RESUMEN
Spinal cerebrospinal fluid-contacting neurons (CSF-cNs) form an evolutionary conserved bipolar cell population localized around the central canal of all vertebrates. CSF-cNs were shown to express molecular markers of neuronal immaturity into adulthood; however, the impact of their incomplete maturation on the chloride (Cl-) homeostasis as well as GABAergic signaling remains unknown. Using adult mice from both sexes, in situ hybridization revealed that a proportion of spinal CSF-cNs (18.3%) express the Na+-K+-Cl- cotransporter 1 (NKCC1) allowing intracellular Cl- accumulation. However, we did not find expression of the K+-Cl- cotransporter 2 (KCC2) responsible for Cl- efflux in any CSF-cNs. The lack of KCC2 expression results in low Cl- extrusion capacity in CSF-cNs under high Cl- load in whole-cell patch clamp. Using cell-attached patch clamp allowing recordings with intact intracellular Cl- concentration, we found that the activation of ionotropic GABAA receptors (GABAA-Rs) induced both depolarizing and hyperpolarizing responses in CSF-cNs. Moreover, depolarizing GABA responses can drive action potentials as well as intracellular calcium elevations by activating voltage-gated calcium channels. Blocking NKCC1 with bumetanide inhibited the GABA-induced calcium transients in CSF-cNs. Finally, we show that metabotropic GABAB receptors have no hyperpolarizing action on spinal CSF-cNs as their activation with baclofen did not mediate outward K+ currents, presumably due to the lack of expression of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Together, these findings outline subpopulations of spinal CSF-cNs expressing inhibitory or excitatory GABAA-R signaling. Excitatory GABA may promote the maturation and integration of young CSF-cNs into the existing spinal circuit.
Asunto(s)
Miembro 2 de la Familia de Transportadores de Soluto 12 , Médula Espinal , Simportadores , Animales , Ratones , Médula Espinal/metabolismo , Femenino , Masculino , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Simportadores/metabolismo , Cotransportadores de K Cl , Transducción de Señal/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Ácido gamma-Aminobutírico/metabolismo , Líquido Cefalorraquídeo/metabolismo , Líquido Cefalorraquídeo/fisiología , Ratones Endogámicos C57BL , Receptores de GABA-A/metabolismo , Cloruros/metabolismo , Cloruros/líquido cefalorraquídeo , Cloruros/farmacología , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/fisiologíaRESUMEN
Leucine-rich glioma inactivated 1 (LGI1) is a glycoprotein secreted by neurons, the deletion of which leads to autosomal dominant lateral temporal lobe epilepsy. We previously showed that LGI1 deficiency in a mouse model (i.e., knock-out for LGI1 or KO-Lgi1) decreased Kv1.1 channel density at the axon initial segment (AIS) and at presynaptic terminals, thus enhancing both intrinsic excitability and glutamate release. However, it is not known whether normal excitability can be restored in epileptic neurons. Here, we show that the selective expression of LGI1 in KO-Lgi1 neurons from mice of both sexes, using single-cell electroporation, reduces intrinsic excitability and restores both the Kv1.1-mediated D-type current and Kv1.1 channels at the AIS. In addition, we show that the homeostatic-like shortening of the AIS length observed in KO-Lgi1 neurons is prevented in neurons electroporated with the Lgi1 gene. Furthermore, we reveal a spatial gradient of intrinsic excitability that is centered on the electroporated neuron. We conclude that expression of LGI1 restores normal excitability through functional Kv1 channels at the AIS.SIGNIFICANCE STATEMENT The lack of leucine-rich glioma inactivated 1 (LGI1) protein induces severe epileptic seizures that leads to death. Enhanced intrinsic and synaptic excitation in KO-Lgi1 mice is because of the decrease in Kv1.1 channels in CA3 neurons. However, the conditions to restore normal excitability profile in epileptic neurons remain to be defined. We show here that the expression of LGI1 in KO-Lgi1 neurons in single neurons reduces intrinsic excitability, and restores both the Kv1.1-mediated D-type current and Kv1.1 channels at the axon initial segment (AIS). Furthermore, the homeostatic shortening of the AIS length observed in KO-Lgi1 neurons is prevented in neurons in which the Lgi1 gene has been rescued. We conclude that LGI1 constitutes a critical factor to restore normal excitability in epileptic neurons.
