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)

McGaughy JA, Rubin S, Stollstorff M, Baxter MG, Eichenbaum HB (2002) 192 IgG-saporin-induced cortical, cholinergic deafferentation in rats produces a dissociation in the function of prelimbic/infralimbic and orbitofrontal cortex in an attentional set-shifting task. Neuroscience 2002 Abstracts 674.4. Society for Neuroscience, Orlando, FL.

Summary: Converging data support the hypothesis that cholinergic afferents to the cortex mediate attentional processes. Rats with selective cholinergic lesions of the nucleus basalis magnocellularis produced by 192 IgG-saporin (SAP) show deficits in attentional performance. These deficits are highly correlated with diminished cholinergic efflux in the infralimbic/prelimbic (IL/PL) cortex during attentional testing. Excitotoxic lesions of the IL/PL in rats trained in an attentional set-shifting task did not impair the initial discimination, a novel discrimination with the previously relevant dimension (intradimensional shift; IDS) or reversal learning, but did impair the ability to shift attention to the previously irrelevant stimulus dimension (extradimensional shifting; EDS). It is not known from the previous study whether the loss of cortical, cholinergic afferents alone would be sufficient to produce the EDS deficit. Consequently, infusions of SAP(0.01 μg/μl; 0.25 μl) were made into either the IL/PL or the orbitofrontal (OF) cortex. Rats were then trained in the same attentional set-shifting task. Subjects had to discriminate between stimuli based on one of two perceptual dimensions, odor or digging media with both dimensions present on all trials. Preliminary analyses show that neither OF nor IL/PL lesions impair the initial discrimination or the IDS. However, IL/PL lesions impair the EDS whereas OF lesions impair reversal learning. These data support dissociable roles of cholinergic afferents to OF and IL/PL in attentional set-shifting.

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

Sarter MF, Draut A, Herzog CD, Bruno JP (2002) Effects of septohippocampal cholinergic deafferentation on attention and learning. Neuroscience 2002 Abstracts 674.8. Society for Neuroscience, Orlando, FL.

Summary: In contrast to the extensively studied attentional functions mediated via basal forebrain corticopetal cholinergic projections, the role of septohippocampal cholinergic projections in attention and memory have remained poorly understood. For example, selective lesions of this system have limited, if any, effects on spatial memory performance. The present experiment initially tested the effects of intraseptal injections of the cholinergic neurotoxin 192-IgG saporin (SAP) on the performance of rats in an operant procedure designed to assess sustained attention. Despite almost complete hippocampal cholinergic deafferentation, the lesioned animals’ attentional performance remained identical to that of controls. Task parameter manipulations designed to further increase the demands on attentional processing also failed to reveal an effect of the lesions. However, lesioned animals were superior in acquiring a version of this task in which the propositional rules of the task were reversed. Lesioned rats achieved >65% hits after 14 training sessions, while intact rats did not reach this level of performance after 50 sessions of training. As the acquisition of the reversal of such extensively practiced response rules suffers from interference from the rather automatic processing of the original rules, loss of hippocampal cholinergic inputs may have reduced the interference that resulted from the processing of the original rules. Thus, hippocampal cholinergic inputs are speculated to modulate, but not to be necessary for, the recall of propositional rules. Collectively, the available data completely dissociate the functions of septo-hippocampal and corticopetal cholinergic projections.

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

Harrell LE, Parsons DS, Conger K, Kolasa K (2002) ß1 adrenergic antagonist effect on brain muscarinic cholinergic receptors. Neuroscience 2002 Abstracts 685.13. Society for Neuroscience, Orlando, FL.

