RESUMEN
Chemogenetic manipulation of neuronal activities has been enabled by a designer receptor (designer receptor exclusively activated by designer drugs, DREADD) that is activated exclusively by clozapine-N-oxide (CNO). Here, we applied CNO as a functional reporter probe to positron emission tomography (PET) of DREADD in living brains. Mutant human M4 DREADD (hM4Di) expressed in transgenic (Tg) mouse neurons was visualized by PET with microdose [11C]CNO. Deactivation of DREADD-expressing neurons in these mice by nonradioactive CNO at a pharmacological dose could also be captured by arterial spin labeling MRI (ASL-MRI). Neural progenitors derived from hM4Di Tg-induced pluripotent stem cells were then implanted into WT mouse brains and neuronal differentiation of the grafts could be imaged by [11C]CNO-PET. Finally, ASL-MRI captured chemogenetic functional manipulation of the graft neurons. Our data provide the first demonstration of multimodal molecular/functional imaging of cells expressing a functional gene reporter in the brain, which would be translatable to humans for therapeutic gene transfers and cell replacements. SIGNIFICANCE STATEMENT: The present work provides the first successful demonstration of in vivo positron emission tomographic (PET) visualization of a chemogenetic designer receptor (designer receptor exclusively activated by designer drugs, DREADD) expressed in living brains. This technology has been applied to longitudinal PET reporter imaging of neuronal grafts differentiated from induced pluripotent stem cells. Differentiated from currently used reporter genes for neuroimaging, DREADD has also been available for functional manipulation of target cells, which could be visualized by functional magnetic resonance imaging (fMRI) in a real-time manner. Multimodal imaging with PET/fMRI enables the visualization of the differentiation of iPSC-derived neural progenitors into mature neurons and DREADD-mediated functional manipulation along the time course of the graft and is accordingly capable of fortifying the utility of stem cells in cell replacement therapies.
Asunto(s)
Encéfalo/citología , Genes Reporteros , Células Madre Pluripotentes Inducidas/citología , Imagen Multimodal/métodos , Células-Madre Neurales/trasplante , Neuronas/citología , Neuronas/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Ratones , Ratones Transgénicos , Células-Madre Neurales/citología , Tomografía de Emisión de Positrones/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Trasplante de Células Madre/métodosRESUMEN
Synaptic rules that may determine the interaction between coexisting forms of long-term potentiation (LTP) at glutamatergic central synapses remain unknown. Here, we show that two mechanistically distinct forms of LTP could be induced in thalamic input to the lateral nucleus of the amygdala (LA) with an identical presynaptic stimulation protocol, depending on the level of postsynaptic membrane polarization. One form of LTP, resulting from pairing of postsynaptic depolarization and low-frequency presynaptic stimulation, was both induced and expressed postsynaptically ("post-LTP"). The same stimulation in the absence of postsynaptic depolarization led to LTP, which was induced and expressed presynaptically ("pre-LTP"). The inducibility of coexisting pre- and postsynaptic forms of LTP at synapses in thalamic input followed a well-defined hierarchical order, such that pre-LTP was suppressed when post-LTP was induced. This interaction was mediated by activation of cannabinoid type 1 receptors by endogenous cannabinoids released in the lateral nucleus of the amygdala in response to activation of the type 1 metabotropic glutamate receptor. These results suggest a previously unknown mechanism by which the hierarchy of coexisting forms of long-term synaptic plasticity in the neural circuits of learned fear could be established, possibly reflecting the hierarchy of memories for the previously experienced fearful events according to their aversiveness level.
