RESUMEN
Cerebrospinal fluid-contacting neurons (CSF-cNS) are considered mechanoreceptors and chemoreceptors involved in detecting changes in CSF circulation. However, considering that recent data suggest that this type of cell could exert an active response when an external stimulus is sensed, identification of CSF-cNS may be relevant. In this regard, some data suggest that a neuronal connection exists between the ventral region of the hypothalamic paraventricular nucleus (PVN) and rostral agranular insular cortex (RAIC); indeed, a potential CSF-cNS is hypothesized. However, a detailed analysis of this connection has not been conducted. Thus, using neuronal tracers (Fluoro-Gold® (FG) and cholera toxin (ChT)) coupled with transmission electron microscopy and immunofluorescence assays against Fluoro-Gold®, oxytocin (OXT), vasopressin (AVP) and oxytocin receptors (OTR), we describe an oxytocinergic or vasopressinergic CSF-cNS between the PVN and RAIC. Our results showed that CSF-cNS along the PVN labelled with oxytocin and/or AVP were present in dendritic projections near the third ventricle. This CSF-cNS in the PVN seems to project to the RAIC. Inside the RAIC, ultrastructural analysis showed that axons immunopositive for oxytocin from the PVN sustained synaptic connections with neurons that expressed OTR. These findings show that the CSF-cNS from the PVN sends projections to the RAIC. To the best of our knowledge, the relevance of CSF-cNS has not been elucidated; however, we hypothesized that the activation of cells could concomitantly release neuropeptides (i.e., oxytocin and AVP) in the CSF and RAIC. Thus, further analysis of the impact of neuropeptides released into the third ventricle and RAIC is warranted.
Asunto(s)
Corteza Cerebral , Neuronas , Oxitocina , Núcleo Hipotalámico Paraventricular , Animales , Neuronas/ultraestructura , Neuronas/metabolismo , Oxitocina/líquido cefalorraquídeo , Oxitocina/metabolismo , Ratas , Masculino , Corteza Cerebral/ultraestructura , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Núcleo Hipotalámico Paraventricular/metabolismo , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/ultraestructura , Ratas Wistar , Receptores de Oxitocina/metabolismo , Líquido Cefalorraquídeo/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Vasopresinas/metabolismo , Vasopresinas/líquido cefalorraquídeo , Vías Nerviosas/ultraestructura , Vías Nerviosas/metabolismo , Microscopía Electrónica de TransmisiónRESUMEN
BACKGROUND: The CLARITY technique enables researchers to visualize different neuronal connections along the nervous system including the somatosensory system. NEW METHOD: The present work describes the antero-lateral and dorsal column pathways until the thalamic and cortical stations, as well as descending oxytocinergic and vasopressinergic innervations by means of combined CLARITY, neuronal tracing, and immunofluorescence techniques. We used male Sprague-Dawley rats of 13, 30, and 60 days. RESULTS: The main results are as follows: A) CLARITY is a reliable technique that can be combined with fluorescent neuronal tracers and immunofluorescence techniques without major procedure modifications; B) at spinal level, some primary afferent fibers were labeled by CGRP, as well as the presence of neuronal populations that simultaneously project to the gracile and ventral posterolateral thalamic nuclei; C) corticothalamic connections were visible when retrograde tracers were injected at thalamic level; D) oxytocin receptors were expressed in the spinal dorsal horn by GABAergic-positive neurons, reinforcing previous outcomes about the possible mechanism for oxytocin blocking the primary afferent sensory input. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: The CLARITY technique lets us observe in a transparent way the entire processed tissue compared with classical histological methods. CLARITY is a potentially useful tool to describe neuroanatomical structures and their neurochemical stratus.
