 Receptor Rabbit Polyclonal, affinity-purified (Cat. #AB-N33AP)

A pivotal role of lumbar spinothalamic cells in the regulation of ejaculation via intraspinal connections.

Staudt MD, Truitt WA, McKenna KE, de Oliveira CV, Lehman MN, Coolen LM (2012) A pivotal role of lumbar spinothalamic cells in the regulation of ejaculation via intraspinal connections. J Sex Med 9(9):2256-2265. doi: 10.1111/j.1743-6109.2011.02574.x

Summary: The authors examined the hypothesis that specific lumbar spinothalamic (LSt) cells control ejaculation through intraspinal connections. Rats received six bilateral injections of SSP-SAP (Cat. #IT-11) into the spinal cord, 48 ng in total. Saporin (Cat. #PR-01) was used as a control. It was found that while erectile function and emission were not affected, the usual rhythmic contractions of the bulbocaveronosus muscle during ejaculation were prevented.

Related Products: SSP-SAP (Cat. #IT-11), Saporin (Cat. #PR-01)

Read the featured article in Targeting Trends.

Cardiovascular dysfunction and cardiac injury result from selective glial damage in the nucleus tractus solitarii

Talman WT, Nitschke Dragon D, Jones S, Moore SA, Lin L-H (2011) Cardiovascular dysfunction and cardiac injury result from selective glial damage in the nucleus tractus solitarii. Neuroscience 2011 Abstracts 664.14. Society for Neuroscience, Washington, DC.

Summary: In man, extensive CNS dysfunction as may occur after subarachnoid hemorrhage may lead to cardiac damage and cardiac arrhythmias. We have shown that highly selective and restricted lesions of the nucleus tractus solitarii (NTS) may lead to similar cardiac and cardiovascular compromise. For example, using conjugates including the cytotoxin saporin (SAP) to selectively damage NTS neurons that express NK1 receptors or those that express tyrosine hydroxylase (TH) leads to cardiac dysfunction and associated lability of arterial pressure. In continuing efforts to better characterize cellular changes produced by introducing into the NTS conjugates containing SAP, we have studied the effect of anti-dopamine-beta-hydroxylase (anti-DBH)-SAP, stabilized substance P (SSP)-SAP, SAP (unconjugated), blank-SAP (non-targeted peptide conjugate), IgG-SAP (non-targeted immunoglobulin conjugate), and 6-hydoxydopamine (6-OHDA) as a control without SAP injected into NTS. We assessed effects of the injected agents both on cellular markers [NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), gamma-aminobutyric acid (GABA) receptor type a and b, neuronal nitirc oxide synthase (nNOS), TH, vesicular glutamate transporters (VGluTs), choline acetyl transferase (ChAT), glial fibrillary acidic protein (GFAP), connexin 43 (Cx43), DBH and protein gene product 9.5 (PGP 9.5)] and on cardiovascular and cardiac function. We have found that each compound containing SAP (including blank-SAP, IgG-SAP, unconjugated SAP) led to loss of GFAP and Cx43 immunofluorescent labeling in the NTS as well as lability of arterial pressure, cardiac arrhythmias, and cardiac myocytolysis. Those outcomes occurred despite neuronal specificity for each of the SAP conjugates. For example, anti-DBH-SAP led to a decrease in TH and DBH staining as well as a profound loss in GFAP and Cx43. In contrast, SSP-SAP led to loss of NK1 as well as GFAP, Cx43, and glutamate receptor markers but did not lead to loss of DBH or GABA. SSP-SAP also caused a loss in PGP9.5 which was not observed in all other agents. SAP and blank-SAP, on the other hand, led to loss of GFAP and Cx43 while 6-OHDA led to loss of TH and DBH, increased GFAP and decreased Cx-43. We are still investigating the effects of 6-OHDA on lability of arterial pressure and cardiac events but preliminary data suggest that, in doses used, it led to loss of TH and DBH but did not lead to either lability or cardiac events that were seen with each of the conjugates containing an SAP moiety. This study suggests that glial dysfunction may alone interefere with cardiovascular control through the NTS and may lead to cardiac damage and cardiovascular dysfunction.

Related Products: Anti-DBH-SAP (Cat. #IT-03), SSP-SAP (Cat. #IT-11), Mouse IgG-SAP (Cat. #IT-18), Blank-SAP (Cat. #IT-21), Saporin (Cat. #PR-01)

Evidence that focal interneuron lesions in the hippocampus may lead to a model of epileptogenesis in the mouse.

Rossi CA, Lehmkuhle MJ, Dudek FE (2011) Evidence that focal interneuron lesions in the hippocampus may lead to a model of epileptogenesis in the mouse. Neuroscience 2011 Abstracts 249.09. Society for Neuroscience, Washington, DC.

Summary: A selective loss of part of the overall population of GABAergic interneurons is a seminal component of many forms of human epilepsy, and is manifest in many animal models of acquired epilepsy, including those based on chemoconvulsant-induced status epilepticus. The current study specifically tests the hypothesis that partial interneuron loss in the dorsal CA1 area of the hippocampus induces epileptiform activity, and the subsequent hypothesis that interictal-like spikes and seizures progressively worsen during the following weeks and months. Focal interneuron lesions were made by intra-hippocampal injection of SSP-Saporin into dorsal CA1 in the hippocampus of GAD67-GFP transgenic mice. Chronic recording electrodes were implanted at the injection site, and local field potentials (LFPs) were monitored continuously during video recording for several weeks. LFP recordings were analyzed for the occurrence of inter-ictal-like paroxysmal events (hippocampal sharp waves of 50-100 msec), and frank seizures. Although interneuron lesions alone were seen to generate inter-ictal-like activity within several days following surgery, full-blown seizure activity was not observed until several weeks later. The current data suggest that disruption of the local GABAergic interneuron population may be a key event that triggers alteration of neural networks in the hippocampus, leading to paroxysmal events and ultimately seizures. The delay in onset suggests other factors besides interneuron loss play a role in the generation of seizures and the development of epilepsy. Thus, loss of local inhibition may be a necessary, but not sufficient condition for epileptogenesis.

Related Products: SSP-SAP (Cat. #IT-11)

Contribution of afferent pathways to nerve injury-induced spontaneous pain and evoked hypersensitivity.

King T, Qu C, Okun A, Mercado R, Ren J, Brion T, Lai J, Porreca F (2011) Contribution of afferent pathways to nerve injury-induced spontaneous pain and evoked hypersensitivity. Pain 152(9):1997-2005. doi: 10.1016/j.pain.2011.04.020

Summary: Whether exaggerated pain response to a normally innocuous tactile stimulus should be defined as allodynia has been debated. Through the use of several techniques, one of which was intrathecal injection of SSP-SAP (Cat. #IT-11, 16.5 pg), the authors examined which pathways were utilized in this type of pain. Blank-SAP (Cat. #IT-21) was used as a control. The data indicate that tactile stimulation may reflect a different pain state than allodynia.

Related Products: SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

Read the featured article in Targeting Trends.

Ventilatory effects of substance P-saporin lesions in the nucleus tractus solitarii of chronically hypoxic rats.

Wilkinson KA, Fu Z, Powell FL (2011) Ventilatory effects of substance P-saporin lesions in the nucleus tractus solitarii of chronically hypoxic rats. Am J Physiol Regul Integr Comp Physiol 301(2):R343-R350. doi: 10.1152/ajpregu.00375.2010

Summary: Interaction of the multiple brainstem areas that have been established as CO2-sensitive is not well understood. In order to investigate chemoreceptor roles in the nucleus tractus solitarii (NTS) the authors injected 2.6 ng of SP-SAP (alternative: SSP-SAP; Cat. #IT-11) into the caudal NTS of rats. Blank-SAP (Cat. #IT-21) was used as a control. The results indicate that neurokinin-1 receptor-expressing cells in the NTS contribute to plasticity during chronic hypoxia.

Related Products: SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

Evidence that focal hippocampal interneuron loss disrupts theta-rhythm activity in dorsal CA1

Rossi CA, Lehmkhule MJ, Kesner RP, Dudek FE (2010) Evidence that focal hippocampal interneuron loss disrupts theta-rhythm activity in dorsal CA1. Neuroscience 2010 Abstracts 811.1/LLL64. Society for Neuroscience, San Diego, CA.

