RESUMEN
BACKGROUND: Opioid analgesics remain widely used for pain treatment despite the related serious side effects. Some of those, such as opioid tolerance and opioid-induced hyperalgesia may be at least partially due to modulation of opioid receptors (OR) function at nociceptive synapses in the spinal cord dorsal horn. It was suggested that increased release of different chemokines under pathological conditions may play a role in this process. The goal of this study was to investigate the crosstalk between the µOR, transient receptor potential vanilloid 1 (TRPV1) receptor and C-C motif ligand 2 (CCL2) chemokine and the involvement of spinal microglia in the modulation of opioid analgesia. METHODS: Patch-clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) and dorsal root evoked currents (eEPSC) in spinal cord slices superficial dorsal horn neurons were used to evaluate the effect of µOR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), CCL2, TRPV1 antagonist SB366791 and minocycline. Paw withdrawal test to thermal stimuli was combined with intrathecal (i.t.) delivery of CCL2 and DAMGO to investigate the modulation in vivo. RESULTS: Application of DAMGO induced a rapid decrease of mEPSC frequency and eEPSC amplitude, followed by a delayed increase of the eESPC amplitude, which was prevented by SB366791. Chemokine CCL2 treatment significantly diminished all the DAMGO-induced changes. Minocycline treatment prevented the CCL2 effects on the DAMGO-induced eEPSC depression, while mEPSC changes were unaffected. In behavioral experiments, i.t. injection of CCL2 completely blocked DAMGO-induced thermal hypoalgesia and intraperitoneal pre-treatment with minocycline prevented the CCL2 effect. CONCLUSIONS: Our results indicate that opioid-induced inhibition of the excitatory synaptic transmission could be severely attenuated by increased CCL2 levels most likely through a microglia activation-dependent mechanism. Delayed potentiation of neurotransmission after µOR activation is dependent on TRPV1 receptors activation. Targeting CCL2 and its receptors and TRPV1 receptors in combination with opioid therapy could significantly improve the analgesic properties of opioids, especially during pathological states.
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Analgésicos Opioides/farmacología , Quimiocina CCL2/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Nocicepción/efectos de los fármacos , Asta Dorsal de la Médula Espinal/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Anilidas/farmacología , Animales , Cinamatos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Masculino , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Neuronas/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Post-tetanic potentiation (PTP) is a form of short-term plasticity that lasts for tens of seconds following a burst of presynaptic activity. It has been proposed that PTP arises from protein kinase C (PKC) phosphorylation of Munc18-1, an SM (Sec1/Munc-18 like) family protein that is essential for release. To test this model, we made a knock-in mouse in which all Munc18-1 PKC phosphorylation sites were eliminated through serine-to-alanine point mutations (Munc18-1SA mice), and we studied mice of either sex. The expression of Munc18-1 was not altered in Munc18-1SA mice, and there were no obvious behavioral phenotypes. At the hippocampal CA3-to-CA1 synapse and the granule cell parallel fiber (PF)-to-Purkinje cell (PC) synapse, basal transmission was largely normal except for small decreases in paired-pulse facilitation that are consistent with a slight elevation in release probability. Phorbol esters that mimic the activation of PKC by diacylglycerol still increased synaptic transmission in Munc18-1SA mice. In Munc18-1SA mice, 70% of PTP remained at CA3-to-CA1 synapses, and the amplitude of PTP was not reduced at PF-to-PC synapses. These findings indicate that at both CA3-to-CA1 and PF-to-PC synapses, phorbol esters and PTP enhance synaptic transmission primarily by mechanisms that are independent of PKC phosphorylation of Munc18-1.SIGNIFICANCE STATEMENT A leading mechanism for a prevalent form of short-term plasticity, post-tetanic potentiation (PTP), involves protein kinase C (PKC) phosphorylation of Munc18-1. This study tests this mechanism by creating a knock-in mouse in which Munc18-1 is replaced by a mutated form of Munc18-1 that cannot be phosphorylated. The main finding is that most PTP at hippocampal CA3-to-CA1 synapses or at cerebellar granule cell-to-Purkinje cell synapses does not rely on PKC phosphorylation of Munc18-1. Thus, mechanisms independent of PKC phosphorylation of Munc18-1 are important mediators of PTP.