Asunto(s)
Amígdala del Cerebelo/fisiología , Potenciación a Largo Plazo/fisiología , Sinapsis/fisiología , Potenciales Sinápticos/fisiología , Amígdala del Cerebelo/citología , Animales , Moduladores de Receptores de Cannabinoides/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Tálamo/citología , Tálamo/fisiologíaRESUMEN
We herein provide a thorough description of new transgenic mouse models for dentatorubral-pallidoluysian atrophy (DRPLA) harboring a single copy of the full-length human mutant DRPLA gene with 76 and 129 CAG repeats. The Q129 mouse line was unexpectedly obtained by en masse expansion based on the somatic instability of 76 CAG repeats in vivo. The mRNA expression levels of both Q76 and Q129 transgenes were each 80% of that of the endogenous mouse gene, whereas only the Q129 mice exhibited devastating progressive neurological phenotypes similar to those of juvenile-onset DRPLA patients. Electrophysiological studies of the Q129 mice demonstrated age-dependent and region-specific presynaptic dysfunction in the globus pallidus and cerebellum. Progressive shrinkage of distal dendrites of Purkinje cells and decreased currents through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and gamma-aminobutyrate type A receptors in CA1 neurons were also observed. Neuropathological studies of the Q129 mice revealed progressive brain atrophy, but no obvious neuronal loss, associated with massive neuronal intranuclear accumulation (NIA) of mutant proteins with expanded polyglutamine stretches starting on postnatal day 4, whereas NIA in the Q76 mice appeared later with regional specificity to the vulnerable regions of DRPLA. Expression profile analyses demonstrated age-dependent down-regulation of genes, including those relevant to synaptic functions and CREB-dependent genes. These results suggest that neuronal dysfunction without neuronal death is the essential pathophysiologic process and that the age-dependent NIA is associated with nuclear dysfunction including transcriptional dysregulations. Thus, our Q129 mice should be highly valuable for investigating the mechanisms of disease pathogenesis and therapeutic interventions.
Asunto(s)
Epilepsias Mioclónicas Progresivas/fisiopatología , Proteínas del Tejido Nervioso/genética , Expansión de Repetición de Trinucleótido , Factores de Edad , Animales , Encéfalo/metabolismo , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Epilepsias Mioclónicas Progresivas/genética , Epilepsias Mioclónicas Progresivas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Fenotipo , Transmisión SinápticaRESUMEN
Input-specific long-term potentiation (LTP) in afferent inputs to the amygdala serves an essential function in the acquisition of fear memory. Factors underlying input specificity of synaptic modifications implicated in information transfer in fear conditioning pathways remain unclear. Here we show that the strength of naive synapses in two auditory inputs converging on a single neuron in the lateral nucleus of the amygdala (LA) is only modified when a postsynaptic action potential closely follows a synaptic response. The stronger inhibitory drive in thalamic pathway, as compared with cortical input, hampers the induction of LTP at thalamo-amygdala synapses, contributing to the spatial specificity of LTP in convergent inputs. These results indicate that spike timing-dependent synaptic plasticity in afferent projections to the LA is both temporarily and spatially asymmetric, thus providing a mechanism for the conditioned stimulus discrimination during fear behavior.
Asunto(s)
Condicionamiento Psicológico/fisiología , Miedo/fisiología , Memoria/fisiología , Vías Nerviosas/fisiología , Plasticidad Neuronal/fisiología , Sinapsis/fisiología , Potenciales de Acción/fisiología , Amígdala del Cerebelo/fisiología , Animales , Corteza Cerebral/fisiología , Electrofisiología , Potenciales Postsinápticos Excitadores/fisiología , Glutamatos/fisiología , Técnicas In Vitro , Interneuronas/fisiología , Potenciación a Largo Plazo/fisiología , Vías Nerviosas/citología , Neuronas Aferentes/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiología , Transmisión Sináptica/fisiología , Tálamo/fisiologíaRESUMEN
Aberrant neurotransmissions via glutamate and dopamine receptors have been the focus of biomedical research on the molecular basis of psychiatric disorders, but the mode of their interaction is yet to be uncovered. In this study, we demonstrated the pharmacological reversal of methamphetamine-stimulated dopaminergic overflow by suppression of group I metabotropic glutamate (mGlu) receptor in living primates and rodents. In vivo positron emission tomography (PET) was conducted on cynomolgus monkeys and rats using a full agonistic tracer for dopamine D(2/3) receptor, [(11)C]MNPA [(R)-2-(11)CH(3)O-N-n-propylnorapomorphine], and fluctuation of kinetic data resulting from anesthesia was avoided by scanning awake subjects. Excessive release of dopamine induced by methamphetamine and abolishment of this alteration by treatment with an antagonist of group I mGlu receptors, 2-methyl-6-(phenylethynyl)pyridine (MPEP), were measured in both species as decreased binding potential because of increased dopamine and its recovery to baseline levels, respectively. Counteraction of MPEP to the methamphetamine-induced dopamine spillover was also supported neurochemically by microdialysis of unanesthetized rat striatum. Moreover, patch-clamp electrophysiological assays using acute brain slices prepared from rats indicated that direct targets of MPEP mechanistically involved in the effects of methamphetamine are present locally within the striatum. Because MPEP alone did not markedly alter the baseline dopaminergic neurotransmission according to our PET and electrophysiological data, the present findings collectively extend the insights on dopamine-glutamate cross talk from extrastriatal localization of responsible mGlu receptors to intrastriatal synergy and support therapeutic interventions in case of disordered striatal dopaminergic status using group I mGlu receptor antagonists assessable by in vivo imaging techniques.
