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1.
Environ Pollut ; 339: 122731, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37839680

RESUMO

Polycyclic aromatic hydrocarbons (PAHs), in particular benzo [a]pyrene (BaP), have been identified as carcinogenic components of tobacco smoke. In mammals, the toxicological response to BaP-diol-epoxide is driven by cytochrome P450 (CYP1A1), a pathway which is absent in Caenorhabditis elegans. In contrast, in worms prominently the CYP-35 enzyme family seems to be induced after BaP exposure. In C. elegans, BaP exposure reduces the accumulation of lysosomal neutral lipids in a dose dependent manner and the deletion of cyp-35A2 results in a significant elevation of neutral lipid metabolism. A cyp-35A2:mCherry;unc-47:GFP dual-labelled reporter strain facilitated the identification of three potential upstream regulators that drive BaP metabolism in worms, namely elt-2, nhr-49 and fos-1. This newly described reporter line is a powerful resource for future large-scale RNAi regarding toxicology and lipid metabolism screens.


Assuntos
Proteínas de Caenorhabditis elegans , Carcinógenos Ambientais , Animais , Benzo(a)pireno/toxicidade , Benzo(a)pireno/metabolismo , Caenorhabditis elegans/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Lipídeos , Mamíferos/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores
2.
Neuro Endocrinol Lett ; 43(2): 88-98, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35933616

RESUMO

OBJECTIVE: To investigate the effects of estrogen on the threshold and temperature of orofacial pain and explore the influence on the function of glutamate and GABA neurons in the orofacial pain temperature perception pathway by observing the expression of vesicular glutamate transporter 2 (Vglut2) and vesicular GABA transporter 1 (Vgat1). METHODS: A total of 24 adult female Sprague-Dawley rats were divided into three groups: sham operation (SHAM), ovariectomized (OVX) and ovariectomized plus estrogen intervention (OVX+E) (n = 8 per group). The threshold of mechanical pain of the orofacial region was assessed with von Frey filaments, and the temperature of the rat orofacial region was monitored by infrared thermography. Changes in the expression of Vglut2 and Vgat1 in glutamatergic and GABAergic neurons in the trigeminal ganglion (TG), spinal trigeminal nucleus (Sp5C), lateral parabrachial nucleus (LPB) and ventral posteromedial nucleus of the thalamus (VPM) were assessed by immunostaining and Western blotting. RESULTS: Under low-estrogen conditions, the mechanical pain threshold of the orofacial region of rats decreased significantly, and the temperature of the orofacial region increased significantly. The expression of Vglut2 and Vgat1 in the TG and Sp5C showed a downward trend, and the decline in Vgat1 was greater than that in Vglut2. Conversely, both proteins were upregulated in the LPB and VPM, and the magnitude of the changes in Vglut2 was greater than that in Vgat1. Estrogen therapy reversed these changes. CONCLUSION: Under low-estrogen conditions, the proportion of glutamate and GABA neurons in the orofacial pain and temperature sensation pathway changes, which leads to the imbalance of neurotransmission function and the enhancement of excitatory transmission of these two kinds of neurons and finally leads to a decrease in the orofacial pain threshold and an increase in temperature.


Assuntos
Dor Facial , Sensação , Animais , Feminino , Ratos , Estrogênios/farmacologia , Glutamatos , Ratos Sprague-Dawley , Temperatura , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores
3.
Environ Pollut ; 304: 119141, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35301029

RESUMO

Fumonisin B1 (FB1) is a neurodegenerative mycotoxin synthesized by Fusarium spp., but the potential neurobehavioral toxicity effects in organisms have not been characterized clearly. Caenorhabditis elegans (C. elegans) has emerged as a promising model organism for neurotoxicological studies due to characteristics such as well-functioning nervous system and rich behavioral phenotypes. To investigate whether FB1 has neurobehavioral toxicity effects on C. elegans, the motor behavior, neuronal structure, neurotransmitter content, and gene expression related with neurotransmission of C. elegans were determined after exposed to 20-200 µg/mL FB1 for 24 h and 48 h, respectively. Results showed that FB1 caused behavioral defects, including body bends, head thrashes, crawling distance, mean speed, mean amplitude, mean wavelength, foraging behavior, and chemotaxis learning ability in a dose-, and time-dependent manner. In addition, when C. elegans was exposed to FB1 at a concentration of 200 µg/mL for 24 h and above 100 µg/mL for 48 h, the GABAergic and serotonergic neurons were damaged, but no effect on dopaminergic, glutamatergic, and cholinergic neurons. The relative content of GABA and serotonin decreased significantly. Furthermore, abnormal expression of mRNA levels associated with GABA and serotonin were found in nematodes treated with FB1, such as unc-30, unc-47, unc-49, exp-1, mod-5, cat-1, and tph-1. The neurobehavioral toxicity effect of FB1 may be mediated by abnormal neurotransmission of GABA and serotonin. This study provides useful information for understanding the neurotoxicity of FB1.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fumonisinas , Serotonina , Transmissão Sináptica , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/farmacologia , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologia
4.
Mol Psychiatry ; 27(4): 2315-2328, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-33190145

RESUMO

The striatum is the main input structure of the basal ganglia. Distinct striatal subfields are involved in voluntary movement generation and cognitive and emotional tasks, but little is known about the morphological and molecular differences of striatal subregions. The ventrolateral subfield of the striatum (VLS) is the orofacial projection field of the sensorimotor cortex and is involved in the development of orofacial dyskinesias, involuntary chewing-like movements that often accompany long-term neuroleptic treatment. The biological basis for this particular vulnerability of the VLS is not known. Potassium channels are known to be strategically localized within the striatum. In search of possible molecular correlates of the specific vulnerability of the VLS, we analyzed the expression of voltage-gated potassium channels in rodent and primate brains using qPCR, in situ hybridization, and immunocytochemical single and double staining. Here we describe a novel, giant, non-cholinergic interneuron within the VLS. This neuron coexpresses the vesicular GABA transporter, the calcium-binding protein parvalbumin (PV), and the Kv3.3 potassium channel subunit. This novel neuron is much larger than PV neurons in other striatal regions, displays characteristic electrophysiological properties, and, most importantly, is restricted to the VLS. Consequently, the giant striatal Kv3.3-expressing PV neuron may link compromised Kv3 channel function and VLS-based orofacial dyskinesias.


Assuntos
Discinesias , Parvalbuminas , Animais , Corpo Estriado/metabolismo , Discinesias/metabolismo , Interneurônios/metabolismo , Parvalbuminas/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio Shaw/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores
5.
Science ; 374(6565): 316-323, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34648327

RESUMO

Although dexterity relies on the constant transmission of sensory information, unchecked feedback can be disruptive. Yet how somatosensory feedback from the hands is regulated and whether this modulation influences movement remain unclear. We found that mouse tactile afferents recruit neurons in the brainstem cuneate nucleus, whose activity is modulated by distinct classes of local inhibitory neurons. Manipulation of these inhibitory circuits suppresses or enhances the transmission of tactile information, which affects manual behaviors. Top-down cortical pathways innervate cuneate in a complementary pattern, with somatosensory cortical neurons targeting the core tactile region of cuneate and a large rostral cortical population driving feed-forward inhibition of tactile transmission through an inhibitory shell. These findings identify a circuit basis for tactile feedback modulation that enables the effective execution of dexterous movement.


Assuntos
Retroalimentação Sensorial , Destreza Motora/fisiologia , Tato/fisiologia , Animais , Tronco Encefálico/citologia , Tronco Encefálico/fisiologia , Feminino , Masculino , Camundongos , Camundongos Mutantes , Movimento , Inibição Neural , Optogenética , Córtex Somatossensorial/fisiologia , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética
6.
J Neurosci ; 41(42): 8790-8800, 2021 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-34470806

RESUMO

Social behaviors, including reproductive behaviors, often display sexual dimorphism. Lordosis, the measure of female sexual receptivity, is one of the most apparent sexually dimorphic reproductive behaviors. Lordosis is regulated by estrogen and progesterone (P4) acting within a hypothalamic-limbic circuit, consisting of the arcuate, medial preoptic, and ventromedial nuclei of the hypothalamus. Social cues are integrated into the circuit through the amygdala. The posterodorsal part of the medial amygdala (MeApd) is involved in sexually dimorphic social and reproductive behaviors, and sends projections to hypothalamic neuroendocrine regions. GABA from the MeApd appears to facilitate social behaviors, while glutamate may play the opposite role. To test these hypotheses, adult female vesicular GABA transporter (VGAT)-Cre and vesicular glutamate transporter 2 (VGluT2)-Cre mice were transfected with halorhodopsin (eNpHR)-expressing or channelrhodopsin-expressing adeno-associated viruses (AAVs), respectively, in the MeApd. The lordosis quotient (LQ) was measured following either photoinhibition of VGAT or photoexcitation of VGluT2 neurons, and brains were assessed for c-Fos immunohistochemistry (IHC). Photoinhibition of VGAT neurons in the MeApd decreased LQ, and decreased c-Fos expression within VGAT neurons, within the MeApd as a whole, and within the ventrolateral part of the ventromedial nucleus (VMHvl). Photoexcitation of VGluT2 neurons did not affect LQ, but did increase time spent self-grooming, and increased c-Fos expression within VGluT2 neurons in the MeApd. Neither condition altered c-Fos expression in the medial preoptic nucleus (MPN) or the arcuate nucleus (ARH). These data support a role for MeApd GABA in the facilitation of lordosis. Glutamate from the MeApd does not appear to be directly involved in the lordosis circuit, but appears to direct behavior away from social interactions.SIGNIFICANCE STATEMENT Lordosis, the measure of female sexual receptivity, is a sexually dimorphic behavior regulated within a hypothalamic-limbic circuit. Social cues are integrated through the amygdala, and the posterodorsal part of the medial amygdala (MeApd) is involved in sexually dimorphic social and reproductive behaviors. Photoinhibition of GABAergic neurons in the MeApd inhibited lordosis, while photoactivation of glutamate neurons had no effect on lordosis, but increased self-grooming. These data support a role for MeApd GABA in the facilitation of social behaviors and MeApd glutamate projections in anti-social interactions.


Assuntos
Complexo Nuclear Corticomedial/metabolismo , Neurônios GABAérgicos/metabolismo , Ácido Glutâmico/metabolismo , Comportamento Social , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Animais , Feminino , Ácido Glutâmico/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Caracteres Sexuais , Comportamento Sexual Animal/fisiologia , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética
7.
Int J Mol Sci ; 22(18)2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34576177

RESUMO

Eye-drop recombinant human nerve growth factor (ed-rhNGF) has proved to recover the retina and optic nerve damage in animal models, including the unilateral optic nerve crush (ONC), and to improve visual acuity in humans. These data, associated with evidence that ed-rhNGF stimulates the brain derived neurotrophic factor (BDNF) in retina and cortex, suggests that NGF might exert retino-fugal effects by affecting BDNF and its receptor TrkB. To address these questions, their expression and relationship with the GABAergic and glutamatergic transmission markers, GAD65 and GAD67, vesicular inhibitory amino acid transporter (VGAT), and vesicular glutamate transporters 1 and 2 (VGLUT-1 and VGLUT-2) were investigated in adult ONC rats contralateral and ipsilateral visual cortex (VCx). Ed-rhNGF recovers the ONC-induced alteration of GABAergic and glutamatergic markers in contralateral VCx, induces an upregulation of TrkB, which is positively correlated with BDNF precursor (proBDNF) decrease in both VCx sides, and strongly enhances TrkB+ cell soma and neuronal endings surrounded by GAD65 immuno-reactive afferents. These findings contribute to enlarging the knowledge on the mechanism of actions and cellular targets of exogenously administrated NGF, and suggest that ed-rhNGF might act by potentiating the activity-dependent TrkB expression in GAD+ cells in VCx following retina damage and/or ONC.


Assuntos
Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/metabolismo , Masculino , Microscopia Confocal , Fator de Crescimento Neural/genética , Fatores de Crescimento Neural/genética , Ratos , Proteínas Recombinantes/metabolismo , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Córtex Visual/metabolismo , Córtex Visual/fisiologia , Ácido gama-Aminobutírico/metabolismo
8.
J Neuroendocrinol ; 33(9): e13021, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34427015

RESUMO

The roles GABAergic and glutamatergic inputs in regulating the activity of the gonadotrophin-releasing hormone (GnRH) neurons at the time of the preovulatory surge remain unclear. We used expansion microscopy to compare the density of GABAergic and glutamatergic synapses on the GnRH neuron cell body and proximal dendrite in dioestrous and pro-oestrous female mice. An evaluation of all synapses immunoreactive for synaptophysin revealed that the highest density of inputs to rostral preoptic area GnRH neurons occurred within the first 45 µm of the primary dendrite (approximately 0.19 synapses µm-1 ) with relatively few synapses on the GnRH neuron soma or beyond 45 µm of the dendrite (0.05-0.08 synapses µm-1 ). Triple immunofluorescence labelling demonstrated a predominance of glutamatergic signalling with twice as many vesicular glutamate transporter 2 synapses detected compared to vesicular GABA transporter. Co-labelling with the GABAA receptor scaffold protein gephyrin and the glutamate receptor postsynaptic density marker Homer1 confirmed these observations, as well as the different spatial distribution of GABA and glutamate inputs along the dendrite. Quantitative assessments revealed no differences in synaptophysin, GABA or glutamate synapses at the proximal dendrite and soma of GnRH neurons between dioestrous and pro-oestrous mice. Taken together, these studies demonstrate that the GnRH neuron receives twice as many glutamatergic synapses compared to GABAergic synapses and that these inputs preferentially target the first 45 µm of the GnRH neuron proximal dendrite. These inputs appear to be structurally stable before the onset of pro-oestrous GnRH surge.


Assuntos
Ácido Glutâmico/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Dendritos/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia/métodos , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo
9.
Neurosci Lett ; 762: 136135, 2021 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-34311052

RESUMO

Pain constitutes the major non-motor symptom in Parkinson's disease (PD). Its mechanism is still poorly understood although an increase in excitation or a decrease in inhibition have been reported in preclinical studies. The aim of this study was to investigate gamma aminobutyric acid (GABA) inhibition in the 6-hydroxydopamine (6-OHDA) PD rat model. Therefore, the expression of three inhibitory markers parvalbumin, glutamate decarboxylase 67 (GAD67) and vesicular GABA transporter (VGAT) was evaluated, besides cold allodynia, in bilateral 6-OHDA lesioned rat. There was a significant increase in the expression of the three markers labeling within the spinal dorsal horn (SDH) of 6-OHDA lesioned rats. In parallel, there was also an increase of the excitatory marker protein kinase C gamma (PKCγ) . PKCγ cells have a crucial role in pain chronicity and are regulated by GABAergic influences. Central dopamine depletion induced an increase in excitation as reveled by an increase in cFOS expression upon acetone stimulus and the presence of cold allodynia. In addition, dopamine depletion induced increased expression in inhibitory markers, which may reflect a disinhibition or a decreased inhibition in 6-OHDA lesioned rats.


Assuntos
Hiperalgesia/metabolismo , Neuralgia/etiologia , Transtornos Parkinsonianos/complicações , Corno Dorsal da Medula Espinal/metabolismo , Animais , Dopamina/deficiência , Glutamato Descarboxilase/metabolismo , Masculino , Neuralgia/metabolismo , Transtornos Parkinsonianos/metabolismo , Parvalbuminas/metabolismo , Ratos , Ratos Sprague-Dawley , Substância Negra/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo
10.
Mol Brain ; 14(1): 96, 2021 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-34174930

RESUMO

Reductions in the GABAergic neurotransmitter system exist across multiple brain regions in schizophrenia and encompass both pre- and postsynaptic components. While reduced midbrain GABAergic inhibitory neurotransmission may contribute to the hyperdopaminergia thought to underpin psychosis in schizophrenia, molecular changes consistent with this have not been reported. We hypothesised that reduced GABA-related molecular markers would be found in the midbrain of people with schizophrenia and that these would correlate with dopaminergic molecular changes. We hypothesised that downregulation of inhibitory neuron markers would be exacerbated in schizophrenia cases with high levels of neuroinflammation. Eight GABAergic-related transcripts were measured with quantitative PCR, and glutamate decarboxylase (GAD) 65/67 and GABAA alpha 3 (α3) (GABRA3) protein were measured with immunoblotting, in post-mortem midbrain (28/28 and 28/26 control/schizophrenia cases for mRNA and protein, respectively), and analysed by both diagnosis and inflammatory subgroups (as previously defined by higher levels of four pro-inflammatory cytokine transcripts). We found reductions (21 - 44%) in mRNA encoding both presynaptic and postsynaptic proteins, vesicular GABA transporter (VGAT), GAD1, and parvalbumin (PV) mRNAs and four alpha subunits (α1, α2, α3, α5) of the GABAA receptor in people with schizophrenia compared to controls (p < 0.05). Gene expression of somatostatin (SST) was unchanged (p = 0.485). We confirmed the reduction in GAD at the protein level (34%, p < 0.05). When stratifying by inflammation, only GABRA3 mRNA exhibited more pronounced changes in high compared to low inflammatory subgroups in schizophrenia. GABRA3 protein was expressed by 98% of tyrosine hydroxylase-positive neurons and was 23% lower in schizophrenia, though this did not reach statistical significance (p > 0.05). Expression of transcripts for GABAA receptor alpha subunits 2 and 3 (GABRA2, GABRA3) were positively correlated with tyrosine hydroxylase (TH) and dopamine transporter (DAT) transcripts in schizophrenia cases (GABRA2; r > 0.630, GABRA3; r > 0.762, all p < 0.001) but not controls (GABRA2; r < - 0.200, GABRA3; r < 0.310, all p > 0.05). Taken together, our results support a profound disruption to inhibitory neurotransmission in the substantia nigra regardless of inflammatory status, which provides a potential mechanism for disinhibition of nigrostriatal dopamine neurotransmission.


Assuntos
Biomarcadores/metabolismo , Neurônios Dopaminérgicos/patologia , Neurônios GABAérgicos/patologia , Mesencéfalo/patologia , Esquizofrenia/patologia , Adulto , Idoso , Estudos de Coortes , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Feminino , Regulação da Expressão Gênica , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Humanos , Inflamação/genética , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Doenças Neuroinflamatórias/genética , Doenças Neuroinflamatórias/patologia , Parvalbuminas/metabolismo , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Esquizofrenia/genética , Somatostatina/genética , Somatostatina/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Adulto Jovem , Ácido gama-Aminobutírico
11.
Cells Dev ; 166: 203682, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33994355

RESUMO

Homeotic genes and their genomic organization show remarkable conservation across bilaterians. Consequently, the regulatory mechanisms, which control hox gene expression, are also highly conserved. The crucial presence of conserved GA rich motifs between Hox genes has been previously observed but what factor binds to these is still unknown. Previously we have reported that the vertebrate homologue of Drosophila Trl-GAF preferentially binds to GA rich regions in Evx2-hoxd13 intergenic region of vertebrate HoxD cluster. In this study, we show that the vertebrate-GAF (v-GAF) binds at known cis-regulatory elements in the HoxD complex of zebrafish and mouse. We further used morpholino based knockdown and CRISPR-cas9 knockout technique to deplete the v-GAF in zebrafish. We checked expression of the HoxD genes and found gain of the HoxD4 gene in GAF knockout embryos. Further, we partially rescued the morphological phenotypes in GAF depleted embryos by providing GAF mRNA. Our results show that GAF binds at intergenic regions of the HoxD complex and is important for maintaining the spatial domains of HoxD4 expression during embryonic development.


Assuntos
Desenvolvimento Embrionário , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Padronização Corporal/genética , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas de Peixe-Zebra/genética
12.
Neurology ; 96(18): e2251-e2260, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-34038384

RESUMO

OBJECTIVE: To identify the causative gene in a large unsolved family with genetic epilepsy with febrile seizures plus (GEFS+), we sequenced the genomes of family members, and then determined the contribution of the identified gene to the pathogenicity of epilepsies by examining sequencing data from 2,772 additional patients. METHODS: We performed whole genome sequencing of 3 members of a GEFS+ family. Subsequently, whole exome sequencing data from 1,165 patients with epilepsy from the Epi4K dataset and 1,329 Australian patients with epilepsy from the Epi25 dataset were interrogated. Targeted resequencing was performed on 278 patients with febrile seizures or GEFS+ phenotypes. Variants were validated and familial segregation examined by Sanger sequencing. RESULTS: Eight previously unreported missense variants were identified in SLC32A1, coding for the vesicular inhibitory amino acid cotransporter VGAT. Two variants cosegregated with the phenotype in 2 large GEFS+ families containing 8 and 10 affected individuals, respectively. Six further variants were identified in smaller families with GEFS+ or idiopathic generalized epilepsy (IGE). CONCLUSION: Missense variants in SLC32A1 cause GEFS+ and IGE. These variants are predicted to alter γ-aminobutyric acid (GABA) transport into synaptic vesicles, leading to altered neuronal inhibition. Examination of further epilepsy cohorts will determine the full genotype-phenotype spectrum associated with SLC32A1 variants.


Assuntos
Epilepsia Generalizada/diagnóstico , Epilepsia Generalizada/genética , Variação Genética/genética , Mutação de Sentido Incorreto/genética , Convulsões Febris/diagnóstico , Convulsões Febris/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Feminino , Estudos de Associação Genética/métodos , Humanos , Masculino , Linhagem
13.
Cereb Cortex ; 31(9): 4053-4067, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-33895810

RESUMO

The BOLD fMRI response in the cortex is often assumed to reflect changes in excitatory neural activity. However, the contribution of inhibitory neurons to BOLD fMRI is unclear. Here, the role of inhibitory and excitatory activity was examined using multimodal approaches: electrophysiological recording, 15.2 T fMRI, optical intrinsic signal imaging, and modeling. Inhibitory and excitatory neuronal activity in the somatosensory cortex were selectively modulated by 20-s optogenetic stimulation of VGAT-ChR2 and CaMKII-ChR2 mice, respectively. Somatosensory stimulation and optogenetic stimulation of excitatory neurons induced positive BOLD responses in the somatosensory network, whereas stimulation of inhibitory neurons produced biphasic responses at the stimulation site, initial positive and later negative BOLD signals, and negative BOLD responses at downstream sites. When the stimulation duration was reduced to 5 s, the hemodynamic response of VGAT-ChR2 mice to optogenetic stimulation was only positive. Lastly, modeling performed from neuronal and hemodynamic data shows that the hemodynamic response function (HRF) of excitatory neurons is similar across different conditions, whereas the HRF of inhibitory neurons is highly sensitive to stimulation frequency and peaks earlier than that of excitatory neurons. Our study provides insights into the neurovascular coupling of excitatory and inhibitory neurons and the interpretation of BOLD fMRI signals.


Assuntos
Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Imageamento por Ressonância Magnética/métodos , Inibição Neural/fisiologia , Neurônios/fisiologia , Oxigênio/sangue , Animais , Circulação Cerebrovascular/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Neurológicos , Acoplamento Neurovascular , Optogenética , Córtex Somatossensorial/diagnóstico por imagem , Córtex Somatossensorial/fisiologia , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética
14.
Mol Brain ; 14(1): 55, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33726812

RESUMO

Our previous study showed the intrinsic ability of descending noradrenergic neurons projecting from the locus coeruleus to the spinal dorsal horn (SDH) to suppress itch-related behaviors. Noradrenaline and α1A-adrenaline receptor (α1A-AR) agonist increase inhibitory synaptic inputs onto SDH interneurons expressing gastrin-releasing peptide receptors, which are essential for itch transmission. However, the contribution of α1A-ARs expressed in SDH inhibitory interneurons to itch-related behavior remains to be determined. In this study, RNAscope in situ hybridization revealed that Adra1a mRNA is expressed in SDH inhibitory interneurons that are positive for Slc32a1 mRNA (known as vesicular GABA transporter). Mice with conditional knock-out of α1A-ARs in inhibitory interneurons (Vgat-Cre;Adra1aflox/flox mice) exhibited an increase in scratching behavior when induced by an intradermal injection of chloroquine, but not compound 48/80, which are known as models of histamine-independent and dependent itch, respectively. Furthermore, knockout of inhibitory neuronal α1A-ARs in the SDH using the CRISPR-Cas9 system also increased the scratching behavior elicited by chloroquine but not compound 48/80. Our findings demonstrated for the first time that α1A-ARs in SDH inhibitory interneurons contribute to the regulation of itch signaling with preference for histamine-independent itch.


Assuntos
Cloroquina/toxicidade , Interneurônios/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células do Corno Posterior/fisiologia , Prurido/fisiopatologia , Receptores Adrenérgicos alfa 1/fisiologia , Animais , Sistemas CRISPR-Cas , Feminino , Edição de Genes , Técnicas de Inativação de Genes , Masculino , Camundongos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Prurido/induzido quimicamente , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Adrenérgicos alfa 1/biossíntese , Receptores Adrenérgicos alfa 1/deficiência , Receptores Adrenérgicos alfa 1/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/biossíntese , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , p-Metoxi-N-metilfenetilamina/farmacologia
15.
Proc Natl Acad Sci U S A ; 118(6)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33526663

RESUMO

The suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, is a network structure composed of multiple types of γ-aminobutyric acid (GABA)-ergic neurons and glial cells. However, the roles of GABA-mediated signaling in the SCN network remain controversial. Here, we report noticeable impairment of the circadian rhythm in mice with a specific deletion of the vesicular GABA transporter in arginine vasopressin (AVP)-producing neurons. These mice showed disturbed diurnal rhythms of GABAA receptor-mediated synaptic transmission in SCN neurons and marked lengthening of the activity time in circadian behavioral rhythms due to the extended interval between morning and evening locomotor activities. Synchrony of molecular circadian oscillations among SCN neurons did not significantly change, whereas the phase relationships between SCN molecular clocks and circadian morning/evening locomotor activities were altered significantly, as revealed by PER2::LUC imaging of SCN explants and in vivo recording of intracellular Ca2+ in SCN AVP neurons. In contrast, daily neuronal activity in SCN neurons in vivo clearly showed a bimodal pattern that correlated with dissociated morning/evening locomotor activities. Therefore, GABAergic transmission from AVP neurons regulates the timing of SCN neuronal firing to temporally restrict circadian behavior to appropriate time windows in SCN molecular clocks.


Assuntos
Relógios Circadianos , Ritmo Circadiano , Neurônios/metabolismo , Núcleo Supraquiasmático/metabolismo , Vasopressinas/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Comportamento Animal , Cálcio/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Regulação da Expressão Gênica , Locomoção , Camundongos , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Fatores de Tempo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/deficiência , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo
16.
Neural Plast ; 2021: 8833087, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33510780

RESUMO

Accumulating evidence implicates a role for brain structures outside the ascending auditory pathway in tinnitus, the phantom perception of sound. In addition to other factors such as age-dependent hearing loss, high-level sound exposure is a prominent cause of tinnitus. Here, we examined how noise exposure altered the distribution of excitatory and inhibitory synaptic inputs in the guinea pig hippocampus and determined whether these changes were associated with tinnitus. In experiment one, guinea pigs were overexposed to unilateral narrow-band noise (98 dB SPL, 2 h). Two weeks later, the density of excitatory (VGLUT-1/2) and inhibitory (VGAT) synaptic terminals in CA1, CA3, and dentate gyrus hippocampal subregions was assessed by immunohistochemistry. Overall, VGLUT-1 density primarily increased, while VGAT density decreased significantly in many regions. Then, to assess whether the noise-induced alterations were persistent and related to tinnitus, experiment two utilized a noise-exposure paradigm shown to induce tinnitus and assessed tinnitus development which was assessed using gap-prepulse inhibition of the acoustic startle (GPIAS). Twelve weeks after sound overexposure, changes in excitatory synaptic terminal density had largely recovered regardless of tinnitus status, but the recovery of GABAergic terminal density was dramatically different in animals expressing tinnitus relative to animals resistant to tinnitus. In resistant animals, inhibitory synapse density recovered to preexposure levels, but in animals expressing tinnitus, inhibitory synapse density remained chronically diminished. Taken together, our results suggest that noise exposure induces striking changes in the balance of excitatory and inhibitory synaptic inputs throughout the hippocampus and reveal a potential role for rebounding inhibition in the hippocampus as a protective factor leading to tinnitus resilience.


Assuntos
Neurônios GABAérgicos/metabolismo , Hipocampo/metabolismo , Ruído/efeitos adversos , Zumbido/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Estimulação Acústica/efeitos adversos , Animais , Vias Auditivas/metabolismo , Vias Auditivas/patologia , Feminino , Neurônios GABAérgicos/química , Ácido Glutâmico/análise , Ácido Glutâmico/metabolismo , Cobaias , Hipocampo/patologia , Masculino , Sinapses/química , Sinapses/metabolismo , Zumbido/patologia , Proteínas Vesiculares de Transporte de Glutamato/análise , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/análise
17.
Mol Neurobiol ; 58(6): 2574-2589, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33471287

RESUMO

Cell adhesion molecules (CAMs) are key players in the formation of neural circuits during development. The γ-protocadherins (γ-Pcdhs), a family of 22 CAMs encoded by the Pcdhg gene cluster, are known to play important roles in dendrite arborization, axon targeting, and synapse development. We showed previously that multiple γ-Pcdhs interact physically with the autism-associated CAM neuroligin-1, and inhibit the latter's ability to promote excitatory synapse maturation. Here, we show that γ-Pcdhs can also interact physically with the related neuroligin-2, and inhibit this CAM's ability to promote inhibitory synapse development. In an artificial synapse assay, γ-Pcdhs co-expressed with neuroligin-2 in non-neuronal cells reduce inhibitory presynaptic maturation in contacting hippocampal axons. Mice lacking the γ-Pcdhs from the forebrain (including the cortex, the hippocampus, and portions of the amygdala) exhibit increased inhibitory synapse density and increased co-localization of neuroligin-2 with inhibitory postsynaptic markers in vivo. These Pcdhg mutants also exhibit defective social affiliation and an anxiety-like phenotype in behavioral assays. Together, these results suggest that γ-Pcdhs negatively regulate neuroligins to limit synapse density in a manner that is important for normal behavior.


Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Interação Social , Sinapses/metabolismo , Animais , Axônios/metabolismo , Comportamento Animal , Células COS , Proteínas Relacionadas a Caderinas , Membrana Celular/metabolismo , Chlorocebus aethiops , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Prosencéfalo/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo
18.
Sleep ; 44(6)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33270105

RESUMO

STUDY OBJECTIVES: Sleep and wake are opposing behavioral states controlled by the activity of specific neurons that need to be located and mapped. To better understand how a waking brain falls asleep it is necessary to identify activity of individual phenotype-specific neurons, especially neurons that anticipate sleep onset. In freely behaving mice, we used microendoscopy to monitor calcium (Ca2+) fluorescence in individual hypothalamic neurons expressing the vesicular GABA transporter (vGAT), a validated marker of GABA neurons. METHODS: vGAT-Cre mice (male = 3; female = 2) transfected with rAAV-FLEX-GCaMP6M in the lateral hypothalamus were imaged 30 days later during multiple episodes of waking (W), non-rapid-eye movement sleep (NREMS) or REMS (REMS). RESULTS: 372 vGAT neurons were recorded in the zona incerta. 23.9% of the vGAT neurons showed maximal fluorescence during wake (classified as wake-max), 4% were NREM-max, 56.2% REM-max, 5.9% wake/REM max, while 9.9% were state-indifferent. In the NREM-max group, Ca2+ fluorescence began to increase before onset of NREM sleep, remained high throughout NREM sleep, and declined in REM sleep. CONCLUSIONS: We found that 60.2% of the vGAT GABA neurons in the zona incerta had activity that was biased towards sleep (NREM and REMS). A subset of vGAT neurons (NREM-max) became active in advance of sleep onset and may induce sleep by inhibiting the activity of the arousal neurons. Abnormal activation of the NREM-max neurons may drive sleep attacks and hypersomnia.


Assuntos
Zona Incerta , Animais , Feminino , Masculino , Camundongos , Sono , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Vigília , Zona Incerta/metabolismo , Ácido gama-Aminobutírico
19.
Behav Brain Res ; 396: 112885, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32860829

RESUMO

Studies with human subjects indicate that ethanol exposure during fetal development causes long-lasting alterations in motor coordination that are, in part, a consequence of cerebellar damage. Studies with rats exposed to ethanol during the neonatal brain growth spurt have consistently recapitulated these deficits. However, studies with mice have yielded mixed results. We hypothesized that the use of highly sensitive motor function tests, such as the Catwalk test, would reliably detect motor function deficits in mice developmentally exposed to ethanol. Venus-vesicular GABA transporter transgenic mice were ethanol exposed during postnatal days 4-9 using vapor inhalation chambers and then subjected to the Catwalk test during adolescence. Catwalk data were rigorously analyzed using an innovative multistep statistical approach. For comparison, motor coordination and strength were assessed with the triple horizontal bar and rotarod tests. Unexpectedly, we found that out of 186 parameters analyzed in the Catwalk test, only one was affected by ethanol exposure (i.e., reduced coupling between left front paw and the right hind paw). In the triple horizontal bar test, ethanol-exposed mice were able to hold to the bars for less time than controls. Surprisingly, ethanol-exposed mice performed better in the rotarod test than controls. These data indicate that neonatal ethanol exposure of mice causes mixed effects on motor function during adolescence. The Catwalk test suggests that gait is generally preserved in these mice, whereas the triple horizontal bar test revealed deficits on motor strength and the rotarod test an increase in motor coordination.


Assuntos
Transtornos do Sistema Nervoso Induzidos por Álcool/fisiopatologia , Depressores do Sistema Nervoso Central/efeitos adversos , Etanol/efeitos adversos , Atividade Motora/fisiologia , Desempenho Psicomotor/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Comportamento Animal/fisiologia , Depressores do Sistema Nervoso Central/administração & dosagem , Modelos Animais de Doenças , Etanol/administração & dosagem , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores
20.
Brain ; 144(2): 665-681, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33367648

RESUMO

Opioids such as morphine are mainstay treatments for clinical pain conditions. Itch is a common side effect of opioids, particularly as a result of epidural or intrathecal administration. Recent progress has advanced our understanding of itch circuits in the spinal cord. However, the mechanisms underlying opioid-induced itch are not fully understood, although an interaction between µ-opioid receptor (MOR) and gastrin-releasing peptide receptor (GRPR) in spinal GRPR-expressing neurons has been implicated. In this study we investigated the cellular mechanisms of intrathecal opioid-induced itch by conditional deletion of MOR-encoding Oprm1 in distinct populations of interneurons and sensory neurons. We found that intrathecal injection of the MOR agonists morphine or DAMGO elicited dose-dependent scratching as well as licking and biting, but this pruritus was totally abolished in mice with a specific Oprm1 deletion in Vgat+ neurons [Oprm1-Vgat (Slc32a1)]. Loss of MOR in somatostatin+ interneurons and TRPV1+ sensory neurons did not affect morphine-induced itch but impaired morphine-induced antinociception. In situ hybridization revealed Oprm1 expression in 30% of inhibitory and 20% of excitatory interneurons in the spinal dorsal horn. Whole-cell recordings from spinal cord slices showed that DAMGO induced outward currents in 9 of 19 Vgat+ interneurons examined. Morphine also inhibited action potentials in Vgat+ interneurons. Furthermore, morphine suppressed evoked inhibitory postsynaptic currents in postsynaptic Vgat- excitatory neurons, suggesting a mechanism of disinhibition by MOR agonists. Notably, morphine-elicited itch was suppressed by intrathecal administration of NPY and abolished by spinal ablation of GRPR+ neurons with intrathecal injection of bombesin-saporin, whereas intrathecal GRP-induced itch response remained intact in mice lacking Oprm1-Vgat. Intrathecal bombesin-saporin treatment reduced the number of GRPR+ neurons by 97% in the lumber spinal cord and 91% in the cervical spinal cord, without changing the number of Oprm1+ neurons. Additionally, chronic itch from DNFB-induced allergic contact dermatitis was decreased by Oprm1-Vgat deletion. Finally, naloxone, but not peripherally restricted naloxone methiodide, inhibited chronic itch in the DNFB model and the CTCL model, indicating a contribution of central MOR signalling to chronic itch. Our findings demonstrate that intrathecal morphine elicits itch via acting on MOR on spinal inhibitory interneurons, leading to disinhibition of the spinal itch circuit. Our data also provide mechanistic insights into the current treatment of chronic itch with opioid receptor antagonist such as naloxone.


Assuntos
Analgésicos/administração & dosagem , Morfina/administração & dosagem , Prurido/induzido quimicamente , Prurido/fisiopatologia , Receptores Opioides mu/fisiologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiopatologia , Animais , Dermatite/fisiopatologia , Feminino , Injeções Espinhais , Interneurônios/efeitos dos fármacos , Interneurônios/fisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Receptores da Bombesina/fisiologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPV/fisiologia , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/fisiologia
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