targeted-toxins

Mazei MS, Wiley RG, Deutch AY (2000) Intracortical injection of DBH-saporin targets noradrenergic axons in the medial prefrontal cortex of the rat. Neuroscience 2000 Abstracts 639.8. Society for Neuroscience, New Orleans, LA.

Summary: The medial prefrontal cortex (mPFC) is innervated by both dopaminergic and noradrenergic neurons. While the densities of the two types of catecholamine axons are different in subregions of the mPFC, axons of both types are present in most of the mPFC. The goal of this study was to selectively lesion noradrenergic axons in the rat mPFC while sparing dopaminergic axons, using microinjection of the immunotoxin dopamine β-hydroxylase (DBH)-saporin. DBH-saporin (10- 100 ng/μ1) was unilaterally injected into three brain regions, the mPFC, caudate-putamen (CP) and cerebellum. Rats were sacrificed 2-10 days post-injection and the lesion was characterized by tyrosine hydroxylase (TH), DBH, and norepinepherine transporter (NET) immunohistochemistry. DBH-saporin administration resulted in discrete lesions. There appeared to be a decrease in the number of immunoreactive (ir) axons after DGH-saporin infusion into the mPFC: surviving axons were swollen and dystrophic. However, the effect of DBH-saporin appears more pronounced when examining DBH-ir than with NET-ir. Intrastriatal injections of DBH-saporin did not decrease TH-ir, suggesting that dopaminergic axons were spared. Since DBH-saporin acts by entry into DBH-containing vesicles, a longer time course may be necessary to observe loss of the NET marker than DBH. Studies are in progress to examine this possibility.

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

Holguin A, Armstrong CB, Twining CM, Milligan ED, Hansen MK, McGorry M, O’Connor KA, Quan N, Martin D, Lappi DA, Maier SF, Watkins LR (2000) Anatomical evidence for glial activation after intrathecal lumbosacral HIV-1 glycoprotein; gp120-induced allodynia. Neuroscience 2000 Abstracts 733.4. Society for Neuroscience, New Orleans, LA.

Summary: Intrathecal (IT) HIV-1 glycoprotein, gp120: (a) produces thermal hyperalgesia & low threshold mechanical allodynia, and (b) increases interleukin-1β (IL1β) protein levels in lumbosacral (LS) spinal cord tissue & surrounding cerebrospinal fluid (CSF). Activated astrocytes & microglia (glia) release IL1β in response to gp120, and IT IL1 receptor antagonist or glial metabolic inhibitors prevent IT gp120-induced allodynia and thermal hyperalgesia. We determined whether IT gp120 produces glial activation and increased expression of glial IL1β as well as allodynia. LS spinal cord was collected 1.5 & 3 hrs after IT gp120 injection & verification of allodynia for immunocytochemistry (ICC) & in situ analysis of IL1β protein & mRNA. ICC for glial activation markers was performed 4,8 & 18 hrs after IT gp120 in LS & cervical spinal cords, as upregulation of these markers is delayed relative to behavioral changes. IT gp120 produced allodynia & increased IL1β protein ICC expression in LS spinal white (astrocytes) & gray matter (cells not identifiable) at 1.5 but not 3 hrs after injection. Increases in in situ IL1β mRNA were not detected. RT-PCR analysis of IL1β mRNA is underway. Glial activation (ICC) was observed in LS tissue 8 & 18 hrs after IT gp120. We are examining IT gp120 allodynia & hyperalgesia after an IT microglia-specific toxin (Saporin-linked Mac-1 antibody) injection that disrupts glial function. ICC procedures will verify Mac-1 Saporin microglial toxicity.

Related Products: Mac-1-SAP mouse/human (Cat. #IT-06)

Prakash N, Cohen-Cory SC, Frostig RD (2000) NGF-induced rapid functional plasticity in the adult rat somatosensory cortex is mediated by fibers originating in the basal forebrain cholinergic system. Neuroscience 2000 Abstracts 722.6. Society for Neuroscience, New Orleans, LA.

