RESUMEN
Despite the known causality of copy-number variations (CNVs) to human neurodevelopmental disorders, the mechanisms behind each gene's contribution to the constellation of neural phenotypes remain elusive. Here, we investigated the 7q11.23 CNV, whose hemideletion causes Williams syndrome (WS), and uncovered that mitochondrial dysfunction participates in WS pathogenesis. Dysfunction is facilitated in part by the 7q11.23 protein DNAJC30, which interacts with mitochondrial ATP-synthase machinery. Removal of Dnajc30 in mice resulted in hypofunctional mitochondria, diminished morphological features of neocortical pyramidal neurons, and altered behaviors reminiscent of WS. The mitochondrial features are consistent with our observations of decreased integrity of oxidative phosphorylation supercomplexes and ATP-synthase dimers in WS. Thus, we identify DNAJC30 as an auxiliary component of ATP-synthase machinery and reveal mitochondrial maladies as underlying certain defects in brain development and function associated with WS.
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Complejos de ATP Sintetasa/metabolismo , Encéfalo/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Mitocondrias/metabolismo , Síndrome de Williams/genética , Animales , Encéfalo/crecimiento & desarrollo , Células Cultivadas , Femenino , Células HEK293 , Proteínas del Choque Térmico HSP40/genética , Humanos , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación OxidativaRESUMEN
The newly evolved circuits in layer III of primate dorsolateral prefrontal cortex (dlPFC) generate the neural representations that subserve working memory. These circuits are weakened by increased cAMP-K+ channel signaling, and are a focus of pathology in schizophrenia, aging, and Alzheimer's disease. Cognitive deficits in these disorders are increasingly associated with insults to mGluR3 metabotropic glutamate receptors, while reductions in mGluR2 appear protective. This has been perplexing, as mGluR3 has been considered glial receptors, and mGluR2 and mGluR3 have been thought to have similar functions, reducing glutamate transmission. We have discovered that, in addition to their astrocytic expression, mGluR3 is concentrated postsynaptically in spine synapses of layer III dlPFC, positioned to strengthen connectivity by inhibiting postsynaptic cAMP-K+ channel actions. In contrast, mGluR2 is principally presynaptic as expected, with only a minor postsynaptic component. Functionally, increase in the endogenous mGluR3 agonist, N-acetylaspartylglutamate, markedly enhanced dlPFC Delay cell firing during a working memory task via inhibition of cAMP signaling, while the mGluR2 positive allosteric modulator, BINA, produced an inverted-U dose-response on dlPFC Delay cell firing and working memory performance. These data illuminate why insults to mGluR3 would erode cognitive abilities, and support mGluR3 as a novel therapeutic target for higher cognitive disorders.
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Memoria a Corto Plazo/fisiología , Neuronas/citología , Densidad Postsináptica/metabolismo , Corteza Prefrontal/metabolismo , Corteza Prefrontal/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Movimientos Oculares/efectos de los fármacos , Movimientos Oculares/fisiología , Femenino , Procesamiento de Imagen Asistido por Computador , Macaca mulatta , Imagen por Resonancia Magnética , Masculino , Memoria a Corto Plazo/efectos de los fármacos , Neuronas/metabolismo , Densidad Postsináptica/ultraestructura , Corteza Prefrontal/diagnóstico por imagen , Corteza Prefrontal/ultraestructura , Ratas , Receptores de Glutamato Metabotrópico/ultraestructura , Aprendizaje Espacial/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacosRESUMEN
Neurons in primary visual cortex (V1) are more resilient than those in dorsolateral prefrontal cortex (dlPFC) in aging, schizophrenia and Alzheimer's disease. The current study compared glutamate and neuromodulatory actions in macaque V1 to those in dlPFC, and found striking regional differences. V1 neuronal firing to visual stimuli depended on AMPA receptors, with subtle NMDA receptor contributions, while dlPFC depends primarily on NMDA receptors. Neuromodulatory actions also differed between regions. In V1, cAMP signaling increased neuronal firing, and the phosphodiesterase PDE4A was positioned to regulate cAMP effects on glutamate release from axons. HCN channels in V1 were classically located on distal dendrites, and enhanced cell firing. These data contrast with dlPFC, where PDE4A and HCN channels are concentrated in thin spines, and cAMP-HCN signaling gates inputs and weakens firing. These regional differences may explain why V1 neurons are more resilient than dlPFC neurons to the challenges of age and disease.
