References

Matchynski JJ, Lowrance S, Rossignol J, Puckett N, Derkorver N, Radwan J, Trainor K, Sandstrom M, Dunbar G (2009) Intracerebroventricular injections of mu-P-75 saporin can produce memory deficits without impairing motor deficits in a mouse model of Alzheimer’s disease. Neuroscience 2009 Abstracts 528.1/H34. Society for Neuroscience, Chicago, IL.

Summary: Intracerebroventricular injections of mu-P-75 saporin (Advanced Targeting Systems, San Diego, CA) effectively and efficiently destroys cholinergic neurons and creates memory deficits in mice, mimicking some of the key symptoms of Alzheimer’s disease. Early attempts to use mu-P-75 saporin in mice required a relatively high mean effective dose (ED50) of 3.6 µg in order to create behavioral deficits (Berger-Sweeney et al., 2001, The Journal of Neuroscience, 21: 8164-8173; Hunter et al, 2004, European Journal of Neuroscience, 19: 3305-3316). Recent advances in producing the saporin have lowered the ED50 to doses to 0.4 µg, although the resulting memory deficits are transient, and doses above 0.8 µg can cause motor deficits (Moreau et al., 2008, Hippocampus, 18: 610-622). In an effort to elucidate the behavioral effects of a higher (0.8 µg) dose, we gave bilateral intracerbroventricular injections of mu-P-75 saporin (n=6) or sterile phosphate buffered saline (n=3) into C57/BL6 mice and assessed their cognitive abilities on both a Morris water maze (MWM) and an object-recognition task, while monitoring their motor abilities using a rotarod task. Mice receiving the mu-P-75 saporin performed significantly worse than sham animals on an object recognition task and tended to have longer latencies and swim paths during the seven days of MWM testing. Importantly, no between-group differences were observed for latency to fall on the rotarod task. Collectively, these results suggest that the 0.8 µg dose of saporin is both safe and effective for mimicking AD-like memory deficits, without causing significant motor deficits.

Related Products: mu p75-SAP (Cat. #IT-16)

Laplante FP, Dufresne M, Lappi DA, Sullivan RM (2009) Depletion of cholinergic neurons in the nucleus accumbens impairs dopamine function in the prefrontal cortex in the rat. Neuroscience 2009 Abstracts 341.7/O16. Society for Neuroscience, Chicago, IL.

Summary: Studies of post mortem schizophrenic brains have revealed a selective loss of cholinergic interneurons, most pronounced in the ventral striatal region. We have previously shown in the rat, that a novel saporin immunotoxin coupled with an antibody targeting choline acetyltransferase (ChAT) and microinjected (0.5 _g/_l; 0.5 _l) into the nucleus accumbens (N. Acc) of adult rats, reduces the number of cholinergic neurons in N. Acc. by 40-50 %. Such lesions result in a markedly heightened response to the locomotor activating effects of amphetamine and impair prepulse inhibition of the acoustic startle response. We proposed that this local cholinergic deficit leads to a hyperresponsiveness in subcortical dopamine (DA) systems of relevance to schizophrenic symptomatology. Presently, we hypothesize that the same local cholinergic defect may trigger broader changes in cortical/subcortical networks, specifically prefrontal cortex (PFC) deficits in DA-mediated functions, also proposed in schizophrenia. Young adult male Srpague-Dawley rats were injected bilaterally in the N. Acc. as described above with either the cholinergic immunotoxin or vehicle. Two weeks later, they were trained in a working memory task dependent on PFC function, using the delayed alternation paradigm in the T-maze. Lesioned rats took significantly longer to reach criterion performance during training than controls. During testing, lesioned rats were significantly impaired in the percentage of correct arm choices across delay intervals, but especially with longer (40 sec) delays. The same animals were then implanted with voltammetric recording electrodes in the ventromedial PFC to examine the increases in in vivo extracellular DA release in response to a brief tail pinch stress. Lesioned rats showed a significantly reduced activation of the mesocortical DA system compared to controls. Taken together, the data suggest that reduction in the density of cholinergic neurons in the N. Acc also triggers deficits in prefontally-mediated function known to be under mesocortical DAergic regulation. This raises the possibility that ventral striatal cholinergic deficits may be causally linked to cortical/subcortical functional imbalances proposed to exist in schizophrenia.

