sfn2001

Gerashchenko D, Kohls MD, Greco MA, Waleh NS, Salin-Pascual R, Kilduff TS, Lappi DA, Shiromani PJ (2001) Hypocretin-saporin (Hcrt-sap) as a tool to examine hypocretin function. Neuroscience 2001 Abstracts 410.7. Society for Neuroscience, San Diego, CA.

Summary: The hypocretin (Hcrt) peptides are linked to narcolepsy. Humans with narcolepsy have decreased numbers of Hcrt neurons and Hcrt-null mice have narcoleptic symptoms. Hcrt neurons are located only in the lateral hypothalamus (LH) but neither lesions of this nor any other brain region have produced narcoleptic-like sleep, suggesting that specific neurons need to be destroyed. To facilitate lesioning the Hcrt neurons, the ribosome inactivating protein, saporin (SAP), was conjugated to Hcrt-2/orexin B. In vitro binding studies indicated specificity of Hcrt-SAP since it preferentially bound to CHO-cells containing the HcrtR2/OX2 receptor compared to the HcrtR1/OX1 receptor, but not to KNRK cells stably transfected with the NK1 receptor. In vivo specificity was confirmed since administration of the toxin to the LH eliminated some neurons (Hcrt, MCH, and histamine) but not others (alpha-MSH). When the toxin was administered to the LH, rats (n=19) had increased slow wave sleep, REM sleep, and sleep-onset REM sleep periods at night. These were negatively correlated with the loss of Hcrt-containing neurons (r=-0.74; p<0.01). Toxin applied to hypothalamic neurons that are not Hcrt positive but contain the Hcrt receptor lesioned the neurons but did not produce narcoleptic-like sleep. These findings indicate the utility of Hcrt-SAP as a tool and also demonstrate that damage to the LH that also includes a substantial loss of Hcrt neurons is likely to produce the sleep disturbances that occur in narcolepsy.

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Hartonian I, de Lacalle S (2001) Long-term plastic changes in galanin innervation in rat basal forebrain. Neuroscience 2001 Abstracts 254.1. Society for Neuroscience, San Diego, CA.

Summary: Galanin (GAL) immunoreactive (-ir) fibers hyperinnervate remaining cholinergic basal forebrain neurons in Alzheimer’s Disease (AD), perhaps exacerbating the cholinergic deficit. The purpose of our study is to determine whether a similar hyperinnervation of GAL-ir fibers occurs following intraparenchymal injection of 192-IgG saporin, a specific cholinergic neurotoxin, within the horizontal diagonal band of Broca (HDB), in 3-month-old rats, and to identify its origin. Immunotoxic lesions produced on average a 31% reduction in cholinergic cell counts on the lesioned side versus the spared side. Hyperinnervation of GAL-ir fibers was observed within and adjacent to the HDB in 28 out of 36 rats, and this effect persisted across time, with 6 months being the longest time examined. Morphometry revealed an increase in the number of GAL-ir cells on the lesioned basal forebrain, as compared to control. A similar change could not be detected in the number of GAL-ir neurons in the amygdala or the bed nucleus of the stria terminalis. Although there was no significant correlation in the amount of cell loss and of GAL hyperinnervation, we suggest that GAL hyperinnervation is triggered by the loss of cells because it is persistent across time. Our data suggests that this hyperinnervation is the result of overexpression of GAL in some cholinergic neurons of the basal forebrain. Since GAL is known to inhibit acetylcholine release, exacerbating the cholinergic dysfunction in AD, this model can be useful to test the efficacy of GAL inhibitors.

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

Mistlberger RE, Antle MC, Jones M, Kilduff TS (2001) Light- and food-entrained circadian rhythms in rats with neurotoxic lesions of hypocretin cells. Neuroscience 2001 Abstracts 410.8. Society for Neuroscience, San Diego, CA.