Asunto(s)
Epilepsia del Lóbulo Frontal , Glioma , Neuronas , Animales , Femenino , Masculino , Ratones , Epilepsia del Lóbulo Frontal/genética , Epilepsia del Lóbulo Frontal/metabolismo , Leucina/metabolismo , Neuronas/fisiología , Convulsiones/genéticaRESUMEN
In animal models of LGI1-dependent autosomal dominant lateral temporal lobe epilepsy, Kv1 channels are downregulated, suggesting their crucial involvement in epileptogenesis. The molecular basis of Kv1 channel-downregulation in LGI1 knock-out mice has not been elucidated and how the absence of this extracellular protein induces an important modification in the expression of Kv1 remains unknown. In this study we analyse by immunofluorescence the modifications in neuronal Kv1.1 and Kv1.2 distribution throughout the hippocampal formation of LGI1 knock-out mice. We show that Kv1 downregulation is not restricted to the axonal compartment, but also takes place in the somatodendritic region and is accompanied by a drastic decrease in Kv2 expression levels. Moreover, we find that the downregulation of these Kv channels is associated with a marked increase in bursting patterns. Finally, mass spectrometry uncovered key modifications in the Kv1 interactome that highlight the epileptogenic implication of Kv1 downregulation in LGI1 knock-out animals.
Asunto(s)
Regulación hacia Abajo , Hipocampo , Péptidos y Proteínas de Señalización Intracelular , Ratones Noqueados , Animales , Hipocampo/metabolismo , Ratones , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Canal de Potasio Kv.1.1/metabolismo , Canal de Potasio Kv.1.1/genética , Proteínas/metabolismo , Proteínas/genética , Ratones Endogámicos C57BL , Canal de Potasio Kv.1.2/metabolismo , Canal de Potasio Kv.1.2/genética , Neuronas/metabolismoRESUMEN
Astrocytes play crucial roles in brain homeostasis and are regulatory elements of neuronal and synaptic physiology. Astrocytic alterations have been found in Major Depressive Disorder (MDD) patients; however, the consequences of astrocyte Ca2+ signaling in MDD are poorly understood. Here, we found that corticosterone-treated juvenile mice (Cort-mice) showed altered astrocytic Ca2+ dynamics in mPFC both in resting conditions and during social interactions, in line with altered mice behavior. Additionally, Cort-mice displayed reduced serotonin (5-HT)-mediated Ca2+ signaling in mPFC astrocytes, and aberrant 5-HT-driven synaptic plasticity in layer 2/3 mPFC neurons. Downregulation of astrocyte Ca2+ signaling in naïve animals mimicked the synaptic deficits found in Cort-mice. Remarkably, boosting astrocyte Ca2+ signaling with Gq-DREADDS restored to the control levels mood and cognitive abilities in Cort-mice. This study highlights the important role of astrocyte Ca2+ signaling for homeostatic control of brain circuits and behavior, but also reveals its potential therapeutic value for depressive-like states.
Asunto(s)
Astrocitos , Trastorno Depresivo Mayor , Humanos , Ratones , Animales , Astrocitos/fisiología , Neuronas Serotoninérgicas , Serotonina , Transducción de Señal/fisiologíaRESUMEN
Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1's neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus. LGI1 is secreted in both neuronal somatodendritic and axonal compartments, and occurs in oligodendrocytic, neuro-oligodendrocytic and astro-microglial protein complexes. Proteomic data support the presence of LGI1-Kv1-MAGUK complexes, but did not reveal LGI1 complexes with postsynaptic glutamate receptors. Our results extend our understanding of regional, cellular and subcellular LGI1 expression profiles and reveal novel LGI1-associated complexes, thus providing insights into the complex biology of LGI1 and its relationship to seizures and memory loss.
