RESUMO
Triple negative tumors are more aggressive than other breast cancer subtypes and there is a lack of specific therapeutic targets on them. Since muscarinic receptors have been linked to tumor progression, we investigated the effect of metronomic therapy employing a traditional anti-cancer drug, paclitaxel plus muscarinic agonists at low doses on this type of tumor. We observed that MDA-MB231 tumor cells express muscarinic receptors, while they are absent in the non-tumorigenic MCF-10A cell line, which was used as control. The addition of carbachol or arecaidine propargyl ester, a non-selective or a selective subtype 2 muscarinic receptor agonist respectively, plus paclitaxel reduces cell viability involving a down-regulation in the expression of ATP "binding cassette" G2 drug transporter and epidermal growth factor receptor. We also detected an inhibition of tumor cell migration and anti-angiogenic effects produced by those drug combinations in vitro and in vivo (in NUDE mice) respectively. Our findings provide substantial evidence about subtype 2 muscarinic receptors as therapeutic targets for the treatment of triple negative tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Agonistas Colinérgicos/administração & dosagem , Paclitaxel/administração & dosagem , Receptor Muscarínico M2/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Administração Metronômica , Animais , Arecolina/administração & dosagem , Arecolina/análogos & derivados , Carbacol/administração & dosagem , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , RNA Interferente Pequeno/metabolismo , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/genética , Neoplasias de Mama Triplo Negativas/irrigação sanguínea , Neoplasias de Mama Triplo Negativas/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The non-neuronal cholinergic system refers to the presence of acetylcholine, choline acetyltransferase, acetylcholinesterase and cholinergic receptors, nicotinic and muscarinic (mAChRs) expressed in non-neuronal cells. The presence of mAChRs has been detected in different type of tumor cells and they are linked with tumorigenesis. We had previously documented the expression of mAChRs in murine and human mammary adenocarcinomas and the absence of these receptors in normal mammary cells of the same origins. We also demonstrated that mAChRs are involved in breast cancer progression, pointing to a main role for mAChRs as oncogenic proteins. Since the long term treatment of breast cancer cells with the muscarinic agonist carbachol promoted cell death, here we investigated the ability of low doses of this agonist combined with paclitaxel (PX), a taxane usually administered to treat breast cancer, to inhibit the progression of human MCF-7 tumor cells. We demonstrated that PX plus carbachol reduced cell viability and tumor growth in vitro probably due to a down-regulation in cancer stem cells population and in the expression of ATP "binding cassette" G2 drug extrusion pump; also a reduction in malignant-induced angiogenesis was produced by the in vivo administration of the mentioned combination in a metronomic schedule to MCF-7 tumor-bearing NUDE mice. Our results confirm that mAChRs could be considered as therapeutic targets for metronomic therapy in breast cancer as well as the usefulness of a muscarinic agonist as repositioning drug in the treatment of this type of tumors.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Carbacol/administração & dosagem , Agonistas Colinérgicos/administração & dosagem , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Paclitaxel/administração & dosagem , Receptores Muscarínicos/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Administração Metronômica , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Interações Medicamentosas , Feminino , Humanos , Glândulas Mamárias Animais/irrigação sanguínea , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