sfn2003

Butt AE, Corley S, Cabrera S, Chavez C, Kitto M, Ochetti D, Renovato A, Salley T, Sarpong A (2003) 192 IgG-saporin lesions of the nucleus basalis magnocellularis in rats fail to disrupt acquisition or retention of differential reinforcement of low rate responding. Neuroscience 2003 Abstracts 425.5. Society for Neuroscience, New Orleans, LA.

Summary: The frontal cortex has been implicated in supporting timing behavior in tests of differential reinforcement of low rate responding (DRL) in rats. DRL performance is similarly influenced by anticholinergic drugs; scopolamine interferes with DRL performance by increasing the number of nonreinforced responses and thus decreasing DRL efficiency. Because the frontal cortex receives significant cholinergic input from the nucleus basalis magnocellularis (NBM) in rats, we hypothesized that NBM lesions would disrupt DRL performance in the current experiment. Male Long-Evans rats were placed first in a DRL 10 s schedule of reinforcement before advancing to a DRL 20 s schedule. Rats received 50 trials per day for 20 consecutive days on both DRL schedules. When rats reached stable performance, they received either bilateral 192 IgG-saporin lesions of the NBM or sham lesions. Upon recovery, rats were reintroduced to the DRL 20 s task for 10 days of post-operative testing. Finally, rats were tested using a novel delay interval in a DRL 30 s task. Testing continued for 10 additional days. Results showed that the NBM lesion group showed no significant change in either the total number of responses or in DRL efficiency (reinforced responses / total responses) between pre- and post-operative DRL 20 s testing. Subsequent acquisition in the DRL 30 s task was similarly not disrupted by NBM lesions. The effectiveness of the lesions was verified by acetylcholinesterase (AChE) staining, which showed pronounced depletion of cortical AChE with normal AChE-positive staining in the hippocampus and medial septal area. These data suggest that the NBM is not critically involved in either the acquisition or retention of DRL performance.

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

Fletcher BR, Baxter MG, Rapp PR, Shapiro ML (2003) 192-IgG saporin lesions of the medial septum and vertical diagonal band impair cognitive flexibility. Neuroscience 2003 Abstracts 425.8. Society for Neuroscience, New Orleans, LA.

Summary: Learning and memory remain largely intact following selective basal forebrain cholinergic lesions. By comparison, single unit recording studies have documented reliable effects of such lesions, including abnormally rigid hippocampal place fields when animals are confronted with changes in the configuration of the testing environment. The present experiment tested the prediction that cholinergic lesions of the basal forebrain would impair performance of tasks requiring cognitive flexibility. Rats received 192-IgG saporin or control vehicle injections into the medial septal nucleus and vertical diagonal band, and were tested on cued and spatial delayed match-to-place tasks in a radial arm water maze. Test sessions consisted of four sample trials in which animals searched for a cued or hidden escape platform located in a fixed position at the end of one arm (60 sec cutoff, inter-trial interval = 15 sec). A memory delay was imposed by returning rats to the home cage for a variable delay (15 sec. – 6 hrs), followed by two test trials. The lesion and control groups learned at similar rates in both versions of the task, and performed comparably on the critical test trials, independent of the length of the retention interval. However, lesioned rats were impaired during the transition from the cued to spatial variants of testing. Specifically, the lesion group made significantly more errors on an early sample trial in the spatial task, returning to the location that was previously correct during cued training. Pending histological confirmation of the extent and selectivity of the experimental lesions, this pattern of results suggests that damage to the basal forebrain cholinergic system spares spatial learning but impairs cognitive flexibility when task contingencies are changed.

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

Nelson LE, Franks NP, Maze M (2003) Discrete lesioning of orexin (hypocretin)-containing neurons potentiates dexmedetomidine- but not pentobarbital-induced hypnosis. Neuroscience 2003 Abstracts 426.13. Society for Neuroscience, New Orleans, LA.

