RESUMO
Mutations in the SCN9A gene leading to deficiency of its protein product, Na(v)1.7, cause congenital indifference to pain (CIP). CIP is characterized by the absence of the ability to sense pain associated with noxious stimuli. In contrast, the opposite phenotype to CIP, inherited erythromelalgia (IEM), is a disorder of spontaneous pain caused by missense mutations resulting in gain-of-function in Na(v)1.7 that promote neuronal hyperexcitability. The primary aim of this study was to demonstrate that Na(v)1.7 antagonism could alleviate the pain of IEM, thereby demonstrating the utility of this opposite phenotype model as a tool for rapid proof-of-concept for novel analgesics. An exploratory, randomized, double-blind, 2-period crossover study was conducted in 4 SCN9A mutation-proven IEM patients. In each treatment period (2days), separated by a 2-day washout period, patients were orally administered XEN402 (400mg twice daily) or matching placebo. In 3 patients, pain was induced by heat or exercise during each treatment arm. A fourth patient, in constant severe pain, required no induction. Patient-reported outcomes of pain intensity and/or relief were recorded, and the time taken to induce pain was measured. The ability to induce pain in IEM patients was significantly attenuated by XEN402 compared with placebo. XEN402 increased the time to maximal pain induction and significantly reduced the amount of pain (42% less) after induction (P=.014). This pilot study showed that XEN402 blocks Na(v)1.7-mediated pain associated with IEM, thereby demonstrating target engagement in humans and underscoring the use of rare genetic disorders with mutant target channels as a novel approach to rapid proof-of-concept.
Assuntos
Eritromelalgia/tratamento farmacológico , Bloqueadores dos Canais de Sódio/uso terapêutico , Canais de Sódio/genética , Adulto , Método Duplo-Cego , Eritromelalgia/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.7 , Medição da Dor , Projetos Piloto , Resultado do TratamentoRESUMO
Recent studies indicate that levels of presynaptic proteins are altered in the post-mortem brain in schizophrenia. In particular, the hippocampus exhibits reduced levels of synaptophysin and the SNARE protein SNAP-25. The effects of treatment with antipsychotic drugs on levels of SNAP-25 in the hippocampus remains unknown. To determine the effects of typical antipsychotic drugs on levels of synaptophysin and SNAP-25 in the hippocampus, rats were treated with chlorpromazine, haloperidol or trifluoperazine for 21 d. Quantitative immunohistochemistry was used to measure immunoreactivity within the trisynaptic circuit of the hippocampus. Trifluoperazine decreased synaptophysin within the Schaffer collateral region of the radiatum lacunosum in CA1, while haloperidol and chlorpromazine increased SNAP-25 throughout the trisynaptic pathway of the hippocampus, with strongest effects in the mossy fibre region of CA3. These results indicate that presynaptic proteins represent a potential molecular substrate for the effects of antipsychotic drugs on hippocampal synaptic connectivity.
Assuntos
Antipsicóticos/farmacologia , Hipocampo/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinaptofisina/biossíntese , Proteína 25 Associada a Sinaptossoma/biossíntese , Animais , Hipocampo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sinapses/metabolismoRESUMO
The mesocortical dopamine (DA) input to the prefrontal cortex (PFC) is crucial for processing short-term working memory (STWM) to guide forthcoming behavior. Short-term plasticity-like post-tetanic potentiation (PTP, < 3 min) and short-term potentiation (STP, < 10 min) may underlie STWM. Using whole-cell voltage-clamp recordings, mixed glutamatergic excitatory postsynaptic currents (EPSCs) evoked by layer III or layer V stimulation (0.5 or 0.067 Hz) were recorded from layer V pyramidal neurons. With 0.5 Hz basal stimulation of layer III, brief tetani (2 x 50 Hz) induced a homosynaptic PTP (decayed: approximately 1 min). The D1-like antagonist SCH23390 (1 microm) increased the PTP amplitude and decay time without inducing changes to the tetanic response. The tetani may evoke endogenous DA release, which activates a presynaptic D1-like receptor to inhibit glutamate release to modulate PTP. With a slower (0.067 Hz) basal stimulation, the same tetani induced STP (lasting approximately 4 min, but only at 2x intensity only) that was insignificantly suppressed by SCH23390. With stimulation of layer-V-->V inputs at 0.5 Hz, layer V tetani yielded inconsisitent responses. However, at 0.067 Hz, tetani at double the intensity resulted in an STP (lasting approximately 6 min), but a long-term depression after SCH23390 application. Endogenous DA released by tetanic stimulation can interact with a D1-like receptor to induce STP in layer V-->V synapses that receive slower (0.067 Hz) frequency inputs, but suppresses PTP at layer III-->V synapses that receive higher (0.5 Hz) frequency inputs. This D1-like modulation of layer- and frequency-specific synaptic responses in the PFC may contribute to STWM processing.
