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1.
Toxicol Appl Pharmacol ; 419: 115512, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33785355

RESUMO

Nerve agents are highly toxic organophosphorus compounds that inhibit acetylcholinesterase resulting in rapid accumulation of the neurotransmitter acetylcholine (ACh) causing a cholinergic syndrome including respiratory failure. In the present study, respiratory responses and antimuscarinic treatment efficacy was evaluated ex vivo using rat precision-cut lung slices (PCLS) exposed to the nerve agent VX. The respiratory effects were evaluated either by adding exogenous ACh directly to the culture medium or by applying electric-field stimulation (EFS) to the PCLS to achieve a release of endogenous ACh from neurons in the lung tissue. The airway contraction induced by both methods was enhanced by VX and resulted in lingering airway recovery, in particular when airways were exposed to a high VX-dose. Both contractions induced by EFS and exogenously added ACh were significantly reduced by administration of the antimuscarinic drugs atropine or scopolamine. Two additions of atropine or scopolamine after maximal ACh-induced airway response was demonstrated effective to reverse the contraction. By adding consecutive doubled doses of antimuscarinics, high efficiency to reduce the cholinergic airway response was observed. However, the airways were not completely recovered by atropine or scopolamine, indicating that non-muscarinic mechanisms were involved in the smooth muscle contractions. In conclusion, it was demonstrated that antimuscarinic treatment reversed airway contraction induced by VX but supplemental pharmacological interventions are needed to fully recover the airways. Further studies should therefore clarify the mechanisms of physiological responses in lung tissue following nerve agent exposures to improve the medical management of poisoned individuals.


Assuntos
Atropina/farmacologia , Fibras Colinérgicas/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Pulmão/inervação , Antagonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/inervação , Compostos Organotiofosforados/toxicidade , Escopolamina/farmacologia , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Acetilcolinesterase/metabolismo , Animais , Fibras Colinérgicas/enzimologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/metabolismo , Ratos Sprague-Dawley
2.
Hear Res ; 388: 107896, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31982642

RESUMO

The development of knockin mice with Cre recombinase expressed under the control of the promoter for choline acetyltransferase (ChAT) has allowed experimental manipulation of cholinergic circuits. However, currently available ChATCre mouse lines are on the C57BL/6J strain background, which shows early onset age-related hearing loss attributed to the Cdh23753A mutation (a.k.a., the ahl mutation). To develop ChATCre mice without accelerated hearing loss, we backcrossed ChATIRES-Cre mice with CBA/CaJ mice that have normal hearing. We used genotyping to obtain mice homozygous for ChATIRES-Cre and the wild-type allele at the Cdh23 locus (ChATCre,Cdh23WT). In the new line, auditory brainstem response thresholds were ∼20 dB lower than those in 9 month old ChATIRES-Cre mice at all frequencies tested (4-31.5 kHz). These thresholds were stable throughout the period of testing (3-12 months of age). We then bred ChATCre,Cdh23WT animals with Ai14 reporter mice to confirm the expression pattern of ChATCre. In these mice, tdTomato-labeled cells were observed in all brainstem regions known to contain cholinergic cells. We then stained the tissue with a neuron-specific marker, NeuN, to determine whether Cre expression was limited to neurons. Across several brainstem nuclei (pontomesencephalic tegmentum, motor trigeminal and facial nuclei), 100% of the tdTomato-labeled cells were double-labeled with anti-NeuN (n = 1896 cells), indicating Cre-recombinase was limited to neurons. Almost all of these cells (1867/1896 = 98.5%) also stained with antibodies against ChAT, indicating that reporter label was expressed almost exclusively in cholinergic neurons. Finally, an average 88.7% of the ChAT+ cells in these nuclei were labeled with tdTomato, indicating that the Cre is expressed in a large proportion of the cholinergic cells in these nuclei. We conclude that the backcrossed ChATCre,Cdh23WT mouse line has normal hearing and expresses Cre recombinase almost exclusively in cholinergic neurons. This ChATCre,Cdh23WT mouse line may provide an opportunity to manipulate cholinergic circuits without the confound of accelerated hearing loss associated with the C57BL/6J background. Furthermore, comparison with lines that do show early hearing loss may provide insight into possible cholinergic roles in age-related hearing loss.


Assuntos
Tronco Encefálico/enzimologia , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/enzimologia , Perda Auditiva/prevenção & controle , Audição , Integrases/metabolismo , Animais , Limiar Auditivo , Tronco Encefálico/fisiopatologia , Caderinas/genética , Colina O-Acetiltransferase/genética , Cruzamentos Genéticos , Proteínas de Ligação a DNA/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Técnicas de Introdução de Genes , Perda Auditiva/enzimologia , Perda Auditiva/genética , Perda Auditiva/fisiopatologia , Integrases/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Especificidade da Espécie
3.
J Neurosci ; 32(1): 170-82, 2012 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-22219280