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Red Nerviosa/fisiología , Neuronas/fisiología , Corteza Prefrontal/citología , Sinapsis/fisiología , Corteza Visual/citología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Fármacos Cardiovasculares/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/ultraestructura , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/ultraestructura , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/farmacología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/ultraestructura , Macaca mulatta , Potenciales de la Membrana/efectos de los fármacos , Microscopía Inmunoelectrónica , Red Nerviosa/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Estimulación Luminosa , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/ultraestructuraRESUMEN
The prefrontal cortex (PFC) elaborates and differentiates in primates, and there is a corresponding elaboration in cortical dopamine (DA). DA cells that fire to both aversive and rewarding stimuli likely project to the dorsolateral PFC (dlPFC), signaling a salient event. Since 1979, we have known that DA has an essential influence on dlPFC working memory functions. DA has differing effects via D1 (D1R) versus D2 receptor (D2R) families. D1R are concentrated on dendritic spines, and D1/5R stimulation produces an inverted U-shaped dose response on visuospatial working memory performance and Delay cell firing, the neurons that generate representations of visual space. Optimal levels of D1R stimulation gate out "noise," whereas higher levels, e.g., during stress, suppress Delay cell firing. These effects likely involve hyperpolarization-activated cyclic nucleotide-gated channel opening, activation of GABA interneurons, and reduced glutamate release. Dysregulation of D1R has been related to cognitive deficits in schizophrenia, and there is a need for new, lower-affinity D1R agonists that may better mimic endogenous DA to enhance mental representations and improve cognition. In contrast to D1R, D2R are primarily localized on layer V pyramidal cell dendrites, and D2/3R stimulation speeds and magnifies the firing of Response cells, including Response Feedback cells. Altered firing of Feedback neurons may relate to positive symptoms in schizophrenia. Emerging research suggests that DA may have similar effects in the ventrolateral PFC and frontal eye fields. Research on the orbital PFC in monkeys is just beginning and could be a key area for future discoveries.
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Trastornos del Conocimiento/tratamiento farmacológico , Dopamina/metabolismo , Corteza Prefrontal/metabolismo , Animales , Trastornos del Conocimiento/fisiopatología , Agonistas de Dopamina/farmacología , Diseño de Fármacos , Humanos , Memoria a Corto Plazo/fisiología , Corteza Prefrontal/fisiopatología , Primates , Receptores Dopaminérgicos/efectos de los fármacos , Receptores Dopaminérgicos/metabolismo , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/fisiopatologíaRESUMEN
INTRODUCTION: An animal model of late-onset Alzheimer's disease is needed to research what causes degeneration in the absence of dominant genetic insults and why the association cortex is particularly vulnerable to degeneration. METHODS: We studied the progression of tau and amyloid cortical pathology in the aging rhesus macaque using immunoelectron microscopy and biochemical assays. RESULTS: Aging macaques exhibited the same qualitative pattern and sequence of tau and amyloid cortical pathology as humans, reaching Braak stage III/IV. Pathology began in the young-adult entorhinal cortex with protein kinase A-phosphorylation of tau, progressing to fibrillation with paired helical filaments and mature tangles in oldest animals. Tau pathology in the dorsolateral prefrontal cortex paralleled but lagged behind the entorhinal cortex, not afflicting the primary visual cortex. DISCUSSION: The aging rhesus macaque provides the long-sought animal model for exploring the etiology of late-onset Alzheimer's disease and for testing preventive strategies.