Asunto(s)
Proteínas Munc18/metabolismo , Plasticidad Neuronal/fisiología , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Sustitución de Aminoácidos , Animales , Femenino , Técnicas de Sustitución del Gen , Hipocampo/fisiología , Masculino , Ratones , Ratones Noqueados , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Proteínas Munc18/deficiencia , Mutación Missense , Ésteres del Forbol/farmacología , Fosforilación , Mutación Puntual , Proteína Quinasa C/deficiencia , Células de Purkinje/fisiología , Proteínas Recombinantes/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
INTRODUCTION: N-Methyl-D-aspartate receptors (NMDARs) play a critical role in different forms of plasticity in the central nervous system. NMDARs are always assembled in tetrameric form, in which two GluN1 subunits and two GluN2 and/or GluN3 subunits combine together. Previous studies focused mainly on the hippocampus. The anterior cingulate cortex (ACC) is a key cortical region for sensory and emotional functions. NMDAR GluN2A and GluN2B subunits have been previously investigated, however much less is known about the GluN2C/2D subunits. RESULTS: In the present study, we found that the GluN2C/2D subunits are expressed in the pyramidal cells of ACC of adult mice. Application of a selective antagonist of GluN2C/2D, (2R*,3S*)-1-(9-bromophenanthrene-3-carbonyl) piperazine-2,3-dicarboxylic acid (UBP145), significantly reduced NMDAR-mediated currents, while synaptically evoked EPSCs were not affected. UBP145 affected neither the postsynaptic long-term potentiation (post-LTP) nor the presynaptic LTP (pre-LTP). Furthermore, the long-term depression (LTD) was also not affected by UBP145. Finally, both UBP145 decreased the frequency of the miniature EPSCs (mEPSCs) while the amplitude remained intact, suggesting that the GluN2C/2D may be involved in presynaptic regulation of spontaneous glutamate release. CONCLUSIONS: Our results provide direct evidence that the GluN2C/2D contributes to evoked NMDAR mediated currents and mEPSCs in the ACC, which may have significant physiological implications.
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Giro del Cíngulo/fisiología , Plasticidad Neuronal/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transmisión Sináptica/fisiología , Animales , Hipocampo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/biosíntesis , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
The GABAA receptor (GABAAR) is the main inhibitory receptor in the adult mammalian brain. GABAAR function is dependent on its expression, distribution, and the chloride (Cl-) transmembrane gradient, which is determined by the potassium-chloride cotransporter 2 (KCC2) in the adult brain. KCC2 and GABAAR are downregulated in an activity-dependent manner during seizure induction. Functionally, KCC2 and GABAAR are closely related membrane proteins which modulate GABAergic inhibition. However, it remains unclear how their downregulation during seizure induction is coordinated. This study aimed to assess this interaction. Our results revealed that KCC2 and GABAAR were simultaneously downregulated in both in vivo and in vitro seizure models induced by the convulsant cyclothazide (CTZ), which was at least partly due to structural coupling in hippocampal neuronal membranes. Immunohistochemistry revealed colocalization of gephyrin with KCC2 and co-immunoprecipitation exhibited a direct coupling between GABAAR α1-subunit and KCC2 protein in hippocampal cell membranes. KCC2 specific short hairpin RNA (KCC2-shRNA) was employed to specifically reduce the expression of KCC2 in cultured hippocampal neurons. This resulted in a significant reduction in KCC2-independent GABAergic miniature inhibitory post-synaptic current (mIPSC) amplitude in shKCC2-transfected neurons. Further, pre-treatment with furosemide, a KCC2 inhibitor, during CTZ stimulation followed by washout significantly prevented convulsant stimulation-induced membrane KCC2 downregulation and significantly attenuated GABAAR downregulation concomitant with recovery of suppressed KCC2-independent GABAergic mIPSC amplitude. Our results suggest that the coordinated downregulation of KCC2 and GABAAR during seizure induction exerts a strong functional impact on GABAAR, highlighting an important regulatory mechanism in epilepsy.
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Receptores de GABA-A/metabolismo , Convulsiones/metabolismo , Simportadores/metabolismo , Animales , Benzotiadiazinas/toxicidad , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/genética , Convulsiones/inducido químicamente , Simportadores/antagonistas & inhibidores , Simportadores/genética , Cotransportadores de K ClRESUMEN
Cannabidiol (CBD) is a major phytocannabinoid in Cannabis sativa. CBD is being increasingly reported as a clinical treatment for neurological diseases. Febrile seizure is one of the most common diseases in children with limited therapeutic options. We investigated possible therapeutic effects of CBD on febrile seizures and the underlying mechanism. Use of a hyperthermia-induced seizures model revealed that CBD significantly prolonged seizure latency and reduced the severity of thermally-induced seizures. Hippocampal neuronal excitability was significantly decreased by CBD. Further, CBD significantly reduced the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) mediated evoked excitatory postsynaptic currents (eEPSCs) and the amplitude and frequency of miniature EPSCs (mEPSCs). Furthermore, CBD significantly accelerated deactivation in GluA1 and GluA2 subunits. Interestingly, CBD slowed receptor recovery from desensitization of GluA1, but not GluA2. These effects on kinetics were even more prominent when AMPAR was co-expressed with γ-8, the high expression isoform 8 of transmembrane AMPAR regulated protein (TARPγ8) in the hippocampus. The inhibitory effects of CBD on AMPAR depended on its interaction with the distal N-terminal domain of GluA1/GluA2. CBD inhibited AMPAR activity and reduced hippocampal neuronal excitability, thereby improving the symptoms of febrile seizure in mice. The putative binding site of CBD in the N-terminal domain of GluA1/GluA2 may be a drug target for allosteric gating modulation of AMPAR.
