TRPV1 receptors are involved in protein nitration and Muller cell reaction in the acutely axotomized rat retina

We report here the protein expression of TRPV1 receptor in axotomized rat retinas and its possible participation in mechanisms involved in retinal ganglion cell (RGC) death. Adult rats were subjected to unilateral, intraorbital axotomy of the optic nerve, and the retinal tissue was removed for further processing. TRPV1 total protein expression decreased progressively after optic nerve transection, reaching 66.2% of control values 21 days after axotomy. The number of cells labeled for TRPV1 in the remnant GCL decreased after 21 days post-lesion (to 63%). Fluoro-jade B staining demonstrated that the activation of TRPV1 in acutely-lesioned eyes elicited more intense neuronal degeneration in the GCL and in the inner nuclear layer than in sham-operated retinas. A single intraocular injection of capsazepine (100 mu M), a TRPV1 antagonist, 5 days after optic nerve lesion, decreased the number of GFAP-expressing Muller cells (72.5% of control values) and also decreased protein nitration in the retinal vitreal margin (75.7% of control values), but did not affect lipid peroxidation. Furthermore, retinal explants were treated with capsaicin (100 mu M), and remarkable protein nitration was then present, which was reduced by blockers of the constitutive and inducible nitric oxide synthases (7-NI and aminoguanidine, respectively). TRPV1 activation also increased GFAP expression, which was reverted by both TRPV1 antagonism with capsazepine and by 7-NI and aminoguanidine. Given that Muller cells do not express TRPV1, we suppose that the increased GFAP expression in these cells might be elicited by TRPV1 activation and by its indirect effect upon nitric oxide overproduction and peroxynitrite formation. We incubated Fluorogold pre-labeled retinal explants in the presence of capsazepine (1 mu M) during 48 h. The numbers of surviving RGCs stained with fluorogold and the numbers of apoptotic cells in the GCL detected with TUNEL were similar in lesioned and control retinas. We conclude that TRPV1 receptor expression decreased after optic nerve injury due to death of TRPV1-containing cells. Furthermore, these data indicate that TRPV1 might be involved in intrinsic protein nitration and Muller cell reaction observed after optic nerve injury. (C) 2010 Elsevier Ltd. All rights reserved.

Projections to the preoptic area from the paraventricular nucleus, acuate nucleus and the bed nucleus of the stria terminalis are unlikely to be involved in stress-induced suppression of GnRH secretion in sheep

Stress compromises reproductive function and the major physiological system activated during stress is the hypothalamo-pituitary-adrenal axis. Corticotrophin-releasing hormone and arginine vasopressin (AVP), which are produced in neurones of the paraventricular nucleus (PVN), drive the hypothalamo-pituitary-adrenal axis and are also implicated in the suppression of the reproductive axis. We used retrograde tracing and Fos labelling to map the projections from the PVN to the preoptic area (POA) where most gonadotrophin releasing hormone (GnRH) neurones are found. Fluorogold (FG) injections were made into the POA of gonadectomised male and female sheep (n = 5/sex), the animals were stressed and the brains recovered for histochemistry. All animals responded to stress with an increase in the number of Fos-labelled nuclei in the PVN. Few retrogradely labelled cells of the PVN were activated by stress. Dual labelling showed that very few FG-labelled cells also stained for corticotrophin-releasing hormone, none for AVP or enkephalin. Dual labelling for FG and Fos in the bed nucleus of the stria terminalis (BNST) and the arcuate nucleus showed that no FG-labelled cells in the BNST and only few in the ARC were activated by stress. No sex differences were observed in the activation of FG-labelled cells in any of the nuclei examined. We conclude that, although cells of the PVN, BNST and/or arcuate nucleus may affect reproduction via the GnRH cells of the POA, this is unlikely to involve direct input to the POA. If cells of these regions are involved in GnRH suppression during stress, this may occur via interneuronal pathways.