RESUMO

Development of neural circuitry relies on precise matching between correct synaptic partners and appropriate synaptic strength tuning. Adaptive developmental adjustments may emerge from activity and calcium-dependent mechanisms. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been associated with developmental synaptic plasticity, but its varied roles in different synapses and developmental stages make mechanistic generalizations difficult. In contrast, we focused on synaptic development roles of CaMKII in a defined sensory-motor circuit. Thus, different forms of CaMKII were expressed with UAS-Gal4 in distinct components of the giant fiber system, the escape circuit of Drosophila, consisting of photoreceptors, interneurons, motoneurons, and muscles. The results demonstrate that the constitutively active CaMKII-T287D impairs development of cholinergic synapses in giant fiber dendrites and thoracic motoneurons, preventing light-induced escape behavior. The locus of the defects is postsynaptic as demonstrated by selective expression of transgenes in distinct components of the circuit. Furthermore, defects among these cholinergic synapses varied in severity, while the glutamatergic neuromuscular junctions appeared unaffected, demonstrating differential effects of CaMKII misregulation on distinct synapses of the same circuit. Limiting transgene expression to adult circuits had no effects, supporting the role of misregulated kinase activity in the development of the system rather than in acutely mediating escape responses. Overexpression of wild-type transgenes did not affect circuit development and function, suggesting but not proving that the CaMKII-T287D effects are not due to ectopic expression. Therefore, regulated CaMKII autophosphorylation appears essential in central synapse development, and particular cholinergic synapses are affected differentially, although they operate via the same nicotinic receptor.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Fibras Colinérgicas/enzimologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Transmissão Sináptica/fisiologia , Animais , Comportamento Animal/fisiologia , Fibras Colinérgicas/ultraestrutura , Drosophila melanogaster/citologia , Feminino , Masculino , Vias Neurais/citologia , Vias Neurais/enzimologia , Vias Neurais/crescimento & desenvolvimento , Fosforilação
4.
Brain ; 133(Pt 7): 2058-68, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20558417

RESUMO

Corticobasal syndrome, progressive supranuclear palsy and frontotemporal dementia are all part of a disease spectrum that includes common cognitive impairment and movement disorders. The aim of this study was to characterize brain cholinergic deficits in these disorders. We measured brain acetylcholinesterase activity by [11C] N-methylpiperidin-4-yl acetate and positron emission tomography in seven patients with corticobasal syndrome (67.6+/-5.9 years), 12 with progressive supranuclear palsy (68.5+/-4.1 years), eight with frontotemporal dementia (59.8+/-6.9 years) and 16 healthy controls (61.2+/-8.5 years). Two-tissue compartment three-parameter model and non-linear least squares analysis with arterial input function were performed. k3 value, an index of acetylcholinesterase activity, was calculated voxel-by-voxel in the brain of each subject. The k3 images in each disease group were compared with the control group by using Statistical Parametric Mapping 2. Volume of interest analysis was performed on spatially normalized k3 images. The corticobasal syndrome group showed decreased acetylcholinesterase activity (k3 values) in the paracentral region, frontal, parietal and occipital cortices (P<0.05, cluster corrected). The group with progressive supranuclear palsy had reduced acetylcholinesterase activity in the paracentral region and thalamus (P<0.05, cluster corrected). The frontotemporal dementia group showed no significant differences in acetylcholinesterase activity. Volume of interest analysis showed mean cortical acetylcholinesterase activity to be reduced by 17.5% in corticobasal syndrome (P<0.001), 9.4% in progressive supranuclear palsy (P<0.05) and 4.4% in frontotemporal dementia (non-significant), when compared with the control group. Thalamic acetylcholinesterase activity was reduced by 6.4% in corticobasal syndrome (non-significant), 24.0% in progressive supranuclear palsy (P<0.03) and increased by 3.3% in frontotemporal dementia (non-significant). Both corticobasal syndrome and progressive supranuclear palsy showed brain cholinergic deficits, but their distribution differed somewhat. Significant brain cholinergic deficits were not seen in frontotemporal dementia, which may explain the unresponsiveness of this condition to cholinergic modulation therapy.


Assuntos
Gânglios da Base/patologia , Córtex Cerebral/patologia , Fibras Colinérgicas/patologia , Demência Frontotemporal/patologia , Tomografia por Emissão de Pósitrons , Paralisia Supranuclear Progressiva/patologia , Acetilcolinesterase , Idoso , Gânglios da Base/enzimologia , Córtex Cerebral/enzimologia , Fibras Colinérgicas/enzimologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos dos Movimentos/enzimologia , Transtornos dos Movimentos/patologia , Tomografia por Emissão de Pósitrons/métodos , Paralisia Supranuclear Progressiva/enzimologia , Síndrome
5.
J Neurosci ; 30(16): 5677-89, 2010 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-20410120

