RESUMEN

Circadian rhythms synchronize to light through the retinohypothalamic tract (RHT), which is a bundle of axons coming from melanopsin retinal ganglion cells, whose synaptic terminals release glutamate to the ventral suprachiasmatic nucleus (SCN). Activation of AMPA-kainate and NMDA postsynaptic receptors elicits the increase in intracellular calcium required for triggering the signaling cascade that ends in phase shifts. During aging, there is a decline in the synchronization of circadian rhythms to light. With electrophysiological (whole-cell patch-clamp) and immunohistochemical assays, in this work, we studied pre- and postsynaptic properties between the RHT and ventral SCN neurons in young adult (P90-120) and old (P540-650) C57BL/6J mice. Incremental stimulation intensities (applied on the optic chiasm) induced much lesser AMPA-kainate postsynaptic responses in old animals, implying a lower recruitment of RHT fibers. Conversely, a higher proportion of old SCN neurons exhibited synaptic facilitation, and variance-mean analysis indicated an increase in the probability of release in RHT terminals. Moreover, both spontaneous and miniature postsynaptic events displayed larger amplitudes in neurons from aged mice, whereas analysis of the NMDA and AMPA-kainate components (evoked by RHT electrical stimulation) disclosed no difference between the two ages studied. Immunohistochemistry revealed a bigger size in the puncta of vGluT2, GluN2B, and GluN2A of elderly animals, and the number of immunopositive particles was increased, but that of PSD-95 was reduced. All these synaptic adaptations could be part of compensatory mechanisms in the glutamatergic signaling to ameliorate the loss of RHT terminals in old animals.

Asunto(s)

Envejecimiento , Ácido Glutámico , Ratones Endogámicos C57BL , Núcleo Supraquiasmático , Transmisión Sináptica , Animales , Ratones , Núcleo Supraquiasmático/fisiología , Núcleo Supraquiasmático/metabolismo , Transmisión Sináptica/fisiología , Envejecimiento/fisiología , Ácido Glutámico/metabolismo , Masculino , Potenciales Postsinápticos Excitadores/fisiología , Vías Visuales/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Técnicas de Placa-Clamp , Receptores de N-Metil-D-Aspartato/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo

RESUMEN

Optogenetic stimulation of the adrenergic C1 neurons produces cardiorespiratory activation, and selective depletion of these cells attenuates breathing responses induced by hypoxia. The preBötzinger complex (preBötC) is a group of neurons located in the intermediate aspect of the ventrolateral medulla, critical for respiratory rhythmogenesis, and is modulated by glutamate and catecholamines. Our hypothesis is that selective activation of C1 neurons leads to breathing responses by excitatory connections with the preBötC neurons. Anatomical connection between C1 cells and preBötC was evaluated using retrograde (Cholera Toxin b; preBötC) and anterograde (LVV-PRSx8-ChR2-eYFP; C1 region) tracers. LVV-PRSx8-ChR2-eYFP (viral vector that expresses channelrhodopsin-2 (ChR2) under the control of the catecholaminergic neuron-preferring promoter (PRSx8) was also injected into the C1 region of male Wistar rats for the functional experiments. Anatomical results demonstrated that preBötC neurons receive projections from C1 cells, and these projections express tyrosine hydroxylase and vesicular glutamate transporter 2. Functional connection between C1 cells and preBötC was evaluated by photostimulation of ChR2-transduced C1 neurons before and after unilateral injection of the ionotropic glutamate antagonist, kynurenic acid (kyn), or cocktail of adrenergic antagonists in the preBötC. Kyn injection into preBötC blocked the increase in DiaEMG frequency without changing the MAP increase elicited by photostimulation of C1 neurons, while the injection of adrenergic antagonists into the preBötC did not change DiaEMG frequency and MAP increase induced by photostimulation of C1 cells. Our results suggest that the increase in breathing produced by photostimulation of C1 neurons can be caused by a direct glutamatergic activation of preBötC neurons.

