RESUMEN
MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as sensors of extracellular sodium concentration ([Na(+)]out). The mechanism underlying Na(+)-sensing involves Na(+)-flow through the NaX channel, directly regulated by the Na(+)/K(+)-ATPase α1-isoform which controls Na(+)-influx by modulating channel permeability. Together, these two partners form a complex involved in the regulation of intracellular sodium ([Na(+)]in). Here we aim to determine whether environmental changes in Na(+) could actively modulate the NaX/Na(+)/K(+)-ATPase complex activity. We investigated the complex activity using patch-clamp recordings from rat MnPO neurons and Neuro2a cells. When the rats were fed with a high-salt-diet, or the [Na(+)] in the culture medium was increased, the activity of the complex was up-regulated. In contrast, drop in environmental [Na(+)] decreased the activity of the complex. Interestingly under hypernatremic condition, the colocalization rate and protein level of both partners were up-regulated. Under hyponatremic condition, only NaX protein expression was increased and the level of NaX/Na(+)/K(+)-ATPase remained unaltered. This unbalance between NaX and Na(+)/K(+)-ATPase pump proportion would induce a bigger portion of Na(+)/K(+)-ATPase-control-free NaX channel. Thus, we suggest that hypernatremic environment increases NaX/Na(+)/K(+)-ATPase α1-isoform activity by increasing the number of both partners and their colocalization rate, whereas hyponatremic environment down-regulates complex activity via a decrease in the relative number of NaX channels controlled by the pump.
RESUMEN
In rat brain, the detection and integration of chemosensory and neural signals are achieved, inter alia, by the median preoptic nucleus (MnPO) during a disturbance of the hydromineral balance. This is allowed through the presence of the sodium (Na(+) ) sensor neurons. Interestingly, enkephalins and mu-opioid receptors (µ-ORs) are known for their role in ingestive behaviors and have previously been shown to regulate the excitability of MnPO neurons following a single Na(+) depletion. However, little is known about the role of these µ-ORs in the response enhancement following repeated Na(+) challenge. Therefore, we used whole-cell recordings in acute brain slices to determine neuronal plasticity in the electrical properties of the MnPO Na(+) sensor-specific neuronal population following multiple Na(+) depletions. Our results show that the population of Na(+) sensor neurons was represented by 80% of MnPO neurons after a single Na(+) depletion and was reduced after three Na(+) depletions. Interestingly, the subpopulation of Na(+) sensors responding to D-Ala(2) ,N-MePhe(4) ,Gly-ol-enkephalin (DAMGO), a specific µ-OR agonist, represented 11% of MnPO neurons after a single Na(+) depletion and the population doubled after three Na(+) depletions. Moreover, Na(+) sensor neurons displayed modifications in the discharge pattern distribution and shape of calcium action potentials after three Na(+) depletions but these changes did not occur in Na(+) sensors responding to DAMGO. Thus, the reinforced µ-OR functionality in Na(+) sensors might take place to control the neuronal hyperexcitability and this plasticity in opioid-sensitive and Na(+) detection MnPO networks might sustain the enhanced salt ingestion induced by repeated exposure to Na(+) depletion.
Asunto(s)
Área Preóptica/fisiología , Receptores Opioides mu/metabolismo , Células Receptoras Sensoriales/fisiología , Sodio/fisiología , Animales , Técnicas In Vitro , Masculino , Osmorregulación , Área Preóptica/metabolismo , Ratas , Ratas Wistar , Células Receptoras Sensoriales/metabolismoRESUMEN
The median preoptic nucleus (MnPO) holds a strategic position in the hypothalamus. It is adjacent to the third ventricle; hence, it can directly access the ionic composition of the CSF. MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as central sensors of extracellular Na(+) concentration ([Na(+)](ext)). The mechanism underlying Na(+) sensing involves the atypical Na(+) channel, Na(X). Here we sought to determine whether Na(+) influx in Na(+) sensors is actively regulated via interaction with other membrane proteins involved in cellular Na(+) homeostasis, such as Na(+)/K(+)-ATPase. The Na(+)/K(+)-ATPase role was investigated using patch-clamp recordings in rat MnPO dissociated neurons. Na(+) current evoked with hypernatriuric solution was diminished in the absence of ATP/GTP, indicating that Na(+)/K(+)-ATPase play a central role in [Na(+)](ext) detection. Specific blockers of α1 and α3 isoforms of Na(+)/K(+)-ATPase, ouabain or strophanthidin, inhibited this Na(+) current. However, strophanthidin, which selectively blocks the α1 isoform, was more effective in blocking Na(+) current, suggesting that the Na(+)/K(+)-ATPase-α1 isoform is specifically involved in [Na(+)](ext) detection. Although strophanthidin did not alter either the membrane resistance or the Na(+) reversal potential, the conductance and the permeability of the Na(X) channel decreased significantly. Our results suggest that Na(+)/K(+)-ATPase interacts with the Na(X) channel and regulates the high [Na(+)](ext)-evoked Na(+) current via influencing the Na(+) influx rate. This study describes a novel intracellular regulatory pathway of [Na(+)](ext) detection in MnPO neurons. The α1 isoform of Na(+)/K(+)-ATPase acts as a direct regulatory partner of the Na(X) channel and influences Na(+) influx via controlling the Na(+) permeability of the channel.
