sfn2001

62 entries

Targeted lesions of Substance P receptor cells in L3-4 lumbar spinal cord severely impair male ejaculatory behavior.

Truitt WA, Stanton LA, Coolen LM (2001) Targeted lesions of Substance P receptor cells in L3-4 lumbar spinal cord severely impair male ejaculatory behavior. Neuroscience 2001 Abstracts 958.18. Society for Neuroscience, San Diego, CA.

Summary: Previously we demonstrated the existence of a spinothalamic pathway in the male rat where neural activation is specifically induced by ejaculation. This pathway consists of the parvocellular subparafascicular thalamic nucleus (SPFp) and the neurons projecting to the SPFp that are located in the lumbar spinal cord, specifically in laminae 10 and 7 of segments L3-L4. This neuron population coexpresses galanin (Gal) and substance P receptors (SPR). To test the hypothesis that these cells relay sensory information related to ejaculation, these neurons were lesioned by targeting the SPR with the neurotoxin SSP-saporin, a high affinity analogue of substance P conjugated to the ribosome inhibitor saporin. SSP-saporin (4 ng/µl) or an equal concentration of unconjugated saporin was injected bilaterally into L3-L4 region in either sexually experienced or naïve male Sprague Dawley rats (n=5 each). Sexual behavior was tested weekly, starting ten days following surgery. Following the sixth behavioral test, rats were sacrificed and spinal cord tissue was immunostained for GAL, SPR, or NeuN to assess specific and nonspecific tissue damage. In rats injected with unconjugated saporin, the Gal/SPR cell population was intact and sexual behavior was not altered. In contrast, in rats receiving SSP-saporin, Gal/SPR cell population was significantly decreased (77%) or absent. Moreover, SSP-saporin lesions severely disrupted ejaculatory behavior. Interestingly, display of mounts and intromissions remained intact. These results suggest that Gal/SPR cells in laminae 10 and 7 of L3-4 lumbar spinal cord may be essential for male ejaculatory behavior.

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

Selective immunotoxin lesion of spinally projecting norepineprhine and epinephrine (NE/E) neurons impairs the glucagon response to 2-deoxy-d-glucose (2DG).

Dinh TT, Sanders NM, Pedrow C, Ritter S (2001) Selective immunotoxin lesion of spinally projecting norepineprhine and epinephrine (NE/E) neurons impairs the glucagon response to 2-deoxy-d-glucose (2DG). Neuroscience 2001 Abstracts 947.1. Society for Neuroscience, San Diego, CA.

Summary: Previous work has shown that the targeted immunotoxin, anti-dopamine ß-hydroxylase conjugated to saporin (DSAP), can be used to selectively destroy subpopulations of hindbrain NE/E neurons projecting to or through a particular DSAP injection site. Using this approach, we have shown that NE/E neurons projecting to the hypothalamus are required for feeding and glucocorticoid responses to 2DG-induced glucoprivation and those projecting spinally are required for the adrenal medullary response. In this study, we injected DSAP or unconjugated saporin (SAP) control solution into the spinal cord at T2-T4 to investigate the role of the spinally projecting NE/E neurons in glucagon secretion induced by 2DG (250 mg/kg). Controls injected spinally with unconjugated saporin (SAP) had a glucagon response that peaked at 308% of pre-2DG levels, while DSAP-injected rats had a significantly blunted response to 2DG, peaking at 197% of pre-2DG levels. 2DG-induced hyperglycemia also was impaired in the DSAP rats, but not in the SAP rats. Both SAP and DSAP rats had normal feeding and glucocorticoid responses to 2DG. Results suggest that spinally-projecting NE/E neurons participate in the neural control of glucagon secretion under conditions of glucose deficit. In combination with other findings, these results indicate that hindbrain NE/E neurons contribute to four major glucoregulatory responses (increased feeding, and increased secretion of glucagon, glucocorticoids and adrenal medullary epinephrine) through their projections to hypothalamic or spinal cord effector sites.

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

Immunotoxin lesion of catecholaminergic neurons innervating the medial hypothalamus elevates basal expression of and attenuates glucoprivaation-induced increases in agouti gene related protein (AGRP) mRNA.

Fraley GS, Dinh TT, Ritter S (2001) Immunotoxin lesion of catecholaminergic neurons innervating the medial hypothalamus elevates basal expression of and attenuates glucoprivaation-induced increases in agouti gene related protein (AGRP) mRNA. Neuroscience 2001 Abstracts 947.2. Society for Neuroscience, San Diego, CA.

