sfn2001

62 entries

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 cytoarchitexture 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)

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)

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)

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)

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)

DL-homocysteic (DLH)-induced tachypnea is eliminated by ablation of neurokinin-1 receptor immunoreactive (NK1R-ir) neurons of the preBötzinger complex (preBötc).

Wang H, Guyenet PG (2001) DL-homocysteic (DLH)-induced tachypnea is eliminated by ablation of neurokinin-1 receptor immunoreactive (NK1R-ir) neurons of the preBötzinger complex (preBötc). Neuroscience 2001 Abstracts 633.4. Society for Neuroscience, San Diego, CA.

Summary: We identified a hot spot of the ventrolateral medulla (VLM) where tachypnea is produced by small injections of the excitatory amino acid DLH in urethane-anesthetized vagotomized rats. We sought to determine its anatomical location relative to a group of NK1R-ir cells that may be a marker of the preBötC. We also determined the location of the hot spot relative to VLM pressor and depressor sites. The VRG was located by recording respiratory units and then 5-10 nl of 10 mM DLH were injected into the VRG at 200 mm interval from Bregma -12.1 to -13.3 mm. DLH produced site-specific changes in respiratory rate and mean arterial blood pressure (MAP). At rostral levels (Bregma -12.1 mm) PND rate decreased and MAP increased (-57 ± 11% and 9.4 ± 0.9%; N= 4-6). At more caudal levels these effects gradually reversed (cross-over point at -12.4 mm). Tachypneic responses were restricted between Bregma -12.5 and -13.1 mm with a sharp peak (88.7 ± 12.8% increase in rate) at -12.7 mm. The hot spot corresponded to where most pre-inspiratory neurons are found. It overlapped with VRG NK1R-ir cells but was more restricted than the distribution of these cells. Depressor responses were maximal at -12.7 mm and stable at more caudal levels. After unilateral ablation of the NK1R-ir cells (N=3) with a saporin-NK1R agonist conjugate, the DLH-induced tachypnea disappeared bilaterally. In conclusion, the tachypnea hot spot corresponds to the defined preBötC. It overlaps with the rostral end of the VLM depressor area and the NK1R-ir neurons of the preBötC may be responsible for DLH-induced tachypnea.

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

Poster: Selective cholinergic denervation inhibits expression of long-term potentiation in the adult but not infant rat hippocampus.

Motooka Y, Kondoh T, Nomura T, Tamaki N, Nishizaki T (2001) Poster: Selective cholinergic denervation inhibits expression of long-term potentiation in the adult but not infant rat hippocampus. Neuroscience 2001 Abstracts 599.7. Society for Neuroscience, San Diego, CA.

Summary: The present study assessed the role of the cholinergic systems on the expression of perforant path long-term potentiation (LTP) in rat hippocampal slices from the infant and adult brain. To denervate the cholinergic systems, 192 1gG-saporin was injected into the lateral ventricle of the infant (2 weeks old) and adult (6 weeks old) rat brain. There, choline acetyltransferase immunoreactive fibers were little detectable two weeks and two months after injection for both the groups. For the infant rats, perforant path LTP was not affected by selective cholinergic denervation; the probability of LTP development was 0.83 (5/six slices) and 0.78 (7/nine slices) at 2 weeks and 2 months later in 192 IgG-saporin-treated slices, as compared with 0.83 at each period in control saline-treated slices. In contrast, the expression of the LTP was blocked by selective cholinergic denervation for the adult rats; the probability of LTP development was 0 (0/ten slices) and 0.38 (3/eight slices) at 2 weeks and 2 months later in 192 IgG-saporin-treated slices, as compared with 0.8 (8/ten slices) and 0.83 (5/six slices) at each period in control saline-treated slices. The results of the present study thus suggest that the cholinergic systems play a crucial role in the expression of LTP in the adult brain and that the denervated systems in the infant brain could be compensated by the sprouting of non-cholinergic fibers.

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

Immunolesioning of identified motoneuron pools following intramuscular injection of the immunotoxin, 192-IgG-saporin, in neonatal rats.