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Anticonvulsivantes/farmacología , Ondas Encefálicas/efectos de los fármacos , Región CA1 Hipocampal/efectos de los fármacos , Cannabidiol/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Hipertermia/complicaciones , Receptores AMPA/antagonistas & inhibidores , Convulsiones Febriles/prevención & control , Animales , Anticonvulsivantes/metabolismo , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/fisiopatología , Cannabidiol/metabolismo , Modelos Animales de Enfermedad , Antagonistas de Aminoácidos Excitadores/metabolismo , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Cinética , Ratones , Ratones Endogámicos C57BL , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Modelos Moleculares , Unión Proteica , Tiempo de Reacción/efectos de los fármacos , Receptores AMPA/genética , Receptores AMPA/metabolismo , Convulsiones Febriles/etiología , Convulsiones Febriles/metabolismo , Convulsiones Febriles/fisiopatologíaRESUMEN
Long-term memory of complex olfactory learning is expressed by wide spread enhancement in excitatory and inhibitory synaptic transmission onto piriform cortex pyramidal neurons. A particularly interesting modification in synaptic inhibition is the hyperpolarization of the reversal potential of the fast post synaptic inhibitory potential (fIPSP). Here we study the mechanism underlying the maintenance of such a shift in the fIPSP. Blocking of the neuronal specific K+-Cl- co-transporter (KCC2) in neurons of trained rats significantly depolarized the averaged fIPSP reversal potential of the spontaneous miniature inhibitory post synaptic currents (mIPSCs), to the averaged pre-training level. A similar effect was obtained by blocking PKC, which was previously shown to upregulate KCC2. Accordingly, the level of PKC-dependent phosphorylation of KCC2, at the serine 940 site, was significantly increased after learning. In contrast, blocking two other key second messenger systems CaMKII and PKA, which have no phosphorylation sites on KCC2, had no effect on the fIPSP reversal potential. Importantly, the PKC inhibitor also reduced the averaged amplitude of the spontaneous miniature excitatory synaptic currents (mEPSCs) in neurons of trained rats only, to the pre-training level. We conclude that learning-induced hyper-polarization of the fIPSP reversal potential is mediated by PKC-dependent increase of KCC2 phosphorylation.
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Aprendizaje Discriminativo/fisiología , Inhibición Neural/fisiología , Proteína Quinasa C/metabolismo , Simportadores/metabolismo , Sinapsis/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Aprendizaje Discriminativo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Masculino , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Olfato/efectos de los fármacos , Olfato/fisiología , Simportadores/antagonistas & inhibidores , Sinapsis/efectos de los fármacos , Cotransportadores de K ClRESUMEN
The United States is experiencing an opioid crisis imposing enormous fiscal and societal costs and driving the staggering overdose death rate. While prescription opioid analgesics are essential for treating acute pain, cessation of use in individuals with a physical dependence induces an aversive withdrawal syndrome that promotes continued drug use to alleviate/avoid these symptoms. Additionally, repeated bouts of withdrawal often lead to an increased propensity for relapse. Understanding the neurobiology underlying withdrawal is essential for providing novel treatment options to alleviate physiological and affective components accompanying the cessation of opiate use. Here, we administered morphine and precipitated withdrawal with naloxone to investigate behavioral and cellular responses in C57BL/6J male and female mice. Following 3 days of administration, both male and female mice demonstrated sensitized withdrawal symptoms. Since the bed nucleus of the stria terminalis (BNST) plays a role in mediating withdrawal-associated behaviors, we examined plastic changes in inhibitory synaptic transmission within this structure 24 hours following the final precipitated withdrawal. In male mice, morphine withdrawal increased spontaneous GABAergic signaling compared with controls. In contrast, morphine withdrawal decreased spontaneous GABAergic signaling in female mice. Intriguingly, these opposing GABAergic effects were contingent upon activity-dependent dynamics within the ex vivo slice. Our findings suggest that male and female mice exhibit some divergent cellular responses in the BNST following morphine withdrawal, and alterations in BNST inhibitory signaling may contribute to the expression of behaviors following opioid withdrawal.
Asunto(s)
Analgésicos Opioides/farmacología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Morfina/farmacología , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Inhibición Neural/efectos de los fármacos , Núcleos Septales/efectos de los fármacos , Síndrome de Abstinencia a Sustancias/fisiopatología , Transmisión Sináptica/efectos de los fármacos , Animales , Femenino , Masculino , Ratones Endogámicos C57BL , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Dependencia de Morfina , Plasticidad Neuronal/efectos de los fármacos , Técnicas de Placa-Clamp , Núcleos Septales/citología , Núcleos Septales/metabolismo , Núcleos Septales/fisiopatología , Síndrome de Abstinencia a Sustancias/etiología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The aim of this study was to compare the acute effects of thrombin and brain-derived neurotrophic factor (BDNF) on spontaneous miniature endplate potentials (MEPPs) and multiquantal evoked endplate potentials (EPPs) in mouse neuromuscular junctions (NMJs) of m. diaphragma and m. EDL. Intracellular microelectrode recordings of MEPPs and EPPs were used to evaluate the changes in acetylcholine (ACh) release in mature and newly-formed mouse NMJs. Thrombin (1 nM) increased the amplitude of MEPPs and EPPs by 25-30% in mature and newly-formed NMJs. This effect was due to an enhanced loading of synaptic vesicles with ACh and increase of ACh quantal size, since it was fully prevented by blocking of vesicular ACh transporter. It was also prevented by tropomyosin-related kinase B (TrkB) receptors inhibitor ANA12. Exogenous BDNF (1 nM) mimicked thrombin effect and increased the amplitude of MEPPs and EPPs by 25-30%. It required involvement of protein kinase A (PKA) and mitogen-activated protein kinase (MEK1/2)-mediated pathway, but not phospholipase C (PLC). Blocking A2A adenosine receptors by ZM241385 abolished the effect of BDNF, whereas additional stimulation of A2A receptors by CGS21680 increased MEPP amplitudes, which was prevented by MEK1/2 inhibitor U0126. At mature NMJs, BDNF enhanced MEPPs frequency by 30-40%. This effect was selectively prevented by inhibition of PLC, but not PKA or MEK1/2. It is suggested that interrelated effects of thrombin/BDNF in mature and newly-formed NMJs are realized via enhancement of vesicular ACh transport and quantal size increase. BDNF-induced potentiation of synaptic transmission involves the functional coupling between A2A receptor-dependent active PKA and neurotrophin-triggered MAPK pathway, as well as PLC-dependent increase in frequency of MEPPs.
