sfn2002

Watts AG, Sanchez-Watts G, Dinh TT, Ritter S (2002) Immunotoxic lesions of ascending catechoalminergic afferents abolish the CRH gene transcriptional response to 2-deoxyglucose in the rat paraventricular nucleus. Neuroscience 2002 Abstracts 865.2. Society for Neuroscience, Orlando, FL.

Summary: CRH neurons in the medial parvicellular (mp) part of the paraventricular nucleus (PVH) are critical for the neural control of the hypothalamo-pituitary-adrenal axis. One of their most prominent afferents sets derives from hindbrain catecholaminergic neurons that are thought to help mediate viscerosensory influences on the PVHmp. Despite the prominence of this input, its precise role in controlling CRH neuronal function remains controversial. Here we report the effect on basal and stimulated CRH gene expression of an immunotoxin that selectively destroys catecholaminergic neurons. Rats were injected in the PVH with either a saporin-anti-dopamine B-hydroxylase (DBH) conjugate (DSAP), which leads to total loss of DBH immunoreactivity in the PVH, or saporin alone (SAP), which does not. Three weeks later, animals were injected either with 250mg/kg of 2-deoxy-D-glucose (2DG) or vehicle. Thirty mins later they were anesthetized and perfused with 4% buffered paraformaldehyde. Fifteen um frozen sections were cut through the hypothalamus and hybridized for CRH mRNA, CRH hnRNA, or c-fos mRNA. DSAP treatment had no effect on CRH mRNA levels in the PVH of vehicle- or 2DG-injected animals, but abolished the CRH hnRNA and c-fos mRNA responses to 2DG. We have reported elsewhere that DSAP lesions selectively abolish the corticosterone response to 2DG, but not to swim stress, or circadian corticosterone release. We now show that catecholaminergic afferents are required for 2DG-induced CRH gene expression, but not for basal expression.

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

Ritter S, Dinh TT, Pedrow C, Roellich K (2002) Immunotoxic lesion of catecholamine afferents to paraventricular hypothalamus (PVH) impairs the corticosterone response to glucoprivation but not the basal secretory rhythm or response to swim stress. Neuroscience 2002 Abstracts 865.4. Society for Neuroscience, Orlando, FL.

Summary: Catecholamine afferents from the hindbrain densely innervate the medial parvicellular part of the PVH, which contains CRH neurons critical for control of corticosterone (CORT) secretion. However, the precise role of these afferents in control of CORT secretion is unclear. Here the immunotoxin, saporin conjugated to anti-dopamine B-hydroxylase(DSAP), which selectively lesions norepinephrine and epinephrine neurons, or unconjugated saporin (SAP) control solution, was microinjected into the PVH. After extensive habituation to testing conditions, DSAP and SAP rats were injected with 2-deoxy-D-glucose (2DG, 250mg/kg) or vehicle or subjected to a 5-min forced swim. Blood was sampled remotely between 0 and 240 min for radioimmunoassay of CORT. In a third test, blood was sampled every 4 hr for 24 hr to assess the basal secretory rhythm of CORT. Subsequently, loss of dopamine B-hydroxylase containing terminals without destruction of CRH neurons in the PVH of DSAP rats was confirmed by immunohistochemistry. In DSAP rats, the CORT response to 2DG was reduced dramatically to 29% of the response in SAP controls. In contrast, DSAP and SAP rats did not differ in their basal secretory rhythm or their CORT response to swim stress, indicating for the first time a stimulus-specific role of catecholamine afferents in control of CORT secretion. This finding is complemented by other work in which we (with A.G. Watts and G. Sanchez-Watts) show that these catecholamine afferents are required for 2DG-induced CRH gene expression, but not basal expression.

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

Wang H, Guyenet PG (2002) Neurokinin-1 receptor immunoreactive (NK1R-ir) neurons control caudal ventrolateral medulla (VLM) gabaergic depressor neurons. Neuroscience 2002 Abstracts 862.4. Society for Neuroscience, Orlando, FL.