Asunto(s)
Glioma , Péptidos y Proteínas de Señalización Intracelular , Animales , Ratones , Leucina , Proteómica , Autoanticuerpos , ConvulsionesRESUMEN
Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of BoNT/B bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside allowing a lipid-binding loop of BoNT/B to interact with the glycone part of the synaptotagmin-associated GT1b. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.
Asunto(s)
Gangliósidos , Sinaptotagmina II , Sitios de Unión , Gangliósidos/química , Gangliósidos/metabolismo , Neuronas/metabolismo , Unión Proteica , Sinaptotagmina II/química , Sinaptotagmina II/genética , Sinaptotagmina II/metabolismo , Sinaptotagminas/genética , Sinaptotagminas/metabolismoRESUMEN
Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b-synaptotagmin cis complex forms the BoNT/B receptor. We identified a consensus glycosphingolipid-binding motif in the extracellular juxtamembrane domain of synaptotagmins 1/2 and confirmed by Langmuir monolayer, surface plasmon resonance, and circular dichroism that GT1b interacts with synaptotagmin peptides containing this sequence, inducing α-helical structure. Molecular modeling and tryptophan fluorescence spectroscopy were consistent with the intertwining of GT1b and synaptotagmin, involving cis interactions between the oligosaccharide and ceramide moieties of GT1b and the juxtamembrane and transmembrane domains of synaptotagmin, respectively. Furthermore, a point mutation on synaptotagmin, located outside of the BoNT/B-binding segment, inhibited GT1b binding and blocked GT1b-induced potentiation of BoNT/B binding to synaptotagmin-expressing cells. Our findings are consistent with a model in which a preassembled GT1b-synaptotagmin complex constitutes the high-affinity BoNT/B receptor.
Asunto(s)
Toxinas Botulínicas Tipo A , Gangliósidos , Sinaptotagmina I , Animales , Sitios de Unión , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/metabolismo , Gangliósidos/química , Gangliósidos/farmacología , Conformación Proteica en Hélice alfa , Dominios Proteicos , Ratas , Sinaptotagmina I/química , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Sinaptotagmina II/química , Sinaptotagmina II/genética , Sinaptotagmina II/metabolismoRESUMEN
Autoantibodies against leucine-rich glioma inactivated 1 (LGI1) are found in patients with limbic encephalitis and focal seizures. Here, we generate patient-derived monoclonal antibodies (mAbs) against LGI1. We explore their sequences and binding characteristics, plus their pathogenic potential using transfected HEK293T cells, rodent neuronal preparations, and behavioural and electrophysiological assessments in vivo after mAb injections into the rodent hippocampus. In live cell-based assays, LGI1 epitope recognition was examined with patient sera (n = 31), CSFs (n = 11), longitudinal serum samples (n = 15), and using mAbs (n = 14) generated from peripheral B cells of two patients. All sera and 9/11 CSFs bound both the leucine-rich repeat (LRR) and the epitempin repeat (EPTP) domains of LGI1, with stable ratios of LRR:EPTP antibody levels over time. By contrast, the mAbs derived from both patients recognized either the LRR or EPTP domain. mAbs against both domain specificities showed varied binding strengths, and marked genetic heterogeneity, with high mutation frequencies. LRR-specific mAbs recognized LGI1 docked to its interaction partners, ADAM22 and ADAM23, bound to rodent brain sections, and induced internalization of the LGI1-ADAM22/23 complex in both HEK293T cells and live hippocampal neurons. By contrast, few EPTP-specific mAbs bound to rodent brain sections or ADAM22/23-docked LGI1, but all inhibited the docking of LGI1 to ADAM22/23. After intrahippocampal injection, and by contrast to the LRR-directed mAbs, the EPTP-directed mAbs showed far less avid binding to brain tissue and were consistently detected in the serum. Post-injection, both domain-specific mAbs abrogated long-term potentiation induction, and LRR-directed antibodies with higher binding strengths induced memory impairment. Taken together, two largely dichotomous populations of LGI1 mAbs with distinct domain binding characteristics exist in the affinity matured peripheral autoantigen-specific memory pools of individuals, both of which have pathogenic potential. In human autoantibody-mediated diseases, the detailed characterization of patient mAbs provides a valuable method to dissect the molecular mechanisms within polyclonal populations.