Summary: Catecholaminergic (CA) innervation of medial hypothalamic structures is necessary for glucoprivation-induced feeding, glucocorticoid secretion and Fos expression in the paraventricular (PVH) and arcuate nuclei of the hypothalamus. In this experiment, we tested the hypothesis that the 2-deoxy-D-glucose (2DG)-induced an increase in AGRP mRNA expression (reported recently by Sergeyev et al., 2000) also requires NE/E neurons. CA neurons innervating the medial hypothalamus were lesioned using the toxin, saporin, targeted for selective entry into NE/E neurons by conjugation with a monoclonal antibody against dopamine beta hydroxylase. This toxin (DSAP), or unconjugated saporin (SAP) control solution, was bilaterally microinjected into the PVH. DSAP rats with confirmed 2DG-induced feeding deficits (DSAP 1.7 +/- 0.29 g; SAP 5.1 +/- 0.31 g; p < 0.05) and controls were injected with 2DG (250 mg/kg), or saline and maintained for 2 hrs without food. Hypothalami were harvested and subjected to Northern blot analysis of AGRP mRNA. Blot analysis revealed that 2DG increased mRNA expression in SAP controls (2DG: 1.0 +/- 0.05 RDU; saline: 0.7 +/- 0.02, p< .05), but not in DSAP lesioned rats (2DG: 1.1 +/- 0.04; saline: 1.0 +/- 0.03). In addition, basal AGRP mRNA expression was significantly elevated in DSAP-lesioned rats compared to SAP controls (p < .05). These data suggest that basal AGRP gene expression is controlled by hindbrain CA neurons and that increased AGRP gene expression induced by glucoprivation also requires these neurons.

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

Selective immunotoxin lesion of hypothalamically-projecting norepinephrine/epinephrine (NE/E) neurons impairs the glucocorticoid response to glucoprivation.

Ritter S, Dinh TT, Sanders NM, Pedrow C (2001) Selective immunotoxin lesion of hypothalamically-projecting norepinephrine/epinephrine (NE/E) neurons impairs the glucocorticoid response to glucoprivation. Neuroscience 2001 Abstracts 947.3. Society for Neuroscience, San Diego, CA.

Summary: Decreased glucose utilization triggers behavioral and neuroendocrine responses that increase blood glucose concentrations and delivery of glucose to the brain. These include stimulation of food intake and increased secretion of glucagon, adrenal E and glucocorticoids. In previous work utilizing the targeted immunotoxin, DSAP (saporin conjugated to a monoclonal antibody against dopamine β-hydroxylase), we demonstrated that hindbrain NE/E neurons that project to the hypothalamus are necessary for glucoprivic feeding and those that project spinally are necessary for glucoprivic control of adrenal medullary secretion. In the present study, we injected DSAP or control solution into the paraventricular nucleus of the hypothalamus (PVH) to investigate the role of NE/E neurons in glucoprivic control of glucocorticoid secretion. DSAP lesions significantly attenuated the magnitude and duration of the glucocorticoid (cortisol) response to 2-deoxy-D-glucose (2DG)-induced glucoprivation, but did not reduce the glucagon response. After 2DG (250 mg/kg), cortisol levels peaked at only 184% of pre-2DG levels in DSAP rats, compared to 440% in controls. Quantitative analysis revealed that DSAP did not destroy CRF-immunoreactive cell bodies in the PVH or terminals in the arcuate/median eminence, but did reduce dopamine β-hydroxylase immunoreactivity in hypothalamus and in hindbrain NE/E cell groups known to innervate the hypothalamus, including those that innervate CRF neurons in the PVH. Results indicate a critical role for hindbrain NE/E neurons in eliciting multiple controls of glucose homeostasis.

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

Hyperphagia and obesity results from the injection of the immunotoxin neuropeptide Y (NPY)-saporin (NPY-sap) into the paraventricular hypothalamus (PVH) of rats.

Bugarith K, Ritter S, Dinh T (2001) Hyperphagia and obesity results from the injection of the immunotoxin neuropeptide Y (NPY)-saporin (NPY-sap) into the paraventricular hypothalamus (PVH) of rats. Neuroscience 2001 Abstracts 948.2. Society for Neuroscience, San Diego, CA.

Summary: NPY is a peptide implicated in the control of numerous physiological processes. A variety of G-protein coupled receptors, Y1, Y2, Y4, and Y5, mediate the actions of NPY. Recently a new immunotoxin, NPY-SAP, has been developed that is potentially of great importance for the study of NPY function. NPY-SAP is a conjugate of the peptide NPY and saporin, a plant toxin that inactivates ribosomes. The proposed mechanism of toxicity involves immunotoxin binding NPY receptors and destruction of neurons containing these receptors. In this study we injected different doses of NPY-sap or unconjugated saporin (SAP) into the PVH to test the effects of this new toxin. We found that at low doses, there was no effect of NPY-SAP on cytoarchitecture or immunoreactivity of select peptides in the PVH. There was also no difference in body weight between the groups. At high doses, there was histologically-detectable damage in the hypothalamus of NPY-SAP animals. We also found a major difference in body weight between the NPY-SAP rats and SAP controls. Ten weeks after the injections, the NPY-SAP rats (654.3g ± 39.04g) were much heavier than the SAP rats (410.6g ± 15.29g). Further, daytime (0800 – 1700H) food intake was about twice as much in NPY-SAP (9.53g ± 0.996g) as in SAP (5.74g ± 0.476g) rats, with no difference in overnight (1700 – 0800H) feeding. Work is ongoing to determine the specificity of the lesion and the mechanism of action of NPY-SAP.