RESUMO

Leak K(+) conductance generated by TASK1/3 channels is crucial for neuronal excitability. However, endogenous modulators activating TASK channels in neurons remained unknown. We previously reported that in the presumed cholinergic neurons of the basal forebrain (BF), activation of NO-cGMP-PKG (protein kinase G) pathway enhanced the TASK1-like leak K(+) current (I-K(leak)). As 8-Br-cGMP enhanced the I-K(leak) mainly at pH 7.3 as if changing the I-K(leak) from TASK1-like to TASK3-like current, such an enhancement of the I-K(leak) would result either from an enhancement of hidden TASK3 component or from an acidic shift in the pH sensitivity profile of TASK1 component. In view of the report that protonation of TASK channel decreases its open probability, the present study was designed to examine whether the activation of PKG increases the conductance of TASK1 channels by reducing their binding affinity for H(+), i.e., by increasing K(d) for protonation, or not. We here demonstrate that PKG activation and inhibition respectively upregulate and downregulate TASK1 channels heterologously expressed in PKG-loaded HEK293 cells at physiological pH, by causing shifts in the K(d) in the acidic and basic directions, respectively. Such PKG modulations of TASK1 channels were largely abolished by mutating pH sensor H98. In the BF neurons that were identified to express ChAT and TASK1 channels, similar dynamic modulations of TASK1-like pH sensitivity of I-K(leak) were caused by PKG. It is strongly suggested that PKG activation and inhibition dynamically modulate TASK1 currents at physiological pH by bidirectionally changing K(d) values for protonation of the extracellular pH sensors of TASK1 channels in cholinergic BF neurons.


Assuntos
Potenciais de Ação/fisiologia , Fibras Colinérgicas/fisiologia , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Prosencéfalo/fisiologia , Potenciais de Ação/genética , Animais , Linhagem Celular , Fibras Colinérgicas/enzimologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação para Baixo/genética , Feminino , Humanos , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Prosencéfalo/enzimologia , Ratos , Ratos Wistar , Regulação para Cima/genética
6.
Neurology ; 73(4): 273-8, 2009 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-19474411

RESUMO

OBJECTIVE: To characterize brain cholinergic deficits in Parkinson disease (PD), PD with dementia (PDD), and dementia with Lewy bodies (DLB). METHODS: Participants included 18 patients with PD, 21 patients with PDD/DLB, and 26 healthy controls. The PD group consisted of nine patients with early PD, each with a disease duration of less than 3 years, five of whom were de novo PD patients, and nine patients with advanced PD, each with a disease duration greater than or equal to 3 years. The PDD/DLB group consisted of 10 patients with PDD and 11 patients with DLB. All subjects underwent PET scans with N-[11C]-methyl-4-piperidyl acetate to measure brain acetylcholinesterase (AChE) activity. Brain AChE activity levels were estimated voxel-by-voxel in a three-compartment analysis using the arterial input function, and compared among our subject groups through both voxel-based analysis using the statistical parametric mapping software SPM5 and volume-of-interest analysis. RESULTS: Among patients with PD, AChE activity was significantly decreased in the cerebral cortex and especially in the medial occipital cortex (% reduction compared with the normal mean = -12%) (false discovery rate-corrected p value <0.01). Patients with PDD/DLB, however, had even lower AChE activity in the cerebral cortex (% reduction = -27%) (p < 0.01). There was no significant difference between early PD and advanced PD groups or between DLB and PDD groups in the amount by which regional AChE activity in the brain was reduced. CONCLUSIONS: Brain cholinergic dysfunction occurs in the cerebral cortex, especially in the medial occipital cortex. It begins in early Parkinson disease, and is more widespread and profound in both Parkinson disease with dementia and dementia with Lewy bodies.


Assuntos
Acetilcolina/deficiência , Acetilcolinesterase/metabolismo , Núcleo Basal de Meynert/enzimologia , Fibras Colinérgicas/enzimologia , Doença por Corpos de Lewy/enzimologia , Doença de Parkinson/enzimologia , Acetilcolinesterase/análise , Idoso , Núcleo Basal de Meynert/diagnóstico por imagem , Núcleo Basal de Meynert/patologia , Biomarcadores/análise , Biomarcadores/metabolismo , Mapeamento Encefálico , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Fibras Colinérgicas/patologia , Estudos de Coortes , Diagnóstico Diferencial , Progressão da Doença , Regulação para Baixo/fisiologia , Humanos , Imuno-Histoquímica , Doença por Corpos de Lewy/diagnóstico por imagem , Doença por Corpos de Lewy/patologia , Pessoa de Meia-Idade , Vias Neurais/diagnóstico por imagem , Vias Neurais/metabolismo , Vias Neurais/patologia , Vias Neurais/fisiopatologia , Lobo Occipital/diagnóstico por imagem , Lobo Occipital/metabolismo , Lobo Occipital/patologia , Lobo Occipital/fisiopatologia , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/patologia , Cintilografia
8.
Am J Physiol Regul Integr Comp Physiol ; 295(6): R1774-81, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18815211