Summary: Hippocampal theta activity (6-12 Hz) is an oscillatory local field potential that is thought to play a critical role in the encoding and storage of new information. As a hypothetical mechanism for theta rhythm generation, interneurons have been proposed to appropriately time the GABAergic inhibition of principal cells, as a means of organizing the theta pattern; however, little experimental work has been done to test this hypothesis directly. The current study aims to test in a relatively direct manner the hypothesis that interneurons synchronize the activity of pyramidal cells into theta-band oscillations. In the current study, SSP-saporin (a selective interneuron-targeting neurotoxic lesioning agent) was infused into six sites located in dorsal CA1 in order to create an interneuron-only lesion confined to that area. Animals were also implanted with chronic field potential recording electrodes aimed at areas CA1, CA3, and dentate gyrus. All animals were then monitored, using video and EEG recordings, 24 h per day for the next 7 to 10 days. In addition, EEG was recorded while animals were allowed to explore a novel open field for 30 min in order to create a situation where theta rhythm activity is highly likely to occur. Local field potentials from animals that received SSP-Saporin injections into the dorsal CA1 area revealed attenuation of theta rhythm activity in the lesioned area. Recordings from controls, however, showed a robust peak of activity in the theta frequency band, similar to what has been traditionally described in the hippocampus of naive rats. Together, these results suggest that local elimination of interneurons disrupts local theta rhythm without induction of seizure activity. These experiments provide evidence concerning the possible organizational role of GABAergic interneurons in theta rhythm, an important component of normal hippocampal function.

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NK-1 Receptors in the RVM: Involvement in hyperalgesia produced by naloxone but not in morphine analgesia

Khasabov SG, Fliss PM, Rao AS, Simone DA (2010) NK-1 Receptors in the RVM: Involvement in hyperalgesia produced by naloxone but not in morphine analgesia. Neuroscience 2010 Abstracts 678.15/QQ2. Society for Neuroscience, San Diego, CA.

Summary: The rostral ventromedial medulla (RVM) is a crucial supraspinal site for opioid analgesia. Descending modulation of nociceptive transmission by the RVM can be antinociceptive, which is associated with increased activity of OFF cells, or pronociceptive, which is related to activation of ON cells. Analgesia produced by opioids at the RVM level is due to direct inhibition of ON cells and the indirect increase in discharge of OFF cells. A subpopulation of neurons in the RVM (approximately 7%) express neurokinin-1 receptors (NK-1R), which are receptors for substance P (SP). We have shown that NK-1R in the RVM are located primarily on ON cells and contribute to descending facilitation of nociception. We suggest that elimination of NK-1R expressing neurons by the specific saporin toxin conjugate SSP-SAP, will reduce the number of ON cells and thereby decrease descending facilitation without affecting antinociception associated with activity of OFF cells. We therefore determined the contribution of NK-1R expressing neurons in the RVM to changes in nocifensive behaviors produced by morphine or the opioid receptor antagonist naloxone by eliminating NK-1R expressing neurons. Adult male Sprague Dawley rats were pretreated with injection of SSP-SAP (1 µM/0.5 µl) or inactive toxin into the RVM. Ablation of NK-1R possessing neurons was determined histologically and did not alter tale flick or paw withdrawal latencies to heat for up to 4 weeks following treatment, indicating that these neurons do not modulate acute nociception. Morphine (30 µg/0.5 µl) injected into the RVM of control rats or rats pretreated with SSP-SAP increased tail flick latencies approximately 133.5 ± 20.8% and 140.4 ± 8.3%, respectively. The increase in paw withdrawal latency following morphine was also similar between groups. However, injection of naloxone (50 µg/0.5 µl) in control rats decreased tail flick latencies for 90 min with a maximal reduction of 32.2 ± 4.1%, whereas in rats treated with SSP-SAP latencies decreased by 17.8 ± 4.9% and for only 30 min. A similar pattern of effects was found on paw withdrawal latencies to heat. These data support the notion that ON cells possess NK-1Rs and contribute to facilitation of nociceptive transmission.

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The role of GABA-ergic interneurons in CA1 and dentate gyrus for sequence learning

Weeden CS, Morris AM, Rossi CA, Roberts JM, Kesner RP (2010) The role of GABA-ergic interneurons in CA1 and dentate gyrus for sequence learning. Neuroscience 2010 Abstracts 99.26/KKK12. Society for Neuroscience, San Diego, CA.

Summary: The hippocampus (HPP) plays an important role in temporal and spatial memory. Lesion investigations of the CA1 region of HPP indicate the region’s importance in temporal processing and lesions of the dentate gyrus (DG) demonstrate an important role in spatial processing. It has been suggested that a subset of GABAergic interneurons that express Substance P mediate the inhibition of pyramidal and granule cells, which further affects the pattern of their output. This synchronizing action may directly affect information processing of CA1 pyramidal and DG granule cells. A form of temporal processing involves learning specific sequences of events for spatial locations, which incorporates both temporal and spatial qualities attributed to CA1 and DG, respectively. In order to investigate whether interneurons mediate CA1 and DG processing of newly learned locations of sequential patterns, Long-Evans male rats were randomly assigned to the following surgical groups: CA1 pyramidal cell (ibotenic acid), CA1 interneuron (peptidase-resistant substance P analog conjugated to the neurotoxin saporin) (SSP-Saporin), DG granule cell (colchicine), DG interneuron (SSP-Saporin) lesions and controls (PBS). Following recovery from surgery, rats were tested on a sequential learning task for spatial locations using an eight-arm radial maze. Six arm locations were pseudo-randomly assigned to a sequence; each of the arms was baited with a food reward. Doors remained closed until the rat oriented in front of the correct door in the sequence, at which time the door was opened and the rat was allowed access to the reward; the choice was scored as a correct response. However, if the rat oriented to an incorrect door in the sequence, the choice was scored as incorrect and the animal was allowed to reorient to the correct door. The same sequence was repeated ten times per day for a total of ten consecutive days. The percentage of correct choices per day was compared across all ten days. The results indicate that subjects with CA1 pyramidal cell, CA1 interneuron, and DG interneuron, but not DG granule cell lesions, had difficulty acquiring the sequential task when compared to controls. These results suggest an important role for CA1 pyramidal cells, and for interneurons in both CA1 and DG subregions of the HPP in temporal processing of spatial locations.

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NK-1-receptor-mediated lesion of spinal post-synaptic dorsal column neurons might improve intractable visceral pain of cancer origin.

Wang Y, Mu X, Liu Y, Zhang X, Wu A, Yue Y (2011) NK-1-receptor-mediated lesion of spinal post-synaptic dorsal column neurons might improve intractable visceral pain of cancer origin. Med Hypotheses 76(1):102-104. doi: 10.1016/j.mehy.2010.08.042

Summary: There is evidence that spinal post-synaptic dorsal column neurons begin to express neurokinin-1 receptors after visceral stimulation. The authors discuss using this expression profile to target SSP-SAP (Cat. #IT-11) to these neurons and eliminate them. This use of ‘molecular neurosurgery’ may be a replacement for traditional neurosurgery for the treatment of cancer-related visceral pain.

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Transplant of GABAergic precursors restores hippocampal inhibitory function in a mouse model of seizure susceptibility.

Zipancic I, Calcagnotto ME, Piquer-Gil M, Mello LE, Alvarez-Dolado M (2010) Transplant of GABAergic precursors restores hippocampal inhibitory function in a mouse model of seizure susceptibility. Cell Transplant 19(5):549-564. doi: 10.3727/096368910X491383

Summary: Although medial ganglionic eminence-derived cells can be grafted into the neonatal brain and become functionally mature GABAergic neurons it is not clear whether the grafted cells can rescue loss of function. The authors injected mice with 1.6-2.0 ng of SSP-SAP (Cat. #IT-11) into the anterior and posterior hippocampus to eliminate GABAergic interneurons. Neuron function in mice receiving the grafts returned to near normal.

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Ablation of NK-1 expressing neurons in the rostral ventromedial medulla attenuates inflammatory hyperalgesia.

Brink TS, Khasabov SG, Fliss PM, Simone DA (2009) Ablation of NK-1 expressing neurons in the rostral ventromedial medulla attenuates inflammatory hyperalgesia. Neuroscience 2009 Abstracts 361.5/BB31. Society for Neuroscience, Chicago, IL.