Summary: Depressor responses to injection of DL-homocysteic acid (DLH) into the caudal VLM are attenuated after selective unilateral lesion of the NK1R-ir cells of the VLM with a saporin-NK1R agonist conjugate (SSP-SAP)(Wang et al., J. Neurosci 2002). Here we tested whether SSP-SAP treatment destroys caudal VLM depressor GABAergic neurons thereby causing loss of DLH-induced sympathoinhibition. Two weeks after unilateral lesion of VLM NK1R-ir cells (97% reduction without loss of catecholaminergic neurons), DLH (5-10 nl, 10mM) was injected into multiple regions of the caudal and rostral VLM on both sides of the brain. The decrease in BP and sympathetic tone (SND) caused by DLH injections into caudal VLM were blunted on the lesioned side vs the intact side (p<.05, N = 7). The rise in BP and SND caused by DLH injection into rostral VLM were normal on both sides. To determine if the GABAergic barosensitive cells of the caudal VLM express NK1R, conscious rats were infused with L-phenylephrine (PE) (7μg/min, for 25 min) or saline. PE infusion raised BP by 25% and decreased HR 27% (mean; N= 4). Saline infusion produced no effect. Fos-ir neurons were mapped throughout the VLM. The caudal VLM of PE-treated rats contained many more Fos-ir cells than that of the saline controls (128.7 ± 4.2 vs. 18.7 ± 1.6, N= 4). Caudal VLM Fos-ir neurons were not NK1R-ir in either group of rats. In conclusion, the baroreceptor-activated GABAergic neurons of the caudal VLM are not NK1R-ir. The data suggests that NK1R-ir cells might provide an excitatory drive to the caudal VLM barosensitive neurons (HL 28785 to PGG).

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

Nagle RA, Liberatore MA, Zombon NJ, Pokala VN, Li PK, Pokala VN, Johnson DA (2002) Effect of selective cholinergic lesioning of basal forebrain with 192 IgG-saporin on neurotransmitter concentrations in hippocampus of rat. Neuroscience 2002 Abstracts 880.6. Society for Neuroscience, Orlando, FL.

Summary: In vivo microdialysis techniques were used to examine the effects of lesioning of cholinergic neurons of the medial septum using the selective cholinergic neurotoxin 192-IgG-Saporin (SAP), on hippocampal acetylcholine (ACh), glutamate and GABA in adult male Sprague Dawley rats. High and low (1.0 and 0.22 μg) doses of SAP were used for infusion into the basal forebrain. SAP treated rats showed a significant dose dependent decrease of 74% and 59% in ACh for the high and low doses respectively, compared to controls. Glutamate decreased 50% in animals treated with 0.22 μg SAP. The data suggest that lesioning of basal forebrain neurons with SAP results in changes in neurotransmitter concentrations in the hippocampus.

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

Fraley GS (2002) Role of hindbrain catecholaminergic afferents to the medial hypothalamus in the regulation of penile reflexes in the rat. Neuroscience 2002 Abstracts 681.4. Society for Neuroscience, Orlando, FL.

Summary: The use of ex copula erections, or reflexive erections, has been used for decades in the study of the central pathways and neuroendocrinology of penile erections. However, the exact neuroendocrine pathways involved in developing penile erections are not known. This study utilized molecular neurosurgical techniques combined with behavioral, histological, and molecular analyses to determine a central link between metabolic state and penis erectile function. Utilizing saporin-conugate immunolesion techniques (DSAP), hindbrain catecholaminergic afferents to the hypothalamus that are reported to be glucoresponsive were eliminated. DSAP-lesioned rats had a significantly attenuated glucoprivic feeding response and significantly attenuated penile reflexes compared to controls. Analysis of Nissl-stained spinal cord sections demonstrated a significant reduction in the size of sexually dimorphic motoneurons. Furthermore, qualitative analysis of calcitonin gene-related immunoreactivity (CGRPir) in alternate spinal sections revealed a decrease in CGRPir in sexually dimorphic motor pools. Analysis of hypothalamic mRNA levels showed a significant increase in both oxytocin and neuropeptide Y mRNA, but not b-actin mRNA. No significant differences were seen, however, in the weight of the perineal muscles, seminal vessicles, or in plasma testosterone levels. These data indicate a novel hindbrain-hypothalamic-spinal cord pathway by which potential glucoresponsive neurons effect the ability to achieve penile erection based upon availability of metabolic fuel.

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

Beach TG, Potter PE, Sue LI, Fisher A, Scott S, Layne KJ, Newell AJ, Roher AE, Walker DG (2002) Cerebral abeta deposition induced by cortical cholinergic deafferentation is reduced by cholinergic therapy. Neuroscience 2002 Abstracts 722.9. Society for Neuroscience, Orlando, FL.