Alternative pathways for catecholamine action in oral motor control

Orofacial movement is a complex function performed by facial and jaw muscles. Jaw movement is enacted through the triggering of motoneurons located primarily in the trigeminal motor nucleus (Mo5). The Mo5 is located in the pontine reticular formation, which is encircled by premotor neurons. Previous studies using retrograde tracers have demonstrated that premotor neurons innervating the Mo5 are distributed in brainstem areas, and electrophysiological studies have suggested the existence of a subcortical relay in the corticofugal-Mo5 pathway. Various neurotransmitters have been implicated in oral movement. Dopamine is of special interest since its imbalance may produce changes in basal ganglia activity, which generates abnormal movements, including jaw motor dysfunction, as in oral dyskinesia and possibly in bruxism. However, the anatomical pathways connecting the dopaminergic systems with Mo5 motoneurons have not been studied systematically. After injecting retrograde tracer fluorogold into the Mo5, we observed retrograde-labeled neurons in brainstem areas and in a few forebrain nuclei, such as the central nucleus of the amygdala, and the parasubthalamic nucleus. By using dual-labeled immunohistochemistry, we found tyrosine hydroxylase (a catecholamine-processing enzyme) immunoreactive fibers in close apposition to retrograde-labeled neurons in brainstem nuclei, in the central nucleus of the amygdala and the parasubthalamic nucleus, suggesting the occurrence of synaptic contacts. Therefore, we suggested that catecholamines may regulate oralfacial movements through the premotor brainstem nuclei, which are related to masticatory control, and forebrain areas related to autonomic and stress responses. (C) 2005 Elsevier B.V.. All rights reserved.

Direct and indirect connections between cochlear root neurons and facial motor neurons: Pathways underlying the acoustic pinna reflex in the albino rat

Cochlear root neurons (CRNs) are involved in the acoustic startle reflex, which is widely used in behavioral models of sensorimotor integration. A short-latency component of this reflex, the auricular reflex, promotes pinna movements in response to unexpected loud sounds. However, the pathway involved in the auricular component of the startle reflex is not well understood. We hypothesized that the auricular reflex is mediated by direct and indirect inputs from CRNs to the motoneurons responsible for pinna movement, which are located in the medial subnucleus of the facial motor nucleus (Mot7). To assess whether there is a direct connection between CRNs and auricular motoneurons in the rat, two neuronal tracers were used in conjunction: biotinylated dextran amine, which was injected into the cochlear nerve root, and Fluoro-Gold, which was injected into the levator auris longus muscle. Under light microscopy, close appositions were observed between axon terminals of CRNs and auricular motoneurons. The presence of direct synaptic contact was confirmed at the ultrastructural level. To confirm the indirect connection, biotinylated dextran amine was injected into the auditory-responsive portion of the caudal pontine reticular nucleus, which receives direct input from CRNs. The results confirm that the caudal pontine reticular nucleus also targets the Mot7 and that its terminals are concentrated in the medial subnucleus. Therefore, it is likely that CRNs innervate auricular motoneurons both directly and indirectly, suggesting that these connections participate in the rapid auricular reflex that accompanies the acoustic startle reflex. © 2008 Wiley-Liss, Inc.

Behavioral and EEG effects of GABAergic manipulation of the nigro-tectal pathway in the Wistar audiogenic rat (WAR) strain II: An EEG wavelet analysis and retrograde neuronal tracer approach

The role of the substantia nigra pars reticulata (SNPr) and superior colliculus (SC) network in rat strains susceptible to audiogenic seizures still remain underexplored in epileptology. In a previous study from our laboratory, the GABAergic drugs bicuculline (BIC) and muscimol (MUS) were microinjected into the deep layers of either the anterior SC (aSC) or the posterior SC (pSC) in animals of the Wistar audiogenic rat (WAR) strain submitted to acoustic stimulation, in which simultaneous electroencephalographic (EEG) recording of the aSC, pSC, SNPr and striatum was performed. Only MUS microinjected into the pSC blocked audiogenic seizures. In the present study, we expanded upon these previous results using the retrograde tracer Fluorogold (FG) microinjected into the aSC and pSC in conjunction with quantitative EEG analysis (wavelet transform), in the search for mechanisms associated with the susceptibility of this inbred strain to acoustic stimulation. Our hypothesis was that the WAR strain would have different connectivity between specific subareas of the superior colliculus and the SNPr when compared with resistant Wistar animals and that these connections would lead to altered behavior of this network during audiogenic seizures. Wavelet analysis showed that the only treatment with an anticonvulsant effect was MUS microinjected into the pSC region, and this treatment induced a sustained oscillation in the theta band only in the SNPr and in the pSC. These data suggest that in WAR animals, there are at least two subcortical loops and that the one involved in audiogenic seizure susceptibility appears to be the pSC-SNPr circuit. We also found that WARs presented an increase in the number of FG + projections from the posterior SNPr to both the aSC and pSC (primarily to the pSC), with both acting as proconvulsant nuclei when compared with Wistar rats. We concluded that these two different subcortical loops within the basal ganglia are probably a consequence of the WAR genetic background. (C) 2012 Elsevier Inc. All rights reserved.

