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Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons.

In vitro, cyclic AMP (cAMP) elevation alters neuronal responsiveness to diffusible growth factors and myelin-associated inhibitory molecules. Here we used an established in vivo model of adult central nervous system injury to investigate the effects of elevated cAMP on neuronal survival and axonal regeneration. We studied the effects of intraocular injections of neurotrophic factors and/or a cAMP analogue (CPT-cAMP) on the regeneration of axotomized rat retinal ganglion cell (RGC) axons into peripheral nerve autografts. Elevation of cAMP alone did not significantly increase RGC survival or the number of regenerating RGCs. Ciliary neurotrophic factor increased RGC viability and axonal regrowth, the latter effect substantially enhanced by coapplication with CPT-cAMP. Under these conditions over 60% of surviving RGCs regenerated their axons. Neurotrophin-4/5 injections also increased RGC viability, but there was reduced long-distance axonal regrowth into grafts, an effect partially ameliorated by cAMP elevation. Thus, cAMP can act cooperatively with appropriate neurotrophic factors to promote axonal regeneration in the injured adult mammalian central nervous system.

Animals↗

Single cell RT-PCR analysis of tyrosine kinase receptor expression in adult rat retinal ganglion cells isolated by retinal sandwiching.

We describe a protocol for analysis of gene expression in single, acutely dissociated adult rat retinal ganglion cells using RT-PCR. Retrograde tracing of retinal ganglion cells from the superior colliculi was conducted using Fluorogold. Retinas were dissected and ganglion cells isolated using retinal layer separation (sandwiching). Single, fluorescently labelled retinal ganglion cells were aspirated using a micropipette and used for PCR. Two PCR protocols are described where single cell cDNA was analysed for TrkB and GAPDH or TrkB, TrkC, Ret, Met, ErbB2 and Beta-actin by multiplex-PCR. All five tyrosine kinase receptors were amplified from single retinal ganglion cells. The method will prove useful for the molecular characterization of adult retinal ganglion cells.

Animals↗

Multiple neuroanatomical tracing in primates.

The present report deals with a multiple tract-tracing procedure in non-human primates enabling the simultaneous visualization of retrogradely transported Fluoro-Gold (FG) and cholera toxin B subunit (CTB) in combination with anterogradely transported biotinylated dextran amine (BDA). Two issues have played key roles on the achievement of this reliable procedure: first, the recent development of a commercial antiserum against FG that allows us to convert the original fluorescent signal of this dye in a permanent precipitate via standard peroxidase-anti-peroxidase methods; second, the introduction of the novel peroxidase substrate Vector(R) VIP (V-VIP), resulting in a purple precipitate. The combination of these neuroanatomical tracers in one and the same histological section opens a possibility for the permanent visualization of the convergence of inputs from a particular brain area onto identified, two different subsets of projection cells of another area. Furthermore, this combination of three tracers emerges as a powerful technical tool for obtaining broad amounts of complementary data regarding the monkey brain connectivity, thus significantly reducing the number of animals needed to complete a particular study.

Animals↗

Substance P and calcitonin gene-related peptide immunoreactive sensory DRG neurons innervating the lumbar facet joints in rats.

The rat L5/6 facet joint is innervated from L1 to L5 dorsal root ganglions (DRGs) multisegmentally. Sensory fibers from L1 and L2 DRGs were reported to innervate nonsegmentally through the paravertebral sympathetic trunks, while those from L3 to L5 DRGs segmentally innervate the L5/6 facet joint. The presence of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive (ir) nerve fibers has been demonstrated in the lumber facet joints, but their ratios have not been determined. Fluoro-gold (F-G) labeled neurons innervating the L5/6 facet joint were distributed throughout the DRGs for levels L1 to L5. Of the F-G labeled neurons, the ratios of SP-ir L1, L2, L3, L4 and L5 DRG neurons were 13, 15, 29, 31 and 30%, respectively, and those of CGRP-ir neurons were 17, 24, 44, 56 and 50%, respectively. The ratios of SP and CGRP-ir neurons in L1 and L2 DRGs were significantly less than those in L3, L4 or L5 DRGs. In conclusion, the neurons of L3, L4 and L5 DRGs may have a more significant role in pain sensation of the facets than L1 and L2 DRG neurons.