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Acetilcolina/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Unión Neuromuscular/fisiología , Trombina/farmacología , Animales , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Tirosina Quinasas/metabolismo , Receptor de Adenosina A2A/metabolismo , Transmisión Sináptica , Fosfolipasas de Tipo C/metabolismoRESUMEN
Surgical ovariectomy has been shown to reduce spine density in hippocampal CA1 pyramidal cells of rodents, and this reduction is reversed by 17ß-estradiol (E2) treatment in a model of human estrogen replacement therapy. Here, we report reduction of spine density in apical dendrites of layer 5 pyramidal neurons of several neocortical regions that is reversed by subsequent E2 treatment in ovariectomized (OVX) female Thy1M-EGFP mice. We also found that OVX-associated reduction of spine density in somatosensory cortex was accompanied by a reduction in miniature EPSC (mEPSC) frequency (but not mIPSC frequency), indicating a change in functional synapses. OVX-associated spine loss in somatosensory cortex was also rescued by an agonist of the G-protein-linked estrogen receptor (GPER) but not by agonists of the classic estrogen receptors ERα/ERß, whereas the opposite selectivity was found in area CA1. Acute treatment of neocortical slices with E2 also rescued the OVX-associated reduction in mEPSC frequency, which could be mimicked by a GPER agonist and abolished by a GPER antagonist. Time-lapse in vivo two-photon imaging showed that OVX-associated reduction in spine density is achieved by both an increase in spine loss rate and a decrease in spine gain rate and that subsequent rescue by E2 reversed both of these processes. Crucially, the spines added after E2 rescue were no more likely to reappear at or nearby the sites of pre-OVX spines than those in control mice treated with vehicle. Thus, a model of estrogen replacement therapy, although restoring spine density and dynamics, does not entirely restore functional connectivity.SIGNIFICANCE STATEMENT Estrogen replacement therapy following menopause or surgical removal of the ovaries is a widespread medical practice, yet little is known about the consequences of such treatment for cells in the brain. Here, we show that estrogen replacement reverses some of the effects of surgical removal of the ovaries on the structure and function of brain cells in the mouse. Yet, importantly, the fine wiring of the brain is not returned to the presurgery state by estrogen treatment, suggesting lasting functional consequences.
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Espinas Dendríticas/efectos de los fármacos , Estradiol/farmacología , Neocórtex/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Animales , Espinas Dendríticas/metabolismo , Estrógenos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Ratones , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Neocórtex/citología , Neocórtex/metabolismo , Ovariectomía , Células Piramidales/citología , Células Piramidales/metabolismoRESUMEN
Lamina X is localized in the spinal cord within the region surrounding the central canal and receives descending projections from the supraspinal nuclei. Norepinephrine (NE) is a neurotransmitter in descending pathways emanating from the brain stem; NE-containing fibers terminate in the spinal dorsal cord, particularly in the substantia gelatinosa (SG). NE enhances inhibitory synaptic transmission in SG neurons by activating presynaptic α1-receptors and hyperpolarizes the membranes of SG neurons by acting on α2-receptors; NE may thus act directly on SG neurons of the dorsal spinal cord and inhibit nociceptive transmission at the spinal level. NE-containing fibers also reportedly terminate in lamina X, suggesting that NE also modulates synaptic transmission in lamina X. However, the cellular mechanisms underlying such action have not been investigated. We hypothesized that NE might directly act on lamina X and enhance inhibitory synaptic transmission therein. Using rat spinal cord slices and in vitro whole-cell patch-clamps, we found that the bath-application of NE to lamina X does not affect the excitatory interneurons but enhances GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) and induces an outward current. NE-induced enhancement of mIPSCs was blocked by α1A-receptor antagonists, and NE-induced outward current was blocked by α2-receptor antagonists. NE did not affect GABA- or glycine- induced outward currents. These findings are similar to those obtained from SG neurons: NE may act at presynaptic terminals of GABAergic and glycinergic interneurons on lamina X to facilitate inhibitory-transmitter release through α1A-receptor activation and directly induce inhibitory interneuron membrane hyperpolarization through α2-receptors activation.