Asunto(s)
Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/fisiología , Dopamina/fisiología , Ácido Glutámico/fisiología , Tomografía de Emisión de Positrones , Transmisión Sináptica/fisiología , Animales , Macaca , Masculino , Tomografía de Emisión de Positrones/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
Long-term synaptic modifications in afferent inputs to the amygdala underlie fear conditioning in animals. Fear conditioning to a single sensory modality does not generalize to other cues, implying that synaptic modifications in fear conditioning pathways are input specific. The mechanisms of pathway specificity of long-term potentiation (LTP) are poorly understood. Here we show that inhibition of glutamate transporters leads to the loss of input specificity of LTP in the amygdala slices, as assessed by monitoring synaptic responses at two independent inputs converging on a single postsynaptic neuron. Diffusion of glutamate ("spillover") from stimulated synapses, paired with postsynaptic depolarization, is sufficient to induce LTP in the heterosynaptic pathway, whereas an enzymatic glutamate scavenger abolishes this effect. These results establish active glutamate uptake as a crucial mechanism maintaining the pathway specificity of LTP in the neural circuitry of fear conditioning.
Asunto(s)
Condicionamiento Psicológico/fisiología , Miedo/fisiología , Ácido Glutámico/metabolismo , Potenciación a Largo Plazo/fisiología , Amígdala del Cerebelo/fisiología , Animales , Corteza Auditiva/fisiología , Calcio/metabolismo , Potenciales Postsinápticos Excitadores , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Vías Nerviosas/fisiología , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/fisiología , Temperatura , Tálamo/fisiologíaRESUMEN
Dopamine D4 receptors (D4R) are localized in the globus pallidus (GP), but their function remains unknown. In contrast, dopamine D2 receptor activation hyperpolarizes medium spiny neurons projecting from the striatum to the GP and inhibits GABA release. However, using slice preparations from D2R-deficient [D2 knock-out (D2KO)] mice, we found that dopamine inhibited GABA(A)-receptor-mediated currents in GP neurons. The paired-pulse ratio was statistically unchanged after dopamine application but was significantly elevated in D2KO wild-type littermates (WT). Furthermore, in D2KO mice, outward currents elicited by iontophoretically applied GABA were suppressed by dopamine. Dopamine (30 microm) decreased the amplitude of miniature IPSCs in both WT and D2KO mice, but the decrease in the frequency was observed only in the former but not significantly in the latter. Dopamine-induced suppression of IPSCs was blocked by selective D4R antagonists (clozapine or 3-[4-(4-iodophenyl)piperazin-1-yl]methyl-1H-pyrrolo[2,3-b]pyridine trihydrochloride), and a D4R-selective agonist N-[[4-(2-cyanophenyl)-1-piperazinyl]methyl]-3-methyl-benzamide reversibly and dose-dependently suppressed IPSCs, whereas agonists [SKF38,393 ((+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride) or (+)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol] or antagonists [SCH23,390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) or sulpiride] of other receptor subtypes had little effect. In GP neurons from D4R-deficient mice, dopamine-induced inhibition of GABAergic outward currents was undetectable. D4R activation suppressed the activity of protein kinase A in GP neurons, resulting in a decrease in the amplitude of GABAergic IPSCs. These findings showed that postsynaptic activation of D4R on the GP neurons reduces GABAergic currents through the suppression of PKA activity.