Animals↗

Projections of the paratrigeminal nucleus to the ambiguus, rostroventrolateral and lateral reticular nuclei, and the solitary tract.

The paratrigerminal nucleus (Pa5), a constituent of the spinal interstitial system, was linked to the pressor effect caused by bradykinin injected in the dorsal lateral medulla of the rat. The nucleus receives primary afferent sensory fibers contained in branches of the trigeminal, glossopharyngeal and vagus nerves. In this investigation connections of the paratrigeminal nucleus to other medullary structures were studied with the use of retrograde and anterograde neuronal tracers. Fluorescent light microscopy analyses of medullary sections of rats injected with the retrograde transport tracer Fluoro-gold in the nucleus of the solitary tract (NTS) or in the pressor area of the rostral ventrolateral medulla (RVLM) revealed labeled neuronal cell bodies in the ipsi- and contralateral Pa5. FluoroGold microinjections in the caudal ventrolateral medulla (CVLM) did not produce fluorescent labeling of Pa5 neurons. Microinjection of the anterograde transport neuronal tracer biocytin in the Pa5 produced bilateral labeling of the solitary tract (sol). rostroventrolateral reticular nucleus (RVL), ambiguus nucleus (Amb), lateral reticular nucleus (LRt) and ipsilateral parabrachial nuclei, but not the contralateral Pa5. Confocal laser microscopy showed fluorescence labeling of fibers and presumptive terminal varicosities in the NTS, RVL, Amb and LRt. The present findings showing the paratrigeminal nucleus interposed between sensory afferent and stuctures associated to cardiovascular and respiratory functions, suggest that the structure may act as a medullary relay nucleus for sensory stimuli directly connecting primary afferents to structures mediating cardiovascular and respiratory reflexes.

Animals↗

Spinal cord interneurones labelled transneuronally from the adrenal gland by a GFP-herpes virus construct contain the potassium channel subunit Kv3.1b.

Interneurones in the spinal cord are likely to play an important role in the generation of activity in sympathetic preganglionic neurones (SPNs) and, therefore, sympathetic outflow. Although the properties of these interneurones have rarely been studied directly, here we show that neurones antecedent to SPNs contain the voltage-gated potassium channel subunit Kv3.1b, while SPNs do not. SPNs and interneurones were labelled by injection of a green fluorescent protein expressing herpes simplex virus (HSV-GFP) into the adrenal gland. SPNs identified by concomitant tracing with Fluorogold did not contain Kv3.1b immunoreactivity. Significantly, neurones that did not contain Fluorogold and which were unlikely to be SPNs were double labelled for Kv3.1b and GFP. This indicates that spinal cord intemeurones antecedent to SPNs contain Kv3.1b. To test the role of Kv3.1b whole cell patch clamp recordings were made from SPNs and interneurones in spinal cord slices. Selective blockade of Kv3.1b containing channels with 30 microM 4-amino-pyridine (4-AP) or 500 microM tetraethylammonium chloride (TEA) revealed that this Kv subunit contributes to fast repolarisation and fast firing frequencies of interneurones in the vicinity of the IML, allowing them to fire action potentials at much higher frequencies than SPNs. This is the first time that transneuronal labelling with this viral construct has been combined with immunohistochemical detection of ion channels. In conjunction with our electrophysiological data, this highlights a role for the Kv3.1b subunit in shaping the activity of intemeurones involved in sympathetic control.

Action Potentials↗

Ionotropic glutamate receptor subunit immunoreactivity of vagal preganglionic neurones projecting to the rat heart.