Summary: Introduction: Recent work suggests that anesthetics putatively modulated by the α2-adrenoceptor (e.g. dexmedetomidine (DEX)) or the GABAA receptor (e.g. pentobarbital (PTB)) elevate and depress c-Fos expression, respectively, in the orexinergic perifornical area (PeF)1. Here the hypnotic effects of DEX and PTB are assessed after selective lesion of the PeF by orexin-B conjugated to saporin (OX-SAP). Methodology: Anesthetized Fischer rats were administered stereotaxic PeF injections of saline (0.5µl/side) or OX-SAP (490ng/0.5μl/side; as described2). Loss of righting reflex (LORR) induced by DEX (150μg/kg, SC), PTB (50mg/kg, SC), and saline was tested 1 day pre- and 1, 4, 8, and 12 days post-surgery, then lesions were assessed histologically. All data are presented as means±SEMs (n=6; comparisons by unpaired t-tests and ANOVA, Newman-Keuls). Results: Bilateral PeF lesions enhanced DEX-induced hypnosis at days 8 (281.2±15.8 min; p<0.05) and 12 (322.7±15.93 min; p<0.001) as compared to naïve (234.5±9.0 min) and saline sham animals (day 8, 236.7±9.8 min; day 12, 242.8±10.80 min). In contrast, PTB-induced LORR remained unaffected at day 12 (124.8±6.8 min) relative to naïve (119.4±4.6 min) and sham (120.8±5.3 min). These results agree with previous reports that by day 12, PeF-microinjected OX-SAP induces roughly 80% cell loss2. Conclusion: The absence of a functional PeF potentiates hypnosis induced by DEX but not PTB, as perturbation of the PeF by microinjected GABAA receptor antagonist gabazine is known to 1. References: 1 Nelson et al. (2002) SfN abstract 776.14/M20; 2 Geraschenko et al. (2001) J Neurosci 21:7273-83.

Related Products: OX7-SAP (Cat. #IT-02)

Sherren N, Pappas BA (2003) Dendritic dysgenesis in midline cortical regions following selective acetylcholine and dopamine lesions in neonatal rats. Neuroscience 2003 Abstracts 457.11. Society for Neuroscience, New Orleans, LA.

Summary: Both acetylcholine (ACh) and dopamine (DA) afferents reach their cortical targets during periods of synaptogenesis, and are perfectly positioned to influence the cytoarchitectural development of cortical neurons. Thus the behavioural outcomes of these lesions may be related to the development of appropriate dendritic morphology in neurons from cortical regions involved in cognition. Previous studies have either used non-specific lesion techniques or have not examined long-term effects. We lesioned rat pups at P7 with either 600 ng of the selective immunotoxin 192 IgG-saporin, or 150 ug of 6-hydroxydopamine preceded by desmethylimipramine, or both, and aged them to four months. One squad of rats was sacrificed for neurochemistry and another was prepared for morphological analysis using Golgi-Cox stain. The ACh lesion caused a 32% decrease in choline acetyltransferase activity in the frontal/cingulate cortex and a 72% reduction in retrosplenial cortex (RSC). This was associated with reductions in total dendritic length of the apical tree of layer V pyramidal cells in the medial prefrontal cortex (mPFC), the apical tree of layer III pyramidal cells in the anterior cingulate cortex (ACC), and the basal tree of layer III pyramidal cells in RSC. The DA lesion caused a 76% reduction in DA levels in frontal/cingulate cortex and no change in RSC levels. This was associated with reductions in total dendritic length of the basal and apical trees of layer V pyramidal cells in mPFC, and the basal tree of layer III pyramidal cells in ACC. No changes in layer III pyramidal cells were noted in RSC following the DA lesion. These data demonstrate that ascending ACh and DA afferents play a vital role in the cytoarchitectural development of the cortex. This is particularly important considering that hypofunction in these systems is a characteristic feature of neurodevelopmental disorders involving mental retardation, such as Rett and Down syndrome.

