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1.
Nat Commun ; 12(1): 781, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33536416

RESUMO

After complete spinal cord injuries (SCI), spinal segments below the lesion maintain inter-segmental communication via the intraspinal propriospinal network. However, it is unknown whether selective manipulation of these circuits can restore locomotor function in the absence of brain-derived inputs. By taking advantage of the compromised blood-spinal cord barrier following SCI, we optimized a set of procedures in which AAV9 vectors administered via the tail vein efficiently transduce neurons in lesion-adjacent spinal segments after a thoracic crush injury in adult mice. With this method, we used chemogenetic actuators to alter the excitability of propriospinal neurons in the thoracic cord of the adult mice with a complete thoracic crush injury. We showed that activating these thoracic neurons enables consistent and significant hindlimb stepping improvement, whereas direct manipulations of the neurons in the lumbar spinal cord led to muscle spasms without meaningful locomotion. Strikingly, manipulating either excitatory or inhibitory propriospinal neurons in the thoracic levels leads to distinct behavioural outcomes, with preferential effects on standing or stepping, two key elements of the locomotor function. These results demonstrate a strategy of engaging thoracic propriospinal neurons to improve hindlimb function and provide insights into optimizing neuromodulation-based strategies for treating SCI.


Assuntos
Dependovirus/genética , Membro Posterior/fisiopatologia , Locomoção/fisiologia , Neurônios/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Animais , Antipsicóticos/administração & dosagem , Clozapina/administração & dosagem , Clozapina/análogos & derivados , Vetores Genéticos/genética , Membro Posterior/inervação , Locomoção/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/terapia
2.
Nat Neurosci ; 23(9): 1157-1167, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32632286

RESUMO

The chemogenetic technology designer receptors exclusively activated by designer drugs (DREADDs) afford remotely reversible control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, sluggish kinetics, metabolic liabilities and potential off-target effects of CNO represent areas for improvement. Here, we provide a new high-affinity and selective agonist deschloroclozapine (DCZ) for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 µg per kg) enhanced neuronal activity via hM3Dq within minutes in mice and monkeys. Intramuscular injections of DCZ (100 µg per kg) reversibly induced spatial working memory deficits in monkeys expressing hM4Di in the prefrontal cortex. DCZ represents a potent, selective, metabolically stable and fast-acting DREADD agonist with utility in both mice and nonhuman primates for a variety of applications.


Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Clozapina/análogos & derivados , Drogas Desenhadas/farmacologia , Neurônios/efeitos dos fármacos , Animais , Clozapina/farmacologia , Técnicas Genéticas , Humanos , Macaca fuscata , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M4/metabolismo
3.
Am J Physiol Heart Circ Physiol ; 319(1): H3-H12, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32412778

RESUMO

Heart failure (HF) is characterized by autonomic imbalance with sympathetic hyperactivity and loss of parasympathetic tone. Intracardiac ganglia (ICG) neurons represent the final common pathway for vagal innervation of the heart and strongly regulate cardiac functions. This study tests whether ICG cholinergic neuron activation mitigates the progression of cardiac dysfunction and reduces mortality that occurs in HF. HF was induced by transaortic constriction (TAC) in male transgenic Long-Evans rats expressing Cre recombinase within choline acetyltransferase (ChAT) neurons. ChAT neurons were selectively activated by expression and activation of excitatory designer receptors exclusively activated by designer receptors (DREADDs) by clozapine-N-oxide (TAC + treatment and sham-treated groups). Control animals expressed DREADDs but received saline (sham and TAC groups). A separate set of animals were telemetry instrumented to record blood pressure (BP) and heart rate (HR). Acute activation of ICG neurons resulted in robust reductions in BP (∼20 mmHg) and HR (∼100 beats/min). All groups of animals were subjected to weekly echocardiography and treadmill stress tests from 3 to 6 wk post-TAC/sham surgery. Activation of ICG cholinergic neurons reduced the left ventricular systolic dysfunction (reductions in ejection fraction, fractional shortening, stroke volume, and cardiac output) and cardiac autonomic dysfunction [reduced HR recovery (HRR) post peak effort] observed in TAC animals. Additionally, activation of ICG ChAT neurons reduced mortality by 30% compared with untreated TAC animals. These data suggest that ICG cholinergic neuron activation reduces cardiac dysfunction and improves survival in HF, indicating that ICG neuron activation could be a novel target for treating HF.NEW & NOTEWORTHY Intracardiac ganglia form the final common pathway for the parasympathetic innervation of the heart. This study has used a novel chemogenetic approach within transgenic ChAT-Cre rats [expressing only Cre-recombinase in choline acetyl transferase (ChAT) neurons] to selectively increase intracardiac cholinergic parasympathetic activity to the heart in a pressure overload-induced heart failure model. The findings from this study confirm that selective activation of intracardiac cholinergic neurons lessens cardiac dysfunction and mortality seen in heart failure, identifying a novel downstream cardiac-selective target for increasing cardioprotective parasympathetic activity in heart failure.