Summary: We have previously shown that cortical cholinergic deafferentation in rabbits results in cerebral Abeta deposition (Neurosci Lett 283:9-12, 2000). We have also shown that cholinergic therapy with acetylcholinesterase inhibitors and muscarinic agonists reduces Abeta concentrations in the CSF and cortex of normal rabbits (Neurosci Lett 310:21-24, 2001; Brain Res 905:220-223, 2001). Here we show that the histologic deposition and biochemical elevations of Abeta induced by cholinergic immunotoxin are reduced by systemic therapy with AF267B, an M1-selective muscarinic agonist, and physostigmine, an acetylcholinesterase inhibitor. Rabbits received i.c.v. injections of an immunotoxin composed of the p75 NTR-directed monoclonal antibody ME20.4 conjugated to saporin, a ribosomal toxin. One group of animals received s.c. AF267B (2 mg/kg/day) while another group received s.c. physostigmine (3 mg/kg/day). Control groups received either i.c.v. immunotoxin or sham lesion (i.c.v. saline) and no treatment. Four weeks after surgery, imunohistochemical staining for Abeta showed frequent positive blood vessels and perivascular diffuse plaques in the control group which received immunotoxin injection and no treatment. This was significantly reduced in animals which received either AF267B or physostigmine. Cerebrospinal fluid Abeta concentrations were also reduced significantly by both drug treatments. These results are directly relevant to humans since cortical cholinergic deafferentation is part of normal human aging.

Related Products: ME20.4-SAP (Cat. #IT-15)

Schaevitz LR, Baxter MG, Stearns NA, Huang YY, Lappi DA, Berger-Sweeney J (2002) The efficacy of intraparenchymal anti-p75 immunotoxin on medial septal cholinergic neurons in mice. Neuroscience 2002 Abstracts 778.11. Society for Neuroscience, Orlando, FL.

Summary: We have shown previously that anti-murine-p75-SAP (saporin conjugated to a rat monoclonal antibody against the mouse p75 nerve growth factor receptor) selectively destroys basal forebrain cholinergic neurons in vivo after intracerebroventricular injections (J. Neurosci. 21:8164-73). Cholinergic neuronal loss was more extensive in the medial septum (MS) than the nucleus basalis magnocellularis; it is unclear whether this distinction is due to toxin diffusion from the ventricles or differential sensitivity of the neuronal populations. Intraparenchymal (IPC) injections to specific targets can help resolve the issue. Here, we examine the efficacy of anti-murine-p75-SAP IPC injections on cholinergic neurons. Saline or different doses of toxin (0.1, 0.2, 0.4, 0.9, 4.7, and 9.4 microg/microL) were injected into the MS of adult male C57BL/6J mice. Ten days post lesion, brain sections were stained for choline acetyltransferase and p75 (cholinergic markers) to determine toxin efficacy, and calbindin and parvalbumin (non-cholinergic markers) to determine toxin specificity. Toxin doses below 1.0 microg/microL had no effect on cholinergic or non-cholinergic neurons, while doses above 4.7 microg/microL resulted in the complete destruction of both cholinergic and non-cholinergic neurons. More thorough testing of doses between 1 and 4 microg/microL will be required to determine the optimal toxin dose for IPC injections.

Related Products: mu p75-SAP (Cat. #IT-16)

Gerashchenko D, Blanco-Centurion C, Shiromani PJ (2002) Hypocretin2-saporin (HCRT2-SAP) lesions of the lateral hypothalamus does not affect the entrained or free-running rhythm of core body temperature. Neuroscience 2002 Abstracts 776.2. Society for Neuroscience, Orlando, FL.