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

Neale JJ, Goodchild AK, Dampney RAL, Pilowsky PM (2003) Destruction of brainstem catecholamine neurons attenuates somatosympathetic reflex and responses to cholecystokinin. Neuroscience 2003 Abstracts 501.11. Society for Neuroscience, New Orleans, LA.

Summary: The integrity of the rostral ventrolateral medulla (RVLM) is essential for the expression of many sympathetic reflexes and the maintenance of vasomotor tone. The RVLM contains bulbospinal neurons, of which about half are catecholaminergic (C1). Destruction of bulbospinal C1 neurons leads to attenuation or abolition of the sympathetic baroreflex and chemoreflex, respectively. This study examines the effects of such destruction on blood pressure (BP), the somatosympathetic reflex and responses to intravenous (i.v) cholecystokinin (CCK) in urethane-anaesthetised, paralysed and ventilated Sprague-Dawley rats. Eighty percent of the spinally projecting C1 neurons in the RVLM were destroyed by bilateral microinjections of the immunotoxin, anti-DBH-saporin (12ng/100nl), into the intermediolateral cell column of the thoracic spinal cord (T1-2). Following treatment with the neurotoxin, systolic BP was measured for 3-5 weeks before testing the reflexes. No significant changes in systolic BP were observed. In the present study destruction of bulbospinal C1 neurons attenuated the baroreflex, replicating the findings of Schreihofer and Guyenet (2000, Am J Physiol 279:R729-R742). Activation of the somatosympathetic reflex by electrical stimulation of the tibial nerve normally elicits two peaks in averaged splanchnic sympathetic nerve activity. Following destruction of C1 neurons, the threshold voltage was reduced and the second peak was either markedly attenuated or abolished at two times threshold voltage. Intravenous injection of CCK (1, 10 and 100mg/kg) elicited depressor and sympathoinhibitory responses that were significantly reduced following destruction of bulbospinal C1 neurons. These results demonstrate a key role of bulbospinal C1 neurons in the somatosympathetic reflex and the sympathetic responses to i.v CCK but not in the tonic control of blood pressure.

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

Harasawa I, Lai J, Porreca F, Fields HL, Meng ID (2003) Selective elimination of mu-opioid receptor expressing neurons in the rostral ventromedial medulla (RVM) does not affect periaqueductal gray (pag) stimulation-produced analgesia. Neuroscience 2003 Abstracts 177.5. Society for Neuroscience, New Orleans, LA.

Summary: PAG stimulation produces antinociception at spinal levels by modulating RVM neuronal activity. Microinjection of saporin conjugated with the mu-opioid receptor agonist dermorphin (DERM-SAP) into the RVM selectively eliminates MOR expressing neurons and diminishes neuropathic pain symptoms (Porreca et al., 2001). The aim of the present study was to determine whether MOR expressing neurons in the RVM are required for PAG stimulation produced analgesia (PAG/SPA). The minimum electrical current required to inhibit the tail flick response was compared in barbiturate-anesthetized rats given a single RVM injection of SAP or DERM-SAP 3-4 weeks prior to testing. Thresholds in SAP and DERM-SAP treated rats were not different. Furthermore, microinjection of the glutamate receptor antagonist kynurenic acid (10 mM, 800 nl) into the RVM disrupted PAG/SPA in both SAP and DERM-SAP treated rats. These results indicate that 1) mu-receptor expressing neurons in the RVM are not necessary for PAG/SPA, and 2) excitatory amino acid transmission in the RVM is critical for PAG/SPA. In additional experiments, inhibition of neurotransmitter release in the RVM by the microinjection of cobalt chloride (CoCl2, 100 mM, 800 nl), produced significant antinociception only in DERM-SAP treated rats. This finding suggests that DERM-SAP injections result in increased tonic inhibition of RVM neurons and that CoCl2 disinhibits these neurons to produce antinociception. Tonic inhibition of off-cells would account for our failure to find off-cells in DERM-SAP treated rats.