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Envejecimiento/patología , Enfermedad de Alzheimer/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Macaca mulatta , Amiloide/metabolismo , Animales , Encéfalo/patología , Corteza Entorrinal/patología , Microscopía Inmunoelectrónica/métodos , Ovillos Neurofibrilares/patología , Fosforilación , Placa Amiloide/patología , Corteza Prefrontal , Proteínas tau/metabolismoRESUMEN
The pattern of neurodegeneration in Alzheimer's disease (AD) is very distinctive: neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau selectively affect pyramidal neurons of the aging association cortex that interconnect extensively through glutamate synapses on dendritic spines. In contrast, primary sensory cortices have few NFTs, even in late-stage disease. Understanding this selective vulnerability, and why advancing age is such a high risk factor for the degenerative process, may help to reveal disease etiology and provide targets for intervention. Our study has revealed age-related increase in cAMP-dependent protein kinase (PKA) phosphorylation of tau at serine 214 (pS214-tau) in monkey dorsolateral prefrontal association cortex (dlPFC), which specifically targets spine synapses and the Ca(2+)-storing spine apparatus. This increase is mirrored by loss of phosphodiesterase 4A from the spine apparatus, consistent with increase in cAMP-Ca(2+) signaling in aging spines. Phosphorylated tau was not detected in primary visual cortex, similar to the pattern observed in AD. We also report electron microscopic evidence of previously unidentified vesicular trafficking of phosphorylated tau in normal association cortex--in axons in young dlPFC vs. in spines in aged dlPFC--consistent with the transneuronal lesion spread reported in genetic rodent models. pS214-Tau was not observed in normal aged mice, suggesting that it arises with the evolutionary expansion of corticocortical connections in primates, crossing the threshold into NFTs and degeneration in humans. Thus, the cAMP-Ca(2+) signaling mechanisms, needed for flexibly modulating network strength in young association cortex, confer vulnerability to degeneration when dysregulated with advancing age.
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Envejecimiento/patología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/patología , Corteza Prefrontal/enzimología , Corteza Prefrontal/patología , Proteínas tau/metabolismo , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Macaca mulatta , Ratones , Modelos Biológicos , Fosforilación , Transporte de Proteínas , Vesículas Transportadoras/metabolismoRESUMEN
The cognitive function of the highly evolved dorsolateral prefrontal cortex (dlPFC) is greatly influenced by arousal state, and is gravely afflicted in disorders such as schizophrenia, where there are genetic insults in α7 nicotinic acetylcholine receptors (α7-nAChRs). A recent behavioral study indicates that ACh depletion from dlPFC markedly impairs working memory [Croxson PL, Kyriazis DA, Baxter MG (2011) Nat Neurosci 14(12):1510-1512]; however, little is known about how α7-nAChRs influence dlPFC cognitive circuits. Goldman-Rakic [Goldman-Rakic (1995) Neuron 14(3):477-485] discovered the circuit basis for working memory, whereby dlPFC pyramidal cells excite each other through glutamatergic NMDA receptor synapses to generate persistent network firing in the absence of sensory stimulation. Here we explore α7-nAChR localization and actions in primate dlPFC and find that they are enriched in glutamate network synapses, where they are essential for dlPFC persistent firing, with permissive effects on NMDA receptor actions. Blockade of α7-nAChRs markedly reduced, whereas low-dose stimulation selectively enhanced, neuronal representations of visual space. These findings in dlPFC contrast with the primary visual cortex, where nAChR blockade had no effect on neuronal firing [Herrero JL, et al. (2008) Nature 454(7208):1110-1114]. We additionally show that α7-nAChR stimulation is needed for NMDA actions, suggesting that it is key for the engagement of dlPFC circuits. As ACh is released in cortex during waking but not during deep sleep, these findings may explain how ACh shapes differing mental states during wakefulness vs. sleep. The results also explain why genetic insults to α7-nAChR would profoundly disrupt cognitive experience in patients with schizophrenia.
