targeted-toxins

Strait TA, Montoya D, Pang KCH (2005) Role of the medial septum in a repeated acquisition task. Neuroscience 2005 Abstracts 647.14. Society for Neuroscience, Washington, DC.

Summary: The medial septum/diagonal band of Broca (MSDB) provides a major afferent pathway to the hippocampus and both regions are important in learning and memory. The two major projection cells of the MSDB are cholinergic and GABAergic neurons. Although nonselective lesions of the MSDB impair spatial memory, the role of the different MSDB neuronal population in memory is an active area of research. In the present study, rats with preferential cholinergic or GABAergic lesions of the MSDB will be tested on the repeated acquisition task. The task assesses how well a rat can learn the location of a reward that changes from day to day. All sessions are performed on an 8-arm radial arm maze using one arm for reward. Each daily session consists of 5 trials with rats starting from a different arm on each trial. Rats are tested for a total of 9 days; each day a new rewarded arm is used. Our preliminary data suggest that rats with selective cholinergic lesions using 192-IgG saporin are not impaired in acquisition of the task. These results with selective cholinergic lesions contrast with our previous work showing that preferential GABAergic MSDB lesions impair performance of a repeated acquisition task in a water maze. We are currently investigating the effects of preferential GABAergic MSDB lesions on this task. Our preliminary data suggests that cholinergic and GABAergic MSDB neurons may have differential roles in acquisition of a spatial memory task.

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

Lohals R, Veng LM (2005) Cholinergic therapy does not rescue spatial learning deficits induced by ICV injection of 192 IgG-saporin. Neuroscience 2005 Abstracts 653.5. Society for Neuroscience, Washington, DC.

Summary: The cholinergic hypothesis states that central cholinergic dysfunction is responsible for age-dependent cognitive decline. To model this in rats, we induced cholinergic basal forebrain loss the neurotoxin IgG192-saporin (SAP). Following ICV infusion of 2.5 or 5 microgram (ug) SAP, or saline, rats were tested in the radial 12-arm water maze (RAWM), a spatial learning and memory task. While saline sham or 2.5 ug SAP lesioned rats showed normal learning over 4 trials in the RAWM, 5 ug SAP rats were impaired. However, when trained over 20 trials, 5 ug SAP rats eventually attained the same level of performance as 2.5 ug SAP or saline sham rats, and 28 days later all rats showed intact memory for this platform location. In the open field, 5 ug SAP rats failed to habituate. However, SAP or saline sham lesioned rats did not differ in basal activity, rotarod, or visually cued RAWM performance. SAP lesion resulted in severe depletion of ChAT activity in hippocampus and cortex, which significantly correlated with learning impairment in the RAWM. In a second experiment, we used 5 ug ICV SAP to investigate the effect of cholinergic therapy on SAP-induced spatial learning deficits in the RAWM. However, neither galantamine (0.30, 1.25, 5.0 mg/kg) nor RJR-2403 (0.08, 0.31, 1.25 mg/kg) could reverse the SAP induced deficit in RAWM learning. In conclusion, we found that spatial learning in the RAWM was consistently impaired following severe (5.0 ug SAP) cholinergic basal forebrain lesion in rats. This learning deficit was not confounded by general behavioral disturbances. However, with excessive training SAP lesioned rats could learn and later recall spatial information, suggesting that recall is intact in cholinergic lesioned animals. Severe SAP lesion also impaired habituation in an open field and this was not due to hyperactivity. Finally, treatment with galantamine and RJR-2403 did not alleviate the cognitive deficit induced by the SAP lesion, likely due to the severity of cholinergic depletion.

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

Knox DK, Berntson GG (2005) The potential role of the corticopetal cholinergic system in mediating anxiogenic states in rats. Neuroscience 2005 Abstracts 659.10. Society for Neuroscience, Washington, DC.