Assuntos
Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Córtex Pré-Frontal/citologia , Receptores de Dopamina D1/fisiologia , Animais , Animais Recém-Nascidos , Área Sob a Curva , Benzazepinas/farmacologia , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Masculino , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Ratos , Ritmo Teta/efeitos dos fármacos , Ritmo Teta/efeitos da radiação , Fatores de TempoRESUMO
BACKGROUND: Converging neuroimaging and postmortem evidence indicates synaptic terminals are abnormal in schizophrenia. A putative molecular mechanism implicates abnormalities of proteins involved in the presynaptic secretory machinery, including the modulator proteins complexin I and complexin II. OBJECTIVES: To determine the amount and distribution of complexin proteins in the hippocampus of subjects with schizophrenia, in parallel with markers for excitatory and inhibitory nerve terminals. The functional implications were also investigated. DESIGN: We used immunocytochemistry to study complexin I and complexin II proteins in hippocampus, as well as the vesicular transporters for gamma-aminobutyric acid (GABA) and for glutamate. Immunocytochemical findings were correlated with cognitive function assessed through medical record review. To further explore the implications of the human findings, we studied rats exposed to haloperidol, amphetamine, and ketamine as well as rats trained in memory tasks. SUBJECTS: We studied hippocampal sections from 12 subjects with schizophrenia and 12 subjects with no known neuropsychiatric disorder. RESULTS: The absolute values and ratio of the hippocampal presynaptic proteins complexin II-complexin I were lower in subjects with schizophrenia. Disturbances in the complexin proteins in subjects with schizophrenia were greater than those observed for vesicular gamma-aminobutyric acid or vesicular glutamate transporters. The lower complexin II-complexin I ratio in several hippocampal subfields in subjects with schizophrenia was inversely correlated with the severity of antemortem cognitive impairment. In contrast, the hippocampal complexin II-complexin I ratio was higher in rats trained in a memory task compared with untrained rats. Treatment of rats with antipsychotic drugs or with the psychotomimetic drugs amphetamine or ketamine did not alter the complexin II-complexin I ratio. CONCLUSIONS: The pathology of hippocampal complexin proteins might play an important role in schizophrenia, especially concerning cognitive disturbances.
