targeted-toxins

King CD, Baker B, Gu JG, Vierck CJ, Yezierski RP (2004) Behavioral evidence for a capsaicin-sensitive inhibitory pathway (CSIP): A novel modulatory role for substance P. Neuroscience 2004 Abstracts 292.18. Society for Neuroscience, San Diego, CA.

Summary: Exposure to noxious thermal stimulation or application of capsaicin cream causes the release of SP from capsaicin-sensitive primary afferent terminals that activate neurokinin-1 receptor (NK1R) expressing neurons in the superficial dorsal horn. Recent evidence suggests the existence of a capsaicin-sensitive inhibitory pathway (CSIP), a novel inhibitory mechanism that involves NK1R expressing neurons in laminae III-V. To determine the functional significance of these NK1R expressing neurons, substance P-saporin neurotoxin (SP-SAP) was used to ablate NK1R neurons in the superficial laminae. Elimination of the NK1R neurons in this region made it possible to evaluate the modulatory effects of NK1R expressing inhibitory neurons in deeper laminae. Reflexive responses were evaluated in rats during a 10-minute trial at 44.5°C before (pre-) and 14 days after (post-) intrathecal injection of 350ng SP-SAP. Testing conditions included: 1) baseline; 2) hindpaw application of 1% capsaicin cream; and, 3) intrathecal injection of the NK1 antagonist CP-97,345 following hindpaw application of 1% capsaicin cream. In normal rats, hindpaw application of capsaicin produced thermal hyperalgesia. In contrast, application of capsaicin produced a hypoalgesia in the same rats after treatment with SP-SAP. The capsaicin-induced hyperalgesia in normal rats was blocked by CP-97,345. The antagonist also blocked the capsaicin-induced hypoalgesia in SP-SAP rats. In conclusion, it is suggested that substance P activates inhibitory interneurons in the deep dorsal horn. The inhibitory effect initiated by substance P on pain transmission neurons represents a novel role of substance P in the spinal processing of nociceptive information.

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

Abe T, Shimoda T, Sugiyo S, Ohshita N, Takao T, Takemura M (2004) Altered effects of systemic bicuculline on intensity-dependent c-Fos expression in rats ablated with NK-1 receptor-bearing neurons in the trigeminal caudal nucleus. Neuroscience 2004 Abstracts 294.12. Society for Neuroscience, San Diego, CA.

Summary: In rats pretreated with saporin conjugated to substance P (SP-Sap: 5 μM, 5 μl) into cisterna magna, the numbers of neurons-immunoreactive for NK-1 receptor (NK-1R) in laminae I and III of trigeminal caudal nucleus (Vc) were significantly decreased compared with rats similarly treated with saline (Sal; 5 μl) or blank-saporin (Bl-Sap; 5 μM, 5 μl). We examined the effects of selective ablation of NK-1R-bearing neurons and systemic administration of bicuculline (2 mg/kg, i.p.) on the expression of c-Fos induced 2 hr after electrical stimulation (5 Hz, 5 ms) of the trigeminal ganglion (TG) at low (0.1 mA) and high intensities (1.0 mA). In Sal- or Bl-Sap treated rats, 10 min stimulation at 0.1 and 1.0 mA of the TG induced c-Fos-immunoreactive (c-FosIR) cells in the ipsilateral superficial layers of the Vc (VcI/II) in a intensity-dependent manner. In rats treated with SP-Sap, and stimulated at 1.0 mA but not at 0.1 mA, the numbers of c-FosIR cells in the Vc were significantly decreased compared to Sal- or Bl-Sap treated rats. In Sal- or Bl-Sap treated rats preadministed with bicuculline and stimulated at 0.1 mA and 1.0 mA, the numbers of c-FosIR cells in VcI/II were significantly increased and decreased, respectively. However, in SP-Sap treated rats preadministered with bicuculline and stimulated at 0.1 mA and 1.0 mA, the numbers of c-FosIR cells in VcI/II were significantly increased. These results indicate that NK-1R-bearing neurons in the Vc have pivotal role in the modality and/or intensity-dependent sensory (nociceptive) processing in the TSN through GABAA receptors.

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

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.