Related Products: Anti-ChAT-SAP (Cat. #IT-42)

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.

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

Li A-J, Wang Q, Dinh TT, Ritter S (2009) Leptin-saporin injection into the arcuate nucleus lesions NPY/AGRP and POMC neurons and produces hyperphagia, obesity and changes in diurnal feeding patterns in rats. Neuroscience 2009 Abstracts 374.5/EE116. Society for Neuroscience, Chicago, IL.

Summary: Leptin-saporin (Lep-SAP), a conjugate of leptin with a ribosomal inactivating toxin, saporin (Advanced Targeting Systems), is a novel toxin designed to destroy leptin receptor-expressing cells selectively in vitro. However, its lesioning properties in vivo are currently unknown. Here, we injected Lep-SAP into the arcuate nucleus (Arc), to examine its effects on feeding behavior and on leptin receptor-expressing NPY/AGRP and POMC neurons in this area. Immunohistochemical studies showed unilateral injection of Lep-SAP into the Arc dramatically reduced numbers of NPY-Y1- and α-MSH- positive neurons compared to the contralateral side injected with SAP control. Real-time PCR revealed only 11-21% of Agrp and Pomc expression remaining in the Arc after Lep-SAP injection into this region. Rats injected bilaterally with Lep-SAP were unresponsive to central leptin administration and showed dramatic increases in feeding, body weight and light-phase feeding, compared pre-injection baseline. Two weeks after injection, total daily feeding was increased by 75%, light phase feeding by 359% and dark phase feeding by 33%. Control SAP injections did not produce these changes. Clock gene expression in homogenates of whole hypothalamus and liver were quantified at ZT 5-7. Bmal1 expression in hypothalamus and liver of Lep-SAP rats was decreased, while hepatic Per1 expression was increased compared to control. Results demonstrate that Lep-SAP effectively lesions Arc leptin receptor-expressing NPY/AGRP and POMC neurons in vivo, and that rats with this lesion are hyperphagic and obese, possibly due to enhanced hunger drive, lack of responsiveness to leptin and/or changes in circadian control of feeding behavior.

Related Products: NPY-SAP (Cat. #IT-28), Leptin-SAP (Cat. #IT-47)

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

Carvalho AF, Reyes AS, Van Bockstaele EJ (2009) The role of limbic norepinephrine in cannabinoid-induced aversion. Neuroscience 2009 Abstracts 449.3/V29. Society for Neuroscience, Chicago, IL.

Summary: The endocannabinoid system has been implicated in diverse physiological mechanisms including modulation of pain and analgesia, learning and memory and feeding, among others. Thus, targeting the cannabinoid system has risen to the forefront in the development of novel treatments for a number of pathophysiological processes. Consistent with this, agonists of the cannabinoid receptor type 1 (CB1R) have been successfully used in the treatment of severe anorexia in patients with AIDS and in alleviating nausea and vomiting in patients undergoing chemotherapy. However, significant side effects have been observed in clinical trials raising concerns regarding the potential clinical utility of cannabinoid-based agents. Disturbances in mood and affect, including paranoia, anxiety and nervousness, have been reported in patients. Understanding the neural circuits and neurochemical substrates impacted by cannabinoids will provide a better means of gauging their actions within the central nervous system that contribute to the expression of unwanted side effects. We have previously shown an increase in anxiety-like behaviors in rats receiving repeated administration of cannabinoid agonists. This increase in anxiety was accompanied by increases in indices of noradrenergic activity. In the present study, we investigated whether norepinephrine in the limbic forebrain of rats is required for cannabinoid-induced aversion using an immunotoxin lesion approach combined with behavioral analysis using a place conditioning paradigm. Male Sprague Dawley rats received bilateral injections of a ribosomal toxin, saporin (SAP) conjugated to an antibody that specifically recognizes the enzyme dopamine-beta-hydroxylase (DSAP), into the limbic forebrain. Control rats received saporin alone. As previously reported, administration of the synthetic cannabinoid receptor agonist, WIN 55,212-2 (3.0mg/kg), induced aversion in a place conditioning paradigm in SAP-only treated rats. The rats’ spatial memory was also evaluated using the Morris Water Maze. Depletion of norepinephrine using DSAP in specific limbic regions impaired cannabinoid-induced aversion to WIN 55,212-2 without affecting learning and memory processes. Taken together, noradrenergic projections to the limbic forebrain may be critical in the manifestation of aversive behaviors associated with cannabinoid agonist exposure.