RESUMO

Microinjection of acetylcholine chloride (ACh) in the nucleus of the solitary tract (NTS) of awake rats caused a transient and dose-dependent hypotension and bradycardia. Because it is known that cardiovascular reflexes are affected by nitric oxide (NO) produced in the NTS, we investigated whether these ACh-induced responses depend on NO in the NTS. Responses to ACh (500 pmol in 100 nl) were strongly reduced by ipsilateral microinjection of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 nmol in 100 nl) in the NTS: mean arterial pressure (MAP) fell by 50 +/- 5 mmHg before L-NAME to 9 +/- 4 mmHg, 10 min after L-NAME, and HR fell by 100 +/- 26 bpm before L-NAME to 20 +/- 10 bpm, 10 min after L-NAME (both P < 0.05). Microinjection of the selective inhibitor of neuronal nitric oxide synthase (nNOS), 1-(2-trifluoromethylphenyl) imidazole (TRIM; 13.3 nmol in 100 nl), in the NTS also reduced responses to ACh: MAP fell from 42 +/- 3 mmHg before TRIM to 27 +/- 6 mmHg, 10 min after TRIM (P < 0.05). TRIM also tended to reduce ACh-induced bradycardia, but this effect was not statistically significant. ACh-induced hypotension and bradycardia returned to control levels 30-45 min after NOS inhibition. Control injections with D-NAME and saline did not affect resting values or the response to ACh. In conclusion, injection of ACh into the NTS of conscious rats induces hypotension and bradycardia, and these effects may be mediated at least partly by NO produced in NTS neurons.


Assuntos
Acetilcolina/metabolismo , Pressão Sanguínea , Sistema Cardiovascular/inervação , Fibras Colinérgicas/metabolismo , Frequência Cardíaca , Neurônios Nitrérgicos/metabolismo , Óxido Nítrico/metabolismo , Núcleo Solitário/metabolismo , Acetilcolina/administração & dosagem , Animais , Pressão Sanguínea/efeitos dos fármacos , Bradicardia/metabolismo , Bradicardia/fisiopatologia , Fibras Colinérgicas/efeitos dos fármacos , Fibras Colinérgicas/enzimologia , Inibidores Enzimáticos/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Hipotensão/metabolismo , Hipotensão/fisiopatologia , Imidazóis/farmacologia , Masculino , Microinjeções , NG-Nitroarginina Metil Éster/farmacologia , Neurônios Nitrérgicos/efeitos dos fármacos , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I , Ratos , Ratos Wistar , Núcleo Solitário/citologia , Núcleo Solitário/efeitos dos fármacos , Fatores de Tempo , Vigília
9.
Neurochem Int ; 53(6-8): 193-8, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18674580

RESUMO

The presence of nicotinic and muscarinic receptors suggests the occurrence of cholinergic neurotransmission in white matter; however no quantitative information exists on acetylcholine formation and breakdown in white matter. We compared white structures of pig brain (fimbria, corpus callosum, pyramidal tracts, and occipital white matter) to gray structures (temporal, parietal and cerebellar cortices, hippocampus, and caudate) and found that sodium-dependent, high-affinity choline uptake in white structures was 25-31% of that in hippocampus. White matter choline acetyltransferase activity was 10-50% of the hippocampal value; the highest activity was found in fimbria. Acetylcholine esterase activity in white structures was 20-25% of that in hippocampus. The caudate, which is rich in cholinergic interneurons, gave values for all three parameters that were 2.8-4 times higher than in hippocampus. The results suggest a certain capacity for cholinergic neurotransmission in central nervous white matter. The white matter activity of pyruvate dehydrogenase, which provides acetyl-CoA for acetylcholine synthesis, ranged between 33 and 50% of the hippocampal activity; the activity in the caudate was similar to that in hippocampus and the other gray structures, which was true also for other enzymes of glucose metabolism: hexokinase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase. Acetylcholine esterase activity in white matter was inhibited by the nerve agent soman, which may help explain the reported deleterious effect of soman on white matter. Further, this finding suggests that acetylcholine esterase inhibitors used in Alzheimer's disease may have an effect in white matter.


Assuntos
Acetilcolina/biossíntese , Encéfalo/enzimologia , Fibras Colinérgicas/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Fibras Nervosas Mielinizadas/enzimologia , Acetilcoenzima A/análise , Acetilcoenzima A/metabolismo , Acetilcolinesterase/análise , Acetilcolinesterase/metabolismo , Animais , Encéfalo/citologia , Mapeamento Encefálico , Colina/análise , Colina/metabolismo , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/efeitos dos fármacos , Fibras Colinérgicas/ultraestrutura , Inibidores da Colinesterase/farmacologia , Feminino , Glucose/metabolismo , Imuno-Histoquímica , Interneurônios/metabolismo , Masculino , Proteínas de Membrana Transportadoras/análise , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/ultraestrutura , Complexo Piruvato Desidrogenase/análise , Complexo Piruvato Desidrogenase/metabolismo , Soman/farmacologia , Sus scrofa , Degeneração Walleriana/induzido quimicamente , Degeneração Walleriana/enzimologia , Degeneração Walleriana/fisiopatologia
10.
J Comp Neurol ; 509(6): 566-79, 2008 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-18543295