Summary: Substance P (SP) is a neuropeptide synthesized by many nociceptive primary sensory neurons and is released into the spinal cord following noxious stimulation where it binds to neurokinin-1 (NK-1) receptors, mostly located on ascending spinal neurons. Spinal NK-1 receptors are involved in the development of hyperalgesia and central sensitization. NK-1 expressing neurons are also present in the rostral ventromedial medulla (RVM), a brainstem area involved in descending modulation of nociceptive transmission in the spinal cord. ON cells in the RVM are involved in facilitation of nociceptive transmission and their activity may be modulated by SP. SP injected into the RVM excites ON cells through NK-1 receptors, and NK-1 receptor antagonists into the RVM attenuate hyperalgesia produced by capsaicin. Here, we studied the role of RVM NK-1 positive neurons in modulating hyperalgesia following acute (intraplantar capsaicin injection) or sustained (complete Freund's adjuvant (CFA) in the hindpaw) inflammation. We used the ribosomal toxin saporin (SAP) conjugated to a stable agonist of SP (SSP) to selectively ablate RVM cells that possess NK-1 receptors. In male Sprague-Dawley rats, withdrawal responses to noxious heat and mechanical stimuli were obtained using the Hargreaves method and a 15 g von Frey monofilament applied to the plantar hindpaw, respectively. Rats were treated with either the SSP-SAP toxin (0.5 µg/0.5 µl) or blank-SAP, and were tested 10-24 days after injection, when NK-1 expressing RVM neurons are ablated. In control rats, injection of capsaicin (10 µl of 0.1%) produced a 63% decrease in withdrawal latency to heat and an increase in withdrawal response frequency evoked by the monofilament from 16% up to 87%. However, SSP-SAP attenuated capsaicin-evoked hyperalgesia to heat (15% decrease in withdrawal latency) and mechanical (increase to 44% withdrawal frequency) stimuli. Elimination of NK-1 positive neurons in the RVM also attenuated the development of hyperalgesia following CFA. Whereas control rats exhibited a 60% decrease in withdrawal latency to heat and an increase in withdrawal frequency the monofilament from 10% up to 78%, withdrawal latency decreased 27% and withdrawal frequency increased to only 46% in rats treated with SSP-SAP. We conclude that neurons in the RVM that contain NK-1 receptors are pronociceptive and contribute to the hyperalgesia produced by capsaicin or CFA.

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The neurokinin-1 receptor modulates the methamphetamine-induced striatal apoptosis and nitric oxide formation in mice.

Zhu J, Xu W, Wang J, Ali SF, Angulo JA (2009) The neurokinin-1 receptor modulates the methamphetamine-induced striatal apoptosis and nitric oxide formation in mice. J Neurochem 111(3):656-668. doi: 10.1111/j.1471-4159.2009.06330.x

Summary: This study examined the role of neurokinin-1 receptors (NK1R) on the methamphetamine-induced apoptosis of striatal neurons. 4 ng of SSP-SAP (Cat. #IT-11) or the control, saporin (Cat. #PR-01), was administered to the striatum of mice. Ablation of NK1R-expressing striatal neurons resulted in a significant reduction of methamphetamine-induced apoptosis. The data suggests that the NK1R circuitry in the striatum may be a target for treatment of methamphetamine abuse.

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Ablation of least shrew central neurokinin NK1 receptors reduces GR73632-induced vomiting.

Ray AP, Chebolu S, Ramirez J, Darmani NA (2009) Ablation of least shrew central neurokinin NK1 receptors reduces GR73632-induced vomiting. Behav Neurosci 123:701-706. doi: 10.1037/a0015733

Summary: In this work the authors investigated the role of central and peripheral nervous systems components that mediate the emetic reflex. Least shrews received an 600-ng injection of SSP-SAP (Cat. #IT-11) into the lateral ventricle. Some animals also received a 4.8-µg intraperitoneal injection of SSP-SAP. Blank-SAP (Cat. #IT-21) and unconjugated saporin (Cat. #PR-01) were used as controls. Lesioned animals displayed reduced emesis, but the data indicate that a minor peripheral nervous system component is also present.

Related Products: SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21), Saporin (Cat. #PR-01)

Anxiety-like behavior is modulated by a discrete subpopulation of interneurons in the basolateral amygdala.

Truitt WA, Johnson PL, Dietrich AD, Fitz SD, Shekhar A (2009) Anxiety-like behavior is modulated by a discrete subpopulation of interneurons in the basolateral amygdala. Neuroscience 160:284-294. doi: 10.1016/j.neuroscience.2009.01.083

Summary: It is thought that the basolateral nucleus of the amygdala (BL) is an anxiety regulator. The authors previously demonstrated that SSP-SAP (Cat. #IT-11) lesions of the BL increase anxiety-like behaviors in rats. Using a series of 6 bilateral injections of SSP-SAP (4 ng per injection), the NK-1 receptor-expressing cells of the BL are further characterized.

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Neural regulation of ejaculation.

Young B, Coolen L, McKenna K (2009) Neural regulation of ejaculation. J Sex Med 6(Suppl 3):229-233. doi: 10.1111/j.1743-6109.2008.01181.x

Summary: This review summarizes that a specific population of lumbar spinothalamic (LSt) cells plays in regulation of the ejaculatory response. One method to study these cells is the injection of SSP-SAP (Cat. #IT-11) into the LSt cells surrounding the central canal. Over 90% of these cells express the NK-1 receptor. This lesion significantly disrupts ejaculation without affecting mounts or intromissions.

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Recent progress in research on ribosome inactivating proteins.

Ng TB, Wong JH, Wang H (2009) Recent progress in research on ribosome inactivating proteins. Curr Protein Pept Sci 1323:94-108. doi: 10.2174/138920310790274662

Summary: This review discusses recent literature on ribosome inactivating proteins including the use of saporin-based conjugates in neuroscience and cancer research. Brief descriptions of research done using 192-IgG-SAP (Cat. #IT-01), OX7-SAP (Cat. #IT-02), dermorphin-SAP (Cat. #IT-12), anti-SERT-SAP (Cat. #IT-23), SSP-SAP (Cat. #IT-11), anti-DBH-SAP (Cat. #IT-03), CTB-SAP (Cat. #IT-14), and other conjugates are provided along with basic information about ribosome inactivating proteins.

Related Products: 192-IgG-SAP (Cat. #IT-01), OX7-SAP (Cat. #IT-02), Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Anti-SERT-SAP (Cat. #IT-23), SSP-SAP (Cat. #IT-11), Anti-DBH-SAP (Cat. #IT-03), CTB-SAP (Cat. #IT-14)

Cardiac damage after lesions of the nucleus tractus solitarii.

Nayate A, Moore SA, Weiss R, Taktakishvili OM, Lin LH, Talman WT (2009) Cardiac damage after lesions of the nucleus tractus solitarii. Am J Physiol Regul Integr Comp Physiol 296:R272-R279. doi: 10.1152/ajpregu.00080.2008

Summary: This work tested the hypothesis that nucleus tractus solitarii (NTS) lesions can lead to fatal cardiac arrhythmias and myocardial lesions. Rats received bilateral injections of 9.4 ng of SSP-SAP (Cat. #IT-11) into the dorsolateral and medial portions of the NTS. Lesioned animals displayed increased arterial blood pressure.

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Selective lesion of retrotrapezoid Phox2b-expressing neurons attenuates the central chemoreflex in rats

Moreira TS, Takakura AC, Stornetta RL, Guyenet PG (2008) Selective lesion of retrotrapezoid Phox2b-expressing neurons attenuates the central chemoreflex in rats. Neuroscience 2008 Abstracts 383.3/RR70. Society for Neuroscience, Washington, DC.

Summary: Injection of the neurotoxin saporin-substance P (SSP-SAP) into the retrotrapezoid nucleus (RTN) attenuates the central chemoreflex in rats. Here we ask whether these deficits are caused by the destruction of a type of pH-sensitive interneuron that expresses the transcription factor Phox2b and is non-catecholaminergic (Phox2b+TH-). We show that RTN contains around 2100 Phox2b+TH- cells. Injections of SSP-SAP into RTN destroyed Phox2b+TH- neurons but spared facial motoneurons, catecholaminergic and serotonergic neurons and the ventral respiratory column caudal to the facial motor nucleus. Two weeks after SSP-SAP, the apneic threshold measured under anesthesia was unchanged when fewer than 57% of the Phox2b+TH- neurons were destroyed. However, destruction of 70 ± 3.5 % of these cells was associated with a large rise of the apneic threshold (from 5.6 to 7.9% end-expiratory pCO2). In anesthetized rats with unilateral lesions of around 70% of the Phox2b+TH- neurons, acute inhibition of the contralateral intact RTN with muscimol instantly eliminated phrenic nerve discharge (PND) but normal PND could usually be elicited by strong peripheral chemoreceptor stimulation (8/12 rats). Muscimol had no effect in rats with an intact contralateral RTN. In conclusion, the destruction of the Phox2b+TH- neurons is a plausible cause of the respiratory deficits caused by injection of SSP-SAP into RTN. At least 70% of these cells must be killed to cause a severe attenuation of the central chemoreflex under anesthesia. The loss of an even greater percentage of these cells would presumably be required to produce significant breathing deficits in the awake state.

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Selective lesion of retrotrapezoid Phox2b-expressing neurons raises the apneic threshold in rats.