Assuntos
Transtornos Cognitivos/diagnóstico , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Esquizofrenia/diagnóstico , Psicologia do Esquizofrênico , Proteínas Adaptadoras de Transporte Vesicular , Idoso , Animais , Antipsicóticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Transtornos Cognitivos/induzido quimicamente , Transtornos Cognitivos/metabolismo , Modelos Animais de Doenças , Feminino , Haloperidol/farmacologia , Hipocampo/fisiopatologia , Humanos , Imuno-Histoquímica , Ketamina/farmacologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/fisiologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Sprague-Dawley , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatologia , Proteína Vesicular 1 de Transporte de Glutamato , Proteínas Vesiculares de Transporte de Aminoácidos InibidoresRESUMO
Post-weaning social isolation-rearing of rats leads to behavioural and neurochemical sequelae that model aspects of schizophrenia, and it may be useful to test hypotheses related to putative molecular mechanisms of the illness. In humans, the presynaptic protein CDCrel-1 represents an interesting candidate molecule for the mechanism and aetiology of schizophrenia. CDCrel-1 modulates dopamine neurotransmission, binds to the SNARE protein syntaxin and maps onto a region of chromosome 22q11 deleted in velo-cardio-facial and DiGeorge syndromes, which are associated with increased prevalence of schizophrenia. Using the isolation-rearing model, we measured immunoreactivity of the synaptic proteins CDCrel-1, synaptophysin and syntaxin. Male, Sprague-Dawley rats were raised in groups or in isolation for 12 weeks from weaning. Synaptic protein immunoreactivities were measured in striatal and hippocampal homogenates, using a sensitive enzyme-linked immunoadsorbent assay with monoclonal antibodies. Isolation-rearing produced region- and protein-specific effects. CDCrel-1 immunoreactivity was significantly lower in the striatum and marginally higher in the hippocampus of isolation-reared compared with socially reared animals. There were no statistically significant differences in synaptophysin immunoreactivity in either region. Confocal microscopy demonstrated a high degree of colocalization between the two presynaptic proteins. In striatum, a robust relationship between CDCrel-1 and syntaxin immunoreactivities was observed in socially reared rats, this was lost in the isolation-reared animals. Altered levels of the septin CDCrel-1 in isolation-reared rats may contribute to changes in neuronal connectivity and neurotransmission, and suggest a potential role for CDCrel-1 in schizophrenia related to chromosome 22q11 deletion syndrome.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Neostriado/metabolismo , Isolamento Social , Animais , Comportamento Animal , Western Blotting/métodos , Proteínas de Ciclo Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Hipocampo/metabolismo , Imuno-Histoquímica/métodos , Masculino , Proteínas de Membrana/metabolismo , Proteínas Qa-SNARE , Ratos , Ratos Sprague-Dawley , Septinas , Sinaptofisina/metabolismoAssuntos
Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Esquizofrenia/metabolismo , Proteínas de Transporte Vesicular , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Proteínas Sensíveis a N-Etilmaleimida , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Análise Serial de Proteínas/métodos , RNA Mensageiro/metabolismoRESUMO
To determine the nature of dopamine modulation of dendritic Ca2+ signaling in layers V-VI prefrontal cortex (PFC) neurons, whole-cell Ca2+ potentials were evoked after blockade of Na+ and K+ channels. Soma-dendritic Ca2+ spikes evoked by suprathreshold depolarizing pulses, which could be terminated by superimposed brief intrasomatic hyperpolarizing pulses, are blocked by the L-type Ca2+ channel antagonist nimodipine (1 microM). The D1/D5 receptor agonist dihydrexidine (DHX) (0.01-10 microM; 5 min) or R-(+)SKF81291 (10 microM) induced a prolonged (>30 min) dose-dependent peak suppression of these Ca2+ spikes. This effect was dependent on [Ca2+]i- and protein kinase C (PKC)-dependent mechanisms because [Ca2+]i chelation by BAPTA or inhibition of PKC by bisindolymaleimide (BiM1), but not inhibition of [Ca2+]i release with heparin or Xestospongin C, prevented the D1-mediated suppression of Ca2+ spikes. Depolarizing pulses subthreshold to activating a Ca2+ spike evoked a nimodipine-sensitive Ca2+ "hump" potential. D1/D5 stimulation induced an N-[2-((o-bromocinamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89)- or internal PKA inhibitory peptide[5-24]-sensitive (PKA-dependent) transient (approximately 7 min) potentiation of the hump potential to full Ca2+ spike firing. Furthermore, application of DHX in the presence of the PKC inhibitor BiM1 or internal PKC inhibitory peptide[19-36] resulted in persistent firing of full Ca2+ spike bursts, suggesting that a D1/D5-PKA mechanism switches subthreshold Ca2+ hump potential to fire full Ca2+ spikes, which are eventually turned off by a D1/D5-Ca2+-dependent PKC mechanism. This depolarizing state-dependent, D1/D5-activated, bi-directional switching of soma-dendritic L-type Ca2+ channels via PKA-dependent potentiation and PKC-dependent suppression may provide spatiotemporal regulation of synaptic integration and plasticity in PFC.