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Neuronas/efectos de los fármacos , Norepinefrina/farmacología , Médula Espinal/efectos de los fármacos , Sustancia Gelatinosa/efectos de los fármacos , Animales , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
Striatal output pathways are known to play a crucial role in the control of movement. One possible component for shaping the synaptic output of striatal neuron is the glutamatergic input that originates from cortex and thalamus. Although reports focusing on quantifying glutamatergic-induced morphological changes in striatum exist, the role of glutamatergic input in regulating striatal function remains poorly understood. Using primary neurons from newborn mice of either sex in a reduced two-neuron microcircuit culture system, we examined whether glutamatergic input modulates the output of striatal neurons. We found that glutamatergic input enhanced striatal inhibition in vitro With a glutamatergic partner from either cortex or thalamus, we attributed this potentiation to an increase in the size of quantal IPSC, suggesting a strengthening of the postsynaptic response to GABAergic signaling. Additionally, a differential effect of cortical and thalamic innervation onto striatal GABAergic neurons output was revealed. We observed that cortical, but not thalamic input, enhanced the number of releasable GABAergic synaptic vesicles and morphological synapses. Importantly, these alterations were reverted by blockade of neuronal activity and glutamate receptors, as well as disruption of BDNF-TrkB signaling. Together, our data indicate, for first time, that GABAergic synapse formation in corticostriatal pairs depends on two parallel, but potentially intersecting, signaling pathways that involve glutamate receptor activation in striatal neurons, as well as BDNF signaling. Understanding how cortical and thalamic inputs refine striatal output will pave the way toward dissecting basal ganglia activity in both physiological and pathological conditions.SIGNIFICANCE STATEMENT Striatal GABAergic microcircuits are critical for motor function. However, the mechanisms controlling striatal output, particularly at the level of synaptic strength, are unclear. Using two-neuron culture system, we quantified the synaptic output of individual striatal GABAergic neurons paired with a glutamatergic partner and studied the influence of the excitatory connections that are known to be interregionally formed in vivo We found that glutamatergic input potentiated striatal inhibitory output, potentially involving an increased feedback and/or feedforward inhibition. Moreover, distinct components of glutamatergic innervation, such as firing activity or release of neurotrophic factors were shown to be required for the glutamatergic-induced phenotype. Investigation, therefore, of two-neuron in vitro microcircuits could be a powerful tool to explore synaptic mechanisms or disease pathophysiology.
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Cuerpo Estriado/fisiología , Neuronas GABAérgicas/fisiología , Ácido Glutámico/fisiología , Sinapsis/fisiología , Ácido gamma-Aminobutírico/fisiología , 2-Amino-5-fosfonovalerato/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Anticuerpos Neutralizantes/farmacología , Factor Neurotrófico Derivado del Encéfalo/antagonistas & inhibidores , Factor Neurotrófico Derivado del Encéfalo/farmacología , Factor Neurotrófico Derivado del Encéfalo/fisiología , Células Cultivadas , Corteza Cerebral/citología , Cuerpo Estriado/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Neuronas GABAérgicas/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Proteínas Tirosina Quinasas/fisiología , Quinoxalinas/farmacología , Proteínas Recombinantes/farmacología , Vesículas Sinápticas/fisiología , Tetrodotoxina/farmacología , Tálamo/citologíaRESUMEN
Although NPY has potent anxiolytic actions within the BLA, selective activation of BLA NPY Y2 receptors (Y2Rs) acutely increases anxiety by an unknown mechanism. Using ex vivo male rat brain slice electrophysiology, we show that the selective Y2R agonist, [ahx5-24]NPY, reduced the frequency of GABAA-mediated mIPSCs in BLA principal neurons (PNs). [ahx5-24]NPY also reduced tonic activation of GABAB receptors (GABABR), which increased PN excitability through inhibition of a tonic, inwardly rectifying potassium current (KIR ). Surprisingly, Y2R-sensitive GABABR currents were action potential-independent, persisting after treatment with TTX. Additionally, the Ca2+-dependent, slow afterhyperpolarizing K+ current (IsAHP ) was enhanced in approximately half of the Y2R-sensitive PNs, possibly from enhanced Ca2+ influx, permitted by reduced GABABR tone. In male and female mice expressing tdTomato in Y2R-mRNA cells (tdT-Y2R mice), immunohistochemistry revealed that BLA somatostatin interneurons express Y2Rs, as do a significant subset of BLA PNs. In tdT-Y2R mice, [ahx5-24]NPY increased excitability and suppressed the KIR in nearly all BLA PNs independent of tdT-Y2R fluorescence, consistent with presynaptic Y2Rs on somatostatin interneurons mediating the above effects. However, only tdT-Y2R-expressing PNs responded to [ahx5-24]NPY with an enhancement of the IsAHP Ultimately, increased PN excitability via acute Y2R activation likely correlates with enhanced BLA output, consistent with reported Y2R-mediated anxiogenesis. Furthermore, we demonstrate the following: (1) a novel mechanism whereby activity-independent GABA release can powerfully dampen BLA neuronal excitability via postsynaptic GABABRs; and (2) that this tonic inhibition can be interrupted by neuromodulation, here by NPY via Y2Rs.SIGNIFICANCE STATEMENT Within the BLA, NPY is potently anxiolytic. However, selective activation of NPY2 receptors (Y2Rs) increases anxiety by an unknown mechanism. We show that activation of BLA Y2Rs decreases tonic GABA release onto BLA principal neurons, probably from Y2R-expressing somatostatin interneurons, some of which coexpress NPY. This increases principal neuron excitability by reducing GABAB receptor (GABABR)-mediated activation of G-protein-coupled, inwardly rectifying K+ currents. Tonic, Y2R-sensitive GABABR currents unexpectedly persisted in the absence of action potential firing, revealing, to our knowledge, the first report of substantial, activity-independent GABABR activation. Ultimately, we provide a plausible explanation for Y2R-mediated anxiogenesis in vivo and describe a novel and modulatable means of damping neuronal excitability.