Takakura AC, Moreira TS, Stornetta RL, West GH, Gwilt JM, Guyenet PG (2008) Selective lesion of retrotrapezoid Phox2b-expressing neurons raises the apneic threshold in rats. J Physiol 586:2975-2991. doi: 10.1113/jphysiol.2008.153163

Summary: It is thought that transcription factor Phox2b-expressing glutamatergic interneurons are involved in the activation of breathing by elevated CO2 in the central nervous system. This activation is otherwise known as the central chemoreflex. Injections of SSP-SAP (Cat. #IT-11) into the retrotrapezoid nucleus eliminated Phox2b+TH- neurons but spared other neuron classes. Several different amounts of the conjugate were used (0.15, 0.3, or 0.6 ng in 1 or 2 injections). Elimination of ≥70% of Phox2b+TH- neurons markedly attenuated the central chemoreflex.

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Utilization of the least shrew as a rapid and selective screening model for the antiemetic potential and brain penetration of substance P and NK1 receptor antagonists.

Darmani NA, Wang Y, Abad J, Ray AP, Thrush GR, Ramirez J (2008) Utilization of the least shrew as a rapid and selective screening model for the antiemetic potential and brain penetration of substance P and NK1 receptor antagonists. Brain Res 1214:58-72. doi: 10.1016/j.brainres.2008.03.077

Summary: This work investigated the role of central tachykinin NK1 receptors in delayed phase vomiting caused by chemotherapeutics. Least shrews received 1.2 mg/kg intraperitoneal injections of SSP-SAP (Cat. #IT-11). Saporin (Cat. #PR-01) and blank-SAP (Cat. #IT-21) were used as controls. In response to administration of a NK1 receptor agonist lesioned animals vomited less than the control group, indicating an important role for NK1 receptors in emesis.

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Substance P receptor-expressing dorsal horn neurons: Lessons from the targeted cytotoxin, substance P-saporin.

Wiley RG (2008) Substance P receptor-expressing dorsal horn neurons: Lessons from the targeted cytotoxin, substance P-saporin. Pain 136:7-10. doi: 10.1016/j.pain.2008.03.010

Summary: This review covers some of the more recent work utilizing SP-SAP (Cat. #IT-07) and SSP-SAP (Cat. #IT-11) in the dorsal horn. Specific answers to experimental questions are discussed, as well as some of the questions generated by the research. The potential of SP-SAP and SSP-SAP as pain therapeutics is also explored, along with potential clinical applications of other targeted toxins in pain therapy.

Related Products: SP-SAP (Cat. #IT-07), SSP-SAP (Cat. #IT-11)

Developmental origins of ventral medullary NK1r neurons

Gray PA, Vandunk C (2007) Developmental origins of ventral medullary NK1r neurons. Neuroscience 2007 Abstracts 187.15/SS7. Society for Neuroscience, San Diego, CA.

Summary: Breathing is a fundamental neural behavior generated by neurons within the brainstem. In the adult rat, bilateral injection of substance P conjugated to saporin (SSP) in the preBötzinger Complex (preBötC) eliminates normal breathing. Unilateral injection produces sleep disordered breathing and eliminates the effects of excitatory amino acid injection on breathing and blood pressure. SSP injection in the RTN region, in contrast, blunts chemosensitivity. The extent these different effects are due to ablation of anatomically, functionally, or genetically distinct populations are unknown. Further, the extent NK1R expression identifies unique populations in neonatal mice is unknown. Transcription factors are fundamental to determining the properties of neurons and it has been proposed a “combinatorial code” of transcription factor expression defines each distinct functional population. From a genome-scale analysis of over 1000 transcription factors and transcriptional co-factors we identified several genes expressed in respiratory regions of the brainstem. Using immunohistochemistry, we analyzed gene expression patterns of NK1R expressing neurons of the ventral medulla in embryonic and neonatal mice. We find there are at least four genetically distinct, partially overlapping populations of NK1R expressing neurons in the ventral medulla. These differ in the transcription factors they express, their onset of NK1R expression, and their co-expression of peptides and g-protein coupled receptors. At the level of the preBötC, there are two populations of NK1R expressing neurons derived from distinct developmental progenitor populations. One population corresponds to the preBötC as defined in rats and co-expresses the peptide somatostatin. A second population is continuous with, and genetically identical to the subCVLM population important for respiratory-cardiovascular coordination. Both populations co-express the somatostatin 2a receptor (SST2aR). At the level of the RTN/pFRG, there are also two populations of NK1R expressing neurons that are derived from similar developmental precursors as the preBötC. Neither of these populations expresses SST2aR. Whether either of these populations corresponds to the RTN or pFRG is currently unknown. These data suggest both the preBötC and RTN/pFRG regions contain developmentally related NK1R expressing populations. Further, they identify the developmental origin of preBötC neurons known to be essential for normal breathing and provide a foundation for understanding the genetic origin of an important neural circuit.

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Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN) and central respiratory chemoreception in rats

Stornetta RL, Takakura AC, Moreira TS, Mulkey DP, Bayliss DA, Guyenet PG (2007) Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN) and central respiratory chemoreception in rats. Neuroscience 2007 Abstracts 230.9. Society for Neuroscience, San Diego, CA.

Summary: The RTN contains glutamatergic interneurons that are strongly activated by CO2 via acidification. These chemosensitive neurons are non-catecholaminergic and they express the transcription factor Phox2b. Although RTN chemoreceptors innervate selectively the brainstem regions that contain the respiratory rhythm and pattern generator (CPG), it is not yet clear whether these neurons drive inspiration or expiration, pump or airway muscles, autonomic circuits or all of the above. To determine whether RTN neurons drive inspiration, we examined whether their selective destruction modifies the CO2 sensitivity of the phrenic nerve discharge (PND) in anesthetized vagotomized rats. Using electrophysiological recordings in vivo and in slices, we found that the chemosensitive neurons of RTN express substance P receptors. We also found that these cells can be destroyed by local injection of a substance P agonist conjugated with saporin (SSP-SAP). The kill rate of RTN chemoreceptors was determined by counting the number of residual Phox2b-expressing non-catecholaminergic neurons present in this structure 15 days after toxin injection. Unilateral injection of 0.6 ng SSP-SAP destroyed 75% of the presumptive chemoreceptors on the injected side only. The lesion was selective because nearby neurons such as facial motoneurons, catecholaminergic and serotonergic cells were spared. SSP-SAP also spared the ventral respiratory column caudal to RTN except for a small amount of damage in the Bötzinger region closest to RTN. Unilateral lesion of the Phox2b-expressing neurons of RTN had no effect on PND and on respiratory chemoreception. However, in such rats, a single injection of the GABA-mimetic muscimol into the contralateral intact RTN instantly eliminated PND. After muscimol, PND did not usually reappear in the presence of hypercapnia up to 10% end-expiratory CO2. However, PND could typically be reactivated by strong stimulation of peripheral chemoreceptors which suggests that the respiratory oscillator had remained functional after muscimol. Unilateral injection of a lower dose of SSP-SAP (0.15 ng) had no effect on the Phox2b-expressing neurons of RTN. In such rats, unilateral injection of muscimol into the contralateral RTN had no detectable effect on PND and central chemoreception.

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Anti-nociceptive effects of selectively destroying substance P receptor-expressing dorsal horn neurons using [Sar(9),Met(O(2))(11)]-substance P-saporin: Behavioral and anatomical analyses.

Wiley RG, Kline IV RH, Vierck Jr CJ (2007) Anti-nociceptive effects of selectively destroying substance P receptor-expressing dorsal horn neurons using [Sar(9),Met(O(2))(11)]-substance P-saporin: Behavioral and anatomical analyses. Neuroscience 146:1333-1345. doi: 10.1016/j.neuroscience.2007.01.066

Summary: While lumbar injections of SP-SAP (Cat. #IT-07) produce specific lesions, use of this targeted conjugate in the forebrain has been problematic. The authors investigated the use of SSP-SAP (Cat. #IT-11), a conjugate of saporin with a stable analog of substance P. The greater stability of SSP-SAP resulted in increased potency as well as better specificity. SSP-SAP is shown to be a highly effective reagent for the removal of NK1 receptor-expressing neurons in the brain and spinal cord.

Related Products: SP-SAP (Cat. #IT-07), SSP-SAP (Cat. #IT-11)

From anxiety to autism: spectrum of abnormal social behaviors modeled by progressive disruption of inhibitory neuronal function in the basolateral amygdala in Wistar rats.

Truitt WA, Sajdyk TJ, Dietrich AD, Oberlin B, McDougle CJ, Shekhar A (2007) From anxiety to autism: spectrum of abnormal social behaviors modeled by progressive disruption of inhibitory neuronal function in the basolateral amygdala in Wistar rats. Psychopharmacology (Berl) 191(1):107-118. doi: 10.1007/s00213-006-0674-y

Summary: The amygdala has been identified as being involved in social behaviors. Six 4 ng injections of SSP-SAP (Cat. #IT-11) were administered bilaterally into the basolateral nucleus (BLA) of the amygdala of rats. Blank-SAP (Cat. #IT-21) was used as a control. Results of a social interaction paradigm suggest that in normal animals social inhibition can be overcome by habituation. In lesioned animals, however, social inhibition is not reversed by habituation, indicating that NK-1 receptor-expressing GABAergic interneurons in the BLA are important in this system.