Peterson WE, Jordan LM, Brownstone RM (2001) Immunolesioning of identified motoneuron pools following intramuscular injection of the immunotoxin, 192-IgG-saporin, in neonatal rats. Neuroscience 2001 Abstracts 626.14. Society for Neuroscience, San Diego, CA. PMID: 0

Summary: Studies have shown that the immunotoxin, 192-IgG-Saporin, can selectively lesion p75-positive cholinergic neurons of the basal forebrain in adult rats. Here we demonstrate the novel use of 192-IgG-Saporin to induce MN loss following intramuscular (I.M.) injection in neonatal rats. Two days following I.M. injection of 192-IgG-Cy3, neonatal rats (but not adult rats or neonatal mice) had Cy3-labeled MNs. This suggests that the 192-IgG antibody and its conjugates can be internalised by receptor-mediated endocytosis and retrogradely transported to spinal motor neurons. To induce MN loss, the left hind limb musculature of anaesthetised Sprague-Dawley rats were exposed, and several muscles injected with 0.5μg of 192-IgG-Saporin (Chemicon). Right hind-limb muscles were injected with DiI. Animals were sacrificed 25 days later. Ten μm coronal sections were obtained using a cryostat and Nissl stained. The neonatal rats showed signs of a locomotor deficit 2.5 weeks post injection with 192-IgG-Saporin, which increased slightly in severity over the next week and a half. Nissl stained coronal sections of the lumbar region showed an obvious MN deficit on the 192-IgG-Saporin treated side compared to control side. The injected muscles were also severely atrophic, a not unexpected finding given that they too express p75 receptors. We conclude that 192-IgG-Saporin can be used to lesion MN pools when IM injected in neonatal rats. This model may prove useful for testing cell replacement therapies for the treatment of MN diseases like amyotrophic lateral sclerosis (ALS).

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

Sound sequence discrimination requires cholinergic inputs and suppression of M-currents in the rat auditory cortex.

Kudoh M, Seki K, Watanabe S, Shibuki K (2001) Sound sequence discrimination requires cholinergic inputs and suppression of M-currents in the rat auditory cortex. Neuroscience 2001 Abstracts 621.2. Society for Neuroscience, San Diego, CA.

Summary: Synaptic potentiation after sequential heterosynaptic stimulation is dependent on the stimulus sequence in the auditory cortex. Atropine or antagonists of M1 receptors block this sequence dependence. However, it is rescued by linopirdine, an inhibitor of M-currents, even in the presence of atropine. In the present study, we investigated the role of cholinergic inputs and the resulting suppression of M-currents in sound sequence discrimination. Rats were trained to discriminate sequence of two sounds. Licking a spout during sound presentation of a particular sequence was rewarded with water. The sounds of the rewarded or unrewarded sequence were randomly presented in a trial, which was repeated every one minute for 12 hours in 4 days. The percentage of the trials with a licking response to sounds was calculated separately for the rewarded and unrewarded sequence. Test performance, estimated from the difference, was significantly increased in the 4 days. Atropine (10 mg/Kg, i.p.) suppressed the increase of test performance. A cholinergic immunotoxin, 192IgG-saporin, was injected into the auditory cortex (62-400 ng/1µl) 1 week before the test. Sound sequence discrimination, but not discrimination between the two sounds, was significantly suppressed in these rats. Linopirdine (5 mg/Kg, i.p.) rescued the test performance of sound sequence discrimination in the rats injected with 192IgG-saporin. These results suggest that sound sequence discrimination requires cholinergic inputs and the resulting suppression of M-currents in the auditory cortex.

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

Destruction by SP-SAP of rat retrotrapezoid nucleus (RTN) neurons expressing the neurokinin 1 receptor (NK1R) decreases breathing at rest and in response to hypercapnia.

Nattie EE, Li A (2001) Destruction by SP-SAP of rat retrotrapezoid nucleus (RTN) neurons expressing the neurokinin 1 receptor (NK1R) decreases breathing at rest and in response to hypercapnia. Neuroscience 2001 Abstracts 573.1. Society for Neuroscience, San Diego, CA.

Summary: Neurons in the RTN are hypothesized to provide both a tonic excitation for breathing and one location for central chemoreception (see Nattie, E., Prog. Neurobiol.1999). Lesions in anesthetized animals support the former while both lesions and focal acidification in unanesthetized animals support the latter. Application of substance P (SP) in the RTN increases respiratory output and immunohistochemistry for the SP (NK1) receptor shows extensive staining in the RTN. To destroy specifically these RTN neurons with NK1Rs we injected unilaterally in the RTN of the rat SP conjugated to the ribosomal toxin, saporin (SP-SAP; 100 nl; 1 uM; Advanced Targeting Systems). We measured ventilation by whole body plethysmography in the unanesthetized rat. At 6 to 15 days following SP-SAP injection, ventilation during air breathing was reduced by 19 to 24% and the response to 7% CO2 inhalation was reduced by 22 to 30%. Subsequent immunohistochemistry showed dramatically reduced NK1R staining in the area of the SP-SAP injection, which is difficult to quantify given the small number of RTN neurons and the extensive NK1R distribution along neuronal processes. RTN neurons with NK1Rs provide both a tonic excitation for breathing and a portion of the response to systemic hypercapnia.

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

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)

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