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Autoanticuerpos/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas ADAM/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/sangre , Autoanticuerpos/metabolismo , Autoantígenos/metabolismo , Encéfalo/metabolismo , Epítopos/inmunología , Células HEK293 , Hipocampo/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Encefalitis Límbica/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Unión Proteica/inmunología , Dominios Proteicos/inmunologíaRESUMEN
The aim of this work is to determine the effect of chronic immobilization stress on kinetics and dosimetry of 67Ga in a mouse model. A control group (CG) and a stress group (SG), each with 15 mice, were included in the study, and the latter group was subjected to a chronic immobilization stress model 2 h daily for 14 consecutive days. At day 13, 67Ga-citrate was administered intraperitoneally (11.24 ± 0.44 MBq) to each mouse. Then, sets of three mice were obtained sequentially at 24, 36, 48, 60 and 72 h, in which the radionuclide activity was measured with an activity counter. The 67Ga biokinetic data showed a fast blood clearance in the SG, with a mean residence time of 0.06 h. The calculated mean radiation absorbed doses were: liver (2.45 × 10-03 Gy), heart (3.17 × 10-04 Gy) and kidney (1.88 × 10-04 Gy) in the SG. The results show that stress reduced weight gain by approximately 13% and also increased adrenal gland weight by 26%. On the other hand, chronic stress accelerates 67Ga clearance after 24 h compared to normal conditions. It is concluded that murine organisms under chronic immobilization stress have higher gallium-67 clearance rates, decreasing the cumulated activity and absorbed dose in all organs.
Asunto(s)
Citratos/administración & dosificación , Radioisótopos de Galio , Galio/administración & dosificación , Radiofármacos/administración & dosificación , Restricción Física , Estrés Fisiológico/fisiología , Estrés Psicológico/metabolismo , Glándulas Suprarrenales/patología , Animales , Citratos/farmacocinética , Modelos Animales de Enfermedad , Galio/farmacocinética , Masculino , Ratones , Dosis de Radiación , Radiofármacos/farmacocinética , Distribución Tisular , Aumento de PesoRESUMEN
Optogenetics has been widely expanded to enhance or suppress neuronal activity and it has been recently applied to glial cells. Here, we have used a new approach based on selective expression of melanopsin, a G-protein-coupled photopigment, in astrocytes to trigger Ca2+ signaling. Using the genetically encoded Ca2+ indicator GCaMP6f and two-photon imaging, we show that melanopsin is both competent to stimulate robust IP3-dependent Ca2+ signals in astrocyte fine processes, and to evoke an ATP/Adenosine-dependent transient boost of hippocampal excitatory synaptic transmission. Additionally, under low-frequency light stimulation conditions, melanopsin-transfected astrocytes can trigger long-term synaptic changes. In vivo, melanopsin-astrocyte activation enhances episodic-like memory, suggesting melanopsin as an optical tool that could recapitulate the wide range of regulatory actions of astrocytes on neuronal networks in behaving animals. These results describe a novel approach using melanopsin as a precise trigger for astrocytes that mimics their endogenous G-protein signaling pathways, and present melanopsin as a valuable optical tool for neuron-glia studies.