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

Leung LS, Shen B, Ma J, Rajakumar N (2003) Cholinergic activity enhances hippocampal CA1 long-term potentiation during walking in rats. Neuroscience 2003 Abstracts 255.5. Society for Neuroscience, New Orleans, LA.

Summary: Long-term potentiation (LTP) at the basal dendrites of CA1 pyramidal cells was induced by a single 200-Hz stimulation train (0.5-1 sec duration) in freely behaving rats during one of four behavioral states – awake-immobility (IMM), walking, slow-wave sleep (SWS) and rapid-eye-movement sleep (REMS). Field excitatory postsynaptic potentials (fEPSPs) generated by basal dendritic excitation of CA1 were recorded before and up to 20 hours after the tetanus. Following a tetanus during any behavioral state, basal dendritic LTP was > 170% of the baseline for the first 30 min after the tetanus and decayed to ~125% at 20 hours after. LTP induced during walking was significantly larger than that induced during IMM, SWS or REMS. LTP induced during IMM, SWS and REMS was not significantly different from each other. To test the hypothesis that septohippocampal cholinergic activity enhanced LTP during walking than during immobility, rats were either pretreated with muscarinic cholinergic antagonist scopolamine (5 mg/kg i.p.) or given selective cholinotoxin IgG192-saporin in the medial septum. Pretreatment with scopolamine decreased the LTP induced during walking but did not affect that induced during IMM, such that the difference between LTP induced during walking and IMM was abolished. In IgG192-saporin injected rats, there was no difference in the LTP induced during walking and during IMM, and scopolamine did not reduce the LTP induced during walking. In contrast, sham-lesioned rats, like other control rats, showed larger LTP induced during walking than during IMM, and LTP induced during walking was attenuated by scopolamine. This appears to be the first demonstration of an enhancement of hippocampal LTP by physiologically activated septal cholinergic inputs. LTP of the CA3 to CA1 synapses may serve important behavioral functions.

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

Muir JL, Harrison FE (2003) H3 receptor antagonists modulate behaviour in a visual spatial attention task in rats with selective lesions of the nucleus basalis magnocellularis. Neuroscience 2003 Abstracts 296.7. Society for Neuroscience, New Orleans, LA.

Summary: It is well known that the cholinergic system suffers a large amount of damage in Alzheimer’s Disease (AD). The histaminergic system is known to interact with the cholinergic system but is thought to be largely spared in AD. Compounds which affect histaminergic transmission therefore offer a new further therapeutic avenue to be considered. The effects of two selective H3 receptor antagonists, Thioperamide (1.0, 3.0, 10.0mg/kg) and Ciproxifan (1.5, 3.0, 5.0mg/kg), were investigated for their ability to modulate visual spatial attention using the Five Choice Serial Reaction Time Task. The animal model used was that of cholinergic lesions of the nucleus basalis Magnocellularis (nbM) in the basal forebrain using the selective immunotoxin IgG Saporin. Thioperamide, without affecting overall accuracy, showed very strong trends towards a reduction in anticipatory responses in both sham and lesion groups and also showed a slight reduction in perseverative responses. The more potent Ciproxifan showed no overall change in accuracy but led to a reduction in the anticipatory responses in nbM lesioned animals at all three doses of the drug, returning them to equivalent levels of responses to the sham group. This effect was independent of any overall decrease in activity levels as there were no concomitant changes in response latencies or number of trials completed. Hence these pharmacological manipulations reduced the levels of impulse behaviours manifest by lesioned animals.

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

Miller BT, Collins TJ (2003) Production and characterization of biotinylated gonadotropin-releasing hormone analogs conjugated to avidinylated saporin. Neuroscience 2003 Abstracts 325.15. Society for Neuroscience, New Orleans, LA.