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Cognición/fisiología , N-Metilaspartato/metabolismo , Corteza Prefrontal/fisiología , Receptores Nicotínicos/metabolismo , Sinapsis/fisiología , Percepción Visual/fisiología , Acetilcolina/metabolismo , Aconitina/análogos & derivados , Análisis de Varianza , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes , Agonistas Colinérgicos/administración & dosificación , Agonistas Colinérgicos/farmacología , Antagonistas Colinérgicos/administración & dosificación , Antagonistas Colinérgicos/farmacología , Femenino , Iontoforesis , Macaca mulatta , Masculino , Mecamilamina , Microscopía Inmunoelectrónica , Fenoles , Piperidinas , Corteza Prefrontal/metabolismo , Quinuclidinas , Conducta Espacial/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase that modulates key signaling molecules involved in synaptic plasticity and neuronal function. Targets include extracellular-regulated kinase 1 and 2 (ERK1/2), stress-activated protein kinase p38 (p38), the Src family tyrosine kinase Fyn, N-methyl-D-aspartate receptors (NMDARs), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). STEP-mediated dephosphorylation of ERK1/2, p38, and Fyn leads to inactivation of these enzymes, whereas STEP-mediated dephosphorylation of surface NMDARs and AMPARs promotes their endocytosis. Accordingly, the current model of STEP function posits that it opposes long-term potentiation and promotes long-term depression. Phosphorylation, cleavage, dimerization, ubiquitination, and local translation all converge to maintain an appropriate balance of STEP in the central nervous system. Accumulating evidence over the past decade indicates that STEP dysregulation contributes to the pathophysiology of several neuropsychiatric disorders, including Alzheimer's disease, schizophrenia, fragile X syndrome, epileptogenesis, alcohol-induced memory loss, Huntington's disease, drug abuse, stroke/ischemia, and inflammatory pain. This comprehensive review discusses STEP expression and regulation and highlights how disrupted STEP function contributes to the pathophysiology of diverse neuropsychiatric disorders.
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Trastornos Mentales , Enfermedades del Sistema Nervioso , Proteínas Tirosina Fosfatasas no Receptoras , Encéfalo/metabolismo , Dimerización , Humanos , Trastornos Mentales/tratamiento farmacológico , Trastornos Mentales/etiología , Trastornos Mentales/metabolismo , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/metabolismo , Fosforilación , Conformación Proteica , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/fisiología , Especificidad por SustratoRESUMEN
Schizophrenia associates with impaired prefrontal cortical (PFC) function and alterations in cyclic AMP (cAMP) signaling pathways. These include genetic insults to disrupted-in-schizophrenia (DISC1) and phosphodiesterases (PDE4s) regulating cAMP hydrolysis, and increased dopamine D1 receptor (D1R) expression that elevates cAMP. We used immunoelectron microscopy to localize DISC1, PDE4A, PDE4B, and D1R in monkey PFC and to view spatial interactions with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that gate network inputs when opened by cAMP. Physiological interactions between PDE4s and HCN channels were tested in recordings of PFC neurons in monkeys performing a spatial working memory task. The study reveals a constellation of cAMP-related proteins (DISC1, PDE4A, and D1R) and HCN channels next to excitatory synapses and the spine neck in thin spines of superficial PFC, where working memory microcircuits interconnect and spine loss is most evident in schizophrenia. In contrast, channels in dendrites were distant from synapses and cAMP-related proteins, and were associated with endosomal trafficking. The data suggest that a cAMP signalplex is selectively positioned in the spines to gate PFC pyramidal cell microcircuits. Single-unit recordings confirmed physiological interactions between cAMP and HCN channels, consistent with gating actions. These data may explain why PFC networks are especially vulnerable to genetic insults that dysregulate cAMP signaling.