Asunto(s)
Neuronas/metabolismo , Canales de Sodio/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/fisiología , Algoritmos , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Inmunohistoquímica , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ouabaína/farmacología , Técnicas de Placa-Clamp , Permeabilidad , Área Preóptica/citología , Área Preóptica/metabolismo , Ratas , Ratas Wistar , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Estrofantidina/farmacologíaRESUMEN
The essential role of the median preoptic nucleus (MnPO) in the integration of chemosensory information associated with the hydromineral state of the rat relies on the presence of a unique population of sodium (Na+) sensor neurons. Little is known about the intrinsic properties of these neurons; therefore, we used whole cell recordings in acute brain slices to determine the electrical fingerprints of this specific neural population of rat MnPO. The data collected from a large sample of neurons (115) indicated that the Na+ sensor neurons represent a majority of the MnPO neurons in situ (83%). These neurons displayed great diversity in both firing patterns induced by transient depolarizing current steps and rectifying properties activated by hyperpolarizing current steps. This diversity of electrical properties was also present in non-Na+ sensor neurons. Subpopulations of Na+ sensor neurons could be distinguished, however, from the non-Na+ sensor neurons. The firing frequency was higher in Na+ sensor neurons, showing irregular spike discharges, and the amplitude of the time-dependent rectification was weaker in the Na+ sensor neurons than in non-Na+ sensor neurons. The diversity among the electrical properties of the MnPO neurons contrasts with the relative function homogeneity (Na+ sensing). However, this diversity might be correlated with a variety of direct synaptic connections linking the MnPO to different brain areas involved in various aspects of the restoration and conservation of the body fluid homeostasis.
Asunto(s)
Área Preóptica/metabolismo , Células Receptoras Sensoriales/metabolismo , Transducción de Señal , Canales de Sodio/metabolismo , Sodio/metabolismo , Equilibrio Hidroelectrolítico , Animales , Técnicas In Vitro , Masculino , Potenciales de la Membrana , Técnicas de Placa-Clamp , Área Preóptica/citología , Ratas , Ratas Wistar , Células Receptoras Sensoriales/clasificación , Factores de TiempoRESUMEN
The Scn7a gene encodes for the specific sodium channel Na(X), which is considered a primary determinant of sodium sensing in the brain. Only partial data exist describing the Na(X) distribution pattern and the cell types that express Na(X) in both the rat and mouse brain. To generate a global view of the sodium detection mechanisms in the two rodent brains, we combined Na(X) immunofluorescence with fluorescent cell markers to map and identify the Na(X)-expressing cell populations throughout the network involved in hydromineral homeostasis. Here, we designed an anti-Na(X) antibody targeting the interdomain 2-3 region of the Na(X) channel's α-subunit. In both the rat and mouse, Na(X) immunostaining was colocalized with vimentin positive cells in the median eminence and with magnocellular neurons immunopositive for neurophysin associated with oxytocin or vasopressin in both the supraoptic and paraventricular nuclei. Na(X) immunostaining was also detected in neurons of the area postrema. In addition to this common Na(X) expression pattern, several differences in Na(X) immunostaining for certain structures and cell types were found between the rat and mouse. Na(X) was present in both NeuN and vimentin positive cells in the subfornical organ and the vascular organ of the lamina terminalis of the rat whereas Na(X) was only colocalized with vimentin positive cells in the mouse circumventricular organs. In addition, Na(X) immunostaining was specifically observed in NeuN immunopositive cells in the median preoptic nucleus of the rat. Overall, this study characterized the Na(X)-expressing cell types in the network controlling hydromineral homeostasis of the rat and mouse. Na(X) expression pattern was clearly different in the nuclei of the lamina terminalis of the rat and mouse, indicating that the mechanisms involved in systemic and central Na(+) sensing are specific to each rodent species.
RESUMEN
Tau hyperphosphorylation is one hallmark of Alzheimer's disease (AD) pathology. Pharmaceutical companies have thus developed kinase inhibitors aiming to reduce tau hyperphosphorylation. One obstacle in screening for tau kinase inhibitors is the low phosphorylation levels of AD-related phospho-epitopes in normal adult mice and cultured cells. We have shown that hypothermia induces tau hyperphosphorylation in vitro and in vivo. Here, we hypothesized that hypothermia could be used to assess tau kinase inhibitors efficacy. Hypothermia applied to models of biological gradual complexity such as neuronal-like cells, ex vivo brain slices and adult non-transgenic mice leads to tau hyperphosphorylation at multiple AD-related phospho-epitopes. We show that Glycogen Synthase Kinase-3 inhibitors LiCl and AR-A014418, as well as roscovitine, a cyclin-dependent kinase 5 inhibitor, decrease hypothermia-induced tau hyperphosphorylation, leading to different tau phosphorylation profiles. Therefore, we propose hypothermia-induced hyperphosphorylation as a reliable, fast, convenient and inexpensive tool to screen for tau kinase inhibitors.