Asunto(s)
Neuronas , Núcleos Talámicos Ventrales , Animales , Axones , Técnica del Anticuerpo Fluorescente , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The rostral agranular insular cortex (RAIC) is a relevant structure in nociception. Indeed, recruitment of GABAergic activity in RAIC promotes the disinhibition of the locus ceruleus, which in turn inhibits (by noradrenergic action) the peripheral nociceptive input at the spinal cord level. In this regard, at the cortical level, oxytocin can modulate the GABAergic transmission; consequently, an interaction modulating nociception could exist between oxytocin and GABA at RAIC. Here, we tested in male Wistar rats the effect of oxytocin microinjection into RAIC during an inflammatory (by subcutaneous peripheral injection of formalin) nociceptive input. Oxytocin microinjection produces a diminution of (1) flinches induced by formalin and (2) spontaneous firing of spinal wide dynamic range cells. The above antinociceptive effect was abolished by microinjection (at RAIC) of the following: (1) L-368899 (an oxytocin receptor [OTR] antagonist) or by (2) bicuculline (a preferent GABAA receptor blocker), suggesting a GABAergic activation induced by OTR. Since intrathecal injection of an α2A-adrenoceptor antagonist (BRL 44408) partially reversed the oxytocin effect, a descending noradrenergic antinociception is suggested. Further, injection of L-368899 per se induces a pronociceptive behavioral effect, suggesting a tonic endogenous oxytocin release during inflammatory nociceptive input. Accordingly, we found bilateral projections from the paraventricular nucleus of the hypothalamus (PVN) to RAIC. Some of the PVN-projecting cells are oxytocinergic and destinate GABAergic and OTR-expressing cells inside RAIC. Aside from the direct anatomic link between PVN and RAIC, our findings provide evidence about the role of oxytocinergic mechanisms modulating the pain process at the RAIC level.SIGNIFICANCE STATEMENT Oxytocin is a neuropeptide involved in several functions ranging from lactation to social attachment. Over the years, the role of this molecule in pain processing has emerged, showing that, at the spinal level, oxytocin blocks pain transmission. The present work suggests that oxytocin also modulates pain at the cortical insular level by favoring cortical GABAergic transmission and activating descending spinal noradrenergic mechanisms. Indeed, we show that the paraventricular hypothalamicnucleus sends direct oxytocinergic projections to the rostral agranular insular cortex on GABAergic and oxytocin receptor-expressing neurons. Together, our data support the notion that the oxytocinergic system could act as an orchestrator of pain modulation.
Asunto(s)
Corteza Cerebral/fisiología , Inflamación/fisiopatología , Neuronas/fisiología , Nocicepción/fisiología , Oxitocina/fisiología , Animales , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Formaldehído/administración & dosificación , Neuronas GABAérgicas/fisiología , Inflamación/inducido químicamente , Masculino , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Nocicepción/efectos de los fármacos , Oxitocina/administración & dosificación , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/fisiología , Ratas WistarRESUMEN
Hypothalamic paraventricular nucleus (PVN) projections to the spinal dorsal horn (SDH) are related to antinociception. Several neuropeptides from this nucleus could be released to the spinal cord after nociceptive stimuli. Indeed, it has been shown that enkephalins, oxytocin and vasopressin could be released at this level. Although the antinociceptive effects of these neuropeptides are well studied, little is known about the potential interaction between these molecules. In this study, we provide anatomical evidence of the interaction between oxytocin (OT), vasopressin (AVP), dynorphin (DYN) and enkephalin (ENK) along the PVN projections to the spinal dorsal horn at L3 level. A retrograde tracer (True Blue®) microinjected at L3 in the SDH and immunofluorescence with antibodies against OT, AVP, DYN and ENK were used. The experiments showed different levels of peptide immunoreactivity distribution along the rostro-caudal area of the PVN. A high percentage of co-localizations between two of the peptides (OT-AVP, OT-DYN, AVP-ENK, DYN-ENK) were present along the PVN. The following co-localizations occupied 4.76-9.62% of the total PVN area. PVN projections to the SDH at L3 level showed similar results. Our results show that different antinociceptive peptides may be interacting with each other to evoke PVN antinociceptive effects as part of the endogenous system of nociceptive modulation.