Summary: Previous research has demonstrated that the corticopetal cholinergic system is important in mediating defensive tachycardia which suggests that this system may be important in mediating aversive states such as fear and anxiety. The aim of this study was to determine how corticopetal cholinergic lesions affect behavioral models of aversive states. The cholinergic immunotoxin 192 IgG saporin (ATS, San Diego CA) was infused into the nucleus basalis of rats in order to accomplish corticopetal cholinergic lesions. Lesioned and control rats were then evaluated on three behavioral models of anxiogenic states: behavior in the elevated plus maze, behavioral suppression induced by classical and contextual fear conditioned stimuli, and heightened arousal induced by a footshock. Lesioned and control rats did not differ on any anxiogenic behavioral measure in the elevated plus maze indicating that corticopetal cholinergic lesions did not affect behavioral indices of unconditional fear. In contrast, both classical and contextual fear conditioned stimuli induced behavioral suppression in control rats, and these effects were attenuated in lesioned rats indicating that corticopetal cholinergic lesions attenuated conditioned fear. Lastly, heightened arousal was evaluated in lesioned and control rats by monitoring behavioral suppression and changes in the electroencephalogram (EEG) over the retrosplenial cortex after a footshock. The footshock induced decreases in the integral area of the delta band and increases in the integral area of the theta band of the EEG in control rats. Both of these effects were attenuated in lesioned rats. In addition, changes in delta and behavioral suppression induced by the footshock were significantly correlated. These results indicated that corticopetal cholinergic lesions attenuated the heightened arousal induced by an aversive event. Taken together, the results of the study suggest that the corticopetal cholinergic system may be important in mediating components of anxiogenic states.

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

Stead S, Trottier N, Doering LC (2005) Characterization of an immunotoxin model of Parkinson’s disease in mice. Neuroscience 2005 Abstracts 664.9. Society for Neuroscience, Washington, DC.

Summary: The primary event underlying the motor deficits of Parkinson’s disease (PD) is degeneration of neurons in the nigro-striatal system. The most widely employed laboratory rodent models of Parkinson’s are the neurotoxin 6-hydroxydopamine (6-OHDA) model that causes acute degeneration of the dopamine neurons in the substantia nigra (SN) and the MPTP mouse model. To date, there is no single model which accurately simulates the pathogenic, histological, biochemical and clinical features relevant for the investigation of PD. Toxins conveyed by axonal transport can be used to make selective lesions in the central nervous system. As previously shown in rats (Wiley et al., Cell. Mol. Biol., 2003), we have found that selective degeneration of the SN can be induced with an immunotoxin consisting of the highly active ribosome inactivating protein Saporin linked to an antibody to the dopamine transporter. A unilateral stereotaxic injection of anti-DAT-Saporin (0.25ug/2ul and 0.05ug/2ul) into the striatum of young (6-8 weeks old) female C57BL6 mice causes a progressive reduction in the number of DA neurons in the SN in comparison to the non-lesioned hemisphere and in various controls. Furthermore, in parallel to the immunohistochemical dopamine neuron death, the animals display a pronounced circling behaviour when challenged with apomorphine (3mg/kg). We are currently examining the affected brain sections for inclusion bodies and changes in astrocytes. This model exhibits the selective deterioration of the nigro-striatal system that occurs in Parkinson’s disease and provides a system to intervene at various stages of dopamine neuron loss and evaluate the effectiveness of stem cell therapy.

Related Products: Anti-DAT-SAP (Cat. #IT-25)

Fargo KN, Sengelaub DR (2005) Testosterone treatment prevents deficits in motor activation caused by partial loss of motoneurons. Neuroscience 2005 Abstracts 672.8. Society for Neuroscience, Washington, DC.

Summary: In male rats, motoneurons of the spinal nucleus of the bulbocavernosus (SNB) project to the bulbocavernosus and levator ani muscles (BC/LA). The SNB system is dependent on androgens for its development, adult morphology, and function. We have previously demonstrated that unilateral depletion of SNB motoneurons induces atrophy of dendrites and somata in contralateral SNB motoneurons, and that this atrophy is prevented by treatment with exogenous testosterone. In the present experiment, we tested the hypothesis that this neuroprotective effect of testosterone on the morphology of SNB motoneurons is accompanied by a neuroprotective effect on the electrophysiological function of the system. We unilaterally depleted right-side SNB motoneurons by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, some of the saporin-injected rats were castrated and immediately given exogenous testosterone in subcutaneous Silastic capsules designed to produce testosterone titers in the high-normal physiological range. Four weeks later, animals were anesthetized and spinally transected. A stimulating electrode was placed on the left L6 dorsal root, which carries motor afferents from the BC/LA, and a recording electrode was placed on the motor branch of the left pudendal nerve, which carries SNB motoneuron axons to the BC/LA. Both nerves were then severed distal to electrode placement, and recruitment curves were generated by stimulating through the entire range of effective intensities. Consistent with our previously reported morphological changes, unilateral motoneuron depletion resulted in an attenuation of the recruitment of motoneurons in the contralateral SNB, and this was completely prevented by treatment with exogenous testosterone. This result provides a functional correlate to the neuroprotective effects of testosterone treatment on SNB morphology following unilateral motoneuron depletion, further supporting a role for testosterone as a neurotherapeutic agent in the injured nervous system.