Related Products: NPY-SAP (Cat. #IT-28)

Monoamine modulation of spinal reflex excitability of the lower limb in the rat: Intrathecal infusion (i.t.) of anti-DBH saporin toxin – time course for behavior.

Bose P, Wang DC, Parmer R, Wiley RG, Thompson FJ (2001) Monoamine modulation of spinal reflex excitability of the lower limb in the rat: Intrathecal infusion (i.t.) of anti-DBH saporin toxin – time course for behavior. Neuroscience 2001 Abstracts 771.3. Society for Neuroscience, San Diego, CA.

Summary: Progressive neurophysiological changes in the excitability of ankle extensor stretch reflexes were observed following T8 spinal cord contusion injury. Our previous study indicated that nonspecific monoamine depletion (reserpine, i.p.)contributed to pathologic hyperreflexia. To test a more specific hypothesis, a longitudinal study was performed to evaluate the time course of changes in reflex excitability after i.t. injection of 250ng of anti-DBH saporin toxin (that specifically lesions descending spinal noradrenergic neurons) into the lumbar spinal cord of normal rats. Measures of ankle torque and time-locked EMGs were used to scale stretch reflex excitability across a broad range of stretch velocities (49-612°/sec) before and at weekly intervals following i.t. injection of toxin (n=12) or vehicle (n=6) using instrumentation and protocol previously reported. An elevated pattern of ankle toque was noted in all velocities tested on day 1 and week 1 of toxin treated animals compared with vehicle controls. By week-2, and for the remaining 5 weeks of testing, significant elevation of the ankle torque was only observed in the faster velocities. Significant increases in hindlimb axis and base of support were also observed from footprint analysis. These findings indicate that selective lesion of spinal noradrenergic fibers produced some of the specific changes in the reflex excitability that were observed following midthoracic spinal contusion injury.

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

Monoamine modulation of spinal reflex excitability of the lower limb in the rat: Intrathecal (i.t.) infusion of anti-DBH saporin toxin – time course for neurophysiology.

Wang DC, Bose P, Parmer R, Wiley RG, Thompson FJ (2001) Monoamine modulation of spinal reflex excitability of the lower limb in the rat: Intrathecal (i.t.) infusion of anti-DBH saporin toxin – time course for neurophysiology. Neuroscience 2001 Abstracts 771.5. Society for Neuroscience, San Diego, CA.

Summary: Progressive neurophysiological changes in the excitability of ankle extensor stretch reflex were identified following T8 spinal cord contusion injury (Thompson et. al., 1992). Previous study suggested that nonspecific monoamine depletion may be a significant contributor to the pathologic hyperreflexia associated with chronic spinal cord injury (Thompson et. al., 1999). To test a more specific hypothesis, a study was performed to evaluate the time course of changes in reflex excitability after i.t. injection of 250ng of anti-DBH saporin toxin to specifically lesion descending spinal noradrenergic neurons into the lumbar spinal cord of normal rats. Measures of H-reflex excitability were obtained prior to and at weekly intervals following toxin injection until a physiological plateau was observed. Significant decreases in rate-depression of H-reflexes were observed by the second week after toxin infusion and were maintained throughout the five weeks of testing. These studies indicate that selective lesioning of noradrenergic fibers produced specific changes in reflex excitability previously observed following midthoracic spinal cord contusion injury in the rat. The results of this study further implicate neurophysiological changes associated with monoamine loss as a contributing factor leading to hyperreflexia derived from chronic spinal cord injury. Correlated behavioral changes are reported in the companion poster, Bose et.al.

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

A monoclonal antibody to an extracellular domain of the serotonin transporter: Characterization and targeting properties.