Asunto(s)
Evaluación Preclínica de Medicamentos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Anestesia , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/metabolismo , Femenino , Glucógeno Sintasa Quinasa 3/metabolismo , Hipotermia/inducido químicamente , Hipotermia/enzimología , Cloruro de Litio/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacosRESUMEN
The goal of this study was to create a realistic and quantitative simulation of vasopressin (AVP) secretion under iso-osmotic and short-term challenged plasma osmolality. The relationship between AVP concentration ([AVP]) and plasma osmolality was computed using a sophisticated and integrated model that chronologically simulates (1) the overall firing rate of the hypothalamus' magnocellular neuronal (MCN) population, (2) the propagation of the spike activity down the axons, (3) the fatigue and facilitation mechanisms of AVP release at the axon terminals and (4) the [AVP] pharmacodynamics based on the trains of AVP release. This global simulation predicted that the differential MCN sensitivity to dynorphin would be the most critical mechanism underlying the individual variability of MCN firing behaviors (silence, irregular, phasic and continuous firing patterns). However, at the level of the MCN population, the simulation predicted that the dynorphin factor must be combined with the distribution of the resting membrane potentials among the MCNs to obtain a realistic overall firing rate in response to a change in osmolality. Moreover, taking advantage of the integrated model, the simulation predicted that the selective removal of the frequency-dependent facilitation of AVP secretion has a major impact on the overall [AVP]-to-osmolality relationship (mean absolute change of 2.59 pg/ml); the action potential propagation failure, while critical, has a smaller quantitative impact on the overall [AVP] (0.58 pg/ml). The present integrated model (from a single MCN to a quantitative plasma [AVP]) improves our knowledge of the mechanisms underlying overall MCN firing and AVP excitation-secretion coupling.
Asunto(s)
Arginina Vasopresina/metabolismo , Núcleo Basal de Meynert/metabolismo , Modelos Neurológicos , Neuronas/metabolismo , Algoritmos , Animales , Arginina Vasopresina/farmacología , Axones/fisiología , Núcleo Basal de Meynert/efectos de los fármacos , Simulación por Computador , Dinorfinas/metabolismo , Fenómenos Electrofisiológicos/efectos de los fármacos , Predicción , Potenciales de la Membrana/fisiología , Neuronas/fisiología , Concentración Osmolar , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Ratas , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inhibidoresRESUMEN
Arginine vasopressin (AVP), one of the most important hormones involved in hydromineral homeostasis, is secreted by hypothalamic magnocellular neurons (MCNs). Here, we implemented two critical parameters for MCN physiology into a Hodgkin-Huxley simulation of the MCN. By incorporating the mechanosensitive channel (MSC) responsible for osmodetection and the synaptic inputs whose frequencies are modulated by changes in ambient osmolality into our model, we were able to develop an improved model with increased physiological relevance and gain new insight into the determinants of the firing patterns of AVP magnocellular neurons. Our results with this MCN model predict that 1) a single MCN is able to display all the firing patterns experimentally observed: silent, irregular, phasic and continuous firing patterns; 2) under conditions of hyperosmolality, burst durations are regulated by the frequency-dependent fatigue of dynorphin secretion; and 3) the transitions between firing patterns are controlled by EPSP and IPSP frequencies (0, 2, 4, 8, 16, 32, 64 and 128 Hz). Moreover, this simulation predicts that EPSPs and IPSPs do not modify the spiking frequency (SF) of phasic firing patterns (0.0034 Hz/Hz [EPSP]; 0.0012 Hz/Hz [IPSP]). Rather, these afferents strongly regulate SF during irregular and continuous firing patterns (0.075 Hz/Hz [EPSP]; 0.027 Hz/Hz [IPSP]). The use of the realistic MCN model developed here allows for an improved understanding of the determinants driving the firing patterns and spiking frequencies of vasopressinergic magnocellular neurons.