Assuntos
Neurônios Colinérgicos/fisiologia , Insuficiência Cardíaca/fisiopatologia , Coração/inervação , Função Ventricular , Animais , Sistema Nervoso Autônomo/fisiopatologia , Pressão Sanguínea , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Clozapina/análogos & derivados , Clozapina/farmacologia , Coração/fisiopatologia , Insuficiência Cardíaca/etiologia , Frequência Cardíaca , Masculino , Ratos , Ratos Long-Evans , Obstrução do Fluxo Ventricular Externo/complicações
4.
Nat Neurosci ; 23(6): 730-740, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393896

RESUMO

Descending command neurons instruct spinal networks to execute basic locomotor functions, such as gait and speed. The command functions for gait and speed are symmetric, implying that a separate unknown system directs asymmetric movements, including the ability to move left or right. In the present study, we report that Chx10-lineage reticulospinal neurons act to control the direction of locomotor movements in mammals. Chx10 neurons exhibit mainly ipsilateral projection, and their selective unilateral activation causes ipsilateral turning movements in freely moving mice. Unilateral inhibition of Chx10 neurons causes contralateral turning movements. Paired left-right motor recordings identified distinct mechanisms for directional movements mediated via limb and axial spinal circuits. Finally, we identify sensorimotor brain regions that project on to Chx10 reticulospinal neurons, and demonstrate that their unilateral activation can impart left-right directional commands. Together these data identify the descending motor system that commands left-right locomotor asymmetries in mammals.


Assuntos
Tronco Encefálico/fisiologia , Vias Eferentes/fisiologia , Locomoção/fisiologia , Neurônios/fisiologia , Animais , Clozapina/análogos & derivados , Clozapina/farmacologia , Proteínas de Homeodomínio/imunologia , Camundongos , Técnicas de Rastreamento Neuroanatômico , Neurônios/efeitos dos fármacos , Toxina Tetânica/farmacologia , Fatores de Transcrição/imunologia
5.
Sci Rep ; 10(1): 6884, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327679

RESUMO

Tremendous individual differences exist in stress responsivity and social defeat stress is a key approach for identifying cellular mechanisms of stress susceptibility and resilience. Syrian hamsters show reliable territorial aggression, but after social defeat they exhibit a conditioned defeat (CD) response characterized by increased submission and an absence of aggression in future social interactions. Hamsters that achieve social dominance prior to social defeat exhibit greater defeat-induced neural activity in infralimbic (IL) cortex neurons that project to the basolateral amygdala (BLA) and reduced CD response compared to subordinate hamsters. Here, we hypothesize that chemogenetic activation of an IL-to-BLA neural projection during acute social defeat will reduce the CD response in subordinate hamsters and thereby produce dominant-like behavior. We confirmed that clozapine-N-oxide (CNO) itself did not alter the CD response and validated a dual-virus, Cre-dependent, chemogenetic approach by showing that CNO treatment increased c-Fos expression in the IL and decreased it in the BLA. We found that CNO treatment during social defeat reduced the acquisition of CD in subordinate, but not dominant, hamsters. This project extends our understanding of the neural circuits underlying resistance to acute social stress, which is an important step toward delineating circuit-based approaches for the treatment of stress-related psychopathologies.


Assuntos
Tonsila do Cerebelo/patologia , Complexo Nuclear Basolateral da Amígdala/patologia , Comportamento Social , Estresse Psicológico/patologia , Agressão , Animais , Clozapina/análogos & derivados , Condicionamento Clássico , Cricetinae , Vetores Genéticos/metabolismo , Masculino , Córtex Pré-Frontal/patologia , Proteínas Proto-Oncogênicas c-fos/metabolismo
6.
Rev. psiquiatr. salud ment. (Barc., Ed. impr.) ; 13(1): 31-35, ene.-mar. 2020. tab, graf
Artigo em Espanhol | IBECS | ID: ibc-194432