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

Wei F, Zou S, Robbins MT, Ren K, Dubner R (2004) Mu-opioid receptor-expressing neurons in the nucleus reticularis gigantocellularis contribute to descending facilitation during the development of inflammatory pain. Neuroscience 2004 Abstracts 297.3. Society for Neuroscience, San Diego, CA.

Summary: We have previously shown that nucleus reticularis giangocellularis (NGC) is involved in descending facilitation of inflammatory hyperalgesia. The cellular mechanisms of descending facilitation from the NGC are unknown. The targeted destruction of the mu-opioid receptor-containing neurons in the rostral ventromedial medulla (RVM) by a dermorphin-saporin conjugate prevents nerve injury-induced hyperalgesia in rats (Porreca et al., J. Neurosci. 21:5281, 2001). We examined the effects of selective deletion of the mu-opioid receptor-expressing neurons in the sub-regions of RVM on nocifensive behaviors in rats. After microinjection of dermorphin-saporin conjugate (1.5 pmol/500 nl) into the RVM, there were no changes in baseline thermal and mechanical sensitivity to noxious stimuli. However, the injection of dermorphin-saporin conjugate into bilateral NGC (n=7) significantly attenuated the thermal hyperalgesia and mechanical allodynia at 30 min to 1 d after hindpaw inflammation produced by injection of complete Freund’s adjuvant, compared to sham (blank-saporin or dermorphin) groups (n=3-6). The lesion of the nucleus raphe magnus (NRM) (n=3) only slightly reduced hyperalgesia at 3 h after inflammation. The loss of NGC mu-opioid receptor-containing neurons also decreased nocifensive behaviors only in phase II of the formalin model. In contrast, NRM lesions were without an effect on formalin-induced phase I/II responses. These findings indicate that selective deletion of the mu-opioid receptor-containing neurons in the nucleus reticularis giangocellularis attenuates inflammatory hyperagesia and allodynia.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)

Fargo KN, Sengelaub DR (2004) Prevention of depletion-induced motoneuron dendritic atrophy requires testosterone effects on target musculature. Neuroscience 2004 Abstracts 310.10. Society for Neuroscience, San Diego, CA.

Summary: Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) in male rats project to the penile muscles bulbocavernosus (BC) and levator ani (LA). These motoneurons share a midline location, have intermingled somata, extensive dendritic overlap, and common afferents, and organize coordinated contractions of the penile musculature. Unilateral depletion of BC-projecting motoneurons causes marked dendritic atrophy in contralateral BC-projecting motoneurons, and this atrophy can be prevented with testosterone (T) treatment. In this experiment, we test whether the depletion-induced atrophy is related to the innervation of homotypic muscles. BC-projecting motoneurons were depleted by unilateral injection with saporin conjugated to the cholera toxin B subunit (SAP); some animals were simultaneously treated with T. Four weeks later, a period demonstrated to be sufficient to observe dendritic atrophy in remaining motoneurons, HRP conjugated to the cholera toxin B subunit (BHRP) was injected into the ipsilateral LA. SAP injection into the BC muscle killed over 40% of ipsilateral SNB motoneurons. Dendritic length in LA-projecting motoneurons was reduced by almost 60%. Because the SAP-induced depletion of motoneurons and the resultant dendritic atrophy occurred across motor populations, this result indicates that the dendritic atrophy we have observed previously is not restricted to motoneurons projecting to homotypic muscles. In previous studies, prevention of dendritic atrophy by T treatment in BC-projecting motoneurons was accompanied by a marked hypertrophy of the BC muscle. In the present experiment, T treatment failed to prevent dendritic atrophy in LA-projecting motoneurons, and further did not result in hypertrophy of the LA ipsilateral to the SAP-injected BC. Thus, it appears the neuroprotective effect of T treatment on SNB motoneurons may be dependent on T effects in the target musculature.

Related Products: CTB-SAP (Cat. #IT-14)

Broussard JI, Sarter M, Givens B (2004) Neuronal correlates of signal detection in rat posterior parietal cortex. Neuroscience 2004 Abstracts 331.6. Society for Neuroscience, San Diego, CA.