Asunto(s)
Potenciales de Acción/fisiología , Hipotálamo/fisiología , Modelos Neurológicos , Neuronas/fisiología , Vasopresinas/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Animales , Membrana Celular/fisiología , Humanos , Neuronas/clasificación , Valor Predictivo de las PruebasRESUMEN
The simultaneous localization of several anatomical markers is often required to understand and analyze the organization of complex brain nuclei or identify neuronal networks recruited during a specific biological stimulus. Gathering such information is usually achieved by the combined detection of both mRNA and proteins. Staining techniques using fluorescence have progressively overtaken the use of radioactive tissue labeling and immunostaining based on the avidin-biotin-peroxidase complex. Despite the promise offered by the combination of fluorescent in situ hybridization (FISH) and immunofluorescence (IF), in terms of reduced bench time and easy visualization of multiple labels at once, some technical hurdles have to be overcome to produce reliable data from these state-of-the-art neuroanatomy techniques. Here, we have adapted a combination of FISH and IF for slices mounted on a microscope slide, using mRNA (GAD65 mRNA) and proteins (NeuN, FosB or TH) widely studied in neuroanatomy, to validate this method. Proteinase K (PK), which is often used to optimize riboprobe penetration, is a major limiting factor in obtaining successful IF labeling. This study demonstrates the inaccuracy of PK and provides appropriate tools to improve the efficiency of the combined FISH-IF procedure to obtain high quality fluorescent multi-labeling.
Asunto(s)
Técnica del Anticuerpo Fluorescente/métodos , Hibridación Fluorescente in Situ/métodos , Neuroanatomía/métodos , Coloración y Etiquetado/métodos , Animales , Microtomía/métodos , Ratas , Ratas WistarRESUMEN
Sodium (Na(+)) ions are of primary importance for hydromineral and cardiovascular homeostasis, and the level of Na(+) in the body fluid compartments [plasma and cerebrospinal fluid (CSF)] is precisely monitored in the hypothalamus. Glial cells seem to play a critical role in the mechanism of Na(+) detection. However, the precise role of neurons in the detection of extracellular Na(+) concentration ([Na(+)](out)) remains unclear. Here we demonstrate that neurons of the median preoptic nucleus (MnPO), a structure in close contact with the CSF, are specific Na(+) sensors. Electrophysiological recordings were performed on dissociated rat MnPO neurons under isotonic [Na(+)] (100 mM NaCl) with local application of hypernatriuric (150, 180 mM NaCl) or hyponatriuric (50 mM NaCl) external solution. The hyper- and hyponatriuric conditions triggered an in- and an outward current, respectively. The reversal potential of the current matched the equilibrium potential of Na(+), indicating that a change in [Na(+)](out) modified the influx of Na(+) in the MnPO neurons. The conductance of the Na(+) current was not affected by either the membrane potential or the [Na(+)](out). Moreover, the channel was highly selective for lithium over guanidinium. Together, these data identified the channel as a Na(+) leak channel. A high correlation between the electrophysiological recordings and immunofluorescent labeling for the Na(X) channel in dissociated MnPO neurons strongly supports this channel as a candidate for the Na(+) leak channel responsible for the Na(+)-sensing ability of rat MnPO neurons. The absence of Na(X) labeling and of a specific current evoked by a change in [Na(+)](out) in mouse MnPO neurons suggests species specificity in the hypothalamus structures participating in central Na(+) detection.
Asunto(s)
Activación del Canal Iónico/fisiología , Neuronas/fisiología , Área Preóptica/fisiología , Canales de Sodio/fisiología , Sodio/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Animales , Ratones , Ratas , Ratas Wistar , Especificidad de la EspecieRESUMEN
BACKGROUND: In mammals, vasopressin (AVP) is released from magnocellular neurons of the hypothalamus when osmotic pressure exceeds a fixed set-point. AVP participates to the hydromineral homeostasis (HH) by controlling water excretion at the level of the kidneys. Our current understanding of the HH and AVP secretion is the result of a vast amount of data collected over the five past decades. This experimental data was collected using a number of systems under different conditions, giving a fragmented view of the components involved in HH. RESULTS: Here, we present a high-level model of the rat HH based on selected published results to predict short-term (hours) to long-term (days) variation of six major homeostatic parameters: (1) the extracellular sodium concentration, (2) the AVP concentration, (3) the intracellular volume, (4) the extracellular volume, (5) the urine volume and (6) the water intake. The simulation generates quantitative predictions like the daily mean of the extracellular sodium concentration (142.2 mmol/L), the AVP concentration, (1.7 pg/ml), the intracellular volume (45.3 ml/100 g body weight--bw), the extracellular volume (22.6 ml/100 g bw), the urine volume (11.8 ml/100 g bw) and the cumulative water intake (18 ml/100 g bw). The simulation also computes the dynamics of all these parameters with a high temporal resolution of one minute. This high resolution predicts the circadian fluctuation of the AVP secretion (5 ± 2 pg/ml) and defines the limits of a restoration and a maintenance phase in the HH (2.1 pg/ml). Moreover, the simulation can predict the action of pharmacological compounds that disrupt the HH. As an example, we tested the action of a diuretic (furosemide) combined with a sodium deficient diet to generate quantitative prediction on the extracellular sodium concentration (134 mmol/L) and the need-induced water intake (20.3 ml/100 g bw). These simulated data are compatible with experimental data (136 ± 3 mmol/L and 17.5 ± 3.5 ml/100 g bw, respectively). CONCLUSION: The quantitative agreement of the predictions with published experimental data indicates that our simplified model of the HH integrates most of the essential systems to predict realistic physiological values and dynamics under a set of normal and perturbed hydromineral conditions.