Asunto(s)
Astrocitos/metabolismo , Red Nerviosa/metabolismo , Neuronas/metabolismo , Optogenética/métodos , Opsinas de Bastones/metabolismo , 2-Amino-5-fosfonovalerato/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Compuestos Azo/farmacología , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/citología , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Luz , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Pirimidinas/farmacología , Opsinas de Bastones/genética , Potenciales Sinápticos/fisiología , Triazoles/farmacología , Xantenos/farmacologíaRESUMEN
Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1-pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors (CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210. Guanine nucleotide exchange proteins directly activated by cAMP (Epac proteins) mediate some of the presynaptic effects of cAMP in the potentiation of synaptic transmission. ESI05, a selective Epac2 inhibitor, and U-73122, the specific inhibitor of phospholipase C (PLC), both augment the number of silent synaptic boutons. Moreover, they abolish the capacity of the Epac activator, 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate, to prevent HU-210-induced silencing consistent with PLC signaling lying downstream of Epac2 proteins. Furthermore, Rab3-interacting molecule (RIM)1α KO cells have many more basally silent synaptic boutons (12.9 ± 3.5%) than wild-type cells (1.1 ± 0.5%). HU-210 induced further silencing in these mutant cells, although 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate only awoke the HU-210-induced silence and not the basally silent synaptic boutons. This behavior can be rescued by expressing RIM1α in RIM1α KO cells, these cells behaving very much like wild-type cells. These findings support the hypothesis that a cAMP/Epac/PLC signaling pathway targeting the release machinery appears to mediate cannabinoid-induced presynaptic silencing.
Asunto(s)
Cerebelo/citología , Neuronas/metabolismo , Receptor Cannabinoide CB1/metabolismo , Transmisión Sináptica/efectos de los fármacos , Animales , Cerebelo/efectos de los fármacos , AMP Cíclico/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Estrenos/farmacología , Femenino , Factores de Intercambio de Guanina Nucleótido/metabolismo , Masculino , Neuronas/efectos de los fármacos , Pirrolidinonas/farmacología , Ratas Wistar , Receptor Cannabinoide CB1/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Fosfolipasas de Tipo C/metabolismoRESUMEN
From the early periods of neurogenesis and migration, up until synaptogenesis, both nitric oxide (NO) and its downstream messenger, cGMP, are thought to influence the development of neurons. The NO/cGMP/cGMP-dependent protein kinase (cGK) pathway regulates the clustering and recruitment of synaptic proteins and vesicles to the synapse, adjusting the exoendocytic cycle to the intensity of activity and accelerating endocytosis following large-scale exocytosis. Here, we show that blockage of the N-methyl-D-aspartate receptor impairs the cycling of synaptic vesicles in a subset of boutons on cerebellar granule cells, an effect that was reversed by increasing cGMP. Furthermore, we demonstrate that presynaptic cGK type II (cGKII) plays a major role in this process. Using the FM1-43 dye to track vesicle recycling, we found that knockdown of cGKII and/or the application of a cGK inhibitor reduced the efficiency of synaptic vesicle recycling to a similar extent. Likewise, in cerebellar granule cells transfected with vGlut1-pHluorin to follow the exoendocytotic cycle, application of a cGK inhibitor slowed vesicle endocytosis when exocytosis was accelerated through strong and sustained stimulation. Additionally, ultrastructural analysis showed that cGKII knockdown or inhibition favored the formation of endosomal-like structures after strong and sustained stimulation. We conclude that cGKII controls the homeostatic balance of vesicle exocytosis and endocytosis in synaptic boutons of rat cerebellar granule cells.
Asunto(s)
Cerebelo/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/metabolismo , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Cerebelo/citología , Cerebelo/efectos de los fármacos , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/genética , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/genéticaRESUMEN
The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. Here, we describe the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na(+) channels with tetrodotoxin. We found that 8-pCPT-2'-O-Me-cAMP, a specific activator of the exchange protein directly activated by cAMP (Epac), mimicked and occluded forskolin-induced potentiation of glutamate release. This Epac-mediated increase in glutamate release was dependent on phospholipase C, and it increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Moreover, the potentiation of glutamate release by Epac was independent of protein kinase C, although it was attenuated by the diacylglycerol-binding site antagonist calphostin C. Epac activation translocated the active zone protein Munc13-1 from soluble to particulate fractions; it increased the association between Rab3A and RIM1α and redistributed synaptic vesicles closer to the presynaptic membrane. Furthermore, these responses were mimicked by the ß-adrenergic receptor (ßAR) agonist isoproterenol, consistent with the immunoelectron microscopy and immunocytochemical data demonstrating presynaptic expression of ßARs in a subset of glutamatergic synapses in the cerebral cortex. Based on these findings, we conclude that ßARs couple to a cAMP/Epac/PLC/Munc13/Rab3/RIM-dependent pathway to enhance glutamate release at cerebrocortical nerve terminals.