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Espinas Dendríticas/metabolismo , Memoria a Corto Plazo/fisiología , Corteza Prefrontal/metabolismo , Esquizofrenia/metabolismo , Animales , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Inmunohistoquímica , Macaca mulatta , Trastornos de la Memoria/etiología , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Microscopía Inmunoelectrónica , Técnicas de Placa-Clamp , Corteza Prefrontal/fisiopatología , Receptores de Dopamina D1/metabolismo , Esquizofrenia/complicaciones , Esquizofrenia/fisiopatologíaRESUMEN
Regulator of G protein signaling 4 (RGS4) regulates intracellular signaling via G proteins and is markedly reduced in the prefrontal cortex (PFC) of patients with schizophrenia. Characterizing the expression of RGS4 within individual neuronal compartments is thus key to understanding its actions on individual G protein-coupled receptors (GPCRs). Here we present an ultrastructural reference map of RGS4 protein in macaque PFC based on immunogold electron microscopic analysis. At the soma, all labeling was asynaptic and affiliated with subsurface cistern microdomains of pyramidal neurons. The nucleus displayed most of immunoreactivity. RGS4 levels were particularly high along proximal apical dendrites and markedly decreased with distance from the soma; clustered label was present at the bifurcation into second-order branches. In distal dendrites and in spines, the protein was found flanking or directly facing the postsynaptic density of symmetric and asymmetric synapses. Axons also expressed RGS4. In fact, the density and distribution of pre- and postsynaptic labeling was correlated with the axon ultrastructure and the type of established synapses. The data indicate that RGS4 is strategically positioned to regulate not only postsynaptic but also presynaptic signaling in response to synaptic and nonsynaptic GPCR activation, having broad yet highly selective influences on multiple aspects of PFC cellular physiology.
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Corteza Prefrontal/metabolismo , Terminales Presinápticos/metabolismo , Proteínas RGS/metabolismo , Transducción de Señal/fisiología , Transmisión Sináptica/fisiología , Animales , Retroalimentación/fisiología , Macaca mulatta , Distribución TisularRESUMEN
Major Vault Protein (MVP), the main constituent of the vault ribonucleoprotein particle, is highly conserved in eukaryotic cells and upregulated in a variety of tumors. Vaults have been speculated to function as cargo transporters in several cell lines, yet no work to date has characterized the protein in neurons. Here we first describe the cellular and subcellular expression of MVP in primate and rodent cerebral cortex, and in cortical neurons in vitro. In prefrontal, somatosensory and hippocampal cortices, MVP was predominantly expressed in pyramidal neurons. Immunogold labeled free and attached ribosomes, and structures reminiscent of vaults on the rough endoplasmic reticulum and the nuclear envelope. The nucleus was immunoreactive in association with nucleopores. Axons and particularly principal dendrites expressed MVP along individual microtubules, and in pre- and postsynaptic structures. Synapses were not labeled. Colocalization with microtubule-associated protein-2, tubulin, tau, and phalloidin was observed in neurites and growth cones in culture. Immunoprecipitation coupled with reverse transcription PCR showed that MVP associates with mRNAs that are known to be translated in response to synaptic activity. Taken together, our findings provide the first characterization of neuronal MVP along the nucleus-neurite axis and may offer new insights into its possible function(s) in the brain.
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Núcleo Celular/metabolismo , Corteza Cerebral/metabolismo , Neuritas/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Animales , Conejos , Distribución TisularRESUMEN
cAMP signaling has powerful, negative effects on cognitive functions of the primate dorsolateral prefrontal cortex (dlPFC), opening potassium channels to reduce firing and impair working memory, and increasing tau phosphorylation in aging neurons. This contrasts with cAMP actions in classic circuits, where it enhances plasticity and transmitter release. PDE4 isozymes regulate cAMP actions, and thus have been a focus of research and drug discovery. Previous work has focused on the localization of PDE4A and PDE4B in dlPFC, but PDE4D is also of great interest, as it is the predominant PDE4 isoform in primate association cortex, and PDE4D expression decreases with aging in human dlPFC. Here we used laser-capture microdissection transcriptomics and found that PDE4D message is enriched in pyramidal cells compared to GABAergic PV-interneurons in layer III of the human dlPFC. A parallel study in rhesus macaques using high-spatial resolution immunoelectron microscopy revealed the ultrastructural locations of PDE4D in primate dlPFC with clarity not possible in human post-mortem tissue. PDE4D was especially prominent in dendrites associated with microtubules, mitochondria, and likely smooth endoplasmic reticulum (SER). There was substantial postsynaptic labeling in dendritic spines, associated with the SER spine-apparatus near glutamatergic-like axospinous synapses, but sparse labeling in axon terminals. We also observed dense PDE4D labeling perisynaptically in astroglial leaflets ensheathing glutamatergic connections. These data suggest that PDE4D is strategically positioned to regulate cAMP signaling in dlPFC glutamatergic synapses and circuits, especially in postsynaptic compartments where it is localized to influence cAMP actions on intracellular trafficking, mitochondrial physiology, and internal calcium release.