Related Products: SSP-SAP (Cat. #IT-11), Blank-SAP (Cat. #IT-21)

Lesions of neurokinin-1 receptor immunoreactive (NK1R-ir) neurons in the ventral medulla decrease chemoreception and ventilation

Li A, Nattie EE (2006) Lesions of neurokinin-1 receptor immunoreactive (NK1R-ir) neurons in the ventral medulla decrease chemoreception and ventilation. Neuroscience 2006 Abstracts 455.5. Society for Neuroscience, Atlanta, GA.

Summary: We injected SSP-saporin, a toxin specific for NK1R-ir neurons, into the cisterna magna in order to lesion a wide aspect of the ventral medulla. NK1R-ir loss after 21 days was 79% in the retrotrapezoid nucleus, 65% in the A5 region, 38% in the medullary raphe and 49% in the pre-Botzinger complex. This resulted in a large reduction in the ventilatory response to 7% CO2 during wakefulness (-61%) and NREM sleep (-57%). The response to 12% O2 was reduced by 35-40% at 8 days but partially recovered by 22 days. We did not measure chemoreception in REM sleep. Ventilation in wakefulness, NREM and REM sleep measured over 4 hrs of air breathing decreased by 12-13% at 21 days compared to baseline values (P< 0.005, one-way repeated measures ANOVA; P< 0.05 post hoc comparison, Dunnett’s test). In REM sleep compared to awake and NREM sleep, the rats breathed with a higher frequency and smaller tidal volume, a pattern that was unaffected by the lesion, and with a greater coefficient of variability, which was further increased by the lesion (71 +/- 4 % vs 34 +/- 6 %). We did not observe any severe rhythm disturbances. We attribute the effect of these lesions, which are greatest in the ventral medullary regions including the retrotrapezoid nucleus and the medullary raphe, to loss of tonic chemoreceptor input. This input seems to have equal weight in wakefulness and in NREM and REM sleep and it seems to minimize the variability of frequency observed in REM sleep.

Related Products: SSP-SAP (Cat. #IT-11)

Neurokinin-1 receptor expressing neurons in the ventral medulla are essential for normal central and peripheral chemoreception in the conscious rat.

Nattie E, Li A (2006) Neurokinin-1 receptor expressing neurons in the ventral medulla are essential for normal central and peripheral chemoreception in the conscious rat. J Appl Physiol 101(6):1596-1606. doi: 10.1152/japplphysiol.00347.2006

Summary: All known chemoreceptor sites in the mammalian brainstem are rich in the neurokinin-1 receptor (NK1r). The authors ask if these cells scattered throughout the ventral medulla are involved in central and peripheral chemoreception. Rats received 250-280 ng of SSP-SAP (Cat. #IT-11) into the cisterna magna, mouse IgG-SAP (Cat. #IT-18) was used as a control. The results indicate that NK1r neurons in the ventral medulla are involved in both central and peripheral chemoreception, during both waking and sleep states.

Related Products: SSP-SAP (Cat. #IT-11), Mouse IgG-SAP (Cat. #IT-18)

Neurokinin 1 receptor containing interneurons of the BLA are putative candidates for the inhibitory component of feed-forward inhibition from the mPFC to the BLA

Truitt WA, Oberlin BG, Dietrich AD, Fitz SD, Shekhar A (2005) Neurokinin 1 receptor containing interneurons of the BLA are putative candidates for the inhibitory component of feed-forward inhibition from the mPFC to the BLA. Neuroscience 2005 Abstracts 796.12. Society for Neuroscience, Washington, DC.

Summary: The amygdala and in particular the basolateral nucleus of the amygdala (BLA) is a central site for fear and anxiety. The BLA is under tonic inhibition by a network of inhibitory interneurons. Additionally, cortical inputs can both excite or inhibit the output of the BLA, resulting in increases or decreases in anxiety/fear-like behaviors. In particular mPFC inputs to the BLA can suppress BLA output and inhibit fear conditioning. However, the mechanism by which this occurs is not fully understood. Evidence from electrophysiological studies suggests a feed forward inhibitory relationship between the mPFC and the BLA. This feed forward inhibition putatively occurs by mPFC-glutamatergic inputs exciting GABAergic interneurons of the BLA, which in turn suppress firing of the BLA projection neurons. However, tracing studies demonstrate that the vast majority of mPFC inputs to the BLA form synapses with dendritic spines, which have been reported to exist only on projection neurons of the BLA. Here we present data that suggests a specific subclass of BLA interneurons, those that express neurokinin 1 receptors (NK-1r) are likely candidates for the inhibitory component of the feed forward inhibition described above. Here we report that, in the rat, the NK-1r containing BLA-interneurons contain dendritic spines and NMDA receptors. Furthermore, we report that in anesthetized rats disinhibition of mPFC neurons by injections of bicuculline methiodide (50 pmol), leads to cFos induction in 25 – 50% of the NK-1r containing interneurons in the BLA, while 0% of the NK-1r interneurons expressed cFos following vehicle injections. Furthermore, selective ablation of these NK-1r containing BLA-interneurons (by use of the targeted toxin, SSP-Saporin) suppressed anxiolytic-like effects of familiarity in the SI test. Collectively, these data suggest the NK-1r containing interneurons of the BLA may respond to glutamatergic inputs from the mPFC and may be involved in a mPFC-BLA anxiety or fear regulating pathway.

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Involvement of lumbar spinothalamic cells in relay of sensory cues related to vaginocervical stimulation in female rats

Coolen LM, Amstalden KAZ, Allard J (2005) Involvement of lumbar spinothalamic cells in relay of sensory cues related to vaginocervical stimulation in female rats. Neuroscience 2005 Abstracts 321.13. Society for Neuroscience, Washington, DC.

Summary: The rat lumbar spinal cord contains a population of galanin containing spinothalamic (LSt) cells, which in male rats play a pivotal role in the control of ejaculation. However, the function of LSt cells in female rats is unknown. LSt cells project to the parvocellular subparafascicular thalamic nucleus (SPFp), where Fos is expressed following vaginocervical stimulation (VCS). Hence, we hypothesize that LSt cells are involved in relay of sensory cues related to VCS to the brain. To test this hypothesis, the effects of LSt lesions were investigated on two parameters that are dependent on relay of VCS-related cues: pseudopregancy and Fos expression in the SPFp. Adult female Sprague Dawley rats received infusions of substance P-conjugated (SSP-SAP) or unconjugated saporin (control) in lumbar levels 3-4. Females were investigated for: estrous cyclicity, expression of sexual behavior, and induction of pseudopregnancy by mating with vasectomized male partners including 5, 10, or 15 intromissions. For the final test, females received 10 or 15 intromissions from male partners, were perfused one hour later, and brains and spinal cords were examined for Fos expression and LSt lesions. SSP-SAP treatment resulted in severe reduction of LSt cells, but did not affect cyclicity or expression of sexual behavior, suggesting that LSt cells are not involved in regulation of these functions. In contrast, LSt lesions significantly reduced mating-induced Fos expression in the SPFp, supporting the involvement of LSt cells in relay of VCS-related sensory information to the SPFp. However, LSt lesions did not prevent mating-induced pseudopregancy and only partly blocked mating-induced neural activation in SPFp, indicating the possible involvement of alternate pathways. Alternatively, the few remaining LSt cells in lesioned females are sufficient for induction of Fos in SPFp and pseudopregnancy.

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Ablation of NK-1 receptor-expressing interneurons prevents methamphetamine-induced apoptosis but not dopamine terminal toxicity in the striatum of mice

Xu, WZhu JPQ, Angulo JA (2005) Ablation of NK-1 receptor-expressing interneurons prevents methamphetamine-induced apoptosis but not dopamine terminal toxicity in the striatum of mice. Neuroscience 2005 Abstracts 337.9. Society for Neuroscience, Washington, DC.