RESUMO

This study provides the first immunohistochemical evidence visualizing cholinergic octopus neurons containing choline acetyltransferase (ChAT), the synthetic enzyme of acetylcholine. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, and initially used for studies in mammals, to validate antibody specificity for the octopus counterpart enzyme we therefore used three methods. Immunoprecipitation using Pansorbin indicated that immunoreactive octopus brain molecules were capable of synthesizing acetylcholine. Western blot analysis after denatured gel electrophoresis of octopus brain extracts revealed a single band at approximately 81 kDa. A gel slice containing the 81-kDa protein after native (nondenatured) gel electrophoresis exhibited high ChAT activity. All findings obtained with these three methods clearly indicated that the antiserum effectively recognizes octopus ChAT. The immunohistochemical use of the antiserum in the retina, optic lobe, and its neighboring peduncle complex detected enzyme-containing neuronal cell bodies in only two regions, the cell islands of the optic lobe medulla and the cortical layer of the posterior olfactory lobule. Immunoreactive fibers and probable nerve terminals were also found in the plexiform layer of the deep retina, within the stroma of the optic gland, and the neuropils of the optic lobe, peduncle lobe, and olfactory lobe. These results provide information on the morphology and distribution patterns of cholinergic neurons in the octopus visual system, a useful invertebrate model for learning and memory where the cholinergic system, as in higher vertebrates including mammals, plays an important role.


Assuntos
Acetilcolina/metabolismo , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/enzimologia , Fibras Nervosas/enzimologia , Lobo Óptico de Animais não Mamíferos/enzimologia , Tegmento Mesencefálico/enzimologia , Animais , Western Blotting , Imuno-Histoquímica , Octopodiformes
11.
Fiziol Zh (1994) ; 54(1): 45-53, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18416183

RESUMO

The distribution of NADPH-diaphorase-reactive (NADPH-dr) neurons and neuronal processes in the cerebral cortex and basal forebrain and their association with parenchymal vessels were studied in normal adult rats using NADPH-d histochemical protocol. The intensely stained cortical interneurons and reactive subcortically originating afferents, and stained microvessels were examined through a light microscope at law (x250) and high (x630) magnifications. NADPH-dr interneurons were concentrated in layers 2-6 of the M1 and M2 areas. However, clear predominance in their concentration (14 +/- 0.8 P < 0.05 per section) was found in layer 6. A mean number of labeled neurons in auditory (AuV), granular and agranular (GI, AIP) areas of the insular cortex was calculated to reach 12.3 +/- 0.7, 18.5 +/- 1.0 and 23.3 +/- 1.7 units per section, respectively (P < 0.05). The distinct apposition of labelled neurons to intracortical vessels was found in the M1, M2. The order of frequency of neurovascular coupling in different zones of the cerebral cortex was as following sequence: AuV (31.2%, n = 1040) > GI (18.0%, n = 640) > S1 (13.3%, n = 720) > M1 (6.3%, n = 1360). A large number of structural associations between labeled cells and vessels in the temporal and insular cortex indicate that NADPH-d-reactive interneurons can contribute to regulation of the cerebral regional blood flow in these areas.


Assuntos
Córtex Cerebral , NADPH Desidrogenase/metabolismo , Neurônios Aferentes , Animais , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/enzimologia , Córtex Cerebral/fisiologia , Córtex Cerebral/ultraestrutura , Fibras Colinérgicas/enzimologia , Fibras Colinérgicas/fisiologia , Fibras Colinérgicas/ultraestrutura , Histocitoquímica , Técnicas In Vitro , Interneurônios/enzimologia , Interneurônios/fisiologia , Interneurônios/ultraestrutura , Masculino , Microcirculação/enzimologia , Microcirculação/fisiologia , Microcirculação/ultraestrutura , Neurônios Aferentes/enzimologia , Neurônios Aferentes/fisiologia , Neurônios Aferentes/ultraestrutura , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Wistar
12.
Neurodegener Dis ; 5(3-4): 228-31, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18322398

RESUMO

BACKGROUND: Fibers containing galanin (GAL) enlarge and hyperinnervate cholinergic basal forebrain (CBF) nucleus basalis (NB) neurons in late-stage Alzheimer's disease (AD), yet the physiological consequences of this phenomenon are unclear. OBJECTIVE: To determine whether GAL hyperinnervation of cholinergic NB neurons modulates the expression of genes critical to cholinergic transmission [e.g. acetylcholine (ACh) metabolism and ACh receptors] in AD. METHODS: Single-cell gene expression profiling was used to compare cholinergic mRNA levels in non-GAL-hyperinnervated NB neurons in tissue autopsied from cases classified as having no cognitive impairment (NCI) or late-stage AD (AD/GAL-) and in GAL-hyperinnervated (AD/GAL+) NB neurons from the same AD subjects. RESULTS: AD/GAL+ cells displayed a significant upregulation in choline acetyltransferase (ChAT) mRNA expression compared to NCI and AD/GAL- cells. CONCLUSION: GAL fiber hyperinnervation of cholinergic NB neurons upregulates the expression of ChAT, the synthetic enzyme for ACh, suggesting that GAL regulates the cholinergic tone of CBF neurons in AD.