The ionotropic glutamate receptor subunits expressed by vagal preganglionic neurones in the rat medulla oblongata were examined by using fluorescence immunolabelling combined with retrograde neuronal tracing. The general population of these neurones in the medulla was identified by intraperitoneal injections of Fluorogold and also with choline acetyltransferase antibodies. Cardiac projecting neurones were specifically identified by applying the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (DiI) to the heart or by injecting cholera toxin B-subunit into the pericardium. Both tracers labelled populations of neurones lying in the dorsal vagal nucleus, intermediate reticular formation and nucleus ambiguus, and when both tracers were applied simultaneously, approximately 50% of cells were dual-labelled. Control experiments established that the labelling was specific for neurones projecting to the heart. Most vagal preganglionic neurones, including those projecting to the heart, irrespective of their location in the medulla, had a similar profile of glutamate receptor immunoreactivity. Labelling of somata for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) subunit GluR1 was weak or absent, while labelling with antibodies directed to GluR2, a common sequence of GluR2 and GluR3, and GluR4 was moderate or intense. All neurones studied appeared to express the N-methyl-D-aspartate (NMDA) receptor subunit NR1, and while antibodies recognising the NR2A and NR2B splice variants gave strong labelling, immunoreactivity with a NR2B specific antibody was weaker. Weak to moderate labelling was seen in some neurones using antibodies to the kainate receptor subunits KA2 and GluR5-7. These results are consistent with neurophysiological data indicating the presence of AMPA, NMDA and kainate responses in cardiac vagal preganglionic neurones, and suggest that these neurones are similar to other vagal parasympathetic preganglionic neurones in expressing mainly AMPA receptor subunits GluR2-4.

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Neurons of the medial terminal accessory optic nucleus of the rat are poorly collateralized.

The vast majority of neurons of the rat medial terminal nucleus (MTN) project to the nucleus of the optic tract (NOT), but the MTN also projects to a lesser degree upon a number of other brainstem nuclei controlling optokinetic nystagmus. Because of the diversity of targets of the MTN, it is possible that individual neurons have branched axons that project to two or more brainstem nuclei. The possibility that axons of MTN-NOT neurons collateralize to innervate other MTN targets is examined in the rat with the fluorescent, double-labeling, retrograde tracer technique. Fluoro-Gold was injected into the NOT while Fast Blue was simultaneously injected into each of five other known targets of the MTN: the supraoculomotor-periaqueductal gray; the dorsal cap of the inferior olive; the visual tegmental relay zone; the dorsolateral nucleus of the basal pons; and the superior/lateral vestibular nuclei. Brainstem sections were processed for fluorescence microscopy and the MTN was examined for single- and double-labeled neurons. Results show that virtually all neurons of the MTN (greater than 97.5%), together with neurons in the visual tegmental relay zone immediately surrounding the MTNd, are single-labeled in all paired injections involving the NOT and the other target nuclei. It was found that about 69% of MTN neurons project exclusively to the NOT, 5-10% project to each one of the other nuclei, and 3% of MTN neurons project to more than one target. Based upon cell counts from the fluorescent material, and previous analysis of Nissl-stained serial sections, the findings show that virtually all MTN neurons are projection neurons. It was concluded that the MTN is comprised of independent projection systems, possibly involved in different aspects of generating optokinetic nystagmus.

Amidines↗

Brain-stem afferents upon retinal projecting isthmo-optic and ectopic neurons of the pigeon centrifugal visual system demonstrated by retrograde transneuronal transport of rhodamine beta-isothiocyanate.