Assuntos
Sinalização do Cálcio , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Córtex Pré-Frontal/fisiologia , Proteína Quinase C/metabolismo , Células Piramidais/fisiologia , Receptores de Dopamina D1/metabolismo , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Dendritos/enzimologia , Dendritos/fisiologia , Ativação Enzimática , Potenciais Evocados , Receptores de Inositol 1,4,5-Trifosfato , Transporte de Íons , Cinética , Masculino , Plasticidade Neuronal , Nimodipina/farmacologia , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Córtex Pré-Frontal/citologia , Células Piramidais/citologia , Células Piramidais/enzimologia , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Dopamina D5RESUMO
The bacterial endotoxin lipopolysaccharide (LPS) produces a host of effects in mammals known collectively as 'sickness' behaviours. Acute treatment with LPS also results in a loss of hedonic capacity in rodents that can be measured by changes in responding for reinforcing electrical stimulation of the lateral hypothalamus. In contrast, repeated exposure to LPS typically leads to the development of tolerance to many of the physiological and behavioural effects of endotoxin, although the effect of chronic treatment with LPS on anhedonia remains unknown. In the present experiment, rats were trained to respond on an ascending-series current-intensity intracranial self-stimulation (ICSS) protocol, and were then treated with either acute or sub-chronic LPS (100 microg). Compared to vehicle-treated subjects, acute exposure to LPS induced a dramatic loss of ICSS responding; however, with repeated exposure to LPS, rats developed a behavioural tolerance to its anhedonic effects. To investigate a potential molecular substrate for the anhedonic effects of LPS, quantitative immunohistochemistry was used to measure levels of the synaptic proteins syntaxin, SNAP-25 and synaptophysin in the dorsal and ventral striatum of rats treated acutely and sub-chronically with LPS. A single injection of LPS produced a significant decrease in syntaxin immunoreactivity in the nucleus accumbens core and shell, while similar treatment in chronically treated rats that displayed behavioural tolerance had no effect. These results demonstrate a novel molecular substrate for the effects of LPS, and imply that the underlying physiology of the transient anhedonic effects of LPS may differ from that involved in chronic psychiatric disorders in humans.
Assuntos
Tolerância a Medicamentos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Núcleo Accumbens/metabolismo , Proteínas Qa-SNARE/metabolismo , Autoestimulação/efeitos dos fármacos , Análise de Variância , Animais , Biofísica , Condicionamento Operante/efeitos dos fármacos , Estimulação Elétrica/métodos , Masculino , Núcleo Accumbens/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteína 25 Associada a Sinaptossoma/metabolismo , Fatores de TempoRESUMO
The role of integrin-linked kinase (ILK), a kinase that is involved in various cellular processes, including adhesion and migration, has not been studied in primary neurons. Using mRNA dot blot and Western blot analysis of ILK in rat and human brain tissue, we found that ILK is expressed in various regions of the CNS. Immunohistochemical and immunocytochemical techniques revealed granular ILK staining that is enriched in neurons and colocalizes with the beta1 integrin subunit. The role of ILK in neurite growth promotion by NGF was studied in rat pheochromocytoma cells and dorsal root ganglion neurons using a pharmacological inhibitor of ILK (KP-392) or after overexpression of dominant-negative ILK (ILK-DN). Both molecular and pharmacological inhibition of ILK activity significantly reduced NGF-induced neurite outgrowth. Survival assays indicate that KP-392-induced suppression of neurite outgrowth occurred in the absence of cell death. ILK kinase activity was stimulated by NGF. NGF-mediated stimulation of phosphorylation of both AKT and the Tau kinase glycogen synthase kinase-3 (GSK-3) was inhibited in the presence of KP-392 and after overexpression of ILK-DN. Consequently, ILK inhibition resulted in an increase in the hyperphosphorylation of Tau, a substrate of GSK-3. Together these findings indicate that ILK is an important effector in NGF-mediated neurite outgrowth.