Expression of trefoil factor 1 in the developing and adult rat ventral mesencephalon

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides with a characteristic tree-looped trefoil structure. TFFs are mainly expressed in the gastrointestinal tract where they play a critical role in the function of the mucosal barrier. TFF1 has been suggested as a neuropeptide, but not much is known about its expression and function in the central nervous system. We investigated the expression of TFF1 in the developing and adult rat midbrain. In the adult ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly found in the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA) and in periaqueductal areas. While around 90% of the TFF1-ir cells in the SNc co-expressed tyrosine hydroxylase (TH), only a subpopulation of the TH-ir neurons expressed TFF1. Some TFF1-ir cells in the SNc co-expressed the calcium-binding proteins calbindin or calretinin and nearly all were NeuN-ir confirming a neuronal phenotype, which was supported by lack of co-localization with the astroglial marker glial fibrillary acidic protein (GFAP). Interestingly, at postnatal (P) day 7 and P14, a significantly higher proportion of TH-ir neurons in the SNc co-expressed TFF1 as compared to P21. In contrast, the proportion of TFF1-ir cells expressing TH remained unchanged during postnatal development. Furthermore, significantly more TH-ir neurons expressed TFF1 in the SNc, compared to the VTA at all four time-points investigated. Injection of the tracer fluorogold into the striatum of adult rats resulted in retrograde labeling of several TFF1 expressing cells in the SNc showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in SNc of 6-hydroxylase-lesioned hemiparkinsonian rats. In conclusion, we show for the first time that distinct subpopulations of midbrain dopaminergic neurons express TFF1, and that this expression pattern is altered in a rat model of Parkinson's disease.

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson’s disease in rats

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson’s disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson’s disease.

Evidence for a hypothalamothalamocortical circuit mediating pheromonal influences on eye and head movements.

A method for simultaneous iontophoretic injections of the anterograde tracer Phaseolus vulgaris leukoagglutinin and the retrograde tracer fluorogold was used to characterize in the rat a hypothalamothalamocortical pathway ending in a region thought to regulate attentional mechanisms by way of eye and head movements. The relevant medial hypothalamic nuclei receive pheromonal information from the amygdala and project to specific parts of the thalamic nucleus reuniens and anteromedial nucleus, which then project to a specific lateral part of the retrosplenial area (or medial visual cortex). This cortical area receives a convergent input from the lateral posterior thalamic nucleus and projects to the superior colliculus. Bidirectional connections with the hippocampal formation suggest that activity in this circuit is modified by previous experience. Striking parallels with basal ganglia circuitry are noted.

Electroretinographical and histological study of mouse retina after optic nerve section: a comparison between wild-type and retinal degeneration 1 mice

Background: Retinal ganglion cell death underlies the pathophysiology of neurodegenerative disorders such as glaucoma or optic nerve trauma. To assess the potential influence of photoreceptor degeneration on retinal ganglion cell survival, and to evaluate functionality, we took advantage of the optic nerve section mouse model. Methods: Surviving retinal ganglion cells were double-stained by exposing both superior colliculi to fluorogold, and by applying dextran-tetramethylrhodamine to the injured optic nerve stump. To assess retinal function in wild-type animals, electroretinograms were recorded on the injured eyes and compared with the contralateral. Similar labelling experiments were carried out on retinal degeneration 1 mice. Surviving retinal ganglion cells were counted 21 days after axotomy and compared with wild-type mice. No functional experiments were performed on retinal degeneration 1 animals because they do not develop normal electroretinographical responses. Results: A significant decrease in retinal ganglion cell density was observed 6 days after axotomy in the wild type. Functional studies revealed that, in scotopic conditions, axotomy induced a significant amplitude decrease in the positive scotopic threshold response component of the electroretinogram. Such decrease paralleled cell loss, suggesting it may be an appropriate technique to evaluate functionality. When comparing retinal ganglion cell densities in wild-type and retinal degeneration 1 mice, a significant greater survival was observed on the latter. Conclusions: After optic nerve section, electroretinographical recordings exhibited a progressive decrease in the amplitude of the positive scotopic threshold response wave, reflecting ganglion cell loss. Interestingly, rod degeneration seemed, at least initially, to protect from axotomy-driven damage.