Assuntos
Doença de Alzheimer/enzimologia , Colina O-Acetiltransferase/biossíntese , Fibras Colinérgicas/enzimologia , Galanina/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Neurônios/enzimologia , Prosencéfalo/enzimologia , Regulação para Cima/fisiologia , Doença de Alzheimer/genética , Colina O-Acetiltransferase/genética , Humanos
13.
Pediatr Dev Pathol ; 11(4): 274-82, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18078369

RESUMO

Variability in cholinergic innervation may contribute to false-negative and false-positive evaluations for Hirschsprung disease (HD). We compared intraspecimen variability of histochemical acetylcholinesterase (ACE) activity in 62 distal rectal mucosal biopsy specimens from 33 patients with short-segment HD (SSHD), 14 patients with short-segment HD/Down syndrome (SSHD/DS), 10 patients with total colonic aganglionosis (HDTC), and 45 ganglionated control specimens obtained because of clinical suspicion for HD. Specimens were evaluated in 1-mm linear segments for abnormal distribution and abnormal intensity of staining reaction in the lamina propria (LP), muscularis mucosae (MM), numerical excess of small submucosal nerves (SSN), and presence or absence of large submucosal nerves (LSN) defined as >35 microns in diameter. Patients with HD and controls were predominantly infant males. Aggregate length of specimens was 186 mm in HD and 136 mm in controls. We defined multiple patterns and relative frequencies of normal and abnormal ACE reactivity in the muscularis mucosae, submucosal nerves, and innervation in the lamina propria. We verified a hyperinnervation pattern more common in neonates within each subset of HD. Large submucosal nerves >35 microns in diameter are relatively less common in SSHD/DS and HDTC. Eleven of 57 patients with HD had at least 1 mm of normal muscularis mucosae accounting for 19/186 mm examined. Ten percent of control mm had at least 1 submucosal nerve >35 microns in diameter. Intraspecimen variability in ACE patterns, when extreme, can usually be resolved by findings elsewhere in an adequate specimen, minimizing the need for repeat procedures.


Assuntos
Acetilcolinesterase/metabolismo , Fibras Colinérgicas/enzimologia , Doença de Hirschsprung/enzimologia , Mucosa Intestinal/inervação , Reto/inervação , Biópsia , Pré-Escolar , Feminino , Doença de Hirschsprung/patologia , Humanos , Lactente , Recém-Nascido , Masculino
14.
Behav Brain Res ; 183(1): 78-86, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17610963

RESUMO

Investigations using selective lesion techniques suggest that the septohippocampal cholinergic system may not be critical for spatial orientation. These studies employ spatial tasks that provide the animal with access to both environmental and self-movement cues; therefore, intact performance may reflect spared spatial orientation or compensatory mechanisms associated with one class of spatial cues. The present study investigated the contribution of the septohippocampal cholinergic system to spatial behavior by examining performance in foraging tasks in which cue availability was manipulated. Thirteen female Long-Evans rats received selective lesions of the medial septum/vertical band with 192 IgG saporin, and 11 received sham surgeries. Rats were trained to forage for hazelnuts in an environment with access to both environmental and self-movement cues (cued condition). Manipulations include altering availability of environmental cues associated with the refuge (uncued probe), removing all visual environmental cues (dark probe), and placing environmental and self-movement cues into conflict (reversal probe). Medial septum lesions disrupted homeward segment topography only under conditions in which self-movement cues were critical for organizing food hoarding behavior (dark and reversal). These results are consistent with medial septum lesions producing a selective impairment in self-movement cue processing and suggest that these rats were able to compensate for deficits in self-movement cue processing when provided access to environmental cues.


Assuntos
Vias Aferentes/fisiologia , Fibras Colinérgicas/fisiologia , Hipocampo/fisiologia , Orientação/fisiologia , Propriocepção/fisiologia , Comportamento Espacial/fisiologia , Acetilcolinesterase/metabolismo , Vias Aferentes/citologia , Vias Aferentes/enzimologia , Animais , Fibras Colinérgicas/enzimologia , Denervação , Meio Ambiente , Comportamento Exploratório/fisiologia , Feminino , Hipocampo/citologia , Hipocampo/enzimologia , Atividade Motora/fisiologia , Distribuição Aleatória , Ratos , Ratos Long-Evans , Núcleos Septais/citologia , Núcleos Septais/enzimologia , Núcleos Septais/fisiologia
15.
J Neurosci ; 27(21): 5633-42, 2007 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-17522308

RESUMO

Acetylcholine is a major neurotransmitter that modulates cortical functions. In addition to basal forebrain neurons that give rise to the principal cholinergic input into the cortex, a second source constituted by intrinsic cholinergic interneurons has been identified. Although these cells have been characterized anatomically, little is known about their functional role in cortical microcircuits. The paucity of this cell population has been a major hindrance for detailed electrophysiological investigations. To facilitate functional studies, we generated transgenic mice that express enhanced green fluorescent protein (EGFP) in choline acetyltransferase (ChAT)-positive neurons. Aided by the transgene expression, the characterization of distinct cholinergic interneurons was possible. These cells were located in layer 2-3, had a bipolar morphology, were calretinin- and vasoactive intestinal peptide positive, but had a non-GABAergic phenotype. Paired recordings showed that EGFP/ChAT-positive neurons receive excitatory and inhibitory input from adjacent principal cells and various types of interneurons. However, EGFP/ChAT-positive neurons do not exert direct postsynaptic responses in neighboring neurons. Interestingly, prolonged activation of EGFP-labeled cholinergic neurons induces an increase in spontaneous EPSCs in adjacent pyramidal neurons. This indirect effect is mediated by nicotinic receptors that are presumably presynaptically localized. Thus, intrinsic bipolar cholinergic neurons can modulate cortical function locally.