Asunto(s)
Corteza Cerebral/metabolismo , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Ácido Glutámico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Adrenérgicos beta/metabolismo , Proteína de Unión al GTP rab3A/metabolismo , Adyuvantes Inmunológicos/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Corteza Cerebral/citología , Colforsina/farmacología , Inhibidores Enzimáticos/farmacología , Isoproterenol/farmacología , Ratones , Naftalenos/farmacología , Terminales Presinápticos/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Membranas Sinápticas/metabolismo , Transmisión Sináptica/fisiología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
Following the exocytosis of neurotransmitter-containing synaptic vesicles, endocytosis is fundamental to re-establishing conditions for synaptic transmission. As there are distinct endocytotic pathways that each differ in their efficiency to generate releasable synaptic vesicles, we used the dye FM1-43 to track vesicle recycling, and to determine whether nerve terminals use multiple pathways of endocytosis. We identified two types of synaptic boutons in cultured cerebellar granule cells that were characterized by weak or strong FM1-43-unloading profiles. Decreasing the extent of exocytosis dramatically increased the proportion of synaptic boutons that exhibited strong FM1-43-unloading and dramatically reduced the number of endosome-like structures. Hence, we concluded that efficient recycling of synaptic vesicles is concomitant with the formation of non-releasable endosomes in both types of synaptic boutons, although to different extents. Furthermore, cell maturation in culture increased the proportion of synaptic boutons that were capable of an intense release response, whereas the chronic blockage of synaptic activity diminished the capacity of boutons to release dye.
Asunto(s)
Endosomas/metabolismo , Exocitosis , Vesículas Sinápticas/metabolismo , Animales , Inhibidores de la Calcineurina , Células Cultivadas , Cerebelo/citología , Cerebelo/metabolismo , Dinaminas/fisiología , Endocitosis , Femenino , Colorantes Fluorescentes , Masculino , Terminales Presinápticos/clasificación , Terminales Presinápticos/metabolismo , Compuestos de Piridinio , Compuestos de Amonio Cuaternario , Ratas , Ratas Wistar , Vesículas Sinápticas/efectos de los fármacos , Tacrolimus/farmacologíaRESUMEN
BACKGROUND: In terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several proteins that govern exo/endocytosis. Upon stimulation, individual terminals in networks of cultured cerebellar granule neurons exhibit heterogeneous exocytic responses, which reflect the distinct states of maturity and plasticity intrinsic to individual synaptic terminals. This dynamic scenario serves as the substrate for processes such as scaling, plasticity and synaptic weight redistribution. Presynaptic strength has been associated with the activity of several types of proteins, including the scaffolding proteins that form the active zone cytomatrix and the proteins involved in presynaptic exocytosis. METHODS: We have combined fluorescence imaging techniques using the styryl dye FM1-43 in primary cultures of cerebellar granule cells with subsequent post-hoc immunocytochemistry in order to study synaptic efficiency in terms of vesicular release. We describe a protocol to easily quantify these results with minimal user intervention. RESULTS: In this study we describe a technique that specifically correlates presynaptic activity with the levels of presynaptic markers. This method involves the use of the styryl dye FM1-43 to estimate the release capacity of a synaptic terminal, and the subsequent post-hoc immunolabelling of thousands of individual nerve terminals. We observed a strong correlation between the release capacity of the nerve terminal and the levels of the RIM1α but not the Munc13-1 protein in the active zone. CONCLUSIONS: Our findings support those of previous studies and point out to RIM1α as a crucial factor in determining synaptic efficiency. These results also demonstrate that this technique is a useful tool to analyse the molecular differences underlying the heterogeneous responses exhibited by neuronal networks.