Summary: The hypocretins (Hcrt1 & 2) are lateral hypothalamic (LH) neuropeptides implicated in the regulation of feeding and sleep-wake states. Hcrt lesions attenuate the amplitude of sleep-wake circadian rhythms in rats entrained to light-dark (LD; Gerashchenko et al, Soc. Neurosci Abst., 2000). We examined whether Hcrt cells also participate in the expression of circadian rhythms entrained by daily feeding schedules. Rats received LH injections of Hcrt2 conjugated to the ribosome- inactivating protein saporin. Drinking was recorded in LD 12:12 for 1-2 months, in DD for 48 h, and in LD 2:2 for 24 h, to assess photic entrainment and masking. The rats were then restricted to a 3 h daily meal beginning 6 h after lights-on (LD 12:12). After 29 days, the rats were deprived of food for 50 h. Lesions, assessed by immunocytochemistry using preprohypocretin antibody (Chemicon Int., Inc.), ranged from 0-100% complete. Complete lesions were associated with attenuated mean level and amplitude of circadian drinking rhythms in LD 12:12 and DD, but photic masking in LD 2:2 was unaffected. All rats exhibited food-entrained activity that anticipated feeding time by 1-3 h. This was of lower magnitude in the rat with the largest lesion. These results are consistent with a recent report that Hcrt2-saporin lesions attenuate sleep-wake circadian rhythms, but differ from earlier reports that electrolytic lesions of the LH may potentiate the masking effects of light on behavior. The results suggest that Hcrt cells do not play an essential role in the regulation of circadian rhythms by scheduled feeding.

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Gibbs RB (2001) Estrogen enhancement of learning requires intact basal forebrain cholinergic neurons. Neuroscience 2001 Abstracts 312.12. Society for Neuroscience, San Diego, CA.

Summary: We have shown that long-term continuous estrogen replacement, or weekly administration of estrogen plus progesterone, enhances acquisition of a delayed matching-to-position (DMP) spatial memory task. The present study examined whether hormone replacement enhances acquisition of the task after destroying basal forebrain cholinergic neurons with the selective immunotoxin 192IgG-saporin (SAP). Ovariectomized Sprague-Dawley rats received either SAP (1.0 Μg in 1.0 ΜL saline) or vehicle injected directly into the medial septum. Two weeks later, animals received either continuous estrogen replacement (E; 3 mm silastic capsule containing 17-β-estradiol, implanted s.c.) or weekly administration of estradiol (10 Μg in oil s.c.) followed two days later with progesterone (500 Μg in oil s.c.). Controls received sham implantation surgery and vehicle injections. DMP training began after 1 month of treatment. SAP injections significantly impaired acquisition of the DMP task (median days to criterion = 21.0 for SAP-treated animals vs. 14.0 for Ovariectomized controls; p<0.05). Neither continuous E replacement nor weekly administration of E+P significantly enhanced acquisition in the SAP-treated animals. Histology and hippocampal ChAT activity confirmed the loss of cholinergic cells in the medial septum and diagonal band of Broca in SAP treated animals. These findings suggest that cholinergic neurons in the medial septum/diagonal band are necessary for hormone-mediated enhancement of DMP acquisition. Studies using lower doses of SAP are currently underway.

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

Blanco-Centurion CA, Salin-Pascual RJ, Gerashchenko D, Greco MA, Shiromani PJ (2001) Hypocretin B-saporin lesions of the brainstem increase rem sleep at night. Neuroscience 2001 Abstracts 410.9. Society for Neuroscience, San Diego, CA.

Summary: Loss of hypocretin (Hcrt) neurons has been linked to narcolepsy. These neurons project widely throughout brain, but it is not known which projection to which target site produces what symptom of narcolepsy. We (Molec Brain Res, 88:176-182,2001) showed that Hcrt receptors are present in brainstem areas implicated in REM sleep. Since abnormal REM sleep triggering characterizes narcolepsy, we have used Hcrt-saporin, a toxin that selectively lesions Hcrt receptor bearing cells, to assess the effects of such lesions on sleep. In the present study, Sprague Dawley rats (n=21) were administered (under anesthesia) Hcrt-sap (100ng/1ul, vol=0.5 ul bilaterally) or saline to the locus subcoeruleus (LSC) or the medullary inhibitory area of Magoun and Rhines. Subsequently, continuous sleep recordings were made for 21 days. Sleep records were scored blind. In the medulla, Hcrt-sap (n=5) increased the length of REM sleep bouts (p<0.039), which produced a trend towards an increase in REM sleep at night. There were no significant changes in SWS or W. Lesions of the LSC (n=5) increased total sleep time at night (p<0.03) and produced a trend towards a REM sleep increase. Data from both target sites were combined and the Hcrt-sap lesioned rats (n=10 versus saline=11) had a significant increase in REM sleep (30%; p<0.015). At neither site, cataplectic attacks were evident. Our studies with Hcrt-sap indicate site-specific effects on sleep and EEG depending on which Hcrt-receptor bearing neurons are lesioned. All of the symptoms of narcolepsy are evident when the Hcrt-containing neurons are lost. Supported by: NS30140, AG09975, AG15853, MH55772, DVA Med Research.