Summary: Pharmacological evidence from our laboratory demonstrates that the neurokinin-1 (NK-1) receptor mediates methamphetamine (METH)-induced toxicity of the dopamine terminals and the apoptosis of some striatal neurons. We have shown that systemic administration of the NK-1 receptor antagonist, WIN 51,708, prior to METH exposure, can protect the striatum from METH-induced damage at pre- and post-synaptic sites. To further assess the role of the NK-1 receptor on METH-induced striatal neural damage, NK-1 receptor-expressing interneurons were selectively ablated by means of intrastriatal injections of [Sar9,Met(O2)11]substance P conjugated to the ribosomal-inactivating cytotoxin saporin (SSP-SAP). TUNEL-labeling showed that ablation of striatal neurons that express NK-1 receptors provided protection against METH-induced apoptosis of some striatal neurons. However, ablation of NK-1 receptor-expressing interneurons did not provide protection against METH-induced depletion of tyrosine hydroxylase, a reliable marker of the dopamine terminals of the strtiatum. These results suggest that METH-induced apoptosis and dopamine terminal toxicity occur via distinct mechanisms in the mouse striatum.

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Molecular neurosurgery with targeted toxins

Wiley RG, Lappi DA (2005) Molecular neurosurgery with targeted toxins. Humana Press, Totowa, New Jersey.

Summary: The idea behind the book was to provide a road map for the users of Molecular Neurosurgery to see how experienced scientists used these exceptional reagents in their work. Experiments with several targeted toxins are described, and readers can get an idea either specifically about a targeted toxin that they’re using, or about how a type of molecule is used and at what dosage, in a paradigm similar to theirs.

Related Products: 192-IgG-SAP (Cat. #IT-01), ME20.4-SAP (Cat. #IT-15), Anti-DBH-SAP (Cat. #IT-03), SSP-SAP (Cat. #IT-11), Dermorphin-SAP / MOR-SAP (Cat. #IT-12), IB4-SAP (Cat. #IT-10), CTB-SAP (Cat. #IT-14)

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Elimination of rat spinal neurons expressing neurokinin 1 receptors reduces bladder overactivity and spinal c-fos expression induced by bladder irritation.

Seki S, Erickson KA, Seki M, Nishizawa O, Igawa Y, Ogawa T, de Groat WC, Chancellor MB, Yoshimura N (2005) Elimination of rat spinal neurons expressing neurokinin 1 receptors reduces bladder overactivity and spinal c-fos expression induced by bladder irritation. Am J Physiol Renal Physiol 288(3):F466-F473. doi: 10.1152/ajprenal.00274.2004

Summary: Substance P is reported to play a role in the micturition reflex as well as in nociceptive responses. The authors investigated the role that neurokinin-1 receptor-expressing cells in the spinal cord play in the micturition reflex of rats. 8 µl of 1.0 or 1.5 µM SSP-SAP (Cat. #IT-11) was injected into the L6-S1 level of the spinal cord, and cystometric parameters were measured before and after capsaicin administration to the bladder. Lesioned animals did not display the bladder overactivity normally seen in the presence of capsaicin.

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Local striatal deletions of neurokinin-1 receptor-expressing neurons protect against methamphetamine-induced neural damage

Xu W, Zhu JPQ, Angulo JA (2004) Local striatal deletions of neurokinin-1 receptor-expressing neurons protect against methamphetamine-induced neural damage. Neuroscience 2004 Abstracts 908.8. Society for Neuroscience, San Diego, CA.

Summary: Recent collective evidence from our laboratory and others has implicated the peptidergic system involving the neuropeptide substance P (SP) and its receptor, neurokinin-1 (NK-1), in mediating METH-induced adverse effects in the neostriatum. Here we test to see if local striatal abolishment of the NK-1 receptor-signaling pathway can protect from METH-induced neural damage in the striatum. Selective striatal knockouts of this pathway was done using an intrastriatal injection of [Sar9,Met(O2)11]substance P conjugated to the ribosomal-inactivating cytotoxin saporin (SSP-SAP). Selective striatal elimination of NK-1 receptor-expressing neurons demonstrated protection against METH-induced apoptosis by TUNEL-labeling. This further confirms the important modulatory effects of this peptidergic receptor in striatum.

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Neurokinin 1 receptor expressing interneurons of the BLA regulate anxiety-like responses in the rat

Truitt WA, Dietrich AD, Fitz SD, Minick PE, Shekhar A (2004) Neurokinin 1 receptor expressing interneurons of the BLA regulate anxiety-like responses in the rat. Neuroscience 2004 Abstracts 782.5. Society for Neuroscience, San Diego, CA.

Summary: The Basolateral Nucleus of the Amygdala (BLA) has been implicated in the regulation and development of anxiety. In general, regarding BLA projection neurons, excitation tends to increase, while inhibition tends to reduce anxiety-like responses. These projection neurons, which comprise approximately 85% of the BLA neurons, are tightly regulated by the activity of local circuit GABAergic interneurons. To date, at least four distinct interneuronal subpopulations have been identified in the BLA, with characteristic morphological and physiological properties suggestive of functional diversity. Yet the in vivo functional selectivity of these subpopulations has not been critically examined. Here we propose to examine the function of one specific interneuronal subpopulation within the BLA by making selective lesions and monitoring anxiety-like behavior. To accomplish this objective the subpopulation of BLA interneurons expressing NK-1r receptors were ablated with the targeted toxin SSP-saporin (SAP). Lesions were made by a series of 6 bilateral, 500nl injections spread throughout the anterior BLA. Control rats were injected with an equal volume of blank-SAP, which does not enter the cells. SSP-SAP injections significantly reduced the number of NK-1r expressing cells compared to blank-SAP treated rats, with little to no nonspecific damage. Lesioning NK-1r expressing cells resulted in increased anxiety-like responses in the social interaction (SI) and elevated plus maze (EPM) tests. Specifically, SI time compared to pre-surgery value was significantly reduced in lesion rats. Lesion rats also had fewer open arm entries in the EPM compared to control rats. Furthermore, lesioned rats failed to recover from this decrease in SI even after 4 weeks of testing. These results suggest that the subpopulation of interneurons within the BLA that express NK-1r is critical in regulating anxiety-like behavior.

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The effects of chronic deafferentation and SSP-saporin on pain responses, spinal cord neurons and on the structure and function of the somatosensory thalamus (VPL) in the macaque monkey

Ralston HJ, Wiley RG, Dougherty PM, Weng HR, Cata J, Chen JH, Hopkins SD, Canchola SA, Galo E, Vierck CJ (2004) The effects of chronic deafferentation and SSP-saporin on pain responses, spinal cord neurons and on the structure and function of the somatosensory thalamus (VPL) in the macaque monkey. Neuroscience 2004 Abstracts 295.1. Society for Neuroscience, San Diego, CA.

Summary: We have used behavioral, physiological and anatomical methods to examine the effects of chronic (> 2 years) lesions of the dorsal column pathway and of the intrathecal administration of the neurotoxin SSP-saporin in adult M. arctoides. Normal animals were evaluated to determine their responses to noxious heat (52 to 58°C) applied to the lower limbs. Subsequently, the monkeys were anesthetized and had unilateral lesions of the dorsal and dorsolateral spinal cord white matter pathways at midthoracic levels. After recovering from the surgery, their pain responses were studied for more than 1 year, following which SSP-saporin was administered to the lumbosacral spinal cord. The animals were found to have a decrease in their responses to noxious heat applied to the lower limbs. Terminal physiological experiments revealed that the neurons within the lower limb representation of VPL on the side contralateral to the thoracic cord lesion did not have normal receptive fields, although some cells responded to stimulation of both upper and lower limbs. Histological sections of lumbar spinal cord were stained for neurokinin 1 receptor (NK-1R) and showed a significant decrease in lamina I NK-1R positive neurons. Electron microscopy of VPL revealed patterns of synaptic terminals that were different than those found in VPL of normal macaques. We will determine whether there is a significant correlation between the altered behaviors and the physiological and anatomical changes in these animals as a consequence of somatosensory deafferentation.

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SSP-saporin decreases formalin induced c-Fos expression throughout the dorsal horn.

Kline IV RH, Wiley RG (2003) SSP-saporin decreases formalin induced c-Fos expression throughout the dorsal horn. Neuroscience 2003 Abstracts 174.7. Society for Neuroscience, New Orleans, LA.

Summary: Substance P (SP) antagonists and SP-saporin have been shown to decrease phase II of the formalin test suggesting an important role for SP in this model of persistent pain. SP antagonists also decrease formalin induced c-fos expression in dorsal horn neurons. A congener of SP-sap that is more stable and has higher affinity for NK-1R, SSP-sap (Sar9Met(02)11-substance P-saporin) has been studied by injection into the striatum and hippocampus where it was more potent and specific than SP-sap. In the present study, this selective and more potent toxin was used to determine the effects of destroying dorsal horn NK-1R on behavior and c-fos induction after intraplantar formalin. Twelve Sprague Dawley male rats were injected intrathecally with 100ng SSP-sap or PBS. After 2 weeks survival, rats underwent hindpaw formalin injections and behavioral scoring, and then were sacrificed after 3 hours and the lumbar spinal cords processed for immunohistochemical demonstration of NK-1R and c-fos. There were significant correlations between the loss of superficial laminae NK-1R neurons, decreased formalin behavior and dorsal horn c-fos expression. Therefore lumbar i.t. SSP-sap 1) decreased NK-1R cells in laminae I but not in the deeper laminae 2) decreased phase II formalin behavior 3) decreased c-fos in both the superficial and deep laminae. Since c-fos expression in the deeper laminae was decreased and NK-1R was spared in these laminae, we conclude that a lesion affecting only laminae I NK-1R lesion alters activation of neurons throughout the dorsal horn suggesting a key role for the missing neurons in the transfer of nociceptive inputs to deeper laminae.