Summary: Hypocretin (HCRT)neurons are present only in the lateral hypothalamus (LH) from where they project heavily to major arousal centers. HCRT neurons are lost in the sleep disorder narcolepsy, an illness characterized by an increased tendency to fall asleep during the normal active period. As such, it is hypothesized that HCRT neurons are responsible for “waking-up” the brain. To test this hypothesis we monitored the rhythm of core body temperature during entrained & free-run conditions after lesions of the HCRT neurons. 23 male Long-Evans rats implanted with sleep recording electrodes and a temperature transmitter were given one of two concentrations (90 ng/0.5 ìl vs 490 ng/0.5 ìl) of the neurotoxin hypocretin2-saporin (HCRT2-SAP) or unconjugated saporin to the LH. Control rats received saline (n=5). After surgery, sleep and temperature were continuously recorded for 21d in entrained conditions followed by 21d in continuous darkness. Both concentrations of the HCRT2-SAP lesioned HCRT neurons (88% vs 91% HCRT loss). However, HCRT lesions did not disrupt the entrained rhythm of core temperature by either advancing or delaying the phase position of the temperature rhythm. In the saline rats, the free-run period of temperature rhythm (tau) was 24.16 (±0.07) and this was not significantly different in the HCRT2-SAP or SAP rats. These results indicate that in the absence of HCRT, the animal wakes up at the correct time of day but then is not able to stay awake.

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

Mania I, Levita L, Rainnie DG (2002) Subtypes of substance P receptor immunoreactive interneurons in the basolateral amygdala. Neuroscience 2002 Abstracts 637.8. Society for Neuroscience, Orlando, FL.

Summary: Neurotoxic lesions of substance P receptor immunoreactive (SPR-IR) interneurons in the basolateral amygdala (BLA) using SP-saporin reduce anxiety related behavior. These lesions might provide a way to study how specific interneuron populations regulate neuronal activity in the BLA. In the hippocampus, SP-saporin lesions result in an ablation of PV-, CCK-, and SOM-IR interneurons, while sparing CB-IR interneurons. However, limited information is available about the type of neurons affected by this lesion in the BLA. In this study SPR-IR interneurons were characterized immunocytochemically using dual-labeling immunofluorescence. SPR-IR interneurons were examined for their colocalization with calcium-binding proteins and NPY in the rat BLA. The majority of SPR-IR (74%) neurons had a small round or multipolar somata that emanated 3-4 thin aspiny dendrites consistent with them being local interneurones. Interestingly, none of the SPR-IR cells colocalized PV, and they represent only 3–6 % of the CB expressing interneuron population. However, those SPR-IR neurons that do colocalize CB represent 25-45% of the total SPR-IR population. In contrast, 94% of the NPY-IR neurons colocalized with SPR-IR. However, only 51% of SPR-IR cells also co-express NPY-IR. These data suggest that SPR-IR cells represent a heterogeneous population comprising of roughly equal proportions of CB and NPY neurons. Moreover, in the rat BLA SPR-IR cells form a distinct and dissociable group from the PV-IR interneuron population, which should remain intact after SP-saporin lesions.

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

Fox K, Wolff I, Curtis A, Pernar L, Van Bockstaele EJ, Valentino RJ (2002) Multiple lines of evidence for the existence of corticotropin-releasing factor (CRF) receptors on locus coeruleus (LC) neurons. Neuroscience 2002 Abstracts 637.9. Society for Neuroscience, Orlando, FL.

Summary: Several physiological and anatomical findings suggest that CRF acts as a neuromodulator of LC neuronal activity. However, in situ hybridization studies have failed to demonstrate the existence of CRF receptor mRNA in LC neurons, arguing against a direct effect on these neurons. Here, a combination of techniques was used to test the hypothesis that LC neurons express CRF receptors. Primers for CRF-R1 and beta-actin were generated and micropunches of the LC were subjected to RT-PCR analysis. Bands at the predicted size for each PCR product were detected in samples obtained from the LC. The presence of CRF-receptor immunolabeling in LC tissue was also examined in Western blots. This revealed a band at 52 kD, consistent with the molecular weight reported in brain and the band was absent in membranes incubated with a combination of the CRF receptor antisera and the blocking peptide. In dual labeling immunohistochemical studies, tyrosine hydroxylase (TH) immunolabeled LC neurons exhibited CRF-receptor immunolabeling and this was absent in sections that were incubated in antisera that was preabsorbed with the blocking peptide. Ultrastructural analysis also revealed co-localization of CRF-receptor immunolabeling and TH in LC dendrites. Finally, intra-LC injection of a CRF-saporin conjugate (40-60 ng in 30 nl), but not unconjugated saporin, resulted in a time dependent neuronal damage that was selective to LC neurons. The present findings provide convergent evidence for the existence of CRF receptors in LC neurons.

Related Products: CRF-SAP (Cat. #IT-13)