Asunto(s)
Inmunohistoquímica/métodos , Neuronas/fisiología , Imagen Óptica/métodos , Transmisión Sináptica/fisiología , Animales , Cerebelo/fisiología , Colorantes Fluorescentes , Proteínas del Tejido Nervioso/metabolismo , Terminales Presinápticos/metabolismo , Compuestos de Piridinio , Compuestos de Amonio CuaternarioRESUMEN
Here we present a deep learning-based image analysis platform (DLAP), tailored to autonomously quantify cell numbers, and fluorescence signals within cellular compartments, derived from RNAscope or immunohistochemistry. We utilised DLAP to analyse subtypes of tyrosine hydroxylase (TH)-positive dopaminergic midbrain neurons in mouse and human brain-sections. These neurons modulate complex behaviour, and are differentially affected in Parkinson's and other diseases. DLAP allows the analysis of large cell numbers, and facilitates the identification of small cellular subpopulations. Using DLAP, we identified a small subpopulation of TH-positive neurons (~5%), mainly located in the very lateral Substantia nigra (SN), that was immunofluorescence-negative for the plasmalemmal dopamine transporter (DAT), with ~40% smaller cell bodies. These neurons were negative for aldehyde dehydrogenase 1A1, with a lower co-expression rate for dopamine-D2-autoreceptors, but a ~7-fold higher likelihood of calbindin-d28k co-expression (~70%). These results have important implications, as DAT is crucial for dopamine signalling, and is commonly used as a marker for dopaminergic SN neurons.
Asunto(s)
Aprendizaje Profundo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Animales , Humanos , Ratones , Dopamina , Neuronas Dopaminérgicas , Sustancia NegraRESUMEN
The physiological responses of AMPA receptors can be modulated through the differential expression of their subunits and by modifying their number at the cell surface. Here we have studied the expression of AMPA receptor subunits (GluR1-4) mRNAs in cerebellar granule cells grown in depolarizing (25mMK(+)) medium, and we have evaluated the effect of decreasing the [K(+)] in the culture medium for 24 h on both GluR1-4 expression (both mRNA and protein) and their presence at the plasma membrane. The expression of the four AMPAR subunits increases as the [K(+)] decreases, although the increase in GluR2 and GluR3 was only observed in the cell soma but not in the dendrites. Calcium entry through L-type calcium channel and CaMKIV activation are responsible for the reduction in the expression of AMPA receptor subunits in cells cultured in depolarizing conditions. Indeed, prolonged reduction of extracellular [K(+)] or blockage of L-type calcium channels enhanced both the surface insertion of the four AMPAR subunits and the AMPA response measured through intracellular calcium increase. These findings reveal a balanced increase in functional AMPA receptors at the surface of cells that can trigger strong increases in calcium in response to the persistent reduction of calcium entry.
Asunto(s)
Cerebelo/metabolismo , Potenciales de la Membrana , Receptores AMPA/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Cerebelo/citología , Femenino , Técnicas para Inmunoenzimas , Masculino , Neuronas/citología , Neuronas/metabolismo , Fosforilación , Subunidades de Proteína , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Receptores AMPA/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismoRESUMEN
Substantia nigra pars compacta (SNc) dopaminergic (DA) neurons display a peculiar electrical phenotype characterized in vitro by a spontaneous tonic regular activity (pacemaking activity), a broad action potential (AP) and a biphasic postinhibitory response. The transient A-type current (IA) is known to play a crucial role in this electrical phenotype, and so far, this current was considered to be carried exclusively by Kv4.3 potassium channels. Using Kv4.3-/- transgenic mice, we demonstrate that the constitutive loss of this channel is associated with increased exploratory behavior and impaired motor learning at the behavioral level. Consistently, it is also associated with a lack of compensatory changes in other ion currents at the cellular level. Using antigen retrieval (AR) immunohistochemistry, we then demonstrate that Kv4.2 potassium channels are also expressed in SNc DA neurons, although their contribution to IA appears significant only in a minority of neurons (â¼5-10%). Using correlative analysis on recorded electrophysiological parameters and multicompartment modeling, we then demonstrate that, rather than its conductance level, IA gating kinetics (inactivation time constant) appear as the main biophysical property defining postinhibitory rebound delay and pacemaking frequency. Moreover, we show that the hyperpolarization-activated current (IH) has an opposing and complementary influence on the same firing features.