RESUMEN
Working memory relies on the dorsolateral prefrontal cortex (dlPFC), where microcircuits of pyramidal neurons enable persistent firing in the absence of sensory input, maintaining information through recurrent excitation. This activity relies on acetylcholine, although the molecular mechanisms for this dependence are not thoroughly understood. This study investigated the role of muscarinic M1 receptors (M1Rs) in the dlPFC using iontophoresis coupled with single-unit recordings from aging monkeys with naturally occurring cholinergic depletion. We found that M1R stimulation produced an inverted-U dose response on cell firing and behavioral performance when given systemically to aged monkeys. Immunoelectron microscopy localized KCNQ isoforms (Kv7.2, Kv7.3, and Kv7.5) on layer III dendrites and spines, similar to M1Rs. Iontophoretic manipulation of KCNQ channels altered cell firing and reversed the effects of M1R compounds, suggesting that KCNQ channels are one mechanism for M1R actions in the dlPFC. These results indicate that M1Rs may be an appropriate target to treat cognitive disorders with cholinergic alterations.
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Canales de Potasio KCNQ/metabolismo , Memoria a Corto Plazo/fisiología , Neuronas/metabolismo , Corteza Prefrontal/metabolismo , Receptor Muscarínico M1/metabolismo , Animales , Femenino , Macaca mulatta , MasculinoRESUMEN
Receptor signaling is mediated by direct protein interaction with various types of cytoskeletal, adapter, effector, and additional receptor molecules. In brain tissue and in cultured neurons, activation of dopamine D2 receptors (D2Rs) has been found to impact cellular calcium signaling. Using a yeast two-hybrid approach, we have uncovered a direct physical interaction between the D2R and the transient receptor potential channel (TRPC) subtypes 1, 4 and 5. The TRPC/D2R interaction was further validated by GST-pulldown assays and coimmunoprecipitation from mammalian brain. Ultrastructural analysis of TRPC1 and D2R expression indicates colocalization of the two proteins within the cell body and dendrites of cortical neurons. In cultured cells, expression of D2Rs was found to increase expression of TRPC1 at the cell surface by 50%. These findings shed new light on the constituents of the D2R signalplex, and support the involvement of D2Rs in cellular calcium signaling pathways via a novel link to TRPC channels.
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Receptores de Dopamina D2/metabolismo , Canales Catiónicos TRPC/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biotinilación , Línea Celular , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Humanos , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/metabolismo , Neuronas/ultraestructura , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Receptores de Dopamina D2/química , Sinapsis/metabolismo , Sinapsis/ultraestructura , Canales Catiónicos TRPC/químicaRESUMEN
The prefrontal cortex (PFC) mediates higher cognition but is impaired by stress exposure when high levels of catecholamines activate calcium-cAMP-protein kinase A (PKA) signaling. The current study examined whether stress and increased cAMP-PKA signaling in rat medial PFC (mPFC) reduce pyramidal cell firing and impair working memory by activating KCNQ potassium channels. KCNQ2 channels were found in mPFC layers II/III and V pyramidal cells, and patch-clamp recordings demonstrated KCNQ currents that were increased by forskolin or by chronic stress exposure, and which were associated with reduced neuronal firing. Low dose of KCNQ blockers infused into rat mPFC improved cognitive performance and prevented acute pharmacological stress-induced deficits. Systemic administration of low doses of KCNQ blocker also improved performance in young and aged rats, but higher doses impaired performance and occasionally induced seizures. Taken together, these data demonstrate that KCNQ channels have powerful influences on mPFC neuronal firing and cognitive function, contributing to stress-induced PFC dysfunction.