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

Freiria-Oliveira AH, Blanch GT, De Paula PM, Colombari E, Menani JV, Colombari DS (2009) Role of A2 noradrenergic neurons and angiotensinergic mechanisms on hypotension induced by hemorrhage. Neuroscience 2009 Abstracts 467.18/DD70. Society for Neuroscience, Chicago, IL.

Summary: The A2 catecholaminergic neurons in the commissural subdivision of the nucleus tractus solitarii (cNTS) are activated by hemorrhage. However, the role of these neurons on the cardiovascular adjustments to hemorrhage is not fully understood. In the present study we investigated the effects of A2 noradrenergic neuron lesion alone or combined with the blockade of angiotensinergic mechanisms on the recovery of blood pressure after hemorrhage. Male Holtzman rats (280-320 g) anesthetized with ketamine combined with xylazine were submitted to lesions of dopamine-beta-hydroxilase (DβH)-containing neurons in the cNTS achieved with injections of anti-DβH-saporin (12.6 ng/60 nl, n=6-8) or sham lesions (injection of immunoglobulin-G-saporin, 12.6 ng/60 nl, n=6). Changes in blood pressure to hemorrhage were tested 30 days after lesions. Immunohistochemistry for tyrosine-hydroxilase was performed to confirm the efficacy of DβH neuron lesion in the cNTS. Two days before tests, femoral artery and vein were cannulated under ketamine and xylazine anesthesia. Hemorrhage consisted in four blood withdrawals (2 ml/300 g body weight, every 10 min) in conscious rats. Immediately after the 4th blood withdrawal, the hypotension was similar in A2-lesioned and sham-lesioned rats (-62 ± 7 mmHg and -73±7 mmHg, respectively). However, A2-lesioned rats rapidly (20 min) recovered from hypotension (-7±2 mmHg), while sham rats did not completely recover from hypotension until the end of experiment (60 min after the 4th blood withdrawn, -20±3 mmHg). The pre-treatment with losartan (angiotensin type 1 receptor antagonist, 10 mg/kg of body weight, iv) impaired the recovery of blood pressure by A2-lesioned rats (-29 ± 4 mmHg and -28 ± 3 mmHg, 20 and 60 min after the 4th blood withdrawal). In sham rats, the treatment with losartan also reduced the partial recovery of blood pressure at the end of the test (-39±6 mmHg, vs. sham control: -20±3mmHg), however, losartan did not affect the hypotension 20 min after the 4th blood withdrawal (-30± 6 mmHg vs. sham control: -35 ± 9 mmHg). The results suggest that A2 noradrenergic neuron lesion in the cNTS facilitates the recovery of hypotension after hemorrhage, probably increasing the action of angiotensinergic mechanisms.

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

Macleod JE, Ackerman CM, Bucci DJ (2009) Contributions of the medial prefrontal cortex to negative occasion setting. Neuroscience 2009 Abstracts 477.2/FF61. Society for Neuroscience, Chicago, IL.