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Tait DS, Latimer M, Woodson W, Brown VJ (2001) Cholinergic lesions of the thalamic reticular nucleus using 192-IgG-saporin. Neuroscience 2001 Abstracts 313.13. Society for Neuroscience, San Diego, CA.

Summary: The TRN is likely to be involved in attention, based on studies of its anatomy, electrophysiological properties and the effects of lesions (see Guillery et al, 1998, TINS 21:28-32). The cholinergic neurons of the basal forebrain (BF) have also been implicated in attention (Sarter and Bruno, 2000, Neuroscience, 95:933-952). Intriguingly, rostral TRN receives cholinergic innervation from the BF (Hallanger et al, 1987, J Comp Neurol, 262:105-124) and yet the role of acetylcholine in the TRN has not been investigated, in part because of difficulty in selectively manipulating the cholineric input to TRN. 192-IgG-saporin is a conjugation of the ribosome-inactivating protein, saporin, with the monoclonal antibody for the p75 neurotrophin receptor (192-IgG). 192-IgG-saporin, injected into BF or cortical BF-terminal regions, can be used to make selective cholinergic BF lesions. The purpose of the study was to investigate whether it would be possible to lesion the BF cholinergic input to TRN, using the immunotoxin 192-IgG-saporin. Male Lister hooded rats (450-500g) were stereotaxically injected with 192-IgG-saporin into TRN. Doses of 1.4, 1.95 or 2.4μg produced lesions of TRN, with a loss of cholinergic cells observed in the BF at all doses. There was also evidence of depletion of cholinergic input to frontal cortex with all doses. At the highest dose, there was cholinergic depletion in hippocampus. These results suggest that 192-IgG-saporin can be used to lesion the TRN. The behavioural effects of these lesions are under investigation.

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

Sauro KM, Sweeney M, Saari MJ (2001) Enriched housing, reticular thalamic nucleus and nucleus basalis: Mediators of attention and learning?. Neuroscience 2001 Abstracts 313.14. Society for Neuroscience, San Diego, CA.

Summary: Housing manipulations have been shown to alter behaviour, neural function and morphology. The Reticular Thalamic Nucleus (Rt) may be involved in thalamocortical relay loops possibly acting as a bi-directional filter for sensory information. Similarly, the Nucleus Basalis (NB) may play a role in attention via cholinergic projections to the cortex. In the current experiment 160 female Wistar rats received bilateral lesions of either the Rt (using ibotenic acid), the NB (using 192 IgG-saporin; Advanced Targeting Systems), or both. Following recovery, the rats were allocated to either an enriched or an isolated housing condition for two weeks. Following open field testing, a modification of Tolman’s latent learning paradigm was used to evaluate the effects of the treatments on attention. Thus, prior to behavioural testing half of the rats from each group were pre-exposed to the Spatial Orientation Task (SPOT) while the other half were not. Following sacrifice, brain slices were stained for metabolic activity using the cytochrome-oxidase method, for morphology using cresyl violet stain, and for a marker of cholinergic function, acetylcholinesterase. Data analysis revealed significant interactions among the lesions, housing and latent learning and provided some support for the suggestion that the Rt may filter incoming sensory information whereas the NB may mediate behaviourally relevant attention. Lesions and housing effects were confirmed by histology. (Approved by the Animal Care Committee; supported by Nipissing University).

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

Isaac M, Pearce JM, Muir JL (2001) Visual orienting response and the nucleus basalis of Meynert. Neuroscience 2001 Abstracts 313.2. Society for Neuroscience, San Diego, CA.