RESUMEN
Dorsolateral prefrontal cortex mediates high-order cognitive functions that are impaired early in the aging process in monkeys and humans. Here, we report pronounced changes in mitochondrial morphology in dendrites of dorsolateral prefrontal cortex neurons from aged rhesus macaques. Electron microscopy paired with 3D reconstruction from serial sections revealed an age-related increase in mitochondria with thin segments that intermingled with enlarged ones, the 'mitochondria-on-a-string' phenotype, similar to those recently reported in patients with Alzheimer's disease. The thin mitochondrial segments were associated with endoplasmic reticulum cisterns, and the mitochondrial proteins Fis1 and Drp1, all of which initiate mitochondrial fission. These data suggest that the 'mitochondria-on-a-string' phenotype may reflect malfunction in mitochondrial dynamics, whereby fission is initiated, but the process is incomplete due to malfunction of subsequent step(s). Thus, aged rhesus monkeys may be particularly helpful in exploring the age-related changes that render higher cortical circuits so vulnerable to degeneration.
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Envejecimiento/patología , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Dinámicas Mitocondriales , Corteza Prefrontal/ultraestructura , Animales , Dendritas/ultraestructura , Imagenología Tridimensional , Macaca mulatta , Microscopía Electroquímica de Rastreo , Mitocondrias/patología , Neuronas/ultraestructura , Corteza Prefrontal/patologíaRESUMEN
Dopaminergic modulation of glutamate neurotransmission in prefrontal cortex (PFC) microcircuits is commonly perceived as a basis for cognitive operations. Yet it appears that although the control of recurrent excitation between deep-layer prefrontal pyramids may involve presynaptic and postsynaptic D1 receptor (D1R) mechanisms, pyramid-to-interneuron communication will engage a postsynaptic D1R component. The substrate underlying such target-specific neuromodulatory patterns was investigated in the infragranular PFC with immunoelectron microscopy for D1R and parvalbumin, a marker for fast-spiking interneurons. In addition to their proverbial postsynaptic expression, gold-labeled D1Rs were distinctly distributed on perisynaptic/extrasynaptic membranes and the axoplasm of 13% of excitatory-like, presumably glutamatergic varicosities. Most importantly, presynaptic D1Rs were highly specific with regard to the cellular compartment and neurochemical identity of the postsynaptic neuron, being present in spine-targeting varicosities but distinctly absent from those synapsing with parvalbumin profiles often coexpressing D1Rs. We define therein an axonal D1 heteroreceptor component, apparently mediating volume neurotransmission, yet strategically positioned to convey target cell-specific modulation of the glutamatergic drive. We also indicate that presynaptic D1R mechanisms may indeed be associated with recurrent excitation in prefrontal microcircuits, consistent with physiological evidence for a role of these receptors in modulating the persistent activity-profile of neurons essential for working memory.
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Ácido Glutámico/fisiología , Interneuronas/fisiología , Corteza Prefrontal/fisiología , Receptores de Dopamina D1/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Biomarcadores , Membrana Celular/ultraestructura , Dopamina/fisiología , Inmunohistoquímica , Interneuronas/química , Interneuronas/ultraestructura , Macaca mulatta , Microscopía Inmunoelectrónica , Parvalbúminas/fisiología , Terminales Presinápticos/química , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Células Piramidales/química , Células Piramidales/fisiología , Células Piramidales/ultraestructuraRESUMEN
The explicit yet enigmatic involvement of dopamine in cortical physiology is in part volumetric (beyond the synapse), as is apparently the action of neuroleptics targeting dopamine receptors. The notion that nonsynaptic neuronal membranes would translate extracellular dopamine into receptor-specific spatiotemporal downstream signaling, similar to the chemical synapse, is intriguing. Here, we report that dopamine D5 (but not D1 or D2) receptors in the perisomatic plasma membrane of prefrontal cortical neurons form discrete and exclusively extrasynaptic microdomains with inositol 1,4,5-trisphosphate-gated calcium stores of subsurface cisterns and mitochondria. These findings introduce a novel dopaminoceptive substratum in the brain and a unique D5 receptor-specific signaling paradigm.