Summary: The medial prefrontal cortex of rats has a role in many aspects of cognitive function, and especially forms of inhibitory learning. Recent research has revealed heterogeneous functions of the prelimbic (PL) and infralimbic (IL) regions of the medial prefrontal cortex in modulating response inhibition. In a recent study, we tested the effects of separate neurotoxic lesions of the PL or IL in a serial feature negative discrimination paradigm (negative occasion setting). Rats received daily training sessions consisting of 16 trials: on 4 trials in each session, a tone was presented and followed by food reward; on the remaining trials, the tone was preceded by a visual stimulus and not reinforced. Our results indicate that PL but not IL is necessary for learning the discrimination. A second study was conducted to investigate the effects of these lesions on rats that were first extensively trained in this task. We found that rats that had been trained for 30 days prior to receiving PL or IL lesions were still able to perform the task as well as controls. Therefore, PL lesions disrupt acquisition but not performance of a serial feature negative discrimination. This same task has been used in our laboratory to investigate the effects of nicotine on learning. We have shown that nicotine-treated rats exhibit greater discrimination between the two trial types as evidenced by less frequent responding during non-reinforced trials, and learn the discrimination in fewer sessions than control rats. In addition, rats receiving nicotine showed an increase in rearing behavior during the presentation of the light, suggesting nicotine enhanced attention to the visual stimulus. One possible critical site of action for nicotine’s effects is the medial prefrontal cortex. Research in other laboratories utilizing other training procedures suggest that cholinergic activity in the medial prefrontal cortex is critical for attending to behaviorally relevant stimuli, and have implicated the rat PL in visual attention as well as inhibiting prepotent, goal oriented responses. We investigated the contribution of the cholinergic PL to learning the serial feature negative discrimination task by training rats that had received infusions of 192-IgG-saporin into PL to remove cholinergic input from the basal forebrain. No differences between control and lesion rats were observed. Taken together, the results suggest that PL is necessary for acquisition of a serial feature negative discrimination, although the basal forebrain cholinergic input into this region is not required to sufficiently learn the task.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Lemons LL, Wiley RG (2009) Role of galanin receptor-expressing dorsal horn neurons in operant nocifensive responses. Neuroscience 2009 Abstracts 170.18/X20. Society for Neuroscience, Chicago, IL.

Summary: Selective destruction of galanin receptor-expressing dorsal horn neurons using the targeted cytotoxin, galanin-saporin (gal-sap), reduced reflex nocifensive hotplate responses, particularly at 44° C (see adjacent poster). The antinociceptive effect of gal-sap was additive with morphine (5 mg/kg, s.c.) in reducing hotplate responses. While these findings are provocative, inferences about analgesia also require information on cerebral processing of nociceptive information, such as obtained from operant nocifensive responses. We therefore sought to determine the effects of lumbar intrathecal gal-sap on nocifensive operant responses. Thirteen Long Evans female rats were injected with either 500 ng gal-sap or 500 ng blank-sap and tested on the escape test at several temperatures. The escape task consists of a two-chambered box; one side is dark with a thermal floor while the other side is brightly lit with a room temperature shelf. Gal-sap treated rats escaped from the thermal plate to the escape shelf less than controls. The difference was particularly striking at 25°, 38°, 44°, 45°, and 47°C. Morphine effects on escape responses was tested at 44°C. Thirty minutes before testing, rats were injected subcutaneously with either 0, 0.5, 1.5 or 4.5 mg/kg morphine. The testing was done over four days such that every rat was tested at each dosage in a counterbalanced fashion. 1.5mg/kg of morphine significantly reduced the escape duration of the blank-sap control rats, but not the gal-sap rats. The 4.5mg/kg dosage completely eliminated escape responding in both control and gal-sap rats. In summary, Gal-sap rats showed reduced nocifensive reflex responding on the hotplate tests, which was further reduced in essentially additive fashion by 5mg/kg of morphine. The Gal-sap treated rats also showed reduced escape behaviors at 44°C in the operant escape test, but were less sensitive to 1.5 mg/kg of morphine than the control rats. These results differ from the effects of NPY-saporin and dermorphin-saporin, cytotoxins also targeted at dorsal horn interneurons, suggesting that selective destruction of galanin receptor-expressing superficial dorsal horn neurons is analgesic and that galanin-receptor-expressing dorsal horn interneurons play a unique role in nociceptive processing.

Related Products: Galanin-SAP (Cat. #IT-34)

Jaime S, Perez Cordova MG, Hernandez S, Colom L (2009) Immunolesions of medial septal GABAergic neurons. Neuroscience 2009 Abstracts 241.8/I15. Society for Neuroscience, Chicago, IL.