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

Fraley GS (2005) Immunolesions of glucoresponsive projections to the arcuate nucleus alter glucoprivic feeding and luteinizing hormone secretion but not sex behavior in adult male rats. Neuroscience 2005 Abstracts 758.7. Society for Neuroscience, Washington, DC.

Summary: Metabolic signals such as insulin, leptin and glucose are known to alter hypothalamic function. Although insulin and leptin are known to directly alter hypothalamic areas that regulate reproduction, the mechanisms by which glucose alters reproductive function are not as clear. Catecholaminergic neurons in the A1/C1 region of the hindbrain are glucose-responsive and project to the arcuate nucleus. To determine if this pathway is involved in the regulation of sex behavior and luteinizing hormone (LH) secretion, this catecholamingergic pathway was lesioned by injecting saporin conjugated to anti-dopamine-β-hydroxalase (DSAP) or unconjugated saporin (SAP) into the arcuate nucleus of adult male rats. Rats were given glucoprivic challenges then feeding and sex behaviors were observed. As was expected, the DSAP treated rats showed a significant decreased in feeding during glucoprivation (250 mg/kg 2-deoxy-D-glucose, 2DG) compared to SAP controls (p < 0.05). Glucoprivation caused a significant reduction in sex behavior (p < 0.05) in both SAP and DSAP animals equally, compared to saline treatments in either treatment group. At the end of the experiment, animals were given a final challenge with 2DG or saline, killed by decapitation and trunk blood was assayed for plasma LH levels. In SAP animals, 2DG elicited a significant decrease in plasma LH levels (p < 0.05). However, in DSAP animals there was a significant increase (p < 0.05) in plasma LH levels compared to saline-treated rats. These data indicate that the A1/C1 efferents to the ventromedial hypothalamus are involved in the glucostatic regulation of feeding behavior and LH secretion, but not sex behavior in the adult male rat.

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

Vijayakumar S, Masood A, Smith M, Clark JP, Nelson DE, Ding JM (2005) Immunolesion of p75 NGF receptor in the mouse SCN attenuated light-induced phase shift of the circadian wheel-running rhythm. Neuroscience 2005 Abstracts 766.18. Society for Neuroscience, Washington, DC.

Summary: Mammalian circadian rhythms are regulated by the suprachiasmatic nucleus (SCN) of the hypothalamus. Alhough the SCN can orchestrate these rhythms in the absence of external cues, it is entrained to environmental cycles through the retinohypothalamic tract. Light-induced phase shifts in circadian rhythm is a classic example of short-term environmental stimuli inducing lasting changes in intracellular signals and gene expression within the SCN cells that alter the phase of the SCN pacemaker and allow entraiment. Since neurotrophins are known to mediate neural plasticity, we investigated the role of brain-derived neurotrophic factor (BDNF) on resetting the phase of the SCN both in vitro and in vivo. We used the SCN brain slice model to study the direct effect of BDNF on the circadian rhythm. Briefly, coronal hypothalamic slices containing the SCN were prepared using 129B/6 mice. The circadian rhythm of the SCN neurons in the brain slice was monitored for 2-3 days by continuously sampling the spike frequencies with single-unit extracellular electrodes. Brief microdrop applications of BDNF (10 uM) induced robust phase delays at CT 16 and phase advances at CT 22. BDNF binds to neurotrophin receptors, including the low affinity p75NTR, which is localized in the SCN. When the toxin Saporin is conjugated to the antibody against p75NTR, it selectively destroys the cells expressing the p75NTR. Three weeks after the stereotaxic injection of the mu p75-Saporin (Advanced Targeting System) into the third ventricle (1 ul over 20 min), the p75NTR immunoreactivity in the mouse SCN is abolished. Immunolesion of the p75NTR in the SCN attenuated light-induced phase delays of the wheel running rhythm at CT16. Consistent with previous findings, our results support a role for BDNF and its receptor p75NTR, in resetting the circadian rhythm of the SCN in mice.