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Potenciales de Acción/efectos de los fármacos , Complejo Nuclear Basolateral/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptores de Neuropéptido Y/agonistas , Animales , Femenino , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , Ratones , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
The discovery of a G-protein-coupled receptor for lactate named hydroxycarboxylic acid receptor 1 (HCAR1) in neurons has pointed to additional nonmetabolic effects of lactate for regulating neuronal network activity. In this study, we characterized the intracellular pathways engaged by HCAR1 activation, using mouse primary cortical neurons from wild-type (WT) and HCAR1 knock-out (KO) mice from both sexes. Using whole-cell patch clamp, we found that the activation of HCAR1 with 3-chloro-5-hydroxybenzoic acid (3Cl-HBA) decreased miniature EPSC frequency, increased paired-pulse ratio, decreased firing frequency, and modulated membrane intrinsic properties. Using fast calcium imaging, we show that HCAR1 agonists 3,5-dihydroxybenzoic acid, 3Cl-HBA, and lactate decreased by 40% spontaneous calcium spiking activity of primary cortical neurons from WT but not from HCAR1 KO mice. Notably, in neurons lacking HCAR1, the basal activity was increased compared with WT. HCAR1 mediates its effect in neurons through a Giα-protein. We observed that the adenylyl cyclase-cAMP-protein kinase A axis is involved in HCAR1 downmodulation of neuronal activity. We found that HCAR1 interacts with adenosine A1, GABAB, and α2A-adrenergic receptors, through a mechanism involving both its Giα and Gißγ subunits, resulting in a complex modulation of neuronal network activity. We conclude that HCAR1 activation in neurons causes a downmodulation of neuronal activity through presynaptic mechanisms and by reducing neuronal excitability. HCAR1 activation engages both Giα and Gißγ intracellular pathways to functionally interact with other Gi-coupled receptors for the fine tuning of neuronal activity.SIGNIFICANCE STATEMENT Expression of the lactate receptor hydroxycarboxylic acid receptor 1 (HCAR1) was recently described in neurons. Here, we describe the physiological role of this G-protein-coupled receptor (GPCR) and its activation in neurons, providing information on its expression and mechanism of action. We dissected out the intracellular pathway through which HCAR1 activation tunes down neuronal network activity. For the first time, we provide evidence for the functional cross talk of HCAR1 with other GPCRs, such as GABAB, adenosine A1- and α2A-adrenergic receptors. These results set HCAR1 as a new player for the regulation of neuronal network activity acting in concert with other established receptors. Thus, HCAR1 represents a novel therapeutic target for pathologies characterized by network hyperexcitability dysfunction, such as epilepsy.
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Proteínas de Unión al GTP Heterotriméricas/fisiología , Lactatos/metabolismo , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Receptores Acoplados a Proteínas G/fisiología , Potenciales de Acción , Animales , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , AMP Cíclico/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Cultivo Primario de Células , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Sistemas de Mensajero Secundario/efectos de los fármacosRESUMEN
The hypothalamic-pituitary-adrenal axis involves timed signaling between the hypothalamus, pituitary, and adrenal glands and back to the brain, causing an inherently oscillating system. Corticosteroids such as corticosterone (CORT) are secreted in a circadian rhythm, characterized by low and high levels at the start of the inactive and active phases, respectively. The circadian rhythm overarches ultradian CORT pulses, with approximate 1-hour interpulse intervals. We examined the physiological relevance of pulsatile CORT exposure for neurons of the basolateral amygdala (BLA), an area important for fear learning. We first applied four pulses of equal, high CORT concentration and measured the frequency of miniature excitatory postsynaptic currents (mEPSCs) reflecting spontaneous glutamate signaling. BLA neurons responded differently to each pulse, showing "metaplasticity," extending earlier studies. Next, we mimicked the progression of the inactive and active phases by four CORT pulses of increasing and decreasing concentrations, respectively. CORT pulses of increasing concentration were necessary and sufficient to gradually increase baseline (between-pulse) mEPSC frequency during the mimicked inactive phase, whereas the opposite was seen with decreasing CORT levels during the mimicked active phase. To study the relevance of changed glutamate transmission on behavior, mice were tested in tone-cued fear conditioning during the active or inactive phase. Animals tested at the inactive compared with the active phase showed efficient fear learning; this was also observed when animals tested during the active phase were treated with the CORT synthesis blocker metyrapone. This suggests that natural CORT rhythms influence electrical activity in the BLA, possibly contributing to altered behavioral function.
