sfn2000

Petruska JC, Johnson RD (2000) Differential input to a nociceptive specific reflex by trka-positive and trka-negative small diameter DRG afferents. Neuroscience 2000 Abstracts 354.7. Society for Neuroscience, New Orleans, LA.

Summary: The cutaneus trunci muscle (CTM) reflex is a nociceptive specific reflex in the rat. We examined whether particular subtypes of afferents may be differentially involved in the reflex. In particular, we wanted to determine if there were differences in input to the reflex between those expressing trkA receptors, and those lacking trkA receptors. We approached this question with two techniques. The selective neurotoxin 192-saporin was injected into the left T13 DRG to destroy the p75 receptor-bearing neurons. Two to three weeks following injection, the ability of the injected DRG to elicit the CTM reflex was examined in contrast to uninjected DRG. The ganglia were also retrieved for histochemical examination. In all cases where histochemical examination revealed a clear depletion of the neurotrophin-bearing neurons the injected DRG lacked the ability to induce the CTM reflex. Histochemical markers for the trkA-negative small diameter afferents appeared normal in all cases. The second approach utilized the trans-synaptic neuronal tracer pseudorabies virus (PRV). PRV was injected into the CTM to generate a retrograde tracing of the reflex circuit, including the afferents involved. This resulted in specific labelling of many small diameter DRG neurons, the vast majority of which expressed trkA. The CTM reflex may therefore not be a suitable monitor for inputs from the trkA-negative small diameter afferents, especially as regards its use in collateral sprouting experiments. These data indicate that inputs from the two populations of afferents are likely processed differently.

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

Vogt LJ, Sim-Selley LJ, Childers SR, Wiley RG, Vogt BA (2000) Distribution of mu-opioid receptors and activated G-proteins in rat cingulate cortex and alterations following removal of noradrenergic afferents. Neuroscience 2000 Abstracts 238.1. Society for Neuroscience, New Orleans, LA.

Summary: Anterior cingulate cortex (ACC) is involved in acute and chronic pain processing. Here we define opioid architecture throughout rat cingulate cortex, relate mu-opioid receptor and G-protein stimulated binding in particular layers, and localize binding to noradrenergic terminals with immunotoxin lesions (anti-DBH-saporin). [3H]DAMGO binding was highest in areas 32 and 24 with a peak in layer I. Midcingulate area 24′ and posterior area 29 had lower and homogeneous binding. DAMGO stimulated [35S]GTPγS binding in area 24′ was similar to that in areas 32 and 24, while area 29 had very low and homogeneous binding. Undercut lesions reduced [3H]DAMGO binding in all layers with greatest loss in layer I, while DAMGO-stimulated [35S]GTPγS binding losses occurred only in layers I-III. Since neurons in the midline thalamic nuclei and locus coeruleus synthesize mu-opioid receptors, noradrenergic afferents were removed with anti-DBH-saporin. This toxin reduced [3H]DAMGO binding only in layer I of areas 32 and 24, while DAMGO-stimulated [35S]GTPγS binding increased in layer II of areas 32 and 24, had no changes in area 24′, and decreased binding in layer I of area 29. Thus, in addition to their actions on ACC neurons, other sites of opiate drug actions are through mu-opioid heteroreceptors on glutamatergic thalamic and noradrenergic locus coeruleus afferents to ACC.

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

Burgess SE, Vanderah TW, Mantyh PW, Malan Jr TP, Ossipov MH, Lappi D, Lai J, Porreca F (2000) Dermorphin-saporin targets tonic descending facilitation in the rostral ventromedial medulla to block and reverse neuropathic pain. Neuroscience 2000 Abstracts 243.6. Society for Neuroscience, New Orleans, LA.