RESUMO

INTRODUCCIÓN: Los niveles plasmáticos de clozapina son útiles para monitorizar el cumplimento farmacológico, pero también para evaluar y prevenir los efectos secundarios. Recientemente, se ha propuesto realizar controles plasmáticos a todos los pacientes para prevenir recaídas. A pesar de ello, también se ha documentado una alta variabilidad intraindividual, aunque los estudios tienen algunas limitaciones. MÉTODOS: Aquí analizamos las diferencias entre 2determinaciones de niveles plasmáticos con al menos un año de diferencias en un subgrupo de 28 pacientes (82% hombres, media de edad=47,9 años) con diagnósticos de psicosis no afectiva y en remisión clínica con dosis de clozapina y niveles de tabaquismo estable. RESULTADOS: Encontramos un incremento no significativo de los niveles de clozapina (0,30mg/l [DE=0,14] vs. 0,32 [DE=0,17]; t=-0,858, p = 0,40) y un descenso significativo de la norclozapina (0,27 [DE=0,11] vs. 0,22 [DE=0,10]; t=3,27; p = 0,003]. Calculamos el coeficiente de variación absoluto (CV) entre la primera y segunda determinación. El CV del 20% en los niveles de clozapina y norclozapina se encontró en el 46 y el 57% de los casos, respectivamente, mientras que el CV 50% se obtuvo en el 20,7 y el 13,8%. Discutimos las causas potenciales de CV tan altos. CONCLUSIONES: Nuestro estudio encontró una alta variación intraindividual incluso en un subgrupo de pacientes especialmente estables, lo que sugiere que la monitorización rutinaria sería adecuada para detectar cambios significativos de los niveles. Creemos que los clínicos deberían ser cautos a la hora de asumir pobre cumplimiento terapéutico a la hora de explicar cambios de los niveles plasmáticos no acompañados de cambios clínicos


INTRODUCTION: Clozapine plasma levels are useful to monitor drug compliance, and also to assess and to prevent some side effects. Recently, routine monitoring to all clozapine-treated patients has been proposed to prevent relapses. However, high intra-individual variability in plasma levels has been reported too, although these studies have some limitations. METHODS: We analysed differences between 2clozapine plasma levels separated by at least one year in a subgroup of 28 outpatients (82% male, mean age 47.9 years-old) with diagnosis of non-affective psychosis in clinical remission whose clozapine doses and smoking habits remained unchanged. RESULTS: We found a non-significant increase in clozapine plasma levels [.30mg/L (SD=.14) vs. .32 (SD=.17); t=-.858, p=.40] and a significant decrease in norclozapine plasma levels [.27 (SD=.11) vs. .22 (SD=.10); t=3.27; p=.003]. Absolute coefficient of variation (CV) between first and second assessment were calculated. Forty-six and fifty-seven percent of cases had CV 20% in clozapine and norclozapine, respectively. CV of 50% was seen in 20.7% and 13.8% of clozapine and norclozapine test respectively. We discussed potential causes of such high CV. CONCLUSIONS: Our study suggest high intra-individual variation even in a subgroup of very stable patients, which suggest that routine monitoring of these levels may be indicated in order to detect significant plasma variations. We think that clinicians should act with caution in case of a sudden decrease in plasma level. In the absence of obvious symptom severity variation, sources of intra-individual fluctuations might be considered first, before assuming poor compliance


Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Antipsicóticos/sangue , Clozapina/análogos & derivados , Clozapina/sangue , Esquizofrenia/sangue , Antipsicóticos/uso terapêutico , Clozapina/uso terapêutico , Monitoramento de Medicamentos , Adesão à Medicação , Estudos Retrospectivos , Esquizofrenia/tratamento farmacológico , Fumar , Fatores de Tempo
7.
Neuron ; 105(6): 1036-1047.e5, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31954621

RESUMO

Dopamine is involved in physiological processes like learning and memory, motor control and reward, and pathological conditions such as Parkinson's disease and addiction. In contrast to the extensive studies on neurons, astrocyte involvement in dopaminergic signaling remains largely unknown. Using transgenic mice, optogenetics, and pharmacogenetics, we studied the role of astrocytes on the dopaminergic system. We show that in freely behaving mice, astrocytes in the nucleus accumbens (NAc), a key reward center in the brain, respond with Ca2+ elevations to synaptically released dopamine, a phenomenon enhanced by amphetamine. In brain slices, synaptically released dopamine increases astrocyte Ca2+, stimulates ATP/adenosine release, and depresses excitatory synaptic transmission through activation of presynaptic A1 receptors. Amphetamine depresses neurotransmission through stimulation of astrocytes and the consequent A1 receptor activation. Furthermore, astrocytes modulate the acute behavioral psychomotor effects of amphetamine. Therefore, astrocytes mediate the dopamine- and amphetamine-induced synaptic regulation, revealing a novel cellular pathway in the brain reward system.


Assuntos
Astrócitos/fisiologia , Dopamina/fisiologia , Núcleo Accumbens/fisiologia , Transmissão Sináptica/fisiologia , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Anfetamina/farmacologia , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Clozapina/análogos & derivados , Clozapina/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Atividade Motora/fisiologia , Optogenética , Receptores de Dopamina D1/genética , Recompensa
8.
Neuron ; 105(6): 1094-1111.e10, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31955944

RESUMO

Interoception, the sense of internal bodily signals, is essential for physiological homeostasis, cognition, and emotions. While human insular cortex (InsCtx) is implicated in interoception, the cellular and circuit mechanisms remain unclear. We imaged mouse InsCtx neurons during two physiological deficiency states: hunger and thirst. InsCtx ongoing activity patterns reliably tracked the gradual return to homeostasis but not changes in behavior. Accordingly, while artificial induction of hunger or thirst in sated mice via activation of specific hypothalamic neurons (AgRP or SFOGLUT) restored cue-evoked food- or water-seeking, InsCtx ongoing activity continued to reflect physiological satiety. During natural hunger or thirst, food or water cues rapidly and transiently shifted InsCtx population activity to the future satiety-related pattern. During artificial hunger or thirst, food or water cues further shifted activity beyond the current satiety-related pattern. Together with circuit-mapping experiments, these findings suggest that InsCtx integrates visceral-sensory signals of current physiological state with hypothalamus-gated amygdala inputs that signal upcoming ingestion of food or water to compute a prediction of future physiological state.