Assuntos
Córtex Cerebral/fisiologia , Fibras Colinérgicas/fisiologia , Interneurônios/fisiologia , Animais , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/biossíntese , Fibras Colinérgicas/enzimologia , Fibras Colinérgicas/metabolismo , Glutamato Descarboxilase/análise , Glutamato Descarboxilase/biossíntese , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Interneurônios/enzimologia , Interneurônios/metabolismo , Isoenzimas/análise , Isoenzimas/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
16.
J Comp Neurol ; 501(6): 854-65, 2007 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-17311313

RESUMO

The distribution patterns of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied in the olfactory bulb (OB) of three species of macaque. AChE was detected by a histochemical method and ChAT immunoreactivity by immunocytochemistry. Similar results were observed in all species analyzed. With the exception of the olfactory nerve layer, all layers of the macaque monkey OB demonstrated a dense innervation of AChE- and ChAT-positive fibers. The distribution patterns of AChE- and ChAT-labeled fibers were similar for both cholinergic markers, although the number of AChE-labeled fibers was clearly higher than the number of ChAT-immunoreactive fibers. The highest density of AChE and ChAT-stained fibers was observed in the interface between the glomerular layer and the external plexiform layer and in the internal plexiform layer. Dense bundles of labeled fibers were observed in the caudal OB, coursing from the olfactory peduncle. All ChAT-immunopositive elements were identified as centrifugal fibers, derived from neurons caudal to the OB. Neither olfactory fibers nor intrinsic neurons were observed after ChAT immunocytochemistry. However, a few AChE-positive cells were observed in the glomerular layer and in both external and internal plexiform layers. These neurons were presumably identified as periglomerular cells, superficial short-axon cells, and/or external tufted cells and deep short-axon cells. Contrary to other neurotransmitters and neuroactive substances, the distribution patterns of ChAT and AChE activities in the macaque monkey OB closely resembled the patterns described in macrosmatic mammals and showed laminar differences with the distribution pattern observed in humans.


Assuntos
Acetilcolinesterase/metabolismo , Mapeamento Encefálico , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/enzimologia , Macaca/metabolismo , Bulbo Olfatório/enzimologia , Animais , Imuno-Histoquímica , Macaca/anatomia & histologia , Masculino , Bulbo Olfatório/citologia , Distribuição Tecidual
17.
Brain Res ; 1139: 68-84, 2007 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-17280650

RESUMO

We investigated the effects of neonatal optic nerve transection on cortical acetylcholinesterase (AChE) activity in hooded rats during postnatal development and following behavioral manipulation after weaning. AChE reaction product was quantified on digitized images of histochemically stained sections in layer IV of primary somatic sensory, primary visual and visual association cortex. Rats with optic nerve transection were compared to sham-operated littermates. In all cortical regions of both types of animal, AChE reaction product was increased to peak 2 weeks after birth and decreased thereafter, reaching adult levels at the end of the third postnatal week. During postnatal development, reaction product in primary visual cortex was lower in rats deprived of retinal input than in sham-operated littermates and the area delineated by reaction product was smaller. However, optic nerve transection did not modify the time course of postnatal development or statistically significantly diminish adult levels of AChE activity. Behavioral manipulations after weaning statistically significantly increased enzyme activity in sham-operated rats in all cortical areas examined. Compared with cage rearing, training in a discrimination task with food reward had a greater impact than environmental enrichment. By contrast, in the rats with optic nerve transection enrichment and training resulted in statistically significantly increased AChE activity only in lateral visual association cortex. Our findings provide evidence for intra- and supramodal influences of the neonatal removal of retinal input on neural activity- and use-dependent modifications of cortical AChE activity. The laminar distribution of the AChE reaction product suggests that the observed changes in AChE activity were mainly related to cholinergic basal forebrain afferents. These afferents may facilitate the stabilization of transient connections between the somatic sensory and the visual pathway.


Assuntos
Acetilcolinesterase/metabolismo , Neocórtex/enzimologia , Plasticidade Neuronal/fisiologia , Traumatismos do Nervo Óptico/enzimologia , Vias Visuais/enzimologia , Animais , Nível de Alerta/fisiologia , Fibras Colinérgicas/enzimologia , Aprendizagem por Discriminação/fisiologia , Meio Ambiente , Feminino , Masculino , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Neurônios/enzimologia , Traumatismos do Nervo Óptico/fisiopatologia , Ratos , Ratos Long-Evans , Vias Visuais/citologia , Vias Visuais/crescimento & desenvolvimento
18.
J Pediatr Surg ; 42(2): 300-4, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17270539