Summary: We have previously demonstrated, by using intrinsic signal optical imaging in vivo, that topical application of nerve growth factor (NGF) to the somatosensory cortex of an adult rat augments the functional representation of a whisker, within minutes after NGF application (Prakash et al. Nature 381:702-6, 1996). In addition, we have shown that the NGF receptor, TrkA is found on fibers projecting to the cortex. We have now tested the hypothesis that these TrkA-positive fibers originate in basal forebrain cholinergic system (BFCS) and that NGF augments the size and amplitude of a cortical representation by enhancing the release of ACh from these fibers. To this end we demonstrate: 1) that BFCS fibers indeed express TrkA receptors by co-localizing TrkA and ChAT immunostaining to single cortical fibers; 2) that removal of these cortical BFCS-fibers by injection of a specific cytotoxin, 192 IgG-saporin, prevented the NGF-induced augmentation; and 3) that topical application of the ACh agonist carbachol induced a rapid augmentation of the whisker functional representation similar to the one observed with NGF. Thus, these results firmly support our hypothesis that NGF-induced effects on cortical functional representations are mediated by the BFCS projection fibers and provide a mechanism for NGF-induced rapid plasticity in vivo.

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

Khasabov SG, Rogers SD, Mantyh PW, Simone DA (2000) Responses of spinal dorsal horn neurons to capsaicin following intrathecal pretreatment with substance p-saporin toxin. Neuroscience 2000 Abstracts 635.13. Society for Neuroscience, New Orleans, LA.

Summary: Intrathecal (i.t.) application of the cytotoxic substance P-saporin (SP-SAP) conjugate is internalized by dorsal horn neurons expressing the SP receptor (SPR) and results in loss of SPR-expressing neurons. Loss of SPR+ neurons attenuates the nocifensive behavior and hyperalgesia produced by intraplantar injection of capsaicin (CAP). Here we determined the effect of SP-SAP on CAP-evoked excitation and sensitization of dorsal horn neurons to heat and mechanical stimuli. Separate groups of rats were given i.t. injection of vehicle (VEH) or SP-SAP (5´10-6mM in 10ml) 10 or 30 days prior to electrophysiological experiments. Extracellular recordings were obtained from nociceptive dorsal horn neurons classed as high threshold (HT) or wide dynamic range (WDR). Responses to mechanical (von Frey monofilaments) and heat (35°C-51°C) stimuli were obtained before and after injection of 10 mg CAP into the receptive field. In VEH-treated animals, CAP produced an intense activation of HT and WDR neurons with a mean peak discharge rate of 52.2±11.2 Hz. In addition, the mean number of impulses evoked by mechanical stimuli increased 267±33% following CAP and mean heat thresholds decreased from 44.7±1.6°C to 37.7±0.7°C. In SP-SAP treated animals, however, the peak response evoked by CAP was decreased by 61±11% as compared to control. Moreover, CAP did not significantly alter responses to mechanical or heat stimuli. These data suggest that dorsal horn neurons that possess the SPR play a critical role in the development of sensitization to mechanical and heat stimuli following CAP.

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

Tarpley JW, Martin WJ, Baldwin BS, Forrest MJ, MacIntyre DE (2000) Contribution of IB4-positive sensory neurons to NGF-induced hyperalgesia in the rat. Neuroscience 2000 Abstracts 633.18. Society for Neuroscience, New Orleans, LA.