Asunto(s)
Neuronas Dopaminérgicas , Sustancia Negra , Potenciales de Acción , Animales , Ratones , Ratones Transgénicos , Porción Compacta de la Sustancia NegraRESUMEN
The co-existence of presynaptic G protein coupled receptors, GPCRs, has received little attention, despite the fact that interplay between the signaling pathways activated by such receptors may affect the neurotransmitter release. Using immunocytochemistry and immuhistochemistry we show that mGlu7 and ß-adrenergic receptors are co-expressed in a sub-population of cerebrocortical nerve terminals. mGlu7 receptors readily couple to pathways that inhibit glutamate release. We found that when mGlu7 receptors are also coupled to pathways that enhance glutamate release by prolonged exposure to agonist, and ß-adrenergic receptors are also activated, a cross-talk between their signaling pathways occurs that affect the overall release response. This interaction is the result of mGlu7 receptors inhibiting the adenylyl cyclase activated by ß adrenergic receptors. Thus, blocking Gi/o proteins with pertussis toxin provokes a further increase in release after receptor co-activation which is also observed after activating ß-adrenergic receptor signaling pathways downstream of adenylyl cyclase with the cAMP analog Sp8Br or 8pCPT-2-OMe-cAMP (a specific activator of the guanine nucleotide exchange protein directly activated by cAMP, EPAC). Co-activation of mGlu7 and ß-adrenergic receptors also enhances PLC-dependent accumulation of IP1 and the translocation of the active zone protein Munc13-1 to the membrane, indicating that release potentiation by these receptors involves the modulation of the release machinery.
Asunto(s)
Terminaciones Nerviosas/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal/fisiología , Transmisión Sináptica/fisiología , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Ionóforos de Calcio/farmacología , Cerebelo/citología , Corteza Cerebral/citología , AMP Cíclico/metabolismo , Ácido Glutámico/metabolismo , Fosfatos de Inositol/farmacología , Ionomicina/farmacología , Ratones , Ratones Endogámicos C57BL , Terminaciones Nerviosas/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Propionatos/farmacología , Transducción de Señal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Sinaptofisina/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Tionucleótidos/farmacologíaRESUMEN
Cannabinoid receptors are the most abundant G protein-coupled receptors in the brain and they mediate retrograde short-term inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at many excitatory synapses. The induction of presynaptically silent synapses is a means of modulating synaptic strength, which is important for synaptic plasticity. Persistent activation of cannabinoid type 1 receptors (CB1Rs) mutes GABAergic terminals, although it is unclear if CB1Rs can also induce silencing at glutamatergic synapses. Cerebellar granule cells were transfected with VGLUT1-pHluorin to visualise the exo-endocytotic cycle. We found that prolonged stimulation (10 min) of cannabinoid receptors with the agonist HU-210 induces the silencing of previously active synapses. However, the presynaptic silencing induced by HU-210 is transient as it reverses after 20 min. cAMP with forskolin prevented CB1R-induced synaptic silencing, via activation of the Exchange Protein directly Activated by cAMP (Epac). Furthermore, Epac activation accelerated awakening of already silent boutons. Electron microscopy revealed that silencing was associated with synaptic vesicle (SV) redistribution within the nerve terminal, which diminished the number of vesicles close to the active zone of the plasma membrane. Finally, by combining functional and immunocytochemical approaches, we observed a strong correlation between the release capacity of the nerve terminals and RIM1α protein content, but not that of Munc13-1 protein. These results suggest that prolonged stimulation of cannabinoid receptors can transiently silence glutamatergic nerve terminals.