Summary: The posterior parietal cortex (PPC) has been shown to be involved in the attentional processing of visual stimuli. Recent evidence has indicated that neuronal activity in the PPC is increased during the detection of signals, and this activation is modulated by visual distractors. We tested the hypothesis that detected signals are associated with increased PPC unit activity. Animals were trained in a sustained attention task using signal and nonsignal trials. After training to criterion (>75% accuracy), we implanted moveable stereotrodes into the PPC. A visual distractor was presented in a block of trials during testing sessions and effects on performance and single unit activity were examined. We also evaluated the effects of varying signal duration on performance and single unit activity. PPC neurons (39/111) exhibited a significantly greater response during signal trials than during nonsignal trials. The presentation of visual signals produced a robust increase in neuronal activity prior to the performance of a hit, but not prior to a miss, both of which required a lever press. Analysis of signal duration indicated that shorter signals resulted in fewer hits. PPC neurons became active when the signal was accurately detected, independent of signal duration. Shorter signals activated the PPC on fewer trials, which was associated with a lower likelihood for detection. The visual distractor reduced both the signal-driven unit activity and the relative number of hits. These findings suggest that activation of the PPC is associated with the detection of visual signals. We are currently investigating the effects of local cholinergic deafferentation (via 192 IgG saporin) on signal driven neuronal activity in the PPC. These studies will elucidate the contribution of basal forebrain cholinergic innervation to attentional processing in the PPC.

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

Saenz C, Dominguez R, de Lacalle S (2004) Estrogen supports structural plasticity of the basal forebrain cholinergic system in vivo. Neuroscience 2004 Abstracts 72.11. Society for Neuroscience, San Diego, CA.

Summary: It is known that estrogen (E2) modulates the structural plasticity of a variety of neurons, involving the activation of second messenger systems. We have previously described a strong E2-induced outgrowth in cholinergic neurons in vitro, and in the present study we follow up those results and examine E2’s ability to enhance cholinergic arborization in vivo, under several conditions. Twenty F344 female rats were used, 10 of them gonadectomized. All the rats received a unilateral lesion (200 nl of 192 IgG-saporin) into the left HDB, and a month later were randomly assigned to receive E2 or placebo via s.c. pellets for 60 days, at which point the rats were sacrificed, the brains prepared for histology and series of sections stained with an antibody against p75NTR. Sections were carefully matched across individuals, 10 neurons selected from both lesioned and intact HDB, and photographed. Neurons were chosen from the same area in all cases, located in the periphery of the HDB, where the neuritic arborization could be easily identified. Image analysis was performed using Metamorph software, on a predetermined set of parameters. Each image was the result of a stack of photographs taken at 2 µm intervals through the depth of the section. We compared mean neurite number per neuron and total neurite length per neuron, and found that in the healthy cholinergic neurons (control side), E2 contributed to a significant increase in neurite length and number. By contrast, no effect was found on cholinergic neurons from the lesioned side, showing that E2 cannot reverse the neuronal degeneration induced by the immunotoxin. These results are important in that they provide additional support to the hypothesis that E2 may be beneficial in preventing cholinergic degeneration, but no longer useful once neuronal damage has occurred.

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

Hawkes CA, Kar S (2004) Loss of basal forebrain cholinergic neurons by 192 igG-Saporin induces increased IGF-II/M6P receptor expression in select brain areas. Neuroscience 2004 Abstracts 92.1. Society for Neuroscience, San Diego, CA.

Summary: Alzheimer’s disease (AD) is characterized neuropathologically by the presence of extracellular amyloid plaques, intracellular neurofibrillary tangles and neuronal loss in selected brain areas, including basal forebrain cholinergic neurons, which project to the hippocampus and neocortex. Increasing evidence supports a role of the endosomal-lysosomal (EL) system in the pathophysiology of AD. A key component of the EL system is the insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor, a single transmembrane domain glycoprotein which functions in the intracellular trafficking of lysosomal enzymes, and in the internalization of extracellular IGF-II and M6P-containing ligands. However, very little is known about the functional significance of this receptor in the brain. We examined expression of the IGF-II/M6P receptor and other markers of the EL system, at different time points following bilateral i.c.v. injection of 192 IgG-saporin. 192 IgG-saporin produced an almost complete loss of ChAT-positive neurons in the basal forebrain, as well as fibers in the hippocampus and frontal cortex, while striatal cholinergic neurons were unaffected. Western blotting and immunocytochemistry results indicate an upregulation of IGF-II/M6P receptor levels in the septum and frontal cortex. A modest increase was also observed in cathepsin D levels. The level of other EL markers, such as Rab5 and LAMP1, showed varied temporal and spatial changes. These results suggest that brain areas innervated by basal forebrain neurons, respond differently to the loss of cholinergic input and that elements of the EL system may be involved in cholinergic degeneration/compensatory responses of surviving neurons.