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Dopamina/fisiología , Microdominios de Membrana/fisiología , Neuronas/fisiología , Corteza Prefrontal/fisiología , Receptores de Dopamina D1/fisiología , Transmisión Sináptica , Animales , Canales de Calcio/fisiología , Dopamina/metabolismo , Técnicas para Inmunoenzimas , Inmunohistoquímica , Receptores de Inositol 1,4,5-Trifosfato , Macaca mulatta , Microdominios de Membrana/metabolismo , Microscopía Inmunoelectrónica , Neuronas/metabolismo , Corteza Prefrontal/citología , Corteza Prefrontal/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/fisiología , Receptores de Dopamina D5 , Transducción de SeñalRESUMEN
BACKGROUND: Psychiatric disorders such as schizophrenia are worsened by stress, and working memory deficits are often a central feature of illness. Working memory is mediated by the persistent firing of prefrontal cortical (PFC) pyramidal neurons. Stress impairs working memory via high levels of dopamine D1 receptor (D1R) activation of cyclic adenosine monophosphate signaling, which reduces PFC neuronal firing. The current study examined whether D1R-cyclic adenosine monophosphate signaling reduces neuronal firing and impairs working memory by increasing the open state of hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels, which are concentrated on dendritic spines where PFC pyramidal neurons interconnect. METHODS: A variety of methods were employed to test this hypothesis: dual immunoelectron microscopy localized D1R and HCN channels, in vitro recordings tested for D1R actions on HCN channel current, while recordings in monkeys performing a working memory task tested for D1R-HCN channel interactions in vivo. Finally, cognitive assessments following intra-PFC infusions of drugs examined D1R-HCN channel interactions on working memory performance. RESULTS: Immunoelectron microscopy confirmed D1R colocalization with HCN channels near excitatory-like synapses on dendritic spines in primate PFC. Mouse PFC slice recordings demonstrated that D1R stimulation increased HCN channel current, while local HCN channel blockade in primate PFC protected task-related firing from D1R-mediated suppression. D1R stimulation in rat or monkey PFC impaired working memory performance, while HCN channel blockade in PFC prevented this impairment in rats exposed to either stress or D1R stimulation. CONCLUSIONS: These findings suggest that D1R stimulation or stress weakens PFC function via opening of HCN channels at network synapses.
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Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/fisiología , Memoria a Corto Plazo/fisiología , Corteza Prefrontal/fisiología , Células Piramidales/fisiología , Receptores de Dopamina D1/fisiología , Estrés Fisiológico , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/antagonistas & inhibidores , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Macaca mulatta , Masculino , Ratones , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/ultraestructura , Células Piramidales/efectos de los fármacos , Células Piramidales/ultraestructura , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
This review describes unique neuromodulatory influences on working memory prefrontal cortical (PFC) circuits that coordinate cognitive strength with arousal state. Working memory arises from recurrent excitation within layer III PFC pyramidal cell NMDA circuits, which are afflicted in aging and schizophrenia. Neuromodulators rapidly and flexibly alter the efficacy of these synaptic connections, while leaving the synaptic architecture unchanged, a process called dynamic network connectivity (DNC). Increases in calcium-cAMP signaling open ion channels in long, thin spines, gating network connections. Inhibition of calcium-cAMP signaling by stimulating α2A-adrenoceptors on spines strengthens synaptic efficacy and increases network firing, whereas optimal stimulation of dopamine D1 receptors sculpts network inputs to refine mental representation. Generalized increases in calcium-cAMP signaling during fatigue or stress disengage dlPFC recurrent circuits, reduce firing and impair top-down cognition. Impaired DNC regulation contributes to age-related cognitive decline, while genetic insults to DNC proteins are commonly linked to schizophrenia.