Summary: Degeneration of basal forebrain cholinergic system and sympathetic ingrowth appear to be pathologic changes in Alzheimer’s Disease (AD). An imbalance between these systems may mediate cognitive deficit in AD. To model this situation, 192-IgG-Saporin, a specific cholinergic immunotoxin, was infused intraventricularly to induce cholinergic denervation and sympathetic ingrowth into cortex and hippocampus. After 8 weeks of intraperitoneal injection of Metoprolol, β1 antagonist, at 2.5 mg/kg and 5 mg/kg, the Kd and Bmax of dorsal hippocampus (DH), anterior (AC) and entorhinal (EC) cortex was determined via [3H]-QNB, muscarinic antagonist, binding. Low dose Metoprolol increased Kd in the sympathetic ingrowth, cholinergic denervation, ganglionectomized groups compared to control and vehicle groups (p<.05). Affinity of AC=DH but was > than EC (p<.02). Bmax was greater in AC than DH (p<.05) > than EC (p<.02). Controls and ganglionectomized had > Bmax in AC and EC (p<.03). EC had > Bmax in control and ganglionectomized animals (p<.04). High dose Metoprolol induced a greater affinity in DH>AC>EC (p<.05). No effect was found on Bmax.The results of our study suggest that a β1 antagonist, which is used clinically, can alter the number and affinity of cholinergic receptors, which in turn could potentially alter the AD patients' response to cholinergic therapy.

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

Rudick CN, Gibbs RB, Woolley CS (2002) Estrogen-induced disinhibition of hippocampal CA1 pyramidal cells depends on basal forebrain cholinergic neurons. Neuroscience 2002 Abstracts 740.6. Society for Neuroscience, Orlando, FL.

Summary: Estrogen (E) increases dendritic spine and synapse density on hippocampal pyramidal cells, both in vivo and in vitro. In both cases, the increase in spine/synapse density is preceded by transient disinhibition. Based on in vitro studies, this transient disinhibition is likely to be involved in the mechanism of the subsequent increase in spine density. In adult female rats, where E increases spine/synapse density on CA1 pyramidal cells, it is unknown whether E acts within the hippocampus itself and/or through hippocampal afferents to regulate synaptic changes. Considerable evidence suggests that the basal forebrain (BF) cholinergic system could be involved in mediating E’s effects in the hippocampus. Therefore, we tested the ability of E to disinhibit CA1 pyramidal cells in adult female rats in which BF cholinergic neurons were eliminated by infusion of 192IgG-saporin toxin (SAP) into the medial septum. Two weeks after SAP or SHAM lesion, rats were ovariectomized and treated with E or oil (O) 3 days later. Synaptically evoked inhibitory postsynaptic currents (eIPSCs) and miniature IPSCs (mIPSCs) in CA1 pyramidal cells were evaluated 24h after E or O, the timepoint at which disinhibition occurs. As previously shown, E decreased eIPSC amplitude and mIPSC frequency at 24h. Additionally, E-induced disinhibition was significantly reduced in SAP lesioned rats, but it was not completely blocked. These data demonstrate that the BF cholinergic system is involved in E-induced disinhibition of CA1 pyramidal cells, but that other cells may also be involved.

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

Sherren N, Pappas BA (2002) Neonatal dopamine lesions: Cognitive impairment or hyper-sensitivity to stress. Neuroscience 2002 Abstracts 782.19. Society for Neuroscience, Orlando, FL.

Summary: Neonatal rat pups given a selective cholinergic immunotoxin exhibit large reductions in cortical (30-70%) and hippocampal (75%) choline acetyltransferase (ChAT) activity which persist into adulthood. However these rats do not show spatial learning deficits in the Morris water maze despite the sensitivity of this task to muscarinic receptor blockade and hippocampal damage. We hypothesized that while the developing brain may be able to compensate for early loss of ACh transmission, it may also become more vulnerable to additional disruptions in other systems. We combined postnatal day 7 i.c.v. administration of 192 IgG-saporin with 6-hydroxydopamine (6-OHDA) to lesion either or both ACh and DA terminals respectively. 6-OHDA treatment produced a 90% loss in striatal and a 75% loss in frontocortical DA levels. No differences in exploratory behaviour were found between ACh, DA and ACh/DA lesioned rats. However upon placement in the Morris water maze, DA depleted rats displayed behaviour suggestive of panic and were unable to search for the hidden platform effectively. In order to determine whether the DA depletion was producing a spatial learning deficit or an exaggerated reaction to a stressor (the hidden platform task), a separate cohort of lesioned rats was tested in the cued platform version of the maze. Just prior to testing, the rats received 4 days of shaping in order to gradually habituate them to the pool and teach them the task. Platform location improved in half of the DA and ACh/DA rats, but never approached control or ACh only levels. Thus rats with neonatal DA lesions may be particularly sensitive to stressful tasks. This sensitivity may be partly dependent on lesion extent.