Assuntos
Córtex Cerebral/fisiologia , Fome/fisiologia , Interocepção/fisiologia , Sede/fisiologia , Proteína Relacionada com Agouti/metabolismo , Animais , Clozapina/análogos & derivados , Clozapina/farmacologia , Sinais (Psicologia) , Feminino , Hipotálamo/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Vias Neurais/fisiologia , Imagem Óptica , Optogenética , Órgão Subfornical/fisiologia
9.
Psychopharmacology (Berl) ; 237(5): 1249-1266, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31980843

RESUMO

BACKGROUND: Pavlovian stimuli can influence instrumental behaviors via phenomena such as Pavlovian-to-instrumental transfer (PIT). PIT arises via dissociable processes as sensory-specific PIT (SS-PIT) and general PIT. The basolateral amygdala (BLA) mediates SS-PIT, but not general PIT. However, the specific BLA neuronal populations involved are unknown. AIMS: To determine the contribution of glutamatergic BLA neurons to the expression of SS-PIT and to the recall of sensory-specific properties of stimulus-outcome associations. METHODS: BLA neurons were transduced with virus containing either GFP or hM4Di, driven by the CamKII promoter. Rats were then tested for SS and general PIT and subsequently for expression of Pavlovian outcome devaluation effects and conditioned taste aversion following injections of vehicle or clozapine-N-oxide (CNO, the hM4Di agonist). RESULTS: CNO selectively blocked SS-PIT in the hM4Di-expressing group, but not controls, without altering expression of Pavlovian outcome devaluation or sensory-specific taste aversion in either group. Unexpectedly, CNO disrupted general PIT in both groups. CONCLUSIONS: CamKII BLA neurons mediate the expression of SS-PIT by enabling Pavlovian stimuli to trigger recall of the correct action-outcome associations rather than by mediating recall of the sensory-specific properties of the stimulus-outcome association. Separately, our data demonstrate that CNO alone is sufficient to disrupt affective, but not sensory-specific processes, an effect that was not due to generalized motor disruption. This non-specific effect on general PIT may be related to CNO-induced shifts in internal state. Together, these data identify BLA CamKII neurons as critical for the expression of SS-PIT and reveal important considerations for using CNO to study general affective motivation.


Assuntos
Complexo Nuclear Basolateral da Amígdala/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Clozapina/análogos & derivados , Condicionamento Clássico/fisiologia , Neurônios/metabolismo , Piperazinas/metabolismo , Animais , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Clozapina/administração & dosagem , Condicionamento Clássico/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologia , Comportamento Alimentar/psicologia , Feminino , Masculino , Motivação/efeitos dos fármacos , Motivação/fisiologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
10.
Exp Neurol ; 326: 113206, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31962128

RESUMO

BACKGROUND AND PURPOSE: Ischemic stroke significantly perturbs neuronal homeostasis leading to a cascade of pathologic events causing brain damage. In this study, we assessed acute stroke outcome after chemogenetic inhibition of forebrain excitatory neuronal activity. METHODS: We generated hM4Di-TG transgenic mice expressing the inhibitory hM4Di, a Designer Receptors Exclusively Activated by Designer Drugs (DREADD)-based chemogenetic receptor, in forebrain excitatory neurons. Clozapine-N-oxide (CNO) was used to activate hM4Di DREADD. Ischemic stroke was induced by transient occlusion of the middle cerebral artery. Neurologic function and infarct volumes were evaluated. Excitatory neuronal suppression in the hM4Di-TG mouse forebrain was assessed electrophysiologically in vitro and in vivo, based on evoked synaptic responses, and in vivo based on occurrence of potassium-induced cortical spreading depolarizations. RESULTS: Detailed characterization of hM4Di-TG mice confirmed that evoked synaptic responses in both in vitro hippocampal slices and in vivo motor cortex were significantly reduced after CNO-mediated activation of the inhibitory hM4Di DREADD. Further, CNO treatment had no obvious effects on physiology and motor function in either control or hM4Di-TG mice. Importantly, hM4Di-TG mice treated with CNO at either 10 min before ischemia or 30 min after reperfusion exhibited significantly improved neurologic function and smaller infarct volumes compared to CNO-treated control mice. Mechanistically, we showed that potassium-induced cortical spreading depression episodes were inhibited, including frequency and duration of DC shift, in CNO-treated hM4Di-TG mice. CONCLUSIONS: Our data demonstrate that acute inhibition of a subset of excitatory neurons after ischemic stroke can prevent brain injury and improve functional outcome. This study, together with the previous work in optogenetic neuronal modulation during the chronic phase of stroke, supports the notion that targeting neuronal activity is a promising strategy in stroke therapy.