Asunto(s)
Simulación por Computador , Modelos Biológicos , Vasopresinas/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Animales , Presión Sanguínea/fisiología , Agua Corporal/fisiología , Sistema Digestivo , Riñón/fisiología , RatasRESUMEN
The median preoptic nucleus (MnPO) is densely innervated by efferent projections from the subfornical organ (SFO) and, therefore, is an important relay for the peripheral chemosensory and humoral information (osmolality and serum levels ANG II). In this context, controlling the excitability of MnPO neuronal populations is a major determinant of body fluid homeostasis and cardiovascular regulation. Using a brain slice preparation and patch-clamp recordings, our study sought to determine whether endogenous ANG II modulates the strength of the SFO-derived GABAergic inputs to the MnPO. Our results showed that the amplitude of the inhibitory postsynaptic currents (IPSCs) were progressively reduced by 44 +/- 2.3% by (Sar(1), Ile(8))-ANG II, a competitive ANG type 1 receptor (AT(1)R) antagonist. Similarly, losartan, a nonpeptidergic AT(1)R antagonist decreased the IPSC amplitude by 40.4 +/- 5.6%. The facilitating effect of endogenous ANG II on the GABAergic input to the MnPO was not attributed to a change in GABA release probability and was mimicked by exogenous ANG II, which potentiated the amplitude of the muscimol-activated GABA(A)/Cl(-) current by 53.1 +/- 11.4%. These results demonstrate a postsynaptic locus of action of ANG II. Further analysis reveals that ANG II did not affect the reversal potential of the synaptic inhibitory response, thus privileging a cross talk between postsynaptic AT(1) and GABA(A) receptors. Interestingly, facilitation of GABAergic neurotransmission by endogenous ANG II was specific to neurons responding to changes in the ambient Na(+) level. This finding, combined with the ANG II-mediated depolarization of non-Na(+)-responsive neurons reveals the dual actions of ANG II to modulate the excitability of MnPO neurons.
Asunto(s)
Angiotensina II/metabolismo , Neuronas Aferentes/metabolismo , Área Preóptica/metabolismo , Sodio/metabolismo , Transmisión Sináptica , Ácido gamma-Aminobutírico/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Cloruros/metabolismo , Agonistas del GABA/farmacología , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores , Masculino , Neuronas Aferentes/efectos de los fármacos , Técnicas de Placa-Clamp , Área Preóptica/citología , Área Preóptica/efectos de los fármacos , Ratas , Ratas Wistar , Receptor Cross-Talk , Receptor de Angiotensina Tipo 1/metabolismo , Receptores de GABA-A/metabolismo , Transmisión Sináptica/efectos de los fármacos , Factores de TiempoRESUMEN
The median preoptic nucleus (MnPO) is an integrator site for the chemosensory and neural signals induced by a perturbation in the hydromineral balance, and it is highly involved in controlling fluid and electrolyte ingestion. Here, we hypothesize that opioid peptides, previously recognized to control ingestive behaviors, may regulate the excitability of MnPO neurons and that this regulatory action may depend on the natriuric (Na(+)) status of body fluid compartments. Our results show that activation of mu-, but not delta-, opioid receptors (OR) triggered a membrane hyperpolarization by recruiting a G-protein-regulated inward-rectifier K(+) (GIRK) conductance in 41% of the neurons tested. Interestingly, 24 h Na(+) depletion strengthened this opioid-mediated control of neuronal excitability. In Na(+)-depleted animals, the neuronal population displaying the mu-OR-induced hyperpolarization expanded to 60% (Z-test, P = 0.012), whereas Na(+) repletion restored this population to the control level (39%; Z-test, P = 0.037). Among the neurons displaying mu-OR-induced hyperpolarization, Na(+) depletion specifically increased the neuronal population responsive to variation in ambient Na(+) (from 27% to 43%; Z-test, P = 0.029). In contrast, Na(+) repletion dramatically reduced the population that was unresponsive to Na(+) (from 17% to 3%; Z-test, P = 0.031). Neither the basic properties of the neurons nor the characteristics of the mu-OR-induced response were altered by the body Na(+) challenge. Our results indicate that an episode of Na(+) depletion/Na(+) repletion modifies the organization of the opioid-sensitive network of the MnPO. Such network plasticity might be related to the avid salt ingestion triggered by repeated Na(+) depletion.
Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Área Preóptica/fisiología , Receptores Opioides mu/fisiología , Sodio en la Dieta/administración & dosificación , Sinapsis/fisiología , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Furosemida/farmacología , Masculino , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Área Preóptica/citología , Área Preóptica/efectos de los fármacos , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sodio en la Dieta/farmacología , Sinapsis/efectos de los fármacosRESUMEN
The present study investigates the influence of a chronic high Na+ diet (8% Na+) on the expression of the angiotensin type 1A (AT1A) receptor gene in the lamina terminalis and paraventricular nucleus of the hypothalamus (PVH) in normotensive Wistar (W) rats, as well as in Dahl salt-resistant (DR) and Dahl salt-sensitive (DS) rats. Three weeks of 8% Na+ diet led to a higher blood pressure in DS rats compared to DR and W rats. Moreover, the high Na+ diet was correlated with a decreased expression of AT1A receptor mRNA in the median preoptic nucleus (MnPO) and in the PVH of DS rats, compared to DR and W rats. Contrastingly, the AT1A receptor mRNA expression was not altered by the high Na+ diet in the forebrain circumventricular organs of all the rat strains. Interestingly, a furosemide-induced Na+ depletion was correlated with an increased expression of AT1A receptor mRNA in the PVH, MnPO and SFO of both the DS and DR rats. It is concluded that chronic high Na+ diet did differently regulate the expression of AT1A receptor mRNA in two hypothalamic integrative centers for hydromineral and cardiovascular balance (the PVH and MnPO) in DS rats, compared to DR and W rats. However, the AT1A receptor mRNA expression was similarly regulated in DS and DR rats in response to an acute Na+ depletion, suggesting a distinct high Na+ -induced regulation of the AT1A receptor gene in the PVH and MnPO of DS rats.
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Hipotálamo/metabolismo , ARN Mensajero/genética , Receptor de Angiotensina Tipo 1/genética , Sodio/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Ingestión de Líquidos/efectos de los fármacos , Furosemida/administración & dosificación , Furosemida/farmacología , Expresión Génica/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Hibridación in Situ , Masculino , Ratas , Ratas Endogámicas Dahl , Ratas Wistar , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Sodio en la Dieta/administración & dosificación , Sodio en la Dieta/farmacologíaRESUMEN
The median preoptic nucleus (MnPO) is an integrative structure of the hypothalamus receiving periphery-derived information pertinent to hydromineral and cardiovascular homeostasis. In this context, excitability of MnPO neurones is controlled by fast GABAergic, glutamatergic and angiotensinergic projection from the subfornical organ (SFO). Taking advantage of a brain slice preparation preserving synaptic connection between the SFO and the MnPO, and appropriate bicarbonate-free artificial cerebrospinal fluid (CSF), we investigated a possible implication of an active outward Cl- transport in regulating efficacy of the GABA(A) receptor-mediated inhibitory response at the SFO-MnPO synapse. When somata of the MnPO neurones was loaded with 18 mm chloride, stimulation of the SFO evoked outward inhibitory postsynaptic currents (IPSCs) in 81% of the MnPO neurones held at -60 mV. Accordingly, E(IPSC) was found 25 mV hyperpolarized from the theoretical value calculated from the Nernst equation, indicating that IPSC polarity and amplitude were driven by an active Cl- extrusion system in these neurones. E(IPSC) estimated with gramicidin-based perforated-patch recordings amounted -89.2 +/- 4.3 mV. Furosemide (100 microm), a pharmacological compound known to block the activity of the neurone-specific K(+)-Cl- cotransporter, KCC2, reversed IPSC polarity and shifted E(IPSC) towards its theoretical value. Presence of the KCC2 protein in the MnPO was further detected with immunohistochemistry, revealing a dense network of KCC2-positive intermingled fibres. In the presence of a GABA(B) receptor antagonist, high-frequency stimulation (5 Hz) of the SFO evoked a train of IPSCs or inhibitory postsynaptic potentials (IPSPs), whose amplitude was maintained throughout the sustained stimulation. Contrastingly, similar 5 Hz stimulation carried out in the presence of furosemide (50 microm) evoked IPSCs/IPSPs, whose amplitude collapsed during the high-frequency stimulation. Similar reduction in inhibitory neurotransmission was also observed in MnPO neurones lacking the functional Cl- extrusion mechanism. We conclude that a majority of MnPO neurones were characterized by a functional Cl- transporter that ensured an efficient activity-dependent Cl- transport rate, allowing sustained synaptic inhibition of these neurones. Pharmacological and anatomical data strongly suggested the involvement of KCC2, as an essential postsynaptic determinant of the inhibitory neurotransmission afferent to the MnPO, a key-structure in the physiology of the hydromineral and cardiovascular homeostasis.