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

Blanco-Centurion CA, Gerashchenko D, Murillo-Rodriguez E, Shiromani PJ (2002) Simultaneous neurotoxic lesions of noradrenergic LC, histaminergic TMN and cholinergic BF neurons do not elicit hypersomnia whereas lesions of the hypocretin-containing LH neurons do. Neuroscience 2002 Abstracts 577.16. Society for Neuroscience, Orlando, FL.

Summary: Wakefulness is believed to be due to activation of neurons in the locus coeruleus (LC), tuberomammillary nucleus (TMN) and the basal forebrain (BF). These neurons receive a heavy projection from hypocretin (HCRT) neurons. It has been proposed that the HCRT neurons maintain wakefulness via their innervation of these three groups of neurons. Here we test this hypothesis by lesioning the LC, TMN and the BF. Sprague-Dawley rats implanted with sleep recording electrodes were given microinjections of the following saporin neurotoxins to lesion specific neurons: α-DBH-saporin (vol=0.4 μL; 1 μg/μL, LC lesion) , HCRT2-saporin (vol=0.4 μL; 0.20 μg/μL, TMN lesion) and 192IgG-saporin (vol=0.5 μL; 0.4 μg/μL, BF lesion). Six rats given saline injections served as controls. Immediately after surgery sleep recordings were made continuously for three weeks. In rats that had double lesions (n=7)(combinations of LC, TMN or BF) sleep was not increased. In two rats the LC, TMN and BF were destroyed (>95%) but there was no hypersomnia either. However, one rat that had a triple lesion and also had 30% loss of HCRT neurons showed significant and persistent hypersomnia. Previously, lesion of a single wake-active neuronal population has not been found to increase sleep. We have now found that double or triple lesions also do not produce hypersomnia. Only when the HCRT neurons are lesioned, sleep is affected. This suggests that HCRT neurons play a primary role in waking and the LC, TMN or BF neurons do not mediate this function.

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

Fernandez M, Giuliani A, Giardino L, Calza L (2002) In vivo strategies for stem cells regulation in the adult brain: A chance for cholinergic neurons. Neuroscience 2002 Abstracts 483.14. Society for Neuroscience, Orlando, FL.

Summary: Degenerative diseases represent a severe problem in view of very limited repair capability of nervous system. In order to use stem cells in the adult CNS for repair purpose, we are exploring the possibility to influence, in vivo, proliferation, migration and phenotype lineage of stem cells in adult brain using a growth factor, hormone and cytokine cocktail. In this study we used substances appropriate for in vitro cholinergic differentiation in animals lesioned with icv administration of the cholinergic neurons immunotoxin 195IgG-saporine (3microg/4,5microl). Four months after lesion, no ChAT-positive neurons were found in the basal forebrain, acetylcolinesterase-reactive fibres and ChAT activity in the cerebral cortex and hippocampus dramatically decrease, and animals are severely impaired in water maze learning task. An Alzet osmotic Minipump for chronic release (over 14 days) of the mitogen EGF (360ng/days) was then implanted and connected to a icv catheter. This treatment increases proliferation rate in SVZ in lesioned and unlesioned animals as indicated by the widespread distribution of BrDU-positive nuclei in the forebrain. Rats were then treated with retinoic acid (2.25 mg/day, orally). This treatment reduces Ki67 protein in the SVZ in lesioned rats, and this could indicate a progression toward differentiation. TrkA-positive innervation also increase in the basal forebrain of EGF+retinoic acid treated rats and ChAT activity is lightly, but significantly raises by combined EGF + retinoid acid treatment in the hippocampus.

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

Fargo KN, Sengelaub DR (2002) Androgen manipulation protects remaining motoneurons from dendritic atrophy after induced motoneuron death. Neuroscience 2002 Abstracts 466.13. Society for Neuroscience, Orlando, FL.

