targeted-toxins

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)

Butt AE, Allen K, Arthur K, Cole C, Cook S, Gerth A, Hoichi M, Long C, Noble M, Rea T, Rogers J (2000) A test of negative patterning reveals selective impairment in configural association learning in rats with 192 IgG-saporin lesions of the nucleus basalis magnocellularis. Neuroscience 2000 Abstracts 563.5. Society for Neuroscience, New Orleans, LA.

Summary: We have previously argued that the nucleus basalis magnocellularis (NBM) is selectively involved in configural and not simple association learning, although the experiments used to support our position have involved comparatively non-selective NBM lesions. In the current experiment, rats with bilateral lesions of the NBM created using the highly selective cholinergic immunotoxin 192 IgG-saporin (n = 6) and sham-operated rats (n = 6) were trained in the negative patterning paradigm. In this task, operant responses made in the presence of a light (L) or a tone (T) are reinforced (+) when either stimulus is presented alone, but responses made when these stimuli are presented in compound (LT) are not reinforced (-). We hypothesized that rats in the NBM lesion group would learn to respond to L+ and T+, which requires the ability to learn simple associations, but would fail to learn to withhold responding to the LT- compound, which requires the ability to form configural associations. Responding to L+ and T+ was normal in the NBM lesion group, suggesting an intact ability to learn simple associations, but these animals failed to learn to discriminate between the reinforced stimulus elements L+ and T+ and the nonreinforced compound LT-, suggesting an impairment in configural association learning. The greater level of LT- responding observed in the NBM lesion group did not reflect an overall response disinhibition, as responding during the inter-trial interval did not differ between groups.

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

Ritter S, Dinh TT, Bugarith K (2000) Anti-dβh-saporin injection into the paraventricular nucleus of the hypothalamus selectively abolishes 2DG-induced feeding without causing nonspecific tissue destruction. Neuroscience 2000 Abstracts 502.8. Society for Neuroscience, New Orleans, LA.

Summary: The toxin-antibody complex, saporin conjugated to a monoclonal antibody against dopamine-β-hydroxylase (anti-dβh-sap), selectively destroys dβh-containing catecholamine neurons. Previously we reported that PVH anti-dβh-sap injections abolish feeding and expression of Fos-immunoreactivity (-ir) in the PVH in response to 2DG-induced glucoprivation and cause selective destruction of dβh-containing neurons innervating the hypothalamus. To test the behavioral and neurochemical specificity of this lesion, anti-dβh-sap or control solution (saline or unconjugated saporin) was injected bilaterally into the PVH (n=6 per group). Beginning three weeks later, rats were tested for stimulation of feeding by 2-deoxy-D-glucose (2DG, 200 mg/kg) and mercaptoacetate (MA, 68 mg/kg), which reduce glucose and fatty acid oxidation, respectively, and for suppression of feeding by cholecystokinin octapeptide (CCK, 4 ug/kg). 2DG-induced feeding was abolished by anti-dβh-sap injection, but was not impaired by unconjugated saporin. Neither MA nor CCK responses were altered by anti-dβh-sap. The nonspecific toxicity of anti-dβh-sap at the injection site was assessed. We found that PVH magnocellular neurons at the injection site express oxytocin-ir and do not appear to be disrupted by the anti-βh-sap injection. Further, the ability of PVH neurons at the injection site to express Fos in response to a non-glucoprivic stimulus was not impaired by anti-dβh-sap since lateral ventricular injection of senktide, an NK3 receptor agonist, induced Fos ir in the PVH. PVH injections of anti-dβh-sap impair 2DG-induced feeding and PVH Fos expression by selective destruction of catecholamine neurons. PHS#DK 40498.

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

Secor AJ, Bishop J, Pang KCH (2000) The role of medial septal cholinergic and GABAergic neurons in social memory. Neuroscience 2000 Abstracts 563.6. Society for Neuroscience, New Orleans, LA.