Summary: The hypothesis that chronic pain from L5/L6 spinal nerve ligation (SNL) is due to tonic activation of descending pain facilitation mechanisms was explored by selective targeting mu (μ) opioid receptor expressing cells in the RVM (i.e., presumably, ON cells). Rats were treated with a single RVM injection of dermorphin (DERM)(μ agonist), saporin (SAP), or dermorphin-saporin conjugate (DERM-SAP) and responses to non-noxious (von Frey filaments) or noxious (Hargreave’s test) stimuli characterized. DERM-SAP retained high affinity for μ receptors and acutely produced antinociception (tail-flick test), indicating agonist actions of the conjugate. Decreased staining of μ receptor-expressing cells was seen in superficial dorsal horn and in dorsal root ganglia 28 days after intrathecal injection of DERM-SAP, but not DERM or SAP. RVM DERM-SAP, DERM or SAP did not significantly alter baseline thresholds to von Frey filaments or noxious heat applied to the paw over 28 days. At day 28, RVM pretreated rats were subjected to sham- or SNL surgery and responses to tactile and heat stimuli monitored 7 days later (i.e., 35 days after the RVM pretreatment). DERM and SAP pretreated SNL rats showed the expected development of tactile allodynia and thermal hyperalgesia, while DERM-SAP pretreated rats did not. The RVM pretreatments did not alter responses in sham-operated controls. Administration of RVM DERM-SAP, but not SAP or DERM, to SNL rats showed full reversal of established allodynia/hyperalgesia by day 14. RVM pretreatment with β-funaltrexamine (β-FNA; opioid μ antagonist) prevented the antiallodynic and antihyperalgesic effects of subsequent DERM-SAP injection. These data, together with findings of blockade of SNL pain with RVM lidocaine or lesions of the dorsolateral funiculus, support the possibility of tonic activation of descending facilitation as a basis for chronic neuropathic pain.

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

Han JS, Bizon JL, Chun HJ, Maus CE, Gallagher M (2000) Feedback HPA axis to stress is impaired in rats with selective removal of hippocampal cholinergic input. Neuroscience 2000 Abstracts 388.16. Society for Neuroscience, New Orleans, LA.

Summary: Activation of intracellular glucocorticoid receptors (GRs) may play a permissive role in mechanisms that lead to degeneration of hippocampal neurons in pathological conditions such as Alzheimer’s disease (AD). A previous study demonstrated that loss of cholinergic input from cells in the basal forebrain, a prominent feature of AD, reduced glucocorticoid receptor mRNA expression in the hippocampus in rats (Bizon et al., 1999). This experiment was conducted to see if reduced GRs after loss of cholinergic input would impair the function of the HPA axis in response to acute restraint stress. The cholinergic lesion was made by microinjections of the immunotoxin 192-IgG-saporin into the medial septal area and the vertical limb of the diagonal band. About 2 weeks later, rats were prepared with intravenous silastic catheters in the right jugular vein. After 5 days recovery, restraint stress for 1 hr was performed at 9:00 (a.m.). Blood (∼|50ul) was sampled repeatedly via the jugular catheter immediately (0 min) and at various times following the termination of the stressor (1 hr, 2 hr, 4 hr). For each group, negative feedback after a peak response to restraint was evident as a general trend of decreasing corticosterone that approached basal values by four hours after the cessation of stress. However, the speed of recovery to baseline differed between groups. Rats with loss of cholinergic input had higher corticosterone concentrations for a longer period after restraint stress than control rats, reflecting a diminished negative feedback function. These results suggest a mechanism whereby loss of basal forebrain cholinergic neurons in AD could contribute to a dysregulation of the HPA axis and more protracted exposure to high amounts of glucocorticoids. As an extension of the glucocorticoid cascade hypothesis, glucocorticoids might then be a factor in endangering hippocampal neurons in this disease.

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

Jasmin L, Arsenault P, Ohara PT, Marchand S (2000) Chronic noradrenergic spinal denervation in rats does not produce long-term hyperalgesia. Neuroscience 2000 Abstracts 243.7. Society for Neuroscience, New Orleans, LA.

Summary: Pharmacological studies have established that noradrenaline tonically inhibits spinal nociceptive transmission. We tested the hypothesis that chronically decreasing spinal noradrenaline would result in a disinhibition of nociceptive afferents resulting in behavioral hyperalgesia. We destroyed noradrenergic cells innervating the spinal cord using dopamine beta-hydroxylase antibodies linked to the neurotoxin saporin (anti-DBH-Sap). Male rats (n=6) were injected intrathecally with 4µg/10µl of anti-DBH-Sap, and their responses to nociceptive and non-nociceptive stimuli was monitored over a period of 65 days. Compared to controls (n=6), a significant (p< 0.05) decrease to hot plate (46oC) nociceptive responses could be observed during the first week post-treatment, but no differences were found at later times. At no point was there any altered response to innocuous stimuli. When tested for response to cold water stress, both treated and control animals showed analgesia, demonstrating that descending pain inhibition could still be activated. At 65 days, a formalin test showed no difference between treated (1.1 +/-0.5) and control (0.8 +/-0.5) groups. Post-mortem immunostaining of spinal cords for DBH, however, confirmed that noradrenergic denervation of the spinal cord had occurred in treated animals. These results suggest that a reorganization of the spinal cord following noradrenergic denervation is sufficient to reestablish normal nociceptive responses.