Kohls MD, Majer KA, Russell BJ, Han Q, Blakely RD, Lappi DA (2001) A monoclonal antibody to an extracellular domain of the serotonin transporter: Characterization and targeting properties. Neuroscience 2001 Abstracts 814.9. Society for Neuroscience, San Diego, CA. PMID: 0

Summary: Using a peptide corresponding to a sequence from an extracellular domain of the rat serotonin re-uptake transporter (SERT), we have created a monoclonal antibody that recognizes the molecule on the cell surface. In FACS analysis, the antibody is able to recognize SERT-expressing human platelets and rat basophilic RBL-2H3 cells. Recognition is inhibited by the peptide immunogen. In western blotting of SERT-transfected cells, the antibody recognizes an approximately 95 kDa band corresponding to SERT, and shows no such recognition in the parent cell line. We have created an immunotoxin with this antibody by conjugation to the ribosome-inactivating protein saporin. This immunotoxin is 50-fold more cytotoxic than saporin to RBL-2H3 cells, according to ED50. The ED50 for the immunotoxin is 10.6 nM, whereas the ED50 for non-conjugated saporin is 477 nM. At 100 nM, the immunotoxin eliminates 72% of cells, whereas saporin alone has no significant difference on cells from control (addition of vehicle). These data demonstrate that antibody binding to SERT results in internalization, a situation we have also seen with the dopamine transporter (R.G. Wiley, M.B. Harrison, A.I. Levey and D.A. Lappi, submitted). Antibodies for the targeting and delivery of molecules to the interior of cells can be created through the use of peptide immunogens derived from the sequences of the extracellular domains.

Related Products: Antibody to Serotonin Transporter (SERT, Cat. #AB-N09)

Bcl-2 improves survival of lesioned cholinergic neurons.

Lee KY, Leos R, Borowski TB, de Lacalle S (2001) Bcl-2 improves survival of lesioned cholinergic neurons. Neuroscience 2001 Abstracts 803.6. Society for Neuroscience, San Diego, CA.

Summary: Alzheimer’s disease is associated with the progressive cell death of cholinergic neurons in the central nervous system (CNS). A current challenge is to develop therapeutic approaches that could prevent atrophy and loss of CNS neurons, and promote regeneration of their processes. The purpose of our study is to determine the therapeutic potential of the antiapoptotic gene B-cell lymphoma 2 (bcl-2) on the cholinergic system of the basal forebrain (BF). Retrograde degeneration in the BF of 3-month-old rats was induced by unilateral injection of the immunotoxin 192 IgG-saporin into the entorhinal cortex. Fifteen minutes later, an injection of 5 µg of the pa22bgala4bcl-2 plasmid was placed into the BF. Similar procedures were followed for BF injections of a control plasmid or vehicle alone. We found that a single injection of bcl-2 plasmid into the BF of the lesioned side increased survival of cholinergic neurons ~50%, as compared to vehicle controls. In addition, cell survival was close to 100% of intact side with bcl-2 injections, compared to a 50% cell loss in animals injected with a control DNA. Our results indicate that an injection of the bcl-2 gene into the BF prevents loss of cholinergic neurons that have been injured by IgG-saporin. Mechanisms by which bcl-2 may protect cholinergic neurons could include retrograde transport of the plasmid to the nucleus and subsequent neuroprotective effects of increased levels of bcl-2 protein. Our results, in conjunction with other studies, suggest that neurodegeneration might be amenable to gene therapy.

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

Selective immunotoxin lesions of hindbrain norepinephrine/epinephrine (NE/E) neurons impair feeding and corticosterone responses and Fos-immunoreactivity in hypothalamic sites during insulin-induced hypoglycemia (IIH).

Sanders NM, Dinh TT, Pedrow C, Ritter S (2001) Selective immunotoxin lesions of hindbrain norepinephrine/epinephrine (NE/E) neurons impair feeding and corticosterone responses and Fos-immunoreactivity in hypothalamic sites during insulin-induced hypoglycemia (IIH). Neuroscience 2001 Abstracts 635.21. Society for Neuroscience, San Diego, CA.

Summary: Previously,we used the targeted immunotoxin saporin, conjugated to a monoclonal antibody against dopamine Beta-hydroxylase(DSAP),to destroy hypothalamic projecting NE/E neurons. Results showed that NE/E neurons are required for 2-deoxy-D-glucose induced feeding and Fos expression in the hypothalamus. In the present study,we used this same technique to determine if NE/E neurons play a similar role in mediating IIH responses.Rats were injected with DSAP or unconjugated saporin (SAP)into the hypothalamic paraventricular nucleus(PVH).Insulin reduced blood glucose to similar values in DSAP and SAP rats(15 and 17mg/dl, respectively). Glucagon responses to hypoglycemia were unaffected by DSAP,peaking at 597% and 504% of pre-drug levels after insulin in DSAP and SAP rats,respectively.In contrast,the corticosterone response was severely diminished in DSAP rats,peaking at only 123% of pre-insulin levels, compared to 353% in SAP rats.DSAP injections also abolished the feeding response to IIH.DSAP rats ate 0.9g of food during IIH while the SAP rats at 6.1g of food.DBH-ir was abolished in the A1/C1 overlap and reduced in A2,C2,C3 and A6 sites in DSAP rats. In the SAP rats,IIH induced Fos-ir in hindbrain NE/E neurons the PVH, LH and ARC.In DSAP rats, Fos-ir was reduced or abolished in these hypothalamic sites but was preserved in the adrenal medulla.These findings further support the role of hindbrain NE/E neurons in transmitting information from hindbrain glucoreceptive sites to hypothalamic circuits coordinating feeding and neuroendocrine responses to glucose deficit.