Summary: Electrolytic lesions of the septum alters social behavior, increasing the number of social contacts with a conspecific. Furthermore, lesions of the septum impair both spatial and non-spatial memory. These findings lead researchers to invetigate the role of the septum in social memory. A recent study has demonstrated that administration of vasopressin antagonists into the lateral septum impairs social memory following a 30 minute delay. Social memory was measured as a decreased amount of exploaration of a familiar juvenile compared to a novel juvenile. The present experiment will investigate the role of the cholinergic and GABAergic projection neurons in the medial septum/diagonal band (MS/DB) in social memory. 192-IgG saporin and kainic acid will be administered into the MS/DB to destroy cholinergic and GABAergic MS/DB neurons, respectively. The effects of the drugs on social recognition will be assessed. Each adult rat will be allowed to explore a juvenile rat for 30 seconds. Following various delays (5, 30, 120 minutes), adult rats will be re-exposed to the same or a novel juvenile rat. The amount of time that the adult rat explores the juvenile rat in 1 minute and 3 minute periods will be measured. Differences in exploration time between the control and drug treated rats will provide evidence for alterations in social memory.

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

Siddiq MM, Tsirka SE (2000) Elimination of microglia suggests their involvement in neuronal plasticity. Neuroscience 2000 Abstracts 507.2. Society for Neuroscience, New Orleans, LA.

Summary: Reorganization of mossy fibers occurs in the mammalian hippocampus during consolidation of learning and memory. Induced low level seizures with kainic acid (KA) result in the development of new synapses and the reorganization of existing ones along the mossy fiber pathway. The serine protease tissue plasminogen activator (tPA) is expressed along the mossy fiber pathway and has been implicated in neurite remodeling after stimulation of neuronal activity. Both neurons and microglia secrete tPA. Microglial cells are thought to function only in pathological situations in the CNS, as they exhibit neurotoxic properties. However, a protective role has been observed in the regenerating optic nerve, where intervening activated microglia were involved in tissue remodeling. To investigate whether there is a role for microglia in mossy fiber remodeling, microglia were eliminated in C57/BL6 mice by immunolesioning. The reorganization of mossy fibers was evaluated. Kainate-injected wild-type mice had pronounced mossy fiber reorganization in the dentate gyrus of the hippocampal formation as detected by Timm staining, while the immunolesioned mice had significantly less and shorter mossy fibers. It is therefore suggested that activated microglia may play a role in active remodeling of mossy fibers in the hippocampus after KA-induced seizures.

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

Mattsson A, Ögren SO, Olson L (2000) 192 IgG-saporin immunolesioning causes marked facilitation of dopamine-mediated locomotor activity in adult rats. Neuroscience 2000 Abstracts 563.7. Society for Neuroscience, New Orleans, LA.

Summary: Recent studies have indicated a possible link between changes in cholinergic mechanisms and schizophrenia. However there is limited information regarding the functional consequences of changes in ACh transmission on DA functions, which in turn are believed to be involved in schizophrenic symptoms. 192 IgG-saporin targets cells that express the low-affinity p75 neurotrophin receptor. Intracerebroventricular injection causes severe lesions of the cholinergic projections and may also damage Purkinje neurons. We have compared intracerebroventricular injections of 192 IgG SAP to adult rats (5 μg) with injections to neonate rats (0.4 μg day 4 or 0.4 μg day 4 + 0.8 μg day 13). As expected, treated adult rats are markedly impaired in the Morris swim maze both in terms of time to find the platform and in search behavior when the platform is removed, i.e. memory impairment. When tested as adults, neonatally treated animals were only modestly impaired. Interestingly, adult treated animals showed increased spontaneous motility and locomotion and markedly increased locomotor responses to amphetamine (1.5 mg/kg) as evidenced by increased rearing, motility and locomotion. These animals also responded to apomorphine (1 mg/kg) with an increased amount of rearing. There were no marked changes of locomotor activity in rats treated with 192 IgG SAP at 4 days or 4 and 13 days of age. Our results suggest that cholinergic denervation of the forebrain causes a marked enhancement of the behavior responses related to increased dopaminergic activity. However, it cannot be fully excluded that damage to non-cholinergic systems, e.g. Purkinje cells, might contribute to the effects. The striking overreaction to dopaminergic stimuli presumably caused by the cholinergic deficit is possibly relevant in relation to the role of cholinergic malfunctioning in schizophrenia.

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