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Complejo Nuclear Basolateral/efectos de los fármacos , Ritmo Circadiano/fisiología , Corticosterona/farmacología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Animales , Complejo Nuclear Basolateral/fisiología , Condicionamiento Clásico/efectos de los fármacos , Condicionamiento Clásico/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Miedo/fisiología , Sistema Hipotálamo-Hipofisario/fisiología , Masculino , Ratones , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Sistema Hipófiso-Suprarrenal/fisiologíaRESUMEN
The Medial Habenular (MHb) and the Lateral Habenular nuclei are 2 main parts of the habenular complex (Hb). Recent studies showed that MHb plays an important role in memory, and in the expression of ErbB4. However, the expression of MHb ErbB4 receptor and its role in fear memory is not well understood. In this study, western blotting and quantitative real-time polymerase chain reaction were used to assess the protein and mRNA levels of ErbB4 in the process of contextual fear conditioning. A pharmacological approach was used to block and stimulate the ErbB4 receptor. Contextual fear conditioning tests induced a significant increase on the expression of ErbB4 at various times in the Hb and the MHb. Moreover, the blockade and stimulation of MHb ErbB4 receptors did not affect the fear formation but impaired and improved the contextual-dependent fear expression. Furthermore, in vitro electrophysiological recordings showed that the blockade of the MHb ErbB4 receptor reduced the presynaptic gamma-amino butyric acid release. ErbB4 is a susceptible gene for schizophrenia and the above findings may provide new insights into the mechanisms of fear-related responses.
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Miedo/fisiología , Habénula/metabolismo , Memoria/fisiología , Receptor ErbB-4/metabolismo , Animales , Escala de Evaluación de la Conducta , Condicionamiento Clásico , Miedo/psicología , Reacción Cataléptica de Congelación/efectos de los fármacos , Habénula/efectos de los fármacos , Habénula/fisiología , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Neurregulina-1/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor ErbB-4/agonistas , Receptor ErbB-4/antagonistas & inhibidores , Receptor ErbB-4/genética , Tirfostinos/farmacologíaRESUMEN
Dextromethorphan (DEX) presynaptically decreases glutamatergic transmission in second-order neurons of the nucleus tractus solitarius (TS). To clarify the inhibitory mechanism of DEX, the present study examined the interaction of DEX with cAMP. The effects of DEX on miniature and TS-evoked excitatory postsynaptic currents (mEPSCs and eEPSCs) were recorded under activation of the cAMP-dependent pathway using the brainstem slices. An increase in cAMP by forskolin counteracted the inhibitory effect of DEX on mEPSCs. Eight-Bromo-cAMP and N-ethylmaleimide also attenuated the DEX effect. However, forskolin had negligible effects on the DEX-induced inhibition of eEPSCs. This suggests that DEX decreases spontaneous glutamate release by inhibiting the cAMP-dependent pathway and synchronous release by another unknown mechanism.
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AMP Cíclico/metabolismo , Dextrometorfano/farmacología , Glutamatos/metabolismo , Neuronas/efectos de los fármacos , Núcleo Solitario/efectos de los fármacos , Núcleo Solitario/fisiología , Transmisión Sináptica/efectos de los fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Colforsina/farmacología , Etilmaleimida/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Cobayas , Masculino , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Núcleo Solitario/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Benzodiazepines are commonly prescribed to treat neurological conditions including epilepsy, insomnia, and anxiety. The discovery of benzodiazepine-specific binding sites on γ-aminobutyric acid type-A receptors (GABAARs) led to the hypothesis that the brain may produce endogenous benzodiazepine-binding site ligands. An endogenous peptide, diazepam binding inhibitor (DBI), which can bind these sites, is thought to be capable of both enhancing and attenuating GABAergic transmission in different brain regions. However, the role that DBI plays in modulating GABAARs in the hippocampus remains unclear. Here, we investigated the role of DBI in modulating synaptic inhibition in the hippocampus using a constitutive DBI knockout mouse. Miniature and evoked inhibitory postsynaptic currents (mIPSCs, eIPSCs) were recorded from CA1 pyramidal cells and dentate gyrus (DG) granule cells. Loss of DBI signaling increased mIPSC frequency and amplitude in CA1 pyramidal cells from DBI knockout mice compared to wild-types. In DG granule cells, conversely, the loss of DBI decreased mIPSC amplitude and increased mIPSC decay time, indicating bidirectional modulation of GABAAR-mediated transmission in specific subregions of the hippocampus. eIPSC paired-pulse ratios were consistent across genotypes, suggesting that alterations in mIPSC frequency were not due to changes in presynaptic release probability. Furthermore, cells from DBI knockout mice did not display altered responsiveness to pharmacological applications of diazepam, a benzodiazepine, nor flumazenil, a benzodiazepine-binding site antagonist. These results provide evidence that genetic loss of DBI alters synaptic inhibition in the adult hippocampus, and that the direction of DBI-mediated modulation can vary discretely between specific subregions of the same brain structure.