Summary: Sensory information is transmitted from the periphery to the spinal cord by distinct subsets of primary afferent neurons, including two major classes of C-fibers that are distinguished by their ability to bind the lectin IB4. IB4-positive neurons are primarily non-peptidergic, express the receptor tyrosine kinase, Ret, and are preferentially sensitive to the neurotrophic factor, GDNF. By contrast, the nerve growth factor (NGF) receptor tyrosine kinase, trkA, is predominantly expressed in IB4-negative neurons that contain substance P. Previous work suggested that depletion of IB4-positive neurons increases acute nociceptive thresholds to noxious thermal stimuli. The extent to which these non-peptidergic neurons mediate changes in nociception after injury is unknown. Here, we examined the contribution of IB4-positive, non-peptidergic, neurons to thermal and mechanical sensitivity after acute tissue injury. Baseline thermal (radiant heat) and mechanical (von Frey) thresholds were measured in rats prior to injection of IB4-saporin (5 ug/5 ul) into the left sciatic nerve. By two weeks, IB4-saporin-treated animals exhibited pronounced increases in their nociceptive thresholds to thermal and mechanical stimuli. At this time, treatment with NGF (5 ug/50 ul, i.pl.) increased paw thickness in both control and IB4-saporin-treated rats. However, nociceptive thresholds were significantly lowered in control rats, but not in those treated with IB4-saporin. This suggests that IB4-positive neurons contribute to injury-induced changes in thermal and mechanical sensitivity and provide insight into the function of this unique set of primary afferent neurons.

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

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.

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

Johnson DA, Zambon NJ, Gibbs RB (2000) Selective destruction of basal forebrain cholinergic neurons impairs acquisition of a spatial memory task. Neuroscience 2000 Abstracts 563.3. Society for Neuroscience, New Orleans, LA.

Summary: The effects of selective cholinergic vs. non-selective lesions of the septum and diagonal band on acquisition of a spatial memory task were studied. Adult male S-D rats received intraseptal injections of either the selective immunotoxin 192 IgG-saporin (SAP; 1.0 μg in 1.0 μl) or the non-selective neurotoxin ibotenic acid (IBO; 5 μg in 1.0 μl). Two weeks following injection, the animals were food deprived, adapted to a T-maze, and trained to perform a delayed matching-to-position (DMP) task. Rats received 8 trial pairs/day until they reached a criterion of 15/16 correct choices. Seven days after reaching criterion, rats were tested for 2 days with no intertrial delay, then 1 day with a 60s delay, then 2 days with a 90s delay. Following euthanasia brain tissues were analyzed for either choline acetyltransferase (ChAT) activity or immunohistochemical detection of cholinergic neurons. Animals treated with SAP, but not IBO, had lowered ChAT activity in cortical, hippocampal, and basal forebrain tissues and a significant impairment in DMP acquisition compared to controls. SAP-treated animals required an average of 23.7 days to reach criterion compared to 13.1 days for controls (P<0.05). IBO-treated animals required 17.8 days to reach criterion which did not differ significantly from controls. There were no significant differences in post-criteria performance between any of the treatment groups. These data suggest that basal forebrain cholinergic projections play an important role during acquisition of the DMP task.

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

Berger-Sweeney JE, Murg SL, Baxter MG, Stearns NA, Lappi DA (2000) A specific cholinergic immunotoxin in mice. Neuroscience 2000 Abstracts 563.13. Society for Neuroscience, New Orleans, LA.

Summary: We have shown previously that 192 IgG-saporin, a p75 (nerve growth factor [NGF] receptor) antibody linked to the ribosome-inactivating protein saporin, is an effective lesioning agent for cholinergic basal forebrain neurons in rats (Berger-Sweeney et al., J. Neurosci. 14:4507-4519). The 192 IgG antibody, however, does not crossreact with the mouse NGF receptor, making it unsuitable for mouse studies. Here, we tested the efficacy of a new immunotoxin targeting the mouse p75 receptor. A rat monoclonal antibody (Advanced Targeting Systems) to the extracellular domain of the mouse p75 receptor, which can be internalized and transported retrogradely in mouse neurons (Rossner et al., Metab. Brain Dis. 15:17-28), was coupled to saporin. Different doses of the toxin, or saline (0.5 µl) were injected into the lateral ventricle (icv) of adult C57BL/6 mice (n = 3–6/ group). Two weeks later, brains were processed for choline acetyltransferase (ChAT) neurochemistry or ChAT immunocytochemistry (to examine lesion efficacy), and glutamic acid decarboxylase (GAD) neurochemistry or calbindin/parvalbumin immunocytochemistry (to examine lesion specificity). Toxin doses ranging from 0.4 – 3.6 µg reduced hippocampal and neocortical ChAT activity in a dose-dependent fashion. Immunocytochemistry confirmed a significant loss of ChAT-positive neurons in the basal forebrain. These same doses did not alter hippocampal or neocortical GAD activity, or alter calbindin or parvalbumin staining (non-cholinergic neurons) in the basal forebrain. These data suggest that we have created a specific cholinergic immunotoxin for use in mice.