Asunto(s)
Cloruros/metabolismo , Área Preóptica/metabolismo , Receptores de GABA-A/metabolismo , Animales , Bicuculina/farmacología , Potenciales Evocados/efectos de los fármacos , Furosemida/farmacología , Antagonistas de Receptores de GABA-A , Homeostasis , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Inhibición Neural , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Área Preóptica/efectos de los fármacos , Ratas , Órgano Subfornical/efectos de los fármacos , Órgano Subfornical/metabolismo , Simportadores/antagonistas & inhibidores , Simportadores/metabolismo , Transmisión Sináptica/efectos de los fármacos , Cotransportadores de K ClRESUMEN
It is widely believed that expression of the vesicular glutamate transporter genes VGLUT1 and VGLUT2 is restricted to glutamatergic neurons and that the two transporters segregate in different sets of neurons. Using single-cell multiplex RT-PCR (sc-RT-mPCR), we show that VGLUT1 and VGLUT2 mRNAs were coexpressed in most of the sampled neurons from the rat hippocampus, cortex, and cerebellum at postnatal Day (P)14 but not P60. In accordance, changes in VGLUT1 and VGLUT2 mRNA concentrations were found to occur in these and other brain areas between P14 and P60, as revealed by semiquantitative RT-PCR and quantitated by ribonuclease protection assay. VGLUT1 and -2 coexpression in the hippocampal formation is supported further by in situ hybridization data showing that virtually all cells in the CA1-CA3 pyramidal and granule cell layers were highly positive for both transcripts until P14. It was revealed using sc-RT-mPCR that transcripts for VGLUT1 and VGLUT2 were also present in neurons of the cerebellum, striatum, and septum that expressed markers for gamma-aminobutyric acid (GABA)ergic or cholinergic phenotypes, as well as in hippocampal cells containing transcripts for the glial fibrillary acidic protein. Our study suggests that VGLUT1 and VGLUT2 proteins may often transport glutamate into vesicles within the same neuron, especially during early postnatal development, and that they are expressed widely in presumed glutamatergic, GABAergic, and cholinergic neurons, as well as in astrocytes. Furthermore, our study shows that such coexpressing neurons remain in the adult brain and identifies several areas that contain them in both young and adult rats.
Asunto(s)
Colina O-Acetiltransferasa/genética , Glutamato Descarboxilasa/genética , Hipocampo/fisiología , Proteínas de Transporte de Membrana/genética , Neuronas/fisiología , Animales , Astrocitos/fisiología , Electrofisiología , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Proteína 1 de Transporte Vesicular de Glutamato , Proteína 2 de Transporte Vesicular de GlutamatoRESUMEN
Whole-cell patch-clamp recordings were performed on acute brain slices of male rats to investigate the ability of the neurons of the median preoptic nucleus (MnPO) to detect fluctuation in extracellular osmolarity and sodium concentration ([Na+]out). Local application of hypotonic and hypertonic artificial CSF hyperpolarized and depolarized the neurons, respectively. Similar responses obtained under synaptic isolation (0.5 microM TTX) highlighted the intrinsic ability of the MnPO neurons to detect changes in extracellular osmolarity and [Na+]out. Manipulating extracellular osmolarity, [Na+]out, and [Cl-]out showed in an independent manner that the MnPO neurons responded to a change in [Na+]out exclusively. The specific Na+ response was voltage insensitive and depended on the driving force for Na+ ions, indicating that a sustained background Na+ permeability controlled the membrane potential of the MnPO neurons. This specific response was not reduced by Gd3+, amiloride, or benzamil, ruling out the participation of mechanosensitive cationic channels, specific epithelial Na+ channels, and Phe-Met-Arg-Phe-gated Na+ channels, respectively. Combination of in situ hybridization, using a riboprobe directed against the atypical Na+ channel (Na(X)), and immunohistochemistry, using an antibody against neuron-specific nuclei protein, revealed that a substantial population of MnPO neurons expressed the Na(X) channel, which was characterized recently as a concentration-sensitive Na+ channel. This study shows that a neuronal population of the MnPO acts as functional Na+ sensors and that the Na(X) channel might represent the molecular basis for the extracellular sodium level sensing in these neurons.