RESUMO

PURPOSE: Cholinergic and nitrergic neurons form 2 main subpopulations of the myenteric neurons, and they have been the targets of detailed morphological investigations in bowel motility disorders. However, little is known regarding the colocalization of neurotransmitters within the same enteric neurons. The aim of this study was to determine the histochemical colocalization of cholinergic and nitrergic neurons in the porcine distal large bowel myenteric plexus from fetal to adulthood. METHODS: Distal large bowel specimens were taken from 6 randomly selected age groups (3 animals in each group) from midway of gestation to adulthood. The myenteric plexus was exposed using whole-mount technique. After nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining, cells per ganglion were counted. Then the specimens were stained with acetylcholinesterase (AChE), and the cells that were stained with individual enzymes and with both enzymes were counted. RESULTS: Colocalization of AChE and NADPH-d was seen in all age groups, and it was highest during the mid part of gestation (30%) and decreased steadily thereafter into adulthood (8%). The individual number of NADPH-d- and AChE-positive neurons per ganglion remained constant till 4 weeks of age and significantly increased thereafter into adulthood. CONCLUSION: The use of double-labeling histochemical technique shows for the first time the colocalization of cholinergic and nitrergic activity in a large population of enteric neurons in the late fetal and newborn period. Age-related loss of cholinergic and nitrergic colocalization in the myenteric plexus is most likely a maturational process.


Assuntos
Fibras Colinérgicas/enzimologia , Colo/enzimologia , Colo/inervação , Plexo Mientérico/enzimologia , Neurônios Nitrérgicos/enzimologia , Acetilcolinesterase , Fatores Etários , Animais , Animais Recém-Nascidos , Fibras Colinérgicas/metabolismo , Embrião não Mamífero , Imuno-Histoquímica , Modelos Animais , Plexo Mientérico/metabolismo , NADPH Desidrogenase , Neurônios Nitrérgicos/metabolismo , Distribuição Aleatória , Sensibilidade e Especificidade , Coloração e Rotulagem , Plexo Submucoso/enzimologia , Plexo Submucoso/metabolismo , Suínos , Técnicas de Cultura de Tecidos
19.
Pediatr Surg Int ; 23(5): 505-8, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17205295

RESUMO

Conventional acetylcholinesterase (AChE) histochemistry is both time consuming and complicated and requires the mixing of reagents that are toxic to the human body. We developed a rapid technique for performing AChE histochemistry, which has already been published, and now present a kit for performing AChE histochemistry that is a further improvement. Rectal suction biopsy specimens taken from 20 constipated patients and three full thickness biopsy specimens taken from 4 Hirschsprung's disease (HD) patients during pull-through surgery from aganglionic, transitional, and ganglionic bowel segments were tested using our rapid technique and the new kit. Each specimen was incubated for only 6 min. All ganglion cells stained clearly for AchE in just 6 min using both techniques. However, the kit was able to stain AchE positive nerve fibers more clearly and did not detect endogenous peroxidase-containing histiocytes, as did the earlier rapid technique. The kit could also detect AchE positive nerve fibers in the circular and longitudinal muscle layers, unlike the earlier rapid technique. The kit allows AChE histochemistry to be performed rapidly with complete accuracy, without any risk for toxicity. Moreover, the kit provides more focused information on AchE distribution in the bowel itself without any extraneous staining and can be used for diagnosing HD and allied disorders as well as establishing the exact level of innervation for pull-through resection.


Assuntos
Acetilcolinesterase/análise , Ensaios Enzimáticos Clínicos/métodos , Doença de Hirschsprung/diagnóstico , Doença de Hirschsprung/enzimologia , Kit de Reagentes para Diagnóstico/normas , Coloração e Rotulagem/métodos , Biópsia , Fibras Colinérgicas/enzimologia , Fibras Colinérgicas/patologia , Colo/enzimologia , Colo/inervação , Colo/patologia , Doença de Hirschsprung/patologia , Histocitoquímica , Técnicas Histológicas/métodos , Humanos , Reto/enzimologia , Reto/inervação , Reto/patologia
20.
Learn Mem ; 13(6): 686-9, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17101876

RESUMO

In Drosophila, calcium/calmodulin-dependent protein kinase II (CaMKII) activity is crucial in associative courtship conditioning for both memory formation and suppression of courtship during training with a mated female. We have previously shown that increasing levels of constitutively active CaMKII, but not calcium-dependent CaMKII, in a subset of neurons can decrease the initial level of courtship and enhance the rate of suppression of courtship in response to a mated female. In this study, we demonstrate that a subpopulation of noncholinergic, nondopaminergic, non-GABAergic neurons can cause CaMKII-dependent reductions in initial courtship, but only cholinergic neurons enhance training-dependent suppression. These data suggest that processing of pheromonal signals in two subpopulations of neurons, likely antennal lobe projection neurons, is critical for behavioral plasticity.


Assuntos
Aprendizagem por Associação/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Corte , Neurônios/enzimologia , Comportamento Sexual Animal/fisiologia , Adaptação Psicológica , Análise de Variância , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/classificação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Fibras Colinérgicas/enzimologia , Condicionamento Clássico/fisiologia , Regulação para Baixo , Drosophila , Feminino , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Neurônios/citologia , Órgãos dos Sentidos/enzimologia , Órgãos dos Sentidos/inervação
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