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Inhibidor de la Unión a Diazepam/deficiencia , Hipocampo/metabolismo , Potenciales Postsinápticos Inhibidores/fisiología , Neuronas/metabolismo , Animales , Fármacos del Sistema Nervioso Central/farmacología , Diazepam/farmacología , Inhibidor de la Unión a Diazepam/antagonistas & inhibidores , Inhibidor de la Unión a Diazepam/genética , Femenino , Flumazenil/farmacología , Hipocampo/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Neuronas/efectos de los fármacos , Receptores de GABA-A/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
It has been widely reported that ketamine rescues chronic stress-induced depression-like behavior, but the underlying cellular mechanisms of the rapid antidepressant actions of ketamine remain largely unclear. Both male and female Sprague-Dawley rats were used and received modified learned helplessness paradigm to induce depression-like behavior. Depression-like behavior was assayed and manipulated using forced swim tests, sucrose preference tests and pharmacological microinjection. We conducted whole-cell patch-clamp electrophysiological recordings in the midbrain ventrolateral periaqueductal gray (vlPAG) neurons. Surface and cytosolic glutamate receptor 1 (GluR1) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression were analyzed using Western blotting. Phosphorylated GluR1 expression was quantified using Western blotting analysis. The results showed that a single systemic administration of a ketamine metabolite (2R,6R)-hydroxynorketamine (2R,6R-HNK) rapidly rescued chronic stress-induced depression-like behavior and persisted for up to 21 days. Consistently, the chronic stress-induced diminished glutamatergic transmission and surface GluR1 expression in the vlPAG were also reversed by a single systemic injection of (2R,6R)-HNK. Furthermore, bath application of (2R,6R)-HNK increased the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in the vlPAG. Further evidence for the antidepressant action of (2R,6R)-HNK is provided by the finding that microinjection of (2R,6R)-HNK into the vlPAG exhibited a rapid-acting and long-lasting antidepressant effect. This antidepressant effect of (2R,6R)-HNK was prevented by the intra-vlPAG microinjection of AMPA receptor antagonist CNQX. Together, the current results provide evidence that (2R,6R)-HNK rescues chronic stress-induced depression-like behavior with rapid-acting and long-lasting antidepressant effects through enhancement of AMPA receptor-mediated transmission in the vlPAG.
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Antidepresivos/envenenamiento , Trastorno Depresivo/tratamiento farmacológico , Ketamina/análogos & derivados , Sustancia Gris Periacueductal/efectos de los fármacos , Animales , Antidepresivos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Trastorno Depresivo/fisiopatología , Modelos Animales de Enfermedad , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Ácido Glutámico/metabolismo , Desamparo Adquirido , Ketamina/farmacología , Masculino , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Sustancia Gris Periacueductal/metabolismo , Ratas Sprague-Dawley , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismo , Estrés Psicológico/tratamiento farmacológico , Estrés Psicológico/fisiopatología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
In cancer cachexia, abnormal metabolism and neuroendocrine dysfunction cause anorexia, tissue damage and atrophy, which can in turn alter body fluid balance. Arginine vasopressin, which regulates fluid homeostasis, is secreted by magnocellular neurosecretory cells (MNCs) of the hypothalamic supraoptic nucleus. Arginine vasopressin secretion by MNCs is regulated by both excitatory and inhibitory synaptic activity, alterations in plasma osmolarity and various peptides, including angiotensin II. In the present study, we used whole-cell patch-clamp recordings of brain slices to determine whether hyperosmotic stimulation and/or angiotensin II potentiate excitatory synaptic input in a rat model of cancer cachexia, similar to their effects in normal (control) rats. Hyperosmotic (15 and 60 mmol L-1 mannitol) stimulation and angiotensin II (0.1 µmol L-1 ) increased the frequency, but not the amplitude, of miniature excitatory postsynaptic currents in normal rats; in model rats, both effects were significantly attenuated. These results suggest that cancer cachexia alters supraoptic MNC sensitivity to osmotic and angiotensin II stimulation.
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Caquexia/fisiopatología , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Miniatura/fisiología , Neoplasias/fisiopatología , Neuronas/fisiología , Núcleo Supraóptico/fisiopatología , Angiotensina II/farmacología , Animales , Caquexia/etiología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Masculino , Manitol/farmacología , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Trasplante de Neoplasias , Neoplasias/complicaciones , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Núcleo Supraóptico/efectos de los fármacosRESUMEN
Cannabinoids exert dynamic control over many physiological processes including memory formation, cognition and pain perception. In the central nervous system endocannabinoids mediate negative feedback of quantal transmitter release following postsynaptic depolarization. The influence of cannabinoids in the peripheral nervous system is less clear and might have broad implications for the therapeutic application of cannabinoids. We report a novel cannabinoid effect upon the mouse neuromuscular synapse: acutely increasing synaptic vesicle volume and raising the quantal amplitudes. In a mouse model of myasthenia gravis the cannabinoid receptor agonist WIN 55,212 reversed fatiguing failure of neuromuscular transmission, suggesting future therapeutic potential. Our data suggest an endogenous pathway by which cannabinoids might help to regulate transmitter release at the neuromuscular junction.