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

Greene DM, Castillo MR, Alexander KA, McMahan A, Raap DK, Bult-Ito A (2005) Neurotoxic lesions of serotonin cells in the dorsal raphe reduce compulsive-like nest building in mice. Neuroscience 2005 Abstracts 796.10. Society for Neuroscience, Washington, DC.

Summary: Bi-directional selection for thermoregulatory nest-building behavior in house mice (Mus musculus) has resulted in a 40-fold difference in the amount of cotton used for nest-building between the high and the low selected lines (big and small nest-builders, respectively). The efficacy of serotonin re-uptake inhibitors (SSRIs), especially fluoxetine, for the treatment of obsessive-compulsive disorder (OCD) indicates a serotonergic involvement in the disorder. The repetitive nest-building behavior, characteristic of the big nest-builders, is a compulsive-like behavior that is responsive to SSRI treatment. We investigated the functional involvement of serotonergic pathways in excessive, repetitive nest-building behavior in mice by lesioning serotonergic cells in the dorsal raphe using the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) or SERT-saporin (Advanced Targeting Systems). 5,7-DHT lesioned mice had a significant decrease in the number of serotonin-stained cells in the dorsal raphe. No decrease was noted in serotonin staining after lesions performed with the SERT-saporin neurotoxin. Mice with successful 5,7-DHT lesions also significantly decreased compulsive-like nest-building as compared to sham and non-surgery controls. These data taken together with additional findings in these mice support the involvement of serotonin pathways in OCD. This data further supports the big nest-builders as a potentially valuable animal model of compulsive behaviors in humans and a means to more clearly identify neurobiological pathways involved in OCD.

Related Products: Anti-SERT-SAP (Cat. #IT-23)

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.

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

Zhang W, Gardell SE, Xie Y, Luo M, Rance NE, Vanderah TW, Porreca F, Lai J (2005) Pain facilitatory cells in the rostral ventromedial medulla coexpress opioid-μ receptors and cholecystokinin type 2 receptors. Neuroscience 2005 Abstracts 394.17. Society for Neuroscience, Washington, DC.

Summary: Pain transmission can be modulated by descending input to the spinal dorsal horn from the rostral ventromedial medulla (RVM). RVM neurons that facilitate nociception are termed “ON-cells”, which are inhibited by mu-opioids, suggesting that they express opioid mu receptors (MOR). Focal application of cholecystokinin (CCK8(s)) into the RVM elicits acute thermal and tactile hypersensitivity and induces ON-cell activity. In situ hybridization using riboprobes for either rat MOR or rat cholecystokinin type-2 receptor (CCK-2) confirms the expression of these receptors in the RVM. Pretreatment with a toxin conjugate, CCK8(s)-saporin results in a significant loss of CCK-2 positive cells in the RVM, concomitant with a blockade of CCK8(s) induced hyperalgesia. The pretreatment also significantly reduces the number of neurons labeled for MOR in the RVM, suggesting that MOR and CCK-2 may be co-localized in some RVM cells. Consistent with these data, similar pretreatment with the toxin conjugate, dermorphin-saporin, which selectively targets MOR expressing neurons, significantly reduces the number of MOR labeled cells in the RVM, blocks RVM CCK8(s) induced hyperalgesia and reduces the number of CCK-2 positive cells in the RVM. In situ hybridization using 35S-labeled CCK-2 riboprobes and Digoxigenin-labeled MOR riboprobes shows that over 80% of labeled RVM neurons co-express both MOR and CCK-2, ~15% express only CCK-2, and very few cells express only MOR. These findings represent the first direct demonstration of the phenotype of pain facilitatory neurons in the RVM. Together with previous studies showing that RVM CCK-2 antagonists reverse nerve injury-induced pain, this phenotype provides strong support for the view that endogenous CCK is a critical mediator of the descending pain facilitation, particularly in the maintenance of experimental neuropathic pain. Support Contributed By: NIDCR R01 DE016458

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), CCK-SAP (Cat. #IT-31)

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