sfn2003

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)

Khasabov SG, Ghilardi JR, Mantyh PW, Simone DA (2003) Spinal neurons that possess the substance P receptor (SPR) modulate descending systems that control excitability of spinal nociceptive neurons. Neuroscience 2003 Abstracts 13.3. Society for Neuroscience, New Orleans, LA.

Summary: We have recently shown that ablation of spinal SPR-expressing spinal neurons by intrathecal application of the cytotoxin conjugate substance P-saporin (SP-SAP) prevents the development of sensitization produced by intraplantar injection of capsaicin (Khasabov et al., 2002) and reduced hyperalgesia produced by inflammation and nerve injury (Mantyh et al., 1997; Nichols et al., 1999). Since the majority of spinal SPR-expressing neurons project to the brain, it is possible that these neurons are an integral part of ascendingdescending circuitry that modulates excitability of spinal nociceptive neurons. Here we studied the contribution of ascending SPR positive neurons in the regulation of brain stem descending pathways that pass through the dorsolateral funiculus (DLF) and modulate spinal cord excitability and sensitization. Rats were given an intrathecal injection of vehicle (0.9% NaCl, 10μl) or SP-SAP (5·10-6M, 10μl) at the lumbar enlargement 30 days prior to electrophysiological recording from lumbar spinal neurons. Spontaneous activity and evoked responses of nociceptive neurons to heat (35-51.°C) and mechanical stimuli (von Frey monofilaments) were obtained before and 1 hour after ipsilateral DLF transection. In vehicle-treated animals, DLF transection produced a 183% increase spontaneous activity, a leftward shift in the temperature-response curve, and a 60% increase in the number of impulses evoked by mechanical stimuli (n=25). In contrast, neurons in the SP-SAP group did not show any changes in spontaneous or evoked activity after DLF transaction (n=29). We conclude that ascending spinal SPR-possessing neurons modulate activity of descending inhibitory systems that pass through the DLF.

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

Woods M, Whiteside G, Pearson M, Pomonis J, Turchin P, Walker K (2003) Ablation of a population of NK-1 expressing neurons in the dorsal horn of the spinal cord does not induce αβ sprouting into lamina II. Neuroscience 2003 Abstracts 64.11. Society for Neuroscience, New Orleans, LA.

Summary: Peripheral nerve injury results in hyperalgesia and allodynia. It has been proposed that sprouting of myelinated touch responsive Aβ-fibers into the innervation territory of pain sensitive C fibers in the spinal cord contributes to these abnormal behaviors. In has further been postulated that excitatory cell death of spinal cord neurons may result in “vacant synapses” that induce sprouting (Woolf et al., 1992). We have investigated whether selectively ablating a population of cells in laminae I and II, using intrathecal (i.t.) SP-saporin (SP-SAP), will induce sprouting from deeper laminae. Male Sprague-Dawley rats were either injected i.t. at the lumbar region with SP-SAP (1 μl, 5 μM) or the sciatic nerve was axotomised at the mid-thigh level. Two weeks later the sciatic nerve was injected with the retrograde tracer, cholera toxin-β subunit (CTB) (2 μl, 2%) which selectively traces Aβ-fibers. Three days post CTB the animals were perfused, the lumbar ganglia and spinal cord harvested, sectioned and stained immunohistochemically for NK-1 and CTB. As previously described axotomy resulted in considerable CTB immunostaining in laminae I, II and III compared to non-axotomised controls in which it was present only in I and III. SP-SAP i.t. resulted in a substantial reduction of NK-1 like immunostaining in the spinal cord compared to saline injected controls. CTB was not detected in lamina II of spinal cords from animals with an ablation of NK-1 expressing cells. These results suggest that the death of dorsal horn neurons does not induce sprouting of Aβ-fibers into lamina II.

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

Herron P, Ismail NS (2003) Progressive effects of cholinergic depletion on cortical functional properties in the somatosensory cortex of rats. Neuroscience 2003 Abstracts 61.11. Society for Neuroscience, New Orleans, LA.

Summary: The amount and duration of cholinergic depletion of basal forebrain input appear to be important for how significant the functional capacity of cortical neurons and behavior are affected. Firstly, it is not known whether there is a correlative relationship between the level of cholinergic depletion and the level of degraded functional properties or whether there is a threshold of depletion, beyond which no further degradation occurs. Secondly, it is not known whether similar levels of cholinergic depletion over different periods cause the similar or different effects on functional capacities and behavior. These experiments were done in the posteromedial barrel subfield (PMBSF) cortex of young adult Sprague-Dawley rats. Selective lesion of cholinergic neurons in the NBM was achieved with cortical or intraventricular injections of the immunotoxin (IT), 192 IgG saporin. Electrophysiological recordings and whisker use in exploratory behavior were monitored for different post-injection survival periods. Results show that cholinergic depletion causes a significant decrease in the magnitude of evoked activity and an increase in the size of receptive fields for different periods. Observations of exploratory behavior showed that animals used whiskers controlled by cholinergic depleted cortex less than the whiskers controlled by non-cholinergic depleted cortex. Thus, cholinergic depletion leads to effects that significantly alter the functional capacity of the cortex and the behavioral use of those whiskers.

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

Nolan BC, Freeman JH (2003) Purkinje cell depletion by ox7-saporin impairs eyeblink conditioned excitation and inhibition in rats. Neuroscience 2003 Abstracts 87.3. Society for Neuroscience, New Orleans, LA.

Summary: The role of the cerebellar cortex in conditioned excitation has been demonstrated by studies that used lesions, inactivation, and electrical stimulation (e.g., Attwell, Rahman, & Yeo, 2001, J Neurosci, 21, 5715-5722). However, very little evidence exists concerning the role of the cerebellar cortex in conditioned inhibition. Moreover, there are multiple blink control zones in the cerebellar cortex (Hesslow, 1994, J Physiol, 476, 229-244), which complicates the interpretation of studies that use localized lesions. In the current study, rats were infused with the immunotoxin OX7-saporin into the lateral ventricles to selectively destroy Purkinje cells throughout the cerebellar cortex (Angner, et.al, 2000, Neurotox, 21, 395-404). The OX7- saporin method provides advantages relative to other methods, including the ability to deplete Purkinje cells after initial training. In Experiment 1, rats were given saline or OX7-saporin prior to excitatory conditioning training, which was established using a tone conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US). Rats given OX7-saporin had nearly complete Purkinje cell loss and acquisition of excitatory conditioning was severely impaired. In Experiment 2, rats were first trained with excitatory conditioning procedures, followed by infusion of either saline or OX7-saporin. After a two-week post-infusion period, the rats were given reacquisition training. After reacquiring excitatory conditioning, the rats were trained using a feature-negative discrimination procedure consisting of trials with CS-US pairings and trials with a non-reinforced tone-light compound stimulus. Rats treated with OX7-saporin showed a significant impairment in reacquisition and acquisition of conditioned inhibition. The results suggest that Purkinje cells are critically involved in the acquisition of both conditioned excitation and inhibition in rats.

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