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

Kar S, Hawkes C, Jhamandas JH (2004) Selective loss of basal forebrain cholinergic neurons by 192 IgG-saporin induces activation of glycogen synthase kinase-3β activity. Neuroscience 2004 Abstracts 92.2. Society for Neuroscience, San Diego, CA.

Summary: Glycogen synthase kinase-3β (GSK-3β) is a multifunctional enzyme involved in a variety of biological events including development, glucose metabolism and cell death. Its activity is negatively regulated by phosphorylation of Ser9 and upregulated by Tyr216 phosphorylation. Activation of GSK-3β induces apoptosis in a variety of cultured neurons and the inhibitory control of its activity by Akt kinase is one of the best characterized cell survival signaling pathways. In the present study, the cholinergic immunotoxin 192-IgG saporin was used to address the potential role of GSK-3β in the degeneration of the basal forebrain cholinergic neurons which are preferentially vulnerable in Alzheimer’s disease (AD) brain. Our results show that GSK-3β colocalizes with a subset of the forebrain cholinergic neurons and that loss of these neurons is accompanied by a transient decrease in phospho-Akt and phospho-Ser9 GSK-3β levels in the basal forebrain, hippocampus and the cortex. Neither total Akt, GSK-3β, nor phospho-Tyr216 GSK-3β levels were significantly altered in the aforesaid brain regions of treated animals. These results provide the very first evidence that increased GSK-3β activity is associated with in vivo degeneration of the forebrain cholinergic neurons and thus may be involved in the loss of these neurons as observed in AD brains.

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

Kolasa K, Parsons D, Conger K, Harrell LE (2004) Neurotrophic modulation of cholinergic denervation and hippocampal sympathetic ingrowth following immunolesioning with 192 IgG-saporin. Neuroscience 2004 Abstracts 92.9. Society for Neuroscience, San Diego, CA.

Summary: Injection of specific cholinotoxin, 192 IgG-saporin into the medial septum (MS)of rat induces not only a selective cholinergic denervation of hippocampus (CD),but an ingrowth of peripheral sympathetic fibers, originating from the superior cervical ganglion,into the hippocampus (HSI).A similar process,in which sympathetic noradrenergic axons invade hippocampus,may also occur in Alzheimer’s disease(AD). The severity of cognitive decline in AD patients has been linked to multiple factors including cholinergic and neurotrophic factors and their receptors, which undergo selective alterations throughout the progression of AD.It is known that the sites of neurotrophin synthesis in the septo-hippocampal system are predominantly hippocampal neurons. By using 192 IgG-saporin we have been able to mimic some of the cardinal features of AD e.x.cholinergic denervation and hippocampal sympathetic ingrowth and study their effect on growth factors in dorsal hippocampus. Thus,12 weeks after injection of 192 IgG-saporin we measured neurotrophic protein and mRNA expression using Western blot and RT-PCR techniques,respectively. Choline acetyltransferase activity(ChAT)and norepinephrine(NE) concentration was also detected.There was no change in NGF,BDNF,NT3,GDNF mRNA expression,but we have found significant decrease in 240 bp and increase in 328 bp of persephin mRNA expression in CD, and “normalization” in HSI group. No significant alteration was found in NGF and persephin protein expression, but significant decrease in mature form of BDNF protein expression was found in CD, with “normalization”in HSI group.Results of the study suggest that growth factors are affected by cholinergic denervation and may play an important role in regulation and development of HSI,which might be a beneficial phenomenon for restoration of at least some cognitive function.

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