Asunto(s)

Neuronas Adrenérgicas/fisiología , Respiración , Centro Respiratorio/fisiología , Antagonistas Adrenérgicos/farmacología , Neuronas Adrenérgicas/efectos de los fármacos , Neuronas Adrenérgicas/metabolismo , Animales , Neuronas Colinérgicas/efectos de los fármacos , Neuronas Colinérgicas/metabolismo , Neuronas Colinérgicas/fisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Quinurénico/farmacología , Masculino , Optogenética , Ratas , Ratas Wistar , Centro Respiratorio/citología , Centro Respiratorio/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo

RESUMEN

The lateral habenula (LHb) has a key role in integrating a variety of neural circuits associated with reward and aversive behaviors. There is limited information about how the different cell types and neuronal circuits within the LHb coordinate physiological and motivational states. Here, we report a cell type in the medial division of the LHb (LHbM) in male rats that is distinguished by: (1) a molecular signature for GABAergic neurotransmission (Slc32a1/VGAT) and estrogen receptor (Esr1/ERα) expression, at both mRNA and protein levels, as well as the mRNA for vesicular glutamate transporter Slc17a6/VGLUT2, which we term the GABAergic estrogen-receptive neuron (GERN); (2) its axonal projection patterns, identified by in vivo juxtacellular labeling, to both local LHb and to midbrain modulatory systems; and (3) its somatic expression of receptors for vasopressin, serotonin and dopamine, and mRNA for orexin receptor 2. This cell type is anatomically located to receive afferents from midbrain reward (dopamine and serotonin) and hypothalamic water and energy homeostasis (vasopressin and orexin) circuits. These afferents shared the expression of estrogen synthase (aromatase) and VGLUT2, both in their somata and axon terminals. We demonstrate dynamic changes in LHbM VGAT+ cell density, dependent upon gonadal functional status, that closely correlate with motivational behavior in response to predator and forced swim stressors. The findings suggest that the homeostasis and reward-related glutamatergic convergent projecting pathways to LHbMC employ a localized neurosteroid signaling mechanism via axonal expression of aromatase, to act as a switch for GERN excitation/inhibition output prevalence, influencing depressive or motivated behavior.

Asunto(s)

Conducta Animal/fisiología , Estrógenos/metabolismo , Neuronas GABAérgicas/fisiología , Hormonas Esteroides Gonadales/metabolismo , Habénula/fisiología , Homeostasis/fisiología , Hipotálamo/fisiología , Mesencéfalo/fisiología , Motivación/fisiología , Transducción de Señal/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismo , Animales , Neuronas GABAérgicas/metabolismo , Habénula/metabolismo , Hipotálamo/metabolismo , Masculino , Mesencéfalo/metabolismo , Ratas , Ratas Wistar

RESUMEN

Synaptic inputs from cortex and thalamus were compared in electrophysiologically defined striatal cell classes: direct and indirect pathways' striatal projection neurons (dSPNs and iSPNs), fast-spiking interneurons (FS), cholinergic interneurons (ChINs), and low-threshold spiking-like (LTS-like) interneurons. Our purpose was to observe whether stimulus from cortex or thalamus had equivalent synaptic strength to evoke prolonged suprathreshold synaptic responses in these neuron classes. Subthreshold responses showed that inputs from either source functionally mix up in their dendrites at similar electrotonic distances from their somata. Passive and active properties of striatal neuron classes were consistent with the previous studies. Cre-dependent adeno-associated viruses containing Td-Tomato or eYFP fluorescent proteins were used to identify target cells. Transfections with ChR2-eYFP driven by the promoters CamKII or EF1.DIO in intralaminar thalamic nuclei using Vglut-2-Cre mice, or CAMKII in the motor cortex were used to stimulate cortical or thalamic afferents optogenetically. Both field stimuli in the cortex or photostimulation of ChR2-YFP cortical fibers evoked similar prolonged suprathreshold responses in SPNs. Photostimulation of ChR2-YFP thalamic afferents also evoked suprathreshold responses. Differences previously described between responses of dSPNs and iSPNs were observed in both cases. Prolonged suprathreshold responses could also be evoked from both sources onto all other neuron classes studied. However, to evoke thalamostriatal suprathreshold responses, afferents from more than one thalamic nucleus had to be stimulated. In conclusion, both thalamus and cortex are capable to generate suprathreshold responses converging on diverse striatal cell classes. Postsynaptic properties appear to shape these responses.

Asunto(s)

Corteza Cerebral/fisiología , Cuerpo Estriado/citología , Vías Nerviosas/fisiología , Neuronas/fisiología , Potenciales Sinápticos/fisiología , Tálamo/fisiología , Análisis de Varianza , Animales , Colina O-Acetiltransferasa/metabolismo , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Lisina/análogos & derivados , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas/efectos de los fármacos , Neuronas/efectos de los fármacos , Potenciales Sinápticos/efectos de los fármacos , Proteína 2 de Transporte Vesicular de Glutamato/genética , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo

RESUMEN

Catecholaminergic C1 cells reside in the rostral and intermediate portions of the ventrolateral medulla (RVLM) and can be activated by hypoxia. These neurons regulate the hypothalamic pituitary axis via direct projections to the hypothalamic paraventricular nucleus (PVH) and regulate the autonomic nervous system via projections to sympathetic and parasympathetic preganglionic neurons. Based on the various effects attributed to the C1 cells and what is currently known of their synaptic inputs, our hypothesis is that acute hypoxia (AH) activates RVLM projecting catecholaminergic neurons to PVH. Anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L) was unilaterally injected into the RVLM and a retrograde tracer Cholera toxin b (CTb) was unilaterally injected into the PVH region. After ten days, male Wistar rats that received CTb injection into the PVH were subjected to AH (8% O2, balanced with N2) or normoxia (21% O2) for 3h. Acute hypoxia significantly increased Fos immunoreactivity in the C1 region (68±2 neurons), and half of the RVLM cells activated are catecholaminergic (35±2 neurons). We observed that 23±4% of the RVLM projecting PVH cells that were activated by AH were also C1 cells. The presence of varicosities containing PHA-L in PVH region was also observed. The present results suggest that catecholaminergic C1-PVH projection is hypoxia-sensitive and the pathway between these two important brain areas can be one more piece in the complex puzzle of neural control of autonomic regulation during hypoxia.

Asunto(s)

Catecolaminas/metabolismo , Hipoxia/patología , Bulbo Raquídeo/patología , Vías Nerviosas/fisiología , Neuronas/fisiología , Análisis de Varianza , Animales , Presión Sanguínea/fisiología , Recuento de Células , Toxina del Cólera/farmacocinética , Modelos Animales de Enfermedad , Esquema de Medicación , Glutamato Descarboxilasa/metabolismo , Frecuencia Cardíaca/fisiología , Hipoxia/fisiopatología , Masculino , Proteínas Oncogénicas v-fos/metabolismo , Núcleo Hipotalámico Paraventricular , Fitohemaglutininas/administración & dosificación , Fitohemaglutininas/farmacocinética , Ratas , Ratas Wistar , Tirosina 3-Monooxigenasa/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo

RESUMEN

The Kölliker-Fuse (KF) region, located in the dorsolateral pons, projects to several brainstem areas involved in respiratory regulation, including the chemoreceptor neurons within the retrotrapezoid nucleus (RTN). Several lines of evidence indicate that the pontine KF region plays an important role in the control of the upper airways for the maintenance of appropriate airflow to and from the lungs. Specifically, we hypothesized that the KF region is involved in mediating the response of the hypoglossal motor activity to central respiratory chemoreflex activation and to stimulation of the chemoreceptor neurons within the RTN region. To test this hypothesis, we combined immunohistochemistry and physiological experiments. We found that in the KF, the majority of biotinylated dextran amine (BDA)-labeled axonal varicosities contained detectable levels of vesicular glutamate transporter-2 (VGLUT2), but few contained glutamic acid decarboxylase-67 (GAD67). The majority of the RTN neurons that were FluorGold (FG)-immunoreactive (i.e., projected to the KF) contained hypercapnia-induced Fos, but did not express tyrosine hydroxylase. In urethane-anesthetized sino-aortic denervated and vagotomized male Wistar rats, hypercapnia (10% CO2) or N-methyl-d-aspartate (NMDA) injection (0.1mM) in the RTN increased diaphragm (DiaEMG) and genioglossus muscle (GGEMG) activities and elicited abdominal (AbdEMG) activity. Bilateral injection of muscimol (GABA-A agonist; 2mM) into the KF region reduced the increase in DiaEMG and GGEMG produced by hypercapnia or NMDA into the RTN. Our data suggest that activation of chemoreceptor neurons in the RTN produces a significant increase in the genioglossus muscle activity and the excitatory pathway is dependent on the neurons located in the dorsolateral pontine KF region.

Asunto(s)

Células Quimiorreceptoras/fisiología , Bulbo Raquídeo/fisiología , Puente/fisiología , Respiración , Lengua/fisiología , Animales , Células Quimiorreceptoras/citología , Diafragma/inervación , Diafragma/fisiología , Glutamato Descarboxilasa/metabolismo , Ácido Glutámico/metabolismo , Hipercapnia/patología , Hipercapnia/fisiopatología , Masculino , Bulbo Raquídeo/citología , Muscimol/farmacología , N-Metilaspartato/farmacología , Neurotransmisores/farmacología , Puente/citología , Puente/efectos de los fármacos , Ratas Wistar , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Respiración/efectos de los fármacos , Lengua/inervación , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo

RESUMEN

The rostroventrolateral medulla contains two functional neuronal populations: (1) the parafacial respiratory group (pFRG) neurons and (2) the chemosensitive retrotrapezoid nucleus (RTN) neurons. Using anatomical and physiological techniques, we investigated the role of the RTN/pFRG in CO2-induced active expiration (AE) in urethane-anesthetized rats. Anterograde tracing using biotinylated dextran amine (BDA) revealed dense neuronal projections emanating from the RTN/pFRG to the caudal ventral respiratory group (cVRG), 60% of which contained vesicular glutamate transporter-2. The minority (16%) of the RTN projections to the cVRG emanated from Phox2b positive neurons. Hypercapnia (10% CO2) increased DiaEMG and elicited AbdEMG activity. Bilateral injections of muscimol (2mM) into the RTN/pFRG reduced the activation of DiaEMG (23±4%) and abolished AE-induced by chemoreflex stimulation. Taken together, these results support the presence of direct excitatory projections from RTN/pFRG neurons to cVRG expiratory premotor neurons, playing a role in the generation/modulation of AE.

Asunto(s)

Espiración/fisiología , Bulbo Raquídeo/citología , Bulbo Raquídeo/fisiología , Neuronas/citología , Neuronas/fisiología , Abdomen/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Diafragma/efectos de los fármacos , Diafragma/fisiología , Espiración/efectos de los fármacos , Agonistas de Receptores de GABA-A/farmacología , Proteínas de Homeodominio/metabolismo , Hipercapnia/patología , Hipercapnia/fisiopatología , Inhalación/efectos de los fármacos , Inhalación/fisiología , Masculino , Bulbo Raquídeo/efectos de los fármacos , Corteza Motora/citología , Corteza Motora/fisiología , Muscimol/farmacología , Neuronas/efectos de los fármacos , Estimulación Física , Ratas Wistar , Reflejo/efectos de los fármacos , Reflejo/fisiología , Factores de Transcripción/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo

RESUMEN

Using immunohistochemical techniques, we characterized changes in the expression of several neurochemical markers in lumbar 4-sacral 2 (L4-S2) dorsal root ganglion (DRG) neuron profiles (NPs) and the spinal cord of BALB/c mice after axotomy of the L6 and S1 spinal nerves, major tributaries of the pelvic (targeting pelvic visceral organs) and pudendal (targeting perineum and genitalia) nerves. Sham animals were included. Expression of cyclic AMP-dependent transcription factor 3 (ATF3), calcitonin gene-related peptide (CGRP), transient receptor potential cation channel subfamily V, member 1 (TRPV1), tyrosine hydroxylase (TH) and vesicular glutamate transporters (VGLUT) types 1 and -2 was analysed seven days after injury. L6-S1 axotomy induced dramatic de novo expression of ATF3 in many L6-S1 DRG NPs, and parallel significant downregulations in the percentage of CGRP-, TRPV1-, TH- and VGLUT2-immunoreactive (IR) DRG NPs, as compared to their expression in uninjured DRGs (contralateral L6-S1-AXO; sham mice); VGLUT1 expression remained unaltered. Sham L6-S1 DRGs only showed a small ipsilateral increase in ATF3-IR NPs (other markers were unchanged). L6-S1-AXO induced de novo expression of ATF3 in several lumbosacral spinal cord motoneurons and parasympathetic preganglionic neurons; in sham mice the effect was limited to a few motoneurons. Finally, a moderate decrease in CGRP- and TRPV1-like-immunoreactivities was observed in the ipsilateral superficial dorsal horn neuropil. In conclusion, injury of a mixed visceral/non-visceral nerve leads to considerable neurochemical alterations in DRGs matched, to some extent, in the spinal cord. Changes in these and potentially other nociception-related molecules could contribute to pain due to injury of nerves in the abdominopelvic cavity.

Asunto(s)

Ganglios Espinales/metabolismo , Neuronas/metabolismo , Nervio Pudendo/metabolismo , Médula Espinal/metabolismo , Factor de Transcripción Activador 3/metabolismo , Animales , Axotomía , Péptido Relacionado con Gen de Calcitonina/metabolismo , Regulación hacia Abajo , Genitales/inervación , Masculino , Ratones , Ratones Endogámicos BALB C , Neuronas Motoras/metabolismo , Pelvis/inervación , Perineo/inervación , Canales Catiónicos TRPV/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Regulación hacia Arriba , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo

RESUMEN

Retinal inputs to the optic tectum (TeO) triggered by moving stimuli elicit synchronized feedback signals from two isthmic nuclei: the isthmi parvocelullaris (Ipc) and isthmi semilunaris (SLu). Both of these nuclei send columnar axon terminals back to the same tectal position receiving the retinal input. The feedback signals from the Ipc seem to act as an attentional spotlight by selectively boosting the propagation of retinal inputs from the tectum to higher visual areas. Although Ipc and SLu nuclei are widely considered cholinergic because of their immunoreactivity for choline acetyltransferase (ChAT), contradictory findings, including the expression of the vesicular glutamate transporter 2 (VGluT2) mRNA in Ipc neurons, have raised doubts about the purely cholinergic nature of this nucleus. In this study, in chicks, we revise the neurochemical identity of the isthmic nuclei by using in situ hybridization assays for VGluT2 along with three cholinergic markers: the vesicular acetylcholine transporter (VAChT), the high-affinity choline transporter (CHT1) and ChAT. We found that neurons in the SLu showed strong mRNA expression of all three cholinergic markers, whereas the expression of VAChT mRNA in the Ipc was undetectable in our essays. Instead, Ipc neurons exhibited a strong expression of VGluT2 mRNA. Immunohistochemistry assays showed VGluT2 immunoreactivity in the TeO codistributing with anterogradely labeled Ipc axon-terminal boutons, further supporting a glutamatergic function for the Ipc nucleus. Therefore, our results strongly suggest that, in the chick, whereas the feedback from the SLu to the TeO is indeed cholinergic, the feedback from the Ipc has a marked glutamatergic component.

Asunto(s)

Pollos/metabolismo , Retroalimentación Fisiológica/fisiología , Colículos Superiores/metabolismo , Vías Visuales/metabolismo , Animales , Proteínas Aviares/metabolismo , Pollos/anatomía & histología , Colina O-Acetiltransferasa/metabolismo , Inmunohistoquímica , Hibridación in Situ , Proteínas de Transporte de Membrana/metabolismo , Técnicas de Trazados de Vías Neuroanatómicas , Neuronas/citología , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Colículos Superiores/anatomía & histología , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Vías Visuales/anatomía & histología

RESUMEN

Using specific riboprobes, we characterized the expression of vesicular glutamate transporter (VGLUT)1-VGLUT3 transcripts in lumbar 4-5 (L4-5) dorsal root ganglions (DRGs) and the thoracolumbar to lumbosacral spinal cord in male BALB/c mice after a 1- or 3-day hindpaw inflammation, or a 7-day sciatic nerve axotomy. Sham animals were also included. In sham and contralateral L4-5 DRGs of injured mice, VGLUT1-, VGLUT2- and VGLUT3 mRNAs were expressed in ∼45%, ∼69% or ∼17% of neuron profiles (NPs), respectively. VGLUT1 was expressed in large and medium-sized NPs, VGLUT2 in NPs of all sizes, and VGLUT3 in small and medium-sized NPs. In the spinal cord, VGLUT1 was restricted to a number of NPs at thoracolumbar and lumbar segments, in what appears to be the dorsal nucleus of Clarke, and in mid laminae III-IV. In contrast, VGLUT2 was present in numerous NPs at all analyzed spinal segments, except the lateral aspects of the ventral horns, especially at the lumbar enlargement, where it was virtually absent. VGLUT3 was detected in a discrete number of NPs in laminae III-IV of the dorsal horn. Axotomy resulted in a moderate decrease in the number of DRG NPs expressing VGLUT3, whereas VGLUT1 and VGLUT2 were unaffected. Likewise, the percentage of NPs expressing VGLUT transcripts remained unaltered after hindpaw inflammation, both in DRGs and the spinal cord. Altogether, these results confirm previous descriptions on VGLUTs expression in adult mice DRGs, with the exception of VGLUT1, whose protein expression was detected in a lower percentage of mouse DRG NPs. A detailed account on the location of neurons expressing VGLUTs transcripts in the adult mouse spinal cord is also presented. Finally, the lack of change in the number of neurons expressing VGLUT1 and VGLUT2 transcripts after axotomy, as compared to data on protein expression, suggests translational rather than transcriptional regulation of VGLUTs after injury.

Asunto(s)

Ganglios Espinales/metabolismo , Neuronas/metabolismo , Médula Espinal/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/análisis , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Axotomía , Miembro Posterior , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/metabolismo , Nervio Ciático/lesiones , Proteína 1 de Transporte Vesicular de Glutamato/análisis , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/análisis , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteínas de Transporte Vesicular de Glutamato/análisis