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

Dermorphin-saporin conjugate relieves inflammatory pain after peripheral application.

Palecek J, Paleckova V, Willis WD (2001) Dermorphin-saporin conjugate relieves inflammatory pain after peripheral application. Neuroscience 2001 Abstracts 508.10. Society for Neuroscience, San Diego, CA.

Summary: Opioid receptors have been shown to exist in specific population of DRG neurons signaling nociceptive information from peripheral tissues. In our study, we attempted to selectively destroy these neurons by using a peripheral application of the mu opioid agonist Dermorphin conjugated to ribosome inactivating toxin Saporin (DERM-SAP, Advanced Targeting Systems) in order to alleviate inflammatory pain. Intraarticular or intraplantar injection of carrageenan or CFA was used to induce inflammation in rats. The DERM-SAP conjugate was injected into the inflamed area 12-48h later. Responses of the animals to mechanical and thermal stimuli were tested before and after the inflammation and up to 21 days after the DERM-SAP application. The rats developed heat hyperalgesia in the affected paw 24 hours after the intraarticular CFA injection. In the saline injected group the hyperalgesia persisted for up to 19 days, but in the DERM-SAP injected group the signs of hyperalgesia were improving from day 7. Also mechanical allodynia tested with a VF filament (1.1g) was alleviated in the DERM-SAP group. In the carrageenan group, the DERM-SAP treatment decreased the heat hyperalgesia and prevented the development of hyperalgesia after repeated carrageenan application, 21days after the DERM-SAP treatment. Postmortem evaluation with a specific antibody showed presence of saporin in the DRG neurons. Our results show that peripheral application of DERM-SAP relieves inflammatory pain and suggest that peripheral application of neuropeptides conjugated to cell toxins or other substances such as antisense probes could be a useful tool for treating pain of peripheral origin. Supported by NIH grants NS09743 and NS11253.

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

Effects of 192-saporin lesion of the basal forebrain on sleep homeostasis and adenosine receptor (A1) mRNA levels.

Greco MA, Salin-Pascual R, Gerashchenko D, Blanco-Centurion C, Shiromani PJ (2001) Effects of 192-saporin lesion of the basal forebrain on sleep homeostasis and adenosine receptor (A1) mRNA levels. Neuroscience 2001 Abstracts 523.12. Society for Neuroscience, San Diego, CA.

Summary: Adenosine is hypothesized to be a mediator of sleep since adenosine levels in the basal forebrain increase after wakefulness and decrease following sleep. The inhibitory effect of adenosine on wake-active cholinergic neurons is thought to be mediated by the A-1 receptor subtype. We hypothesized that if adenosine inhibition of cholinergic neurons takes place via A-1A receptors on cholinergic neurons, the elimination of cholinergic cells should affect sleep homeostasis. To test this hypothesis, 192-saporin was used to selectively lesion basal forebrain cholinergic cells. 48h baseline sleep was recorded from male Sprague Dawley rats. Subsequently, the rats were kept awake for 12h and 24h recovery sleep was recorded. 192-saporin (4 ug) was then administered ICV. The rats were again continuously recorded for 3 weeks after the injection, a 12h prolonged waking period and during a 24h recovery sleep period. Brain sections processed for visualization of A1 mRNA and/or immunohistochemistry revealed that both ChAT- and parvalbumin-positive cells contained A-1A mRNA. 192-SAP eliminated ChAT immunoreactive cells in the basal forebrain. There were no differences in sleep-wakefulness up to 3 weeks after drug administration, a finding consistent with previous reports. In addition, there were no changes in recovery sleep following prolonged waking in lesioned rats. These results indicate that the cholinergic basal forebrain groups are not the primary mediators of wakefulness or of sleep homeostasis. We suggest that the effects of adenosine are mediated via binding to non-cholinergic neurons.

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

Basal forebrain cholinergic system: Cortical activation, sleep/waking EEG and evoked potentials.

Shafi R, Berntson GG, Sarter M, Saurer T, Spino M (2001) Basal forebrain cholinergic system: Cortical activation, sleep/waking EEG and evoked potentials. Neuroscience 2001 Abstracts 533.16. Society for Neuroscience, San Diego, CA.