Summary: Androgen treatment facilitates axon regrowth after axotomy of facial and sciatic motoneurons, and reverses castration-induced dendritic atrophy in motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in rats. We assessed whether a similar therapeutic effect of androgen would be seen in dendrites following partial depletion of SNB motoneurons. We injected the toxin saporin, conjugated to choleratoxin (β-saporin), unilaterally into the SNB target muscles, bulbocavernosus (BC) and levator ani (LA), of two groups of adult male rats. One group had been castrated six weeks earlier to induce dendritic atrophy, and received testosterone-filled Silastic capsules coincident with β-saporin injection (SAP+T). The other group had no castration or androgen treatment (SAP-only). Four weeks after β-saporin injection, we injected choleratoxin conjugated HRP into the contralateral (non-saporin injected) BC muscle to label SNB motoneurons. A group of untreated normal males was also included. Cell counts were performed, and dendrites of HRP-labeled SNB motoneurons were reconstructed in three dimensions. β-saporin killed ~65% of motoneurons in the SNB ipsilateral to the saporin-injected muscles; contralateral SNB motoneuron numbers were not affected. SNB dendritic arbors on the non-saporin injected side were ~60% shorter in SAP-only animals compared to those of untreated males; in contrast, dendritic arbors in SAP+T animals were unaffected. These results indicate that a) motoneuron death causes dendritic atrophy in remaining SNB motoneurons, and that b) previous castration and concurrent testosterone replacement protects against this atrophy.

Related Products: CTB-SAP (Cat. #IT-14)

Kanold PO, Shatz CJ (2002) Developmental regulation of GABA Receptor subunits requires subplate neurons. Neuroscience 2002 Abstracts 530.11. Society for Neuroscience, Orlando, FL.

Summary: Subplate neurons (SP) are required for formation and patterning of thalamocortical connections. In visual cortex, SP ablation before the onset of the critical period, but when LGN axons are already in layer 4, prevents segregation into ocular dominance columns (ODCs) and emergence of functional orientation columns. Recent studies have linked ODC plasticity with maturation of inhibitory circuitry, which requires the appropriate expression and developmental regulation of GABA receptor subunits. We therefore wondered if SP ablation alters GABA-R subunit expression. Focal injections of kainic acid or immunotoxin were made into cat SP between P7-P10, just prior to the onset of ODC formation. 3 weeks later, in situ hybridization revealed that expression of several subunits of the GABA-A receptor was reduced in the ablated region, especially in layer 4. Other genes involved in synaptic function such as Homer and mGluR subunits also decreased in expression. These changes in gene expression suggest that the efficacy of inhibition in layer 4 is reduced. A reduction in postsynaptic GABA receptors is consistent with previously observed increases in BDNF and GAD expression after SP ablation. These observations imply that SP neurons are essential for the maturation of cortical inhibition, which in turn may be needed for the formation of ODCs and for the functional maturation of thalamocortical connections.

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Sheehan JJ, Tsirka SE (2002) Reduction of microglia cell populations before induction of excitotoxicity reduces neurodegeneration. Neuroscience 2002 Abstracts 606.9. Society for Neuroscience, Orlando, FL.

Summary: Excitotoxicity is thought to be a component of many neurodegenerative diseases including Alzheimer’s and stroke. In excitotoxicity, as well as other injury models, microglia have been found to have both neuroprotective and neurodegenerative roles. To lend further insight into this controversy we utilized an immunotoxin selective for monocyte derived cell populations including microglia. The immunotoxin will selectively kill microglia and is not toxic to neurons or other glia populations in culture. In addition, infusion of the immunotoxin into the hippocampus of C57/Bl mice results in a selective reduction in endogenous microglial cell populations in this region. Furthermore, this reduction occurs without any perturbation of other cell types or the extracellular matrix. If depletion of microglia in this manner precedes excitotoxic injury, then hippocampal neurodegeneration is significantly reduced. These results agree with other work in our lab, which suggests that microglial cells exhibit neurotoxic properties in excitotoxicity.

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