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A group of glutaminergic interneurons expressing high levels of both neurokinin-1 receptors and somatostatin identifies the region of the pre-Bötzinger complex.

Stornetta RL, Rosin DL, Wang H, Sevigny CP, Weston MC, Guyenet PG (2003) A group of glutaminergic interneurons expressing high levels of both neurokinin-1 receptors and somatostatin identifies the region of the pre-Bötzinger complex. J Comp Neurol 455(4):499-512. doi: 10.1002/cne.10504

Summary: Study of the pre-Bötzinger complex (pre-BötC) has been hindered by the lack of a specific marker. Using SSP-SAP (Cat. #IT-11, three 0.313-ng unilateral injections in the rostral part of the ventral respiratory group) coupled with in situ hybridization and the labeling of selected markers, the authors examined whether somatostatin (SST) might be a marker for this region. The data suggest that a subgroup of cells containing high levels of SST and neurokinin-1 receptor immunoreactivity may identify the pre-BötC.

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Effect of intrathecal substance P-saporin conjugate on bladder hyperactivity induced by intravesical capsaicin

Seki S, Erickson KA, Sasaki K, Sugaya K, Chancellor MB, de Groat WC, Yoshimura N (2002) Effect of intrathecal substance P-saporin conjugate on bladder hyperactivity induced by intravesical capsaicin. Neuroscience 2002 Abstracts 68.2. Society for Neuroscience, Orlando, FL.

Summary: Substance P and neurokinin 1 (NK1) receptors in the spinal cord reportedly play an important role in the micturition reflex as well as in nociceptive responses. We investigated the effect of elimination of NK 1 receptor-expressing spinal cord neurons using the substance P-saporin conjugate (SSP-SAP) on the micturition reflex. Using female rats, an intrathecal catheter was implanted at the level of the L6-S1 spinal cord for injection of either 8 µl of saporin (1.5 µM) or SSP-SAP (1.0 µM). Awake cystometry performed 3 weeks after injection showed no changes in normal bladder function in saporin and SSP-SAP-treated rats. When bladder hyperactivity was induced by intravesical instillation of capsaicin (15 µM), the reduction of intercontraction interval (ICI) was significantly smaller (43.0 ± 6.2% of reduction) in SSP-SAP-treated rats than in saporin-treated rats (59.3 ± 3.1%). Immunohistochemical staining revealed that the area positively stained with NK1 receptor antibodies in the lamina I of the dorsal horn was significantly reduced by 34% in SSP-SAP treated rats, compared with saporin-treated rats. These results suggest that NK1 receptor-expressing neurons in the dorsal horn of the spinal cord play an important role in inducing bladder hyperactivity elicited by intravesical capsaicin. Thus elimination of NK1 receptor-expressing neurons in the spinal cord using SSP-SAP at this concentration could be effective to treat bladder hyperactivity induced by bladder irritation without affecting normal bladder function.

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Lesions of spinothalamic neurons in lumbar spinal cord disrupt ejaculatory reflexes in male rats

Truitt WA, McKenna KE, Coolen LM (2002) Lesions of spinothalamic neurons in lumbar spinal cord disrupt ejaculatory reflexes in male rats. Neuroscience 2002 Abstracts 69.2. Society for Neuroscience, Orlando, FL.

Summary: Previously we tested the significance of a population of lumbar spinothalamic (LUST) cells for male sexual behavior in rats. Anatomically, LUST cells are positioned to relay ejaculation-related sensory signals from reproductive organs to the brain and express substance P receptors as well as several neuropeptides including galanin. Ablation of LUST neurons by the selective toxin SSP-saporin resulted in a complete disruption of ejaculatory behavior. These results suggested that LUST cells play a pivotal role in generation of ejaculatory behavior and may be part of a spinal ejaculation generator. To test this hypothesis, we investigated ejaculatory reflexes in male rats with LUST lesions, using the urethrogenital reflex model. SSP-saporin (4 ng/µl) was injected bilaterally into L3-L4 region in sexually experienced male Sprague Dawley rats. Ten days following surgery, animals were deeply anesthetized and spinal cords were transected at upper thoracic levels. Next, urethral stimulation was provided and muscle contractions were recorded in the bulbocavernous muscle (BCM). Following the experiment, animals were sacrificed and lesions were confirmed using immunostaining for galanin, a marker for LUST cells. In non-lesioned animals (n=5), urethral stimulation produced stereotypical reflex contraction of the BCM, and penile reflexes were observed. In contrast, in animals with complete lesions of LUST cells (n=5) the urogenital reflex was severely attenuated. These results indicate that LUST cells are involved in control of ejaculatory reflexes and are part of a spinal ejaculation generator. Supported by: NIH R01 MH60781(LMC)

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Identification of a potential ejaculation generator in the spinal cord.

Truitt WA, Coolen LM (2002) Identification of a potential ejaculation generator in the spinal cord. Science 297(5586):1566-1569. doi: 10.1126/science.1073885

Summary: The authors lesioned a specific population of rat spinothalamic neurons using 6-8 injections of 4 ng SSP-SAP (Cat. #IT-11). Whereas the treated rats exhibited no change in sexual behavior such as mounts and intromissions, ejaculatory behavior was completely abolished. The data suggest that this population of neurons may function as an ejaculation generator in the spinal cord.

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Role of lamina I neurons expressing the substance P receptor in the prevention and treatment of a spontaneous pain-like behavior following excitotoxic spinal cord injury (SCI).

Yezierski RP, Yu C, Lappi DA, Mantyh PW, Wiley RG (2002) Role of lamina I neurons expressing the substance P receptor in the prevention and treatment of a spontaneous pain-like behavior following excitotoxic spinal cord injury (SCI). IASP 2002 Abstracts International Association for the Study of Pain, San Diego, CA.

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Depressor and tachypneic responses to chemical stimulation of the ventral respiratory group are reduced by ablation of neurokinin-1 receptor-expressing neurons.

Wang H, Germanson TP, Guyenet PG (2002) Depressor and tachypneic responses to chemical stimulation of the ventral respiratory group are reduced by ablation of neurokinin-1 receptor-expressing neurons. J Neurosci 22(9):3755-3764. doi: 10.1523/JNEUROSCI.22-09-03755.2002

Summary: The pre-Bötzinger complex is a region of the ventral respiratory group (VRG) in the brain. Injection of excitatory amino acids into this region can cause a variety of responses such as rapid breathing, hypotension, and elevated arterial pressure. The authors used SSP-SAP (Cat. #IT-11) to eliminate the neurokinin-1 receptor (NK-1r) positive neurons in the VRG to determine their role in control of respiration and arterial pressure. Intraparenchymal injection of 0.313 ng/50 nl SSP-SAP produced several abnormal respiratory effects in rats treated with excitatory amino acids. The results indicate that NK-1r positive neurons in the ventrolateral medulla play an important role in respiratory rhythm and blood pressure.

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Targeted lesions of Substance P receptor cells in L3-4 lumbar spinal cord severely impair male ejaculatory behavior.

Truitt WA, Stanton LA, Coolen LM (2001) Targeted lesions of Substance P receptor cells in L3-4 lumbar spinal cord severely impair male ejaculatory behavior. Neuroscience 2001 Abstracts 958.18. Society for Neuroscience, San Diego, CA.

Summary: Previously we demonstrated the existence of a spinothalamic pathway in the male rat where neural activation is specifically induced by ejaculation. This pathway consists of the parvocellular subparafascicular thalamic nucleus (SPFp) and the neurons projecting to the SPFp that are located in the lumbar spinal cord, specifically in laminae 10 and 7 of segments L3-L4. This neuron population coexpresses galanin (Gal) and substance P receptors (SPR). To test the hypothesis that these cells relay sensory information related to ejaculation, these neurons were lesioned by targeting the SPR with the neurotoxin SSP-saporin, a high affinity analogue of substance P conjugated to the ribosome inhibitor saporin. SSP-saporin (4 ng/µl) or an equal concentration of unconjugated saporin was injected bilaterally into L3-L4 region in either sexually experienced or naïve male Sprague Dawley rats (n=5 each). Sexual behavior was tested weekly, starting ten days following surgery. Following the sixth behavioral test, rats were sacrificed and spinal cord tissue was immunostained for GAL, SPR, or NeuN to assess specific and nonspecific tissue damage. In rats injected with unconjugated saporin, the Gal/SPR cell population was intact and sexual behavior was not altered. In contrast, in rats receiving SSP-saporin, Gal/SPR cell population was significantly decreased (77%) or absent. Moreover, SSP-saporin lesions severely disrupted ejaculatory behavior. Interestingly, display of mounts and intromissions remained intact. These results suggest that Gal/SPR cells in laminae 10 and 7 of L3-4 lumbar spinal cord may be essential for male ejaculatory behavior.