Brain-stem afferents to the n. isthmo-opticus (NIO) and ectopic neurons (EN) of the centrifugal visual system (CVS) were determined in the pigeon using the retrograde transneuronal transport of the fluorescent dye Rhodamine beta-isothiocyanate (RITC) after its intraocular injection. In other experiments, either RITC was injected into various periocular tissues (controls) or the retrograde tracer Fluoro-gold (FG) was injected stereotaxically in the NIO. Following intraocular injections, the RITC retrograde labeling of cell bodies was observed contralaterally in the NIO and EN and transneuronally in layers 9/10 of the optic tectum, area ventralis-Tsai, zona peri-NIII, mesencephalic and pontine reticular formation (PRF), n. linearis caudalis-raphe, and bilaterally within a region referred to as zona peri-n.NVI (Zp-n.NVI) immediately underlying the abducens nerve nucleus. None of the above structures were labeled after RITC periocular injections. The FG labeling revealed that the tectal efferent neurons were mainly medium-sized, multipolar cells whose dendrites extended superficially to retino-recipient tectal layers 6 and 5. Quantitative measurements of the distribution of layers 9/10 RITC-labeled neurons indicated the highest densities to be localized within the ventral tectum corresponding to the representation of the dorsal retina and inferior visual field. We suggest that visual and nonvisual brain-stem afferents upon NIO and EN may play a role in the proposed mechanism of the avian CVS in attention, ground-feeding behavior, and modulation of retinal sensitivity.

Animals↗

Detection of retrogradely transported WGA-HRP in axotomized adult hamster facial motoneurons occurs after initiation of the axon reaction.

We have previously shown that facial nerve transection at the stylomastoid foramen activates ribosomal RNA transcription in injured facial motoneurons (FMN) of the adult hamster within 30 minutes postoperative. The signal for the initiation of the nerve cell body response to injury in vertebrates is currently unknown. It has been hypothesized that the signal for initiating the injury response is dependent on retrograde transport, where the signal itself is either the loss of a repressor substance from the periphery or the loss of retrogradely transported target-derived factors. To examine if a retrograde transport-mediated signal would be sufficient to produce the rapid ribosomal effects observed in hamster FMN following injury, adult hamsters were subjected to right facial nerve axotomies, with the neuronal tracer wheat germ agglutinin horseradish peroxidase (WGA-HRP; M.W. 80,000) applied at the proximal stump of the transected nerve. At time points ranging from 0.5 to 24 hours postoperative (hpo), the animals were killed and brainstem sections containing bilateral facial nuclei processed for WGA-HRP label using the TMB method. The earliest time point at which WGA-HRP was detected in the axotomized facial nucleus occurred at 3 hpo. To eliminate molecular weight as a confounding factor, an additional retrograde transport study was performed using the smaller tracer, Fluoro-Gold (M.W. 532.59). Fluoro-Gold was not detected until well after the 3 hpo time point. Thus, it appears that initiation of the axon reaction in hamster FMN is likely to be independent of the retrograde transport properties of the injured neuron.

Animals↗

Topographic representation of the sciatic nerve motor neurons in the spinal cord of the adult rat correlates to region-specific activation patterns of microglia.

The frequent use of the adult rat sciatic nerve as a model to study the neuronal responses to injury, nerve regeneration and in transplantation studies, requires a detailed knowledge of the projection pattern of motor neurons into this nerve. Thus, as a first goal we determined this topographical projection of motor neurons and labelled small contingents by applying the fluorescent dye DiI in localised incisions made in the dorsal, rostral, ventral or caudal quadrants of the nerve. As a second goal we analysed with immunohistochemical methods the response of microglial cells within the topographical area corresponding to the incision and within areas outside this location. Uptake of the dye occurred only within the area confined to the incision, thus allowing the identification of the corresponding motor neuron perikarya within the ventral horn, eight to ten days later. In serial transverse sections of the lumbosacral spinal cord the number of labelled cells, their position within the ventral horn, and their longitudinal extent have been determined. The data suggest that the gross projection of the lumbosacral motor neuron column at the mid-thigh level of the sciatic nerve is topographic. In accordance, microglial cells showed fast activation within the injured topographic area, and a less pronounced and delayed response within the non-injured areas of the ventral horn. The graded response of microglial cells suggests that these cells possess a potential of local activation by sensing whether neurons are axotomised or just irritated by axotomy of their neighbours. The topographic organisation proves to be useful in studies on local injuries to the sciatic nerve and when analysing retrograde responses within the lumbosacral spinal cord.