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

Ricceri L, Baxter MG, Frick KM, Berger-Sweeney J (2000) Preservation of reactivity to spatial novelty in adult rats after specific basal forebrain 192 IgG-saporin lesions. Neuroscience 2000 Abstracts 563.4. Society for Neuroscience, New Orleans, LA.

Summary: We have shown previously that neonatal intracerebroventricular (icv) injections of the selective cholinergic immunotoxin 192 IgG-saporin induce marked cholinergic loss in both hippocampus and cortex. These lesions also have long-term behavioral effects in adulthood, impairing reactivity to spatial novelty in a spatial open field test with five objects. In the present study, we analyzed behavioral and neurochemical effects of intraparenchymal injections of 192 IgG-saporin in the medial septal area (MS, 175 ng) or nucleus basalis magnocellularis area (nBM, 70 ng per side) of adult Wistar rats. Animals were then tested in the spatial open field test. NBM cholinergic lesions significantly reduced object exploration in the initial phase of the test, whereas locomotor activity, spatial and object novelty responses were unaffected by either the MS or nBM lesion. A loss in cortical (-61%) and hippocampal (-92%) choline acetyl-transferase activity was found following nBM and MS lesions, respectively. These data show that, although interrupting cholinergic basal forebrain innervation of neocortex and hippocampus in the first postnatal week induces long-term deficits in reaction to spatial rearrangement of familiar objects, the removal of the cholinergic inputs in adulthood does not compromise the same behavioral responses. These data suggest that the same MS and nBM neurons play differential roles in regulating reactivity to spatial changes at different maturational stages.

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

Gerashchenko D, Greco MA, Salin-Pascual R, Kilduff TS, Lappi DA, Shiromani PJ (2000) Orexin-B conjugated to saporin lesions LH and TMN neurons and produces narcoleptic-like sleep in rats. Neuroscience 2000 Abstracts 566.27. Society for Neuroscience, New Orleans, LA.

Summary: A dysfunction of the hypocretin/orexin (Hcrt/Ox) system was recently linked with the sleep disorder, narcolepsy. To provide an experimental method that could be used to inactivate Hcrt/Ox receptor bearing neurons, we linked the toxin, saporin, to the orexin receptor binding ligand, orexin-B. Eighteen male Sprague-Dawley rats (400-450 g) were administered orexin-saporin (0.5 ul; 490 ng) to the lateral hypothalamus (LH) (where Hcrt/Ox containing neurons are located) or tuberomammillary nucleus (TMN) (where Hcrt/Ox receptor containing neurons are present) and sleep was recorded for 3 weeks. A significant reduction in the numbers of TMN and Hcrt/Ox neurons in the LH was detected 3 to 5 days after toxin administration and complete loss occurred by 2 weeks. Rats with extensive cell loss exhibited more REM sleep, nonREM sleep, and multiple sleep onset REM periods during the night. In the only two available animal models of human narcolepsy, the dysfunction in the orexin system is inherited and in the entire animal which makes it difficult to localize specific brain regions or circuits underlying narcolepsy. Orexin-saporin provides a method of determining the contribution of a specific Hcrt/Ox innervation in the regulation of behavior.

Related Products: Orexin-B-SAP (Cat. #IT-20)