Asunto(s)
Amilorida/análogos & derivados , Neuronas/fisiología , Área Preóptica/fisiología , Canales de Sodio/metabolismo , Sodio/metabolismo , Amilorida/farmacología , Animales , Biomarcadores , Líquido Cefalorraquídeo/metabolismo , Gadolinio/farmacología , Inmunohistoquímica , Hibridación in Situ , Técnicas In Vitro , Masculino , Potenciales de la Membrana/fisiología , Neuronas/metabolismo , Concentración Osmolar , Técnicas de Placa-Clamp , Área Preóptica/citología , Área Preóptica/metabolismo , Prosencéfalo/fisiología , Ratas , Ratas Wistar , Canales de Sodio/efectos de los fármacos , Canales de Sodio Activados por VoltajeRESUMEN
The present study investigates co-localization of AT(1A) receptor subtype and Fos protein in neuronal populations of the lamina terminalis (LT) that have been recruited during acute Na(+) and water depletion mediated by furosemide injections. For that purpose, we combined high cellular resolution of in situ hybridization technique to reveal neurons expressing AT(1A) receptor gene (AT(1A) mRNA) with the specificity of Fos protein immunoreactivity as a marker of neuronal activation (Fos-ir). As expected, furosemide treatment dramatically increased the density of Fos-immunoreactive neuronal population in all the regions of the LT compared to control (saline-injected animals). Distribution analysis of Fos-ir neurons and AT(1A) receptor-expressing neurons performed consecutively to furosemide-induced Na(+) and water depletion indicated that double-labeled neurons (AT(1A) mRNA+Fos-ir) represented the majority (67%) of the neuronal population that expressed AT(1A) receptor in the rim of the vascular organ of the lamina terminalis (OVLT). Double-labeled neurons amounted about 60% of the neurons that expressed AT(1A) receptor in the core of the subfornical organ (SFO) and 34% in the periphery of the SFO. In the median preoptic nucleus (MnPO), the density of the double-labeled neuronal population observed in the furosemide-treated animals remained weak compared to the control group of animals. Double-labeled neuronal population estimated in the MnPO of the furosemide-treated group of animals represented 17% of the neurons that express AT(1A) receptor gene. Our results report a heterogeneous distribution of the neuronal populations that co-localize AT(1A) receptor and Fos protein in the lamina terminalis after an acute Na(+) and water depletion. This study gives anatomical support to a direct action of endogenous AngII on c-fos transcription via binding on AT(1A) receptor in specific areas of the circumventricular organs (rim of the OVLT and core of the SFO). In the MnPO, our data indicate that intracellular signaling pathways unlikely couple AT(1A) receptor with c-fos transcription. The expression of Fos protein in this nucleus might be therefore secondary to the recruitment of excitatory inputs different from AngII. This observation underlines the complexity of molecules and neurocircuits in the preoptic region that are involved in the control of acute Na(+) and water deficit.
Asunto(s)
Neuronas/metabolismo , Proteínas Oncogénicas v-fos/metabolismo , Prosencéfalo/citología , Receptor de Angiotensina Tipo 1/metabolismo , Sodio/fisiología , Desequilibrio Hidroelectrolítico/metabolismo , Análisis de Varianza , Animales , Recuento de Células , Furosemida/farmacología , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Masculino , Neuronas/efectos de los fármacos , Proteínas Oncogénicas v-fos/genética , Prosencéfalo/metabolismo , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1/genética , Desequilibrio Hidroelectrolítico/inducido químicamenteRESUMEN
In this short review, we hypothesize that the central renin-angiotensin system might participate to the initiation of compensatory responses to a stressor agent. Regulation of the expression of the brain angiotensin receptors might constitute a primary molecular mechanism by which this protecting action would take place. We illustrate this possibility by investigating the expression of the angiotensin type 1 receptor in the hypothalamus in response to systemic and neurogenic stressors.
Asunto(s)
Encéfalo/fisiología , Receptores de Angiotensina/fisiología , Sistema Renina-Angiotensina/fisiología , Estrés Psicológico , Adaptación Fisiológica , HumanosRESUMEN
Magnocellular neurons of the supraoptic nucleus release the neuropeptides oxytocin and vasopressin from their dendrites to regulate their synaptic inputs. This study aims to determine the cellular mechanism by which vasopressin modulates excitatory synaptic transmission. Presumably by electroporation through perforated patch, we were able to successfully introduce biocytin into cells in which we performed an electrophysiological study. This method enabled us to determine that roughly half of the recorded neurons were immunoreactive to oxytocin-associated neurophysin and showed two characteristic features: an inward rectification and a sustained outward rectification. The remaining half showed a linear voltage-current relationship and was immunoreactive to vasopressin-associated neurophysin. Using these electrophysiological characteristics and post hoc immunohistochemistry to identify vasopressin or oxytocin neurons, we found that vasopressin decreased evoked EPSCs in vasopressin neurons while increasing EPSCs in oxytocin neurons. In both types of neurons, EPSC decay constants were not affected, indicating that desensitization of non-NMDA receptors did not underlie the EPSC amplitude change. In vasopressin neurons, both vasopressin and a V1a receptor agonist, F-180, decreased AMPA-induced currents, an effect blocked by a V1a receptor antagonist SR49059. In oxytocin neurons, AMPA-induced currents were facilitated by vasopressin, whereas F-180 had no effect. An oxytocin receptor antagonist blocked the facilitatory effect of vasopressin. Thus, we conclude that vasopressin inhibits EPSCs in vasopressin neurons via postsynaptic V1a receptors, whereas it facilitates EPSCs in oxytocin neurons through oxytocin receptors.