Summary: The nucleus basalis of Meynert (nbM), within the basal forebrain, contains a mass of large cholinergic neurones that send axons throughout the cortex. Previous studies have shown the nbM may be involved in attentional processing. The current study attempted to look at the effects of a cholinergic specific nbM lesion (using 192 IgG-saporin) on an attentional task involving two different serial conditioning schedules. In the Consistent condition a light (10s) was followed by a tone (10s), that signaled food. In the Inconsistent condition the light was presented alone for some trials and for other trials the light was followed by the reinforced tone. The orienting response towards the light was measured at three points throughout its presentation: within the first two seconds (light onset) and then twice within the post-onset period (2s-10s). According to the Pearce-Hall theory (1980), animals pay more attention to the light when it is followed by unpredictable events (Inconsistent condition) rather than predictable events (Consistent condition). Accordingly, the orienting response directed towards the post-onset light (2-10s), was stronger for sham operated rats that were trained with the Inconsistent than the Consistent condition. By contrast by the lesioned group was weak and at a similar level for both groups. These results suggest that the nbM lesions prevented enhanced attention to the light in the Inconsistent condition. These findings support the claim that the nbM is important for enhancing attention to stimuli (Chiba et al, 1995).

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

Gadd CA, Murtra P, Hall CN, Gana M, Webber MJ, De Felipe C, Hunt SP (2001) NK1-expressing neurons critical for morphine reward behaviors in mice: C-fos expression and ablation of NK1-expressing neurons. Neuroscience 2001 Abstracts 224.13. Society for Neuroscience, San Diego, CA.

Summary: We have previously shown using conditioned place preference (CPP) that mice lacking the preferred receptor for substance P (NK1) show an absence of the rewarding response to morphine as well as reduced conditioned place aversion and physical withdrawal signs following chronic opiate treatment (Nature 405, 180-183). To locate those regions of the brain in which NK1-expressing neurons are crucial for opiate-mediated reward behavior, we examined the expression of c-Fos following acute (10 mg/kg IP) and chronic (increasing doses from 10 to 100 mg/kg IP) morphine administration, and following CPP to morphine (7.5 mg/kg) in wild-type and NK1 knockout mice. The expression of c-Fos in the brains of mice treated with chronic or acute morphine treatment was similar in both genotypes. Moreover, NK1-expressing neurons in the striatum and nucleus accumbens (NAc) were never seen to co-express c-Fos immunoreactivity. In contrast, the expression of c-Fos following the CPP protocol was significantly different between genotypes with a reduced number of c-Fos positive neurons in NK1 knockout mice in the amygdala and hippocampus but not in the NAc or dorsomedial striatum (DMS). We next investigated the effects of selective ablation of NK1 expressing neurons by injecting substance P-saporin into these regions. Our results suggest that destruction of these cells in the amygdala but not in the NAc or DMS causes a reduction in CPP to morphine without affecting anxiety levels or locomotor activity.

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

Camara AL, Pereira EF, Alkondon M, Randall WR, Castro NG, Cintra WM, Albuquerque EX (2001) Septal innervation of the hippocampus regulates expression α7 nicotinic receptors in CA1 and CA3 pyramidal neurons. Neuroscience 2001 Abstracts 145.1. Society for Neuroscience, San Diego, CA.

Summary: To investigate the effects of septal innervation on expression of α7 nicotinic receptors (nAChRs) in CA1 and CA3 pyramidal neurons in the hippocampus, the patch-clamp technique and confocal microscopy were applied to organotypic hippocampal cultures and septal-hippocampal co-cultures. In the co-cultures, septal fibers labeled with DiI were visualized in the hippocampus. Field stimulation of septal fibers also resulted in postsynaptic currents that could be recorded from CA1 and CA3 pyramidal neurons in the hippocampus. These currents had glutamatergic, GABAergic and cholinergic components. The latter originated most likely from the septal cholinergic neurons that were labeled in situ with the cholinergic marker Cy3-192 IgG. α7 nAChRs in the somatodendritic region of CA1 and CA3 pyramidal neurons in the hippocampus in cultures and co-cultures were activated by the α7 nAChR agonist choline, which elicited type IA currents, and were visualized by labeling with rhodamine-conjugated α-bungarotoxin (Rho-α-BGT). After 21 days in vitro, the amplitude of type IA currents was substantially smaller in pyramidal neurons in septal-hippocampal co-cultures than in hippocampal oragnotypic cultures. Labeling of the somatodendritic region of hippocampal pyramidal neurons with Rho-α-BGT was also less intense in the organotypic co-cultures than in cultures. These results suggest that functional septal innervation of the hippocampus regulates the expression of α7 nAChRs in hippocampal pyramidal neurons.

Related Products: 192-IgG Mouse Monoclonal, Cy3-labeled (Cat. #AB-N43FL3)