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

Miller SA, Lappi DA, Wiley RG (2000) Intrathecal dermorphin-saporin decreases morphine effect in hotplate algesia testing. Neuroscience 2000 Abstracts 212.8. Society for Neuroscience, New Orleans, LA.

Summary: The targeted cytotoxin, dermorphin-saporin, selectively destroys cells expressing MOR. In the present study, we gave dermorphin-saporin by lumbar i.t. injection and sought to determine if destroying dorsal horn neurons expressing MOR would alter thermal sensitivity and/or response to systemic morphine (MS) using hotplate testing under various conditions. 16 male Sprague-Dawley rats were tested on constant temperature (0.3, 44 and 47 C) and incremental (0.1 C/sec from 28 to 57 C) hotplates. Then 8 rats received lumbar intrathecal injections of derm-sap (465 ng) and 8 received vehicle using a subarachnoid PE-10 catheter that was removed 10 mins after injection. Retesting rats after toxin/vehicle injection showed no change in responses to any of the hotplate conditions. However, vehicle but not derm-sap rats showed increased lick latency on the incremental hotplate 20 mins after MS, 2.5 mg/kg, s.c. At 5 mg/kg of MS, vehicle and dermorphin-saporin rats showed identical responses. Capsaicin cream (0.94%) applied to the plantar surface of both hindpaws 3 hrs before testing on the 44 C hotplate produced decreased lick latencies in both groups of rats. MS, 5 mg/kg, s.c., produced increased lick latencies in capsaicin treated vehicle but not derm-sap rats. At 10 mg/kg, MS produced identical effects in capsaicin treated vehicle and toxin rats. These results indicate that i.t. derm-sap produced no change in baseline thermal sensitivity but did diminish the effect of low dose MS under conditions that preferentially test C nociceptor function suggesting that MOR-expressing dorsal horn neurons play a role in the analgesic action of low dose MS.

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

Gunhan-Agar E, Choudary P, Landerholm TE, Chalupa LM (2000) Depletion of cholinergic amacrine cells does not perturb the segregation of on and off cone bipolar cell projections. Neuroscience 2000 Abstracts 119.3. Society for Neuroscience, New Orleans, LA.

Summary: The pathways signaling onset and offset of light are segregated in the retina with On-cone and Off-cone bipolar cells terminating on the stratified dendrites of On and Off retinal ganglion cells (RGCs). During development the axons of On and Off cone bipolar cells form two strata in the IPL in a remarkably precise manner, without any obvious refinements. Moreover, such a precise ingrowth pattern occurs even after RGCs have been depleted (J. Neurosci., 2000, 201:306-314). Here we show by immunostaining that two bands of cholinergic processes are present in the rat retina as early as P1, some 7 days before the formation of segregated bipolar inputs. Double labeling of retinal sections with the antibody to recoverin (that recognizes On and Off cone bipolar cells) and the antibody for VACHT (which labels cholinergic processes) revealed that the segregated terminals of cone bipolar cells are juxtaposed with the two bands of cholinergic fibers. These observations suggested that the cholinergic fibers could serve as a scaffold for the later ingrowing bipolar cell axons. To test this hypothesis, we devised a novel method for depleting retinal cholinergic amacrine cells with a VACHT-saporin immunotoxin. A single treatment of the developing retina with this immunotoxin was found to eliminate virtually all cholinergic cells and processes. Recoverin labeling of bipolar cells showed that the axons of these neurons still form two stratified terminal bands within the IPL. Thus, neither RGCs nor cholinergic amacrine cell processes are required for the formation of segregated ON and Off cone bipolar cell projections.

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Bitner RS, Nikkel AL, Decker MW (2000) Immunohistochemical detection of alpha-7 nicotinic receptor expression by two distinct cell types in the dorsal raphe and locus coeruleus of rat. Neuroscience 2000 Abstracts 41.7. Society for Neuroscience, New Orleans, LA.