Summary: The role of the basal forebrain cholinergic system in cognitive functions such as arousal, attention and memory has been well documented. The purpose of the present study was to further elucidate the role of the basal forebrain in regulating cortical states and processes that may underlie these functions. Selective lesions of the cholinergic neurons of the basal forebrain were made using the immunotoxin 192 IgG-saporin, which selectively targets the p75 receptor on cholinergic neurons. The effects of these lesions on sleep structure and EEG activity and on afferent priming of cortical reactivity was evaluated. Specifically, we monitored behavioral activity and sleep states and examined the frequency distribution of power distribution in EEG frequency bands during these states. In separate sessions, we also recorded cerebral event-related potentials to auditory stimuli (100 ms, 1 K Hz at 60, 70 and 80 db) after intraperitoneal administration of saline or epinephrine (0.5 mg/kg, which we have previously found to result in priming or enhancement of the auditory evoked response). Compared to controls, lesioned animals showed a reduction in spontaneous activity, reduced power in higher frequency (primarily gamma) EEG bands during both sleep and waking, and altered sleep structure. In addition, lesioned animals displayed lower amplitude auditory evoked potentials and a loss of epinephrine-priming of the evoked response. Results support the view that the basal forebrain cholinergic system may play an important role in cortical activation and the regulation of sleep/waking states, as well as in cortical processing and its enhancement by visceral priming.

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

Low dose 192 IgG-saporin selectively destroys basal forebrain cholinergic neurons and impairs acquisition of a spatial memory task.

Zambon NJ, Nagle R, Pokala V, Gibbs RB, Johnson DA (2001) Low dose 192 IgG-saporin selectively destroys basal forebrain cholinergic neurons and impairs acquisition of a spatial memory task. Neuroscience 2001 Abstracts 534.13. Society for Neuroscience, San Diego, CA.

Summary: We previously showed that a high dose (1 μg) of the selective cholinergic immunotoxin 192 IgG-saporin (SAP), injected into the medial septum (MS) of Sprague-Dawley rats, impeded acquisition of a delayed matching-to-position (DMP) spatial memory task, whereas injections of ibotenate (5 μg in 1 μL) did not. The present study examined the effects of lower doses of SAP (0.22 and 0.45 μg in 1 μl) on DMP acquisition. Animals received either SAP or vehicle injected directly into the MS. Two weeks later, animals were food deprived and trained to the DMP task. Rats received 8 trial pairs/day until they reached a criterion of 15/16 correct choices. Seven days later, post-criteria testing for retention was performed. Brain tissues were analyzed for choline acetyltransferase (ChAT) activity, or were processed for immunohistochemical detection of ChAT and parvalbumin. Control rats required significantly fewer days (13.1) to reach criterion than rats that received 0.22 (22.0 days) or 0.45 (20.1 days) μg SAP. There was no effect of SAP treatment on post-criteria testing. Injections of SAP produced marked depletion of ChAT-positive cells and ChAT activity, but no apparent depletion of parvalbumin staining in the MS. In contrast, ibotenate injections used in the previous study were shown to produce marked depletion of parvalbumin staining in the MS, but no significant cognitive impairment. The data suggest that selective destruction of cholinergic neurons in the MS significantly impairs acquisition of the DMP task.

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

Loss of histaminergic neurons does not produce hypersomnolence.

Chou TC, Gerashchenko D, Saper CB, Shiromani PJ (2001) Loss of histaminergic neurons does not produce hypersomnolence. Neuroscience 2001 Abstracts 522.21. Society for Neuroscience, San Diego, CA.

Summary: Electrolytic lesions of the posterior hypothalamus (PH) produce long-lasting hypersomnolence (1,2). The PH contains histaminergic neurons in the tuberomammillary nucleus (TMN) that project diffusely throughout the brain. Because histamine promotes wakefulness while antihistamines are sedating, the TMN is thought to be critically involved in maintaining wakefulness. To test this hypothesis, we placed cell-specific lesions in the PH and TMN of rats and measured sleep-wake behavior. Lesions were produced using either the conventional excitotoxin ibotenic acid, or the novel toxin orexin (hypocretin) conjugated to the ribosomal toxin saporin (ORX/HCRT-SAP). Ibotenic acid injections were ineffective at lesioning the TMN; most histaminergic neurons were selectively spared while neurons in surrounding regions such as the mammillary bodies and supramammillary area were completely lesioned. In contrast, ORX/HCRT-SAP injections into the TMN lesioned up to 95% of histaminergic neurons, as determined by adenosine-deaminase immunostaining, with a similar loss of neurons in adjacent areas. Surprisingly, neither group of rats showed changes in NREM or REM sleep time or circadian distribution of sleep relative to saline-injected controls for up to 2 weeks after surgery. Thus, the waking state may not be critically dependent on the PH or TMN in rats. Further research is needed to reconcile the sedating effects of antihistamines with the current findings. 1. Ranson 1939, Archiv Neurol and Psychiatry 41(1):1-23. 2. Swett and Hobson 1968, Arch Ital Biol 106(3):283-293.