Asunto(s)
Dopamina/farmacología , Antagonistas del GABA/farmacología , Globo Pálido/fisiología , Neuronas/fisiología , Receptores de Dopamina D2/fisiología , Transmisión Sináptica , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Conductividad Eléctrica , Globo Pálido/citología , Ratones , Ratones Noqueados , Inhibición Neural , Neuronas/efectos de los fármacos , Neuronas/enzimología , Técnicas de Placa-Clamp , Receptores de Dopamina D2/genética , Receptores de Dopamina D4 , Receptores de GABA-A/metabolismo , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
It has been well known that long-term potentiation (LTP) of synaptic transmission in the lateral nucleus of the amygdala (LA) constitutes an essential cellular mechanism contributing to encoding of conditioned fear. Nitric oxide (NO), produced by activation of the postsynaptic N-methyl-D-aspartate receptors (NMDAR) in thalamic input to the LA, has been thought to promote LTP, contributing to the establishment of conditioned fear. However, it is not known whether and how NO, released from cortical input to the LA, plays the role on the plasticity induction and fear memory. Here we report that the diffusion of NO, released in response to activation of presynaptic NMDAR on cortical afferent fibers in the LA, could suppress heterosynaptically a form of presynaptic kainate receptor (KAR) dependent LTP (pre-LTP) in thalamic input, which was induced by low-frequency presynaptic stimuli without postsynaptic depolarization. We also confirmed that NO, produced by activation of postsynaptic NMDAR in thalamic input, can promote postsynaptic NMDAR-dependent LTP (post-LTP), which was induced by pairing protocol. These LTPs were occluded following fear conditioning, indicating that they could contribute to encoding of conditioned fear memory. However, their time courses are different; Post-LTP was more rapidly formed than pre-LTP in the course of fear conditioning. NO, produced by activation of presynaptic NMDAR in cortical input and postsynaptic NMDAR in thalamic input, may control conditioned fear by suppressing pre-LTP and promoting post-LTP, respectively, in thalamic input to the LA.
Asunto(s)
Amígdala del Cerebelo/fisiología , Plasticidad Neuronal/fisiología , Óxido Nítrico/metabolismo , Transducción de Señal , Animales , Condicionamiento Psicológico/fisiología , GMP Cíclico/metabolismo , Difusión , Miedo/fisiología , Potenciación a Largo Plazo/fisiología , Masculino , Memoria/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Ácido Kaínico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Tálamo/metabolismoRESUMEN
Little is known about the molecular mechanisms of learned and innate fear. We have identified stathmin, an inhibitor of microtubule formation, as highly expressed in the lateral nucleus (LA) of the amygdala as well as in the thalamic and cortical structures that send information to the LA about the conditioned (learned fear) and unconditioned stimuli (innate fear). Whole-cell recordings from amygdala slices that are isolated from stathmin knockout mice show deficits in spike-timing-dependent long-term potentiation (LTP). The knockout mice also exhibit decreased memory in amygdala-dependent fear conditioning and fail to recognize danger in innately aversive environments. By contrast, these mice do not show deficits in the water maze, a spatial task dependent on the hippocampus, where stathmin is not normally expressed. We therefore conclude that stathmin is required for the induction of LTP in afferent inputs to the amygdala and is essential in regulating both innate and learned fear.
Asunto(s)
Amígdala del Cerebelo/fisiología , Condicionamiento Psicológico/fisiología , Miedo/fisiología , Estatmina/fisiología , Amígdala del Cerebelo/metabolismo , Animales , Animales Recién Nacidos , Conducta Animal/fisiología , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiología , Electrofisiología , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Hipocampo/fisiología , Inmunohistoquímica , Hibridación in Situ , Técnicas In Vitro , Potenciación a Largo Plazo/fisiología , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/genética , Trastornos de la Memoria/fisiopatología , Ratones , Ratones Noqueados , Microtúbulos/metabolismo , Vías Nerviosas/fisiología , Neuronas/metabolismo , Receptores de GABA-A/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Estatmina/genética , Transmisión Sináptica/fisiología , Tálamo/metabolismo , Tálamo/fisiología , Factores de Tiempo , Tubulina (Proteína)/análisisRESUMEN
Mutant Cu/Zn-superoxide dismutase (SOD1) protein aggregation has been suggested as responsible for amyotrophic lateral sclerosis (ALS), although the operative mediating factors are as yet unestablished. To evaluate the contribution of motoneuronal Ca2+-permeable (GluR2 subunit-lacking) alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors to SOD1-related motoneuronal death, we generated chat-GluR2 transgenic mice with significantly reduced Ca2+-permeability of these receptors in spinal motoneurons. Crossbreeding of the hSOD1G93A transgenic mouse model of ALS with chat-GluR2 mice led to marked delay of disease onset (19.5%), mortality (14.3%) and the pathological hallmarks such as release of cytochrome c from mitochondria, induction of cox2 and astrogliosis. Subcellular fractionation analysis revealed that unusual SOD1 species first accumulated in two fractions dense with neurofilaments/glial fibrillary acidic protein/nuclei and mitochondria long time before disease onset, and then concentrated into the former fraction by disease onset. All these processes for unusual SOD1 accumulation were considerably delayed by GluR2 overexpression. Ca2+-influx through atypical motoneuronal AMPA receptors thus promotes a misfolding of mutant SOD1 protein and eventual death of these neurons.