Summary: Epilepsy is a neurodegenerative condition characterized by spontaneous recurrent seizures that are triggered by excessive electrical activity due to changes in neurological functions. One of the most common forms of epilepsy is Temporal Lobe Epilepsy (TLE) in which seizures originate in limbic structures as hippocampal and/or para-hippocampal areas. Principal cell (i.e. pyramidal cells) activity is indirectly regulated by rhythmic inputs from GABAergic neurons in the septal region of the basal forebrain which selectively innervate inhibitory hippocampal interneurons. In previous studies, using the pilocarpine model of TLE, we have demonstrated that the septum plays an antiepileptic role and that medial septum GABAergic neurons degenerate in the epilepsy process. Thus, damage of medial septum GABAergic neurons may contribute to epileptogenesis. The purpose of this study is to investigate the role of medial septum GABAergic neurons in excitability control and epileptic activity generation. For this purpose, anti-GAT1-SAP (3µL at 325ng/µL) was stereotaxically injected in the medial septum of Sprague Dawley male rats to selectively destroy this neuronal population and investigate the subsequent functional changes. Analysis was performed using stereological approaches which revealed a significant reduction in cell count between treated (anti-GAT1-SAP) and saline-injected control rats (8591.38±941.65 and 25609.87±407.73 respectively; (Student’s t-test; p<0.05). In conclusion, our preliminary results show that the single injections of anti-GAT1-SAP selectively lesions most of the medial septum GABAergic neurons, providing a powerful tool to study the role of these neurons in the control of hyperexcitability states. Studies underway involve the investigation of the functional alterations produced by the selective destruction of MS GABAergic neurons.

Related Products: GAT1-SAP (Cat. #IT-32)

Datta S, Chatterjee K, Kline IV RH, Wiley RG (2009) CCK receptor- expressing dorsal horn neurons: Role in pain and morphine analgesia. Neuroscience 2009 Abstracts 265.13/Z37. Society for Neuroscience, Chicago, IL.

Summary: Spinal intrathecal cholecystokinin (CCK) has anti-opiate activity, and the CCK antagonist, proglumide potentiates opiate analgesia. In the present study, we sought to determine the effects of selectively destroying CCK receptor-expressing lumbar dorsal horn neurons using the targeted cytotoxin, CCK-saporin on reflex and operant nocifensive responses to heat, and on the actions of systemic morphine and naloxone. Exp. 1: Adult, female rats were injected into the lumbar CSF with either 1500 ng of CCK-sap (n=7) or blank (control nonsense peptide)-saporin (n=6). Exp. 2: rats were pre-injected intrathecally with 1 ug of proglumide (CCK antagonist) followed by 1500 ng CCK-sap (n=4) or only CCK-sap (1500 ng; n=4). Rats were then tested on the hotplate at 44°C and 47°C and on an operant thermal preference task (TPT) using a shuttle box where the floor on one side was 15°C and the other 45°C. Morphine was tested in the TPT using 0, 0.5, 1.5 and 2.5 mg/kg s.c. 4-8 weeks post-toxin. Naloxone (0 vs 0.8 mg/kg s.c) was also tested in the TPT. In Exp. 1, the CCK- sap group showed decreased hotplate reflex responses, but decreased time on the 45°C side in the TPT. In Exp. 2, CCK-sap only rats also showed greater heat aversion in the TPT. In both Exps, CCK-sap groups demonstrated greater heat aversion (less analgesia) than either control group after morphine in the TPT. After naloxone, both control groups, but not the CCK-sap rats, showed increased heat aversion (hyperalgesia). We interpret these results as showing that selective destruction of CCK receptor- expressing superficial dorsal horn neurons increases nocifensive reflex responses to aversive heat and produces thermal hyperalgesia while decreasing the effects of both morphine and naloxone suggesting a complex role for CCK receptor-expressing dorsal horn neurons in modulation of nociception and opiate drug action.

Related Products: CCK-SAP (Cat. #IT-31)