Related Products: Orexin-B-SAP (Cat. #IT-20)

Behavioural and neurochemical changes associated with single and combined acetylcholine and dopamine lesions in neonatal rats.

Sherren N, Pappas BA (2001) Behavioural and neurochemical changes associated with single and combined acetylcholine and dopamine lesions in neonatal rats. Neuroscience 2001 Abstracts 539.5. Society for Neuroscience, San Diego, CA.

Summary: The functional outcomes of neonatal ACh or DA lesions are frequently less severe or qualitatively different from those seen in adult rats, and may be due to compensatory neurochemical changes. Given that these transmitter systems interact in the adult brain and that ACh and DA hypofunction may underlie the cognitive and motor disabilities seen in Rett syndrome, we hypothesized that combined neonatal ACh/DA lesions may produce a profile of neurochemical changes and behavioural impairments which are more severe or distinct from that caused by either lesion alone. Rats were lesioned at postnatal day 7 with 192 IgG-saporin (ACh rats), 6-OHDA with NE receptor blockade (DA rats), or both (ACh/DA rats). Behavioural testing occurred at 4 months of age. In the open field, only ACh/DA rats exhibited locomotor hyperactivity whereas all lesioned groups exhibited reduced exploratory behaviour. Neither DA nor ACh/DA rats were able to solve the Morris water maze, however ACh rats were indistinguishable from controls. 192 IgG-saporin treatment produced a 75% decrease in hippocampal ChAT activity, and cortical decreases of 30%, 70% and 40% in the frontal/cingulate (FC), retrosplenial (RS) and partietotemporal (PT) regions respectively. 6-OHDA treatment produced a 90% decrease in striatal DA levels and a 75% decrease in FC cortex. Interestingly PT DA levels were 68% higher in ACh rats but 47% lower in ACh/DA rats compared to control, while DA rats showed a decrease which was not significantly different from control. Thus sparing of spatial learning ability in ACh rats may be mediated by increases in PT DA levels, whereas combined ACh/DA lesions exacerbate DA loss in this region.

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

Immunolesioning of brainstem DBH neurons on the mating-induced LH and prolactin surge in the rabbit.

Pau K (2001) Immunolesioning of brainstem DBH neurons on the mating-induced LH and prolactin surge in the rabbit. Neuroscience 2001 Abstracts 466.7. Society for Neuroscience, San Diego, CA.

Summary: Coitus induces a surge release of norepinephrine (NE) that is accompanied by a preovulatory gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) surge. Prazosin, an alpha-1 adrenergic antagonist, attenuates the GnRH/LH surge, and tyrosine hydroxylase (TH) gene expression in brainstem NE areas increases within 30 min after coitus. Here, we determined the coitus-induced LH/prolactin surge after specific lesioning of dopamine beta-hydroxylase (DBH) neurons in the brainstem with monoclonal anti-DBH sera conjugated with the ribosomal cytotoxin saporin (DBH-SAP). Female NZW rabbits received 3rd cerebroventricular injection (Day 0) of either DBH-SAP (20 µg, n=4) or SAP (3 µg, n=4). On day 14, the four DBH-SAP females were paired with stud males, but none of them mated. After daily injection of estradiol benzoate (EB, 3 µg) for 3 days, all eight females mated. Blood samples were taken once before, and at 10-min intervals for 4 hours after, coitus. Brainstems were prepared for immunocytochemical detection of DBH and TH. Coitus increased both LH and prolactin release in either DBH-SAP or SAP animals. However, postcoital LH and prolactin levels were 55% lower and 50% higher, respectively, in DBH-SAP rabbits than in SAP animals. The number of DBH neurons was near zero in the A6 and reduced by 80% in the A1 and 70% in the A2 noradrenergic areas in DBH-SAP animals. The number of TH neurons was reduced by 95% and 30% in the A6 and A1 areas, respectively, and did not change in the A2 area. The results suggest that the presence of intact brainstem NE neurons are critical for sexual performance and production of normal LH/prolactin surge after coitus in female rabbits.

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

Neonatal cholinergic lesions alter reactivity to a GABAergic agonist in 18-day-old rats.

Ricceri L, Scattoni ML, Calamandrei G (2001) Neonatal cholinergic lesions alter reactivity to a GABAergic agonist in 18-day-old rats. Neuroscience 2001 Abstracts 541.14. Society for Neuroscience, San Diego, CA.