Assuntos
Prosencéfalo/patologia , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/genética , Animais , Células Cultivadas , Clozapina/análogos & derivados , Clozapina/farmacologia , Depressão Alastrante da Atividade Elétrica Cortical , Fenômenos Eletrofisiológicos , Potenciais Evocados , Masculino , Camundongos , Camundongos Transgênicos , Córtex Motor/patologia , Neuroproteção , Desempenho Psicomotor , Traumatismo por Reperfusão/patologia , Acidente Vascular Cerebral/patologia , Sinapses/efeitos dos fármacos , Resultado do Tratamento
11.
Rev Psiquiatr Salud Ment ; 13(1): 31-35, 2020.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-31122757

RESUMO

INTRODUCTION: Clozapine plasma levels are useful to monitor drug compliance, and also to assess and to prevent some side effects. Recently, routine monitoring to all clozapine-treated patients has been proposed to prevent relapses. However, high intra-individual variability in plasma levels has been reported too, although these studies have some limitations. METHODS: We analysed differences between 2clozapine plasma levels separated by at least one year in a subgroup of 28 outpatients (82% male, mean age 47.9 years-old) with diagnosis of non-affective psychosis in clinical remission whose clozapine doses and smoking habits remained unchanged. RESULTS: We found a non-significant increase in clozapine plasma levels [.30mg/L (SD=.14) vs. .32 (SD=.17); t=-.858, p=.40] and a significant decrease in norclozapine plasma levels [.27 (SD=.11) vs. .22 (SD=.10); t=3.27; p=.003]. Absolute coefficient of variation (CV) between first and second assessment were calculated. Forty-six and fifty-seven percent of cases had CV 20% in clozapine and norclozapine, respectively. CV of 50% was seen in 20.7% and 13.8% of clozapine and norclozapine test respectively. We discussed potential causes of such high CV. CONCLUSIONS: Our study suggest high intra-individual variation even in a subgroup of very stable patients, which suggest that routine monitoring of these levels may be indicated in order to detect significant plasma variations. We think that clinicians should act with caution in case of a sudden decrease in plasma level. In the absence of obvious symptom severity variation, sources of intra-individual fluctuations might be considered first, before assuming poor compliance.


Assuntos
Antipsicóticos/sangue , Clozapina/análogos & derivados , Clozapina/sangue , Esquizofrenia/sangue , Antipsicóticos/uso terapêutico , Clozapina/uso terapêutico , Monitoramento de Medicamentos , Feminino , Humanos , Masculino , Adesão à Medicação , Pessoa de Meia-Idade , Estudos Retrospectivos , Esquizofrenia/tratamento farmacológico , Fumar , Fatores de Tempo
13.
J Clin Invest ; 130(1): 83-93, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31738186

RESUMO

The mineralocorticoid aldosterone is produced in the adrenal zona glomerulosa (ZG) under the control of the renin-angiotensin II (AngII) system. Primary aldosteronism (PA) results from renin-independent production of aldosterone and is a common cause of hypertension. PA is caused by dysregulated localization of the enzyme aldosterone synthase (Cyp11b2), which is normally restricted to the ZG. Cyp11b2 transcription and aldosterone production are predominantly regulated by AngII activation of the Gq signaling pathway. Here, we report the generation of transgenic mice with Gq-coupled designer receptors exclusively activated by designer drugs (DREADDs) specifically in the adrenal cortex. We show that adrenal-wide ligand activation of Gq DREADD receptors triggered disorganization of adrenal functional zonation, with induction of Cyp11b2 in glucocorticoid-producing zona fasciculata cells. This result was consistent with increased renin-independent aldosterone production and hypertension. All parameters were reversible following termination of DREADD-mediated Gq signaling. These findings demonstrate that Gq signaling is sufficient for adrenocortical aldosterone production and implicate this pathway in the determination of zone-specific steroid production within the adrenal cortex. This transgenic mouse also provides an inducible and reversible model of hyperaldosteronism to investigate PA therapeutics and the mechanisms leading to the damaging effects of aldosterone on the cardiovascular system.