Summary: Saporin is a potent protein cytotoxin derived from the seeds of the plant Saponaria officinalis. Although unmodified saporin cannot permeate cellular membranes in appreciable amounts, this toxin can be chemically conjugated to various compounds that can enter cells by receptor-mediated uptake. When such conjugates are internalized, saporin can exert a lethal effect by inactivating cellular ribosomes. Thus, when conjugated to compounds that bind to specific cellular receptors, saporin can potentially be used as a relatively precise, targeted toxin. A major challenge in employing saporin-based technologies resides in the construction of conjugates of saporin and various receptor-binding biomolecules. Linking a small, bioactive peptide to saporin must be carried out in such a way as to retain the specific receptor-binding properties of the peptide. Although the successful direct conjugation of a small number of bioactive peptides to saporin has been reported, not all such conjugate preparations are necessarily homogeneous. In theory, the recent commercial availability of avidinylated saporin could allow investigators to utilize the numerous, well-characterized, bioactive, biotinylated peptides that have been developed over the past 25 years. Consequently, we explored the ability of conjugates fashioned from biotinylated analogs of gonadotropin-releasing hormone (GnRH) and avidinylated saporin to selectively target GnRH receptor-bearing pituitary cells. The biotinylated GnRH analogs all contained biotin moieties chemically linked to the epsilon amino group of [D-Lys6]GnRH; single and double spacer arms of aminohexanoic acid were included between the peptide and the biotin group. Some biotinylated GnRH analogs contained disulfide bonds between the peptide and biotin moieties. After conjugating the biotinylated peptides to avidinylated saporin, we tested the specific cytotoxic effect of the conjugates in cultures of dispersed rat pituitary cells.

Related Products: Avidinylated-SAP (Cat. #IT-09)

Kohls MD, Lappi DA (2003) Cholera toxin B-saporin cytotoxicity is correlated with the extent of GM1 expression on the cell surface. Neuroscience 2003 Abstracts 325.16. Society for Neuroscience, New Orleans, LA.

Summary: Cholera toxin is composed of five B-subunits that mediate binding to the cell surface monosialoganglioside GM1, and one A-subunit that irreversibly activates adenylate cyclase. GM1 is found in high concentrations on astrocytes and the myelin of oligodendrocytes, as well as on many other cell types. A targeted toxin was developed consisting of the ribosome-inactivating protein saporin coupled to the B-subunit of cholera toxin (CTB) for the purpose of eliminating cells that express GM1. This targeted toxin (CTB-SAP) binds to cell-surface GM1 and the entire molecule is then internalized. Saporin separates from CTB and is released from the endosome to inactivate ribosomes. The cytoxicity of CTB-SAP in a cell-based in vitro assay can be inhibited by the addition of free CTB, indicating binding specificity. CTB-SAP has been tested on a variety of cell lines and the effectiveness of the targeted toxin correlates with the extent of GM1 cell surface expression as demonstrated by fluorescence-activated cell sorting (FACS) analysis. For example, using FACS analysis, RBL-2H3 cells exhibit an 88% shift when labeled with CTB-FITC. The ED50 of CTB-SAP in a cytoxicity assay on these cells is 380 fM, which corresponds to 9150 CTB-SAP molecules per cell. In comparison, HS294T cells produce a 48% shift in FACS, and the ED50 in the cytotoxicity assay is 5.5 pM. The correlation of surface GM1 number with ED50 indicates that the number of saporin molecules internalized is of primary importance in the process of cytotoxicity. CTB-SAP has been used to demyelinate the lumbar spinal cord (Jasmin et al.), eliminate sympathetic preganglionic neurons, and eliminate facial motoneurons in the rat (Llewellyn-Smith et al.). CTB-SAP is an effective and specific tool for the in vitro and in vivo elimination of cells that express GM1 on the cell surface.

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