Animals↗

The role of claustrum in Pavlovian heart rate conditioning in the rabbit (Oryctolagus cuniculus): anatomical, electrophysiological, and lesion studies.

The role of the claustrum in Pavlovian heart rate (HR) conditioning was studied in the rabbit (Oryctolagus cuniculus) by (a) mapping claustral projections to the prefrontal cortex (PFC), (b) recording claustral single-unit discharge to sensory stimulation and conditioning stimuli during HR conditioning, and (c) assessing the effects of claustral damage on HR conditioning. Contralateral and ipsilateral claustral projections to the PFC were found. Claustral cells responded to nonsignal stimulation with increased discharge and also showed conditioned stimulus-evoked increases in discharge during Pavlovian HR conditioning. Moreover, claustral lesions diminished the magnitude of the HR-conditioned response without affecting the cardiac-orienting response to the conditioned stimulus or the cardiac-unconditioning response to the unconditioned stimulus, suggesting a role for the claustrum in associative learning.

Action Potentials↗

Melanopsin in cells of origin of the retinohypothalamic tract.

All known eukaryotic organisms exhibit physiological and behavioral rhythms termed circadian rhythms that cycle with a near-24-hour period; in mammals, light is the most potent stimulus for entraining endogenous rhythms to the daily light cycle. Photic information is transmitted via the retinohypothalamic tract (RHT) to the suprachiasmatic nucleus (SCN) in the hypothalamus, where circadian rhythms are generated, but the retinal photopigment that mediates circadian entrainment has remained elusive. Here we show that most retinal ganglion cells (RGCs) that project to the SCN express the photopigment melanopsin.

Animals↗

Electrical stimulation of the medial prefrontal cortex increases dopamine release in the striatum.

Exogenous and endogenous glutamate has been shown to evoke dopamine (DA) release in the striatum using both in vitro and in vivo techniques. We hypothesized that stimulation of the prefrontal cortex (PFC) would phasically enhance striatal DA release via the glutamatergic corticostriatal pathway. To test this hypothesis, in vivo brain microdialysis was employed to measure extracellular concentrations of DA in the striatum during electrical stimulation of the PFC. Five rats were implanted with bilateral electrodes located in the medial PFC and dialysis probes in the dorsal striatum. Two days later the PFC of these awake, freely moving rats was stimulated first at 50 microA and then at 100 microA for 20 minutes at 2-hour intervals. Both currents significantly increased DA release. Extracellular DA rose rapidly during stimulation, peaked immediately afterward, and then slowly returned to baseline values. Dopamine reached 118% of baseline values with 50 microA stimulation and 138% with 100 microA stimulation. Histologic analysis using the fluorescent retrograde dye Fluoro Gold confirmed that cells projecting to the vicinity of the striatal dialysis probe originated in the vicinity of the PFC electrodes. These results provide direct evidence for phasic, excitatory modulation of striatal DA release by the PFC.

Animals↗

Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury.

STUDY DESIGN: An animal model of transected spinal cord injury (SCI) was used to test the hypothesis that cografted neural stem cells (NSCs) and NT-3-SCs promote morphologic and functional recoveries of injured spinal cord. OBJECTIVE: To explore whether cotransplant of NSCs and NT-3-SCs could promote the injured spinal cord repair. SETTING: Zhongshan Medical College, Sun Yat-sen University, PR China. METHODS: Female Sprague-Dawley (SD) rats weighing on 200-220 g were used to prepare SCI models. The spinal cord was transected between T(9) and T(10), then NSCs, SCs+NSCs, LacZ-SCs+NSCs, or NT-3-SCs+NSCs were grafted into the transected site. RESULTS: (1) Part of NSCs could differentiate to neuron-like cells in the transected site and the percentage of differentiation was NT-3-SCs+NSCs group>SCs+NSCs group>NSCs group. (2) In the grafted groups, there were 5-HT, CGRP, and SP positive nerve fibres within the transected site. Some fluorogold (FG)-labeled cells were found in the spinal cord rostral to the transected site, the red nuclei and the inner pyramidal layer of sensorimotor cortex. (3) The cells grafted could enhance the injured neurons survival in inner pyramidal layer of sensorimotor cortex, red nuclei of midbrain, and Clark's nuclei of spinal cord's L1 segment, could decrease the latency and increase the amplitude of cortical somatosensory evoked potential (CSEP) and cortical motor evoked potential (CMEP), and could promote partly structural and functional recovery of the SCI rats. CONCLUSION: These results demonstrate that cografted NT-3-SCs and NSCs is a potential therapy for SCI. SPONSORSHIP: This research was supported by Chinese National Key Project for Basic Research (G1999054009), Chinese National Natural Science Foundation (30270700) and Social Developmental Foundation of Guangdong Province (2003C33808) to YS Zeng; Natural Science Foundation of Guangdong Province (04300468) and Medical Science Research Grant of Guangdong Province (A2004081) to JS Guo.