Summary: We have shown previously that neonatal intracerebroventricular (icv) injections of the selective cholinergic immunotoxin 192 IgG-saporin on postnatal day (pnd) 7induces learning impairments on pnd 15 and disruption of reactivity to spatial novelty on pnd 54. The same neonatal treatment also induces a permanent cholinergic loss in both hippocampus and neocortex. In the present study we analyzed behavioral effects induced by a GABAergic drug (muscimol, a GABAa receptor agonist) in rats neonatally lesioned with 192 IgG-saporin (icv on pnd 7). On pnd 18 192 IgG-saporin lesioned and sham rats were injected with muscimol (0.1, 0.5 mg/kg ip) and placed in an open field arena for 20 min; locomotion, wall rearing and rearing responses were measured. In sham animals, as expected, 0.1 muscimol decreased locomotion, wall rearing and rearing responses. In saporin lesioned animals 0.1 muscimol increased locomotion, left wall rearing responses unchanged and decreased only rearing responses. In a 60s hot-plate test, 0.1 muscimol induced comparable analgesic responses in both sham and saporin-lesioned animals. The 0.5 muscimol dose resulted cataleptic for both saporin and sham lesioned rats. Neonatal saporin per se also reduced wall rearing and rearing responses. These data suggest that only in selective behavioral patterns — associated with locomotion and exploration of the environment — reactivity to a GABAergic agonist is reduced following neonatal cholinergic lesions, probably because of a decrease of GABAa receptors in the medial septal nucleus.

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

α-1 adrenergic agonist effect on cholinergic muscarinic receptors.

Harrell LE, Kolasa K, Parsons DS, Conger K (2001) α-1 adrenergic agonist effect on cholinergic muscarinic receptors. Neuroscience 2001 Abstracts 549.16. Society for Neuroscience, San Diego, CA.

Summary: Degeneration of the basal forebrain cholinergic system and sympathetic ingrowth appear to be pathological changes in Alzheimer’s Disease patients (AD), leading to an alteration in the balance between both systems and may mediate cognitive deficits in AD. In an attempt to model this situation, intraventricular injection (ivc) of a specific cholinergic immunotoxin, 192-IgG-saporin, has been used to induce peripheral noradrenergic fibers to grow into cortex and hippocampus after cholinergic denervation of rat cortex (CCD) and hippocampus (HCD). This adrenergic reorganization has been termed cortical (CSI) and hippocampal (HSI) sympathetic ingrowth. 192-IgG-saporin ivc injection was followed by intraperitoneal (ip) treatment with α1 agonist methoxamine. Thus the effects on choline acetyltransferase (ChAT) activity, norepinephrine (NE) level and muscarinic acetylcholine receptors (mAChR) were studied in rat hippocampal and cortical brain tissue. We found that 192-IgG-saporin produced significant decrease in ChAT activity in all experimental groups and areas. Methoxamine (3 and 6 mg/kg ip) did not affect NE levels. It produced significant decrease in mAChR affinity in the cholinergic denervation group and no significant increase in mAChR density in cholinergically denervation groups of dorsal hippocampal and cortical areass. Results of the present study indicate the influence of α1 agonist treatment on mAChR and may provide new concepts for the future combination drug therapy for AD patients.

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

α-1 adrenergic antagonist effect on cholinergic muscarinic receptors.

Kolasa K, Harrell LE, Parsons DS, Conger K (2001) α-1 adrenergic antagonist effect on cholinergic muscarinic receptors. Neuroscience 2001 Abstracts 549.17. Society for Neuroscience, San Diego, CA.

Summary: Cholinergic denervation of cortex and hippocampus in rat causes an unusual neuronal rearrangement, in which peripheral sympathetic fibers, originating from superior cervical ganglia, grow into the cholinergically denervated areas. This process has been termed cortical (CSI) and hippocampal sympathetic ingrowth (HSI). A similar process may occur in Alzheimer’s Disease (AD). Recent studies suggest that the balance between central adrenergic and cholinergic systems may be important for normal learning and memory, while the alterations of these systems may play a critical role in cognitive deficits of AD. To better understand this situation specific cholinotoxin, 192-IgG-saporin, was intraventricularly (ivc) injected to produce a selective loss of cholinergic cells in rat basal forebrain nuclei, cholinergically denervating hippocampal (HCD) and cortical areas (CCD). This effect was confirmed by significant decrease in choline acetyltransferase activity in all groups and brain structures.192-IgG-saporin injection was followed by a treatment with α1-adrenergic antagonist prazosin to determine the effect on hippocampal and cortical muscarinic acetylcholine receptors (mAChR) and norepinephrine (NE) level. Prazosin (0.5 & 2 mg/kg ip) produced decreases in NE levels of HSI and CSI and induced no significant increase in mAChR affinity in HSI and CSI groups in dorsal hippocampus,anterior and entorhinal cortex. Injected at the dose of 2 mg/kg it increased mAChR density in CSI of both cortical areas. The present results began to define the interaction between adrenergic and cholinergic systems, as α1 antagonist treatment affects mAChR,a potential therapeutic target in AD.

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

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