Summary: The α7 nicotinic acetylcholine receptor (nAChR) subunit can be assembled to form a homomeric-pentamer with high permeability to calcium, in contrast to other neuronal nAChR subunit-comprised ligand-gated cation channels. Although the expression of the α7-nAChR has been demonstrated throughout the CNS, the neurochemical phenotype of neurons expressing α7 remains to a large extent unknown. Using a polyclonal antibody raised against the carboxyl terminus (amino acids 460-479) of the α7 nAChR subunit (goat IgG, Santa Cruz Biotech.), immunohistochemical staining was observed in rat dorsal raphe (DR) and locus coeruleus (LC), serotonergic and noradrenergic brainstem nuclei, respectively. In both the DR and LC, there appeared to be two histologically distinct α7-expressing cell types as distinguished by size, i.e. large vs. small diameter. In rats treated with either a selective serotonergic (5,7-dihydroxytryptamine 150 μg i.c.v.) or noradrenergic (anti-dopamine-β-hydroxylase saporin 5 μg i.c.v.) neurotoxin, α7 immunostaining was seen only in small diameter cells, suggesting that the large diameter α7-expressing cells were serotonergic DR and noradrenergic LC neurons. Indeed, double-labeling experiments revealed in the large, but not small, cell types coexpression of α7 with tryptophan hydroxylase in the DR and tyrosine hydroxylase in the LC of saline-treated rats. In contrast, there was no coexpression in the neurotoxin-treated rats due to the loss of serotonergic or noradrenergic neurons, with only small diameter α7-expressing cells remaining. The results of these studies suggest that both serotonergic and noradrenegic neurons express α7 nAChRs. In addition, there appears to be a small diameter, non-serotonergic/noradrenergic cell-type in both the DR and LC that also expresses α7.

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

Rahimi O, Tatham D, Juliano SL (2000) Sensory training improves the ability to process stimuli in barrel cortex after basal forebrain lesion. Neuroscience 2000 Abstracts 51.22. Society for Neuroscience, New Orleans, LA.

Summary: Lesions of the basal forebrain deplete the cerebral cortex of acetylcholine and result in decreased ability to process stimuli. Using a model of unilateral basal forebrain lesion (BFL), we previously determined that the ability to perceive simple touch to the whiskers is impaired after BFL, but improves over time. Functional responses in barrel cortex, however, as measured by 2-deoxyglucose uptake (2DG) or electrophysiological recordings, remain reduced even after long survival times. We questioned whether the impaired cortical responses could be improved with behavioral training involving sensory discrimination using the whiskers. To do this, one group of rats was trained to discriminate between different textures using the whiskers on one side of the face, which projected to the lesioned hemisphere. After learning the task, this group of rats received a BFL using the immunotoxin, 192-IgG Saporin. They then continued the sensory discrimination task for at least 2 months. The second group of rats received a BFL, but no sensory training. They survived after the lesion for comparable periods of time; each rat of both groups then underwent a 2DG experiment. During the 2DG study, 1-4 matched sets of whiskers on both sides of the face were stimulated using an electromagnetic device. The magnitude of the response was measured in barrel cortex by preparing 2-dimensional maps of the label evoked by whisker stimulation. The area of barrel cortex activated in each hemisphere by whisker stimulation was measured and expressed as a ratio of the lesioned to normal hemisphere. We found that the evoked response in the lesioned hemisphere remained diminished compared to the normal side regardless of training. When the magnitude of response was compared between the trained and untrained group, however, the area of 2DG uptake in each barrel in response to stimulation was significantly increased in the animals receiving sensory discrimination training. These findings suggest that sensory training plays a role in improving cortical responses to stimulation after lesion of the basal forebrain.

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

Daniels D, Miselis RR, Flanagan-Cato LM (2000) Local immunotoxin treatment prevents transneuronal labeling of the intermediolateral column; but not the ventral horn; of the spinal cord after tracer injection into lumbar epaxial muscle. Neuroscience 2000 Abstracts 77.13. Society for Neuroscience, New Orleans, LA.

Summary: Pseudorabies virus (PRV) has been used as a transneuronal tracer to study central neural circuits that control various peripheral targets. Our laboratory has injected PRV into the lumbar epaxial muscles that produce the lordosis posture to label sequentially specific brain regions along the neuraxis. However, concomitant uptake of PRV through sympathetic innervation of nearby vasculature has made the interpretation of higher-order labeling problematic. To avoid this confound, we have designed a procedure for focal sympathetic denervation using dopamine-β-hydroxylase immunotoxin (DHIT). Five days after injecting DHIT (5 μg) into the medial portion of lateral longissimus, the Bartha strain of PRV was injected into the pre-treated area. After survival times of 72 or 96 h, animals were sacrificed and the spinal cords were immunostained for PRV. In preliminary studies, DHIT treatment was not effective in all animals, as determined by PRV labeling in the sympathetic preganglionic neurons that reside in IML. However, at each survival time, in 50% of the animals DHIT virtually eliminated PRV-labeling in cells within IML of the thoracic and lumbar spinal cord, whereas robust labeling of motoneurons in the ventral horn was retained. These preliminary results suggest that this procedure for local sympathectomy may allow for selective transneuronal labeling of somatic motor pathways.

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