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Dose-dependent effects of intrathecal substance P-saporin and SSP-saporin.

Wiley RG, Kline IV RH, Lappi DA (2001) Dose-dependent effects of intrathecal substance P-saporin and SSP-saporin. Neuroscience 2001 Abstracts 281.11. Society for Neuroscience, San Diego, CA.

Summary: Selective destruction of lamina I dorsal horn neurons expressing the neurokinin-1 receptor (NK-1R) can attenuate responses to capsaicin injection and thermal hyperalgesia/mechanical allodynia in models of inflammatory, persistent or neuropathic pain. In the present study, we sought to determine the relationships between spinal intrathecal dose of substance P-saporin or the related toxin, SSP-saporin, the loss of NK-1R neurons and reduction of phase II formalin responses. Rats were injected intrathecally with 10 ul of either vehicle, 175 ng, 350 ng or 700 ng of SP-sap. Others were injected with either vehicle, 25 ng, 50 ng or 100 ng of SSP-sap. After 2 weeks, nocifensive behavior was scored for 90 min after a unilateral hindpaw injection of dilute formaldehyde. The amount of phase II nocifensive behavior from 20-90 min post injection was totaled for each animal. Rats were sacrificed and transverse lumbosacral spinal cord sections were stained for NK-1R using indirect immunoperoxidase technique. Digital micrographs of the superficial dorsal horn were captured and the number of pixels in the darkest intensity values were expressed as percent of the analysis area for each dorsal horn. Significant correlations were noted for dose vs dark pixel percentage and for dark pixel percentage vs phase II formalin behavior. The greater the toxin dose the greater the loss of NK-1R staining and the greater the attenuation of phase II formalin behavior. These results indicate that the toxin effects on pain behavior are proportional to the degree of loss of lamina I NK-1R expressing neurons.

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Focal inhibitory interneuron loss and principal cell hyperexcitability in the rat hippocampus after microinjection of a neurotoxic conjugate of saporin and a peptidase-resistant analog of substance P.

Martin JL, Sloviter RS (2001) Focal inhibitory interneuron loss and principal cell hyperexcitability in the rat hippocampus after microinjection of a neurotoxic conjugate of saporin and a peptidase-resistant analog of substance P. J Comp Neurol 436:127-152. doi: 10.1002/cne.1065

Usage: The authors used SSP-SAP (0.4 ng/10 nl; Cat. #IT-11).

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Read the featured article in Targeting Trends.

The molecular dynamics of pain control.

Hunt SP, Mantyh PW (2001) The molecular dynamics of pain control. Nature Rev Neurosci 2:83-91. doi: 10.1038/35053509

Summary: Over the last twenty years a great deal of progress has been made in the understanding of how pain is processed and transmitted by the CNS. The authors of this review highlight advances in systems neurobiology, behavioral analysis, genetics, and cell and molecular techniques. One method discussed is the use of the targeted toxin substance P-saporin (SP-SAP, Cat. #IT-07, also available with a more stable analog of substance P, SSP-SAP, Cat. #IT-11). This targeted toxin selectively lesions neurons expressing the NK1 receptor. Injection of SP-SAP into the spinal cord of rats dramatically attenuates the response to chronic pain stimuli, yet leaves acute pain response intact.

Related Products: SP-SAP (Cat. #IT-07), SSP-SAP (Cat. #IT-11)

Prevention and treatment of a spontaneous pain-like behavior following excitotoxic spinal cord injury (SCI) by ablation of neurons expressing the substance P receptor.

Yezierski RP, Yu CG, Wiley RG (2000) Prevention and treatment of a spontaneous pain-like behavior following excitotoxic spinal cord injury (SCI) by ablation of neurons expressing the substance P receptor. Neuroscience 2000 Abstracts 733.9. Society for Neuroscience, New Orleans, LA.

Summary: Intraspinal injection of the AMPA/metabotropic agonist quisqualic acid (QUIS) leads to the onset of excessive grooming behavior with an average onset time of 11-15 days. This behavior has been proposed as a model of chronic central pain following SCI (Yezierski et al., 1998). An important histological correlate of this behavior is a pattern of neuronal loss that includes the neck of the dorsal horn with sparing of the superficial laminae. Previously, we speculated that laminae I projection neurons might be part of the substrate responsible for the onset and progression of injury induced excessive grooming behavior. To test this hypothesis we evaluated the effects of the [Sar9,Met(OH)11]substance P-saporin (SSP-SAP) neurotoxin delivered directly to the dorsal surface of the cord in 'prevention' and 'treatment' protocols. Two groups of animals were injected with 125mM QUIS. One group received a treatment of SSP-SAP (10μl; 15 or 30ng/μl) for ten minutes immediately after QUIS injection. The second group was treated with 30ng/μl within 5 days after the onset of excessive grooming behavior. The results showed that only 30% (3/10) of the animals receiving SSP-SAP in the prevention protocol developed excessive grooming behavior compared to a norm of 80-90%, and those that developed the behavior had a delayed onset (18-26 days) and small skin area targeted for grooming. Animals receiving SSP-SAP treatment after the onset of grooming had significantly less grooming than animals not receiving treatment. Staining for the NK-1R receptor showed that animals with minimal grooming behavior had a significant decrease in lamina I staining with normal staining around the central canal and IML. In conclusion the results have shown that ablation of lamina I substance P receptive neurons significantly delayed the onset and progression of a spontaneous pain-like behavior induced by excitotoxic SCI.

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Focal hippocampal hyperexcitability after focal interneuron ablation in the rat by substance P-saporin.

Martin JL, Sloviter RS (2000) Focal hippocampal hyperexcitability after focal interneuron ablation in the rat by substance P-saporin. Neuroscience 2000 Abstracts 389.13. Society for Neuroscience, New Orleans, LA.

Summary: Hyperexcitability after prolonged seizures or head trauma may result from interneuron malfunction or loss; but a causal relationship is in doubt because global insults produce widespread brain damage and other effects. We have therefore sought to destroy interneurons selectively using stable Substance P-saporin (SSPsap; ATS); a neurotoxin internalized by SP receptor (SPR)-expressing neurons. Improved immunofluorescent methods revealed that most GABA-; parvalbumin (PV)-; and somatostatin (SS)-positive (+) cells of all hippocampal regions (dentate gyrus and areas CA1-CA3) are SPR+; but that granule cells; mossy cells; and CA1-3 pyramidal cells are not. Intrahippocampal injections of SSPsap or vehicle were made under urethane anesthesia in 3 sites (20nl/site) of the dorsal hippocampus of 6 male Sprague-Dawley rats/group. After 5-90 days; rats were blindly evaluated in two sites for CA1 pyramidal cell and dentate granule cell responses to perforant path stimulation (PPS). SSPsap-treated rats exhibited relatively normal responses in some sites; but pathophysiology at other sites that was virtually identical to that seen after prolonged PPS or kainate (multiple population spikes and paired-pulse disinhibition in response to 0.1-2.0Hz perforant path (PP) stimuli). Abnormal responses were observed at the earliest time tested (5 days); and at 90 days. Anatomical analysis revealed selective loss of SPR+; PV+; SS+; and GABA+ neurons; and survival of principal cells and extrinsic afferents. Importantly; "epileptic" pathophysiology was observed exclusively in areas of interneuron loss. These data indicate that the pathophysiology produced by status epilepticus or head trauma can be replicated focally by selective interneuron loss alone; and provide the first direct evidence that highly focal interneuron loss per se is capable of replicating "epileptic" disinhibition and hyperexcitability. In addition; the pathophysiology is restricted to the region of the affected interneuron somata; suggesting a highly localized influence of inhibitory interneurons. Supported by: NIH grant NS18201.

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ATS Poster of the Year Winner. Read the featured article in Targeting Trends.

Targeting neurokinin-1 receptor-expressing neurons with [Sar9, Met(O2)11] substance P-saporin.

Wiley RG, Lappi DA (1999) Targeting neurokinin-1 receptor-expressing neurons with [Sar9, Met(O2)11] substance P-saporin. Neurosci Lett 277(1):1-4. doi: 10.1016/s0304-3940(99)00846-0

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