Assuntos
Córtex Suprarrenal/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Hiperaldosteronismo/etiologia , Zona Glomerulosa/fisiologia , Animais , Clozapina/análogos & derivados , Clozapina/farmacologia , Citocromo P-450 CYP11B2/fisiologia , Drogas Desenhadas/farmacologia , Feminino , Hiperaldosteronismo/tratamento farmacológico , Hipertensão/etiologia , Camundongos , Camundongos Transgênicos , Receptor Muscarínico M3/fisiologia , Transdução de Sinais/fisiologia
14.
Expert Opin Drug Saf ; 19(1): 43-57, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31770500

RESUMO

Introduction: Clozapine remains the most effective antipsychotic for treatment-refractory schizophrenia. However, ~40% of the patients respond insufficiently to clozapine. Clozapine's effects, both beneficial and adverse, have been proposed to be partially attributable to its main metabolite, N-desmethylclozapine (NDMC). However, the relation of the clozapine to norclozapine ratio (CLZ:NDMC; optimally defined as ~2) to clinical response and metabolic outcomes is not clear.Areas covered: This narrative review comprehensively examines the clinical utility of the CLZ:NDMC ratio to reduce metabolic risk and increase treatment efficacy. The association of the CLZ:NDMC ratio with changes in psychopathology, cognitive functioning, and cardiometabolic burden will be explored, as well as adjunctive treatments and their effects.Expert opinion: The literature suggests a positive association between the CLZ:NDMC ratio and better cardiometabolic outcomes. Conversely, the CLZ:NDMC ratio appears inversely associated with better cognitive functioning but less consistently with other psychiatric domains. The CLZ:NDMC ratio may be useful for predicting and monitoring cardiometabolic adverse effects and optimizing potential cognitive benefits of clozapine. Future studies are required to replicate these findings, which if substantiated, would encourage examination of adjunctive treatments aiming to alter the CLZ:NDMC ratio to best meet the needs of the individual patient, thereby broadening clozapine's clinical utility.


Assuntos
Antipsicóticos/administração & dosagem , Clozapina/administração & dosagem , Esquizofrenia/tratamento farmacológico , Antipsicóticos/efeitos adversos , Antipsicóticos/farmacocinética , Clozapina/efeitos adversos , Clozapina/análogos & derivados , Clozapina/sangue , Clozapina/farmacocinética , Cognição/efeitos dos fármacos , Monitoramento de Medicamentos/métodos , Humanos
15.
J Pharm Biomed Anal ; 180: 113056, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31887669

RESUMO

OBJECTIVE: Clozapine is one of the most effective drugs for resistant schizophrenia, but its severe metabolic and hematological side effects limit the use of clozapine. It has been reported that clozapine blood concentrations should be maintained between 350-600 ng/mL. Our aim was to develop a determination method for clozapine and its main metabolites norclozapine and clozapine-N-oxide, to perform validation studies and to investigate the change of various biochemical parameters in patients using clozapine. METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for clozapine measurement. Thus, blood samples were collected from 38 patients with schizophrenia and 32 healthy volunteers. Biochemical and hematological parameters were measured by Beckman-Coulter AU 5800 (Beckman Coulter, Brea, USA) and Beckman Coulter LH 780 analyzer (Beckman Coulter, Miami, FL, USA), respectively. Hormone levels were analyzed using Cobas 6000 analyzer (Roche Diagnostics, Germany). RESULTS: The LCMS/MS method was linear between 1.22-2500 ng/mL (r2 = 0.9971) for clozapine. The retention times of clozapine, norclozapine and clozapine-N-oxide were 0.92, 0.89 and 0.95, respectively. Blood glucose (GLU) (p = 0.025), low density lipoprotein (LDL-cholesterol) (p = 0.015), triglyseride (TG) (p = 0.042) and total cholesterol (TC) (p = 0.024) levels were higher; hemoglobin (HGB) (0.015), mean corpuscular hemoglobin (MCH) (0.036), red blood cell count (RBC) (0.020), neutrophil (NEU) (0.034), and platelet (PLT) (P = 0.005) levels were lower in the clozapine group. CONCLUSIONS: This LC-MS/MS method was rapid, simple, cost-effective and suitable for the routine clozapine monitoring. Furthermore, norclozapine and clozapine-N-oxide were also determined. Monitoring of metabolic and hematological parameters with clozapine levels is very important. However, the limitations of the study were that the method was not validated for norclozapine and clozapine-N-oxide, so the validation parameters were not evaluated for these two metabolites.


Assuntos
Antipsicóticos/sangue , Clozapina/análogos & derivados , Monitoramento de Medicamentos/métodos , Esquizofrenia/sangue , Adulto , Antipsicóticos/efeitos adversos , Contagem de Células Sanguíneas , Glicemia/análise , Estudos de Casos e Controles , Colesterol/sangue , Cromatografia Líquida , Clozapina/efeitos adversos , Clozapina/sangue , Hemoglobinas/análise , Humanos , Limite de Detecção , Reprodutibilidade dos Testes , Esquizofrenia/tratamento farmacológico , Espectrometria de Massas em Tandem , Triglicerídeos/sangue
16.
Mol Brain ; 12(1): 87, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666100

RESUMO

Parkinson's disease (PD), classically defined as a progressive motor disorder accompanied with dopaminergic neuron loss and presence of Lewy bodies, is the second most common neurodegenerative disease. PD also has various non-classical symptoms, including cognitive impairments. In addition, inflammation and astrogliosis are recognized as an integral part of PD pathology. The hippocampus (Hipp) is a brain region involved in cognition and memory, and the neuropeptide orexin has been shown to enhance learning and memory. Previous studies show impairments in Hipp-dependent memory in a transgenic mouse model of Parkinson's disease (A53T mice), and we hypothesized that increasing orexin tone will reverse this. To test this, we subjected 3, 5, and 7-month old A53T mice to a Barnes maze and a contextual object recognition test to determine Hipp dependent memory. Inflammation and astrogliosis markers in the Hipp were assessed by immuno-fluorescence densitometry. The data show that early cognitive impairment is coupled with an increase in expression of inflammatory and astrogliosis markers. Next, in two separate experiments, mice were given intra-hippocampal injections of orexin or chemogenetic viral injections of an orexin neuron specific Designer Receptor Exclusively Activated by Designer Drug (DREADD). For the pharmacological approach mice were intracranially treated with orexin A, whereas the chemogenetic approach utilized clozapine N-oxide (CNO). Both pharmacological orexin A intervention as well as chemogenetic activation of orexin neurons ameliorated Hipp-dependent early memory impairment observed in A53T mice. This study implicates orexin in PD-associated cognitive impairment and suggests that exogenous orexin treatment and/or manipulation of endogenous orexin levels may be a potential strategy for addressing early cognitive loss in PD.


Assuntos
Hipocampo/metabolismo , Hipocampo/patologia , Transtornos da Memória/genética , Orexinas/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Contagem de Células , Clozapina/análogos & derivados , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/complicações , Gliose/patologia , Inflamação/complicações , Inflamação/patologia , Injeções , Masculino , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Doença de Parkinson/complicações , Reprodutibilidade dos Testes
17.
Nat Neurosci ; 22(12): 1986-1999, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31719672

RESUMO

The importance of neuronal ensembles, termed engram cells, in storing and retrieving memory is increasingly being appreciated, but less is known about how these engram cells operate within neural circuits. Here we tagged engram cells in the ventral CA1 region of the hippocampus (vCA1) and the core of the nucleus accumbens (AcbC) during cocaine conditioned place preference (CPP) training and show that the vCA1 engram projects preferentially to the AcbC and that the engram circuit from the vCA1 to the AcbC mediates memory recall. Direct activation of the AcbC engram while suppressing the vCA1 engram is sufficient for cocaine CPP. The AcbC engram primarily consists of D1 medium spiny neurons, but not D2 medium spiny neurons. The preferential synaptic strengthening of the vCA1→AcbC engram circuit evoked by cocaine conditioning mediates the retrieval of cocaine CPP memory. Our data suggest that the vCA1 engram stores specific contextual information, while the AcbC D1 engram and its downstream network store both cocaine reward and associated contextual information, providing a potential mechanism by which cocaine CPP memory is stored.


Assuntos
Região CA1 Hipocampal/fisiologia , Cocaína/farmacologia , Condicionamento Psicológico/fisiologia , Rememoração Mental/fisiologia , Núcleo Accumbens/fisiologia , Animais , Comportamento Animal/fisiologia , Clozapina/análogos & derivados , Clozapina/farmacologia , Condicionamento Psicológico/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Camundongos Transgênicos , Vias Neurais/fisiologia , Optogenética , Receptores Dopaminérgicos/fisiologia
18.
Nat Neurosci ; 22(11): 1771-1781, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31636449

RESUMO

Microglia dynamically survey the brain parenchyma. Microglial processes interact with neuronal elements; however, what role neuronal network activity plays in regulating microglial dynamics is not entirely clear. Most studies of microglial dynamics use either slice preparations or in vivo imaging in anesthetized mice. Here we demonstrate that microglia in awake mice have a relatively reduced process area and surveillance territory and that reduced neuronal activity under general anesthesia increases microglial process velocity, extension and territory surveillance. Similarly, reductions in local neuronal activity through sensory deprivation or optogenetic inhibition increase microglial process surveillance. Using pharmacological and chemogenetic approaches, we demonstrate that reduced norepinephrine signaling is necessary for these increases in microglial process surveillance. These findings indicate that under basal physiological conditions, noradrenergic tone in awake mice suppresses microglial process surveillance. Our results emphasize the importance of awake imaging for studying microglia-neuron interactions and demonstrate how neuronal activity influences microglial process dynamics.


Assuntos
Microglia/fisiologia , Neurônios/fisiologia , Norepinefrina/fisiologia , Córtex Somatossensorial/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Receptor 1 de Quimiocina CX3C/genética , Clozapina/análogos & derivados , Clozapina/farmacologia , Isoflurano/farmacologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microinjeções , Muscimol/farmacologia , Norepinefrina/farmacologia , Optogenética , Propanolaminas/farmacologia , Propranolol/farmacologia , Receptores Purinérgicos P2Y12/genética , Privação Sensorial/fisiologia , Córtex Somatossensorial/efeitos dos fármacos , Tetrodotoxina/farmacologia , Vigília
19.
Dis Model Mech ; 12(11)2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31601549

RESUMO

Germinal matrix hemorrhage (GMH), affecting about 1 in 300 births, is a major perinatal disease with lifelong neurological consequences. Yet despite advances in neonatal medicine, there is no effective intervention. GMH is characterized by localized bleeding in the germinal matrix (GM), due to inherent vessel fragility unique to this developing brain region. Studies have shown that reduced TGFß signaling contributes to this vascular immaturity. We have previously shown that a region-specific G-protein-coupled receptor pathway in GM neural progenitor cells regulates integrin ß8, a limiting activator of pro-TGFß. In this study, we use mice to test whether this regional pathway can be harnessed for GMH intervention. We first examined the endogenous dynamics of this pathway and found that it displays specific patterns of activation. We then investigated the functional effects of altering these dynamics by chemogenetics and found that there is a narrow developmental window during which this pathway is amenable to manipulation. Although high-level activity in this time window interferes with vessel growth, moderate enhancement promotes vessel maturation without compromising growth. Furthermore, we found that enhancing the activity of this pathway in a mouse model rescues all GMH phenotypes. Altogether, these results demonstrate that enhancing neurovascular signaling through pharmacological targeting of this pathway may be a viable approach for tissue-specific GMH intervention. They also demonstrate that timing and level are likely two major factors crucial for success. These findings thus provide critical new insights into both brain neurovascular biology and the intervention of GMH.


Assuntos
Modelos Animais de Doenças , Hemorragias Intracranianas/prevenção & controle , Neostriado/irrigação sanguínea , Células-Tronco Neurais/fisiologia , Animais , Vasos Sanguíneos/fisiologia , Circulação Cerebrovascular , Clozapina/análogos & derivados , Clozapina/farmacologia , Feminino , Cadeias beta de Integrinas/fisiologia , Hemorragias Intracranianas/etiologia , Camundongos , Receptores Acoplados a Proteínas-G/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/fisiologia
20.
Epilepsia ; 60(11): 2314-2324, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31608439

RESUMO

OBJECTIVE: More than one-third of patients with temporal lobe epilepsy (TLE) continue to have seizures despite treatment with antiepileptic drugs, and many experience severe drug-related side effects, illustrating the need for novel therapies. Selective expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) allows cell-type-specific reduction of neuronal excitability. In this study, we evaluated the effect of chemogenetic suppression of excitatory pyramidal and granule cell neurons of the sclerotic hippocampus in the intrahippocampal mouse model (IHKA) for temporal lobe epilepsy. METHODS: Intrahippocampal IHKA mice were injected with an adeno-associated viral vector carrying the genes for an inhibitory DREADD hM4Di in the sclerotic hippocampus or control vector. Next, animals were treated systemically with different single doses of clozapine-N-oxide (CNO) (1, 3, and 10 mg/kg) and clozapine (0.03 and 0.1 mg/kg) and the effect on spontaneous hippocampal seizures, hippocampal electroencephalography (EEG) power, fast ripples (FRs) and behavior in the open field test was evaluated. Finally, animals received prolonged treatment with clozapine for 3 days and the effect on seizures was monitored. RESULTS: Treatment with both CNO and clozapine resulted in a robust suppression of hippocampal seizures for at least 15 hours only in DREADD-expressing animals. Moreover, total EEG power and the number of FRs were significantly reduced. CNO and/or clozapine had no effects on interictal hippocampal EEG, seizures, or locomotion/anxiety in the open field test in non-DREADD epileptic IHKA mice. Repeated clozapine treatment every 8 hours for 3 days resulted in almost complete seizure suppression in DREADD animals. SIGNIFICANCE: This study shows the potency of chemogenetics to robustly and sustainably suppress spontaneous epileptic seizures and pave the way for an epilepsy therapy in which a systemically administered exogenous drug selectively modulates specific cell types in a seizure network, leading to a potent seizure suppression devoid of the typical drug-related side effects.


Assuntos
Anticonvulsivantes/administração & dosagem , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/prevenção & controle , Convulsões/genética , Convulsões/prevenção & controle , Animais , Clozapina/administração & dosagem , Clozapina/análogos & derivados , Eletroencefalografia/efeitos dos fármacos , Eletroencefalografia/métodos , Epilepsia do Lobo Temporal/fisiopatologia , Vetores Genéticos/administração & dosagem , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Ácido Caínico/administração & dosagem , Ácido Caínico/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Convulsões/fisiopatologia
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