Animals↗

Monosynaptic pathway between the arcuate nucleus expressing glial type II iodothyronine 5'-deiodinase mRNA and the median eminence-projective TRH cells of the rat paraventricular nucleus.

Recent evidence suggests that the thyroid regulation of thyrotropin-releasing hormone (TRH)-containing neurons in the paraventricular nucleus of the hypothalamus involves the activation of other hypothalamic neural circuits. For example, the arcuate nucleus and not the paraventricular nucleus contains the highest enzyme activity of 5'-deiodinase type II, an enzyme that is pivotal for the local synthesis of T3. This experiment was undertaken to demonstrate whether a monosynaptic pathway exists between the arcuate nucleus and those TRH cells of the paraventricular nucleus that are neuroendocrine, i.e. project to the external layer of the median eminence. A specific cRNA probe derived from the coding region of deiodinase type II was used for the in situ hybridization histochemistry which was combined with immunocytochemistry for a specific marker of glial cells, glial fibrillary acidic protein (GFAP). The hybridization signals were present within the hypothalamus in the arcuate nucleus-median eminence region and in the periventricular area. The periventricular labeling was localized to the ependymal layer of the third ventricle and no hybridization product was detected in the paraventricular nucleus and other hypothalamic nuclei adjacent to the third ventricle. Within the median eminence, numerous cells containing the hybridization product were located in the internal layer adjacent to the floor of the third ventricle and in the external layer adjacent to the surface of the brain. In the dorso- and ventromedial regions of the arcuate nucleus, deiodinase type II mRNA-containing cells were also detected. Numerous type II deiodinase mRNA-containing cells in the median eminence and arcuate nucleus were also found to be immunopositive for GFAP. The abundance of arcuate cells expressing the hybridization product was lower than those in the periventricular region or in the median eminence. The anterograde tracer, Phaseolus vulgaris leucoagglutinin, was injected into the medial parts of the arcuate nucleus where the in situ hybridization experiment detected deiodinase type II mRNA. Simultaneously with the anterograde tracing, the retrograde tracer, Fluoro-Gold, was injected into either the median eminence or the general circulation. Light and electron microscopic double and triple immunolabeling experiments on vibratome sections of colchicine-pretreated animals revealed that arcuate fibers innervate TRH cells within the parvicellular region of the paraventricular nucleus. Populations of these TRH cells receiving afferents from the arcuate nucleus were also retrogradely labelled from either the median eminence or the general circulation indicating their direct role in the regulation of thyrotropin secretion from the anterior pituitary. The majority of arcuate nucleus efferents on TRH cells were found to establish symmetrical synaptic connections. The present results provided direct evidence of a monosynaptic pathway between the hypothalamic site of local thyroid hormone production, the arcuate nucleus, and neuroendocrine TRH cells in the paraventricular nucleus. This signalling modality may play an important role in thyroid feedback on TRH cells. Since the arcuate nucleus is involved in the regulation of central mechanisms controlling diverse homeostatic functions, including reproduction and feeding, the pathway described in this study may also carry integrated signals related to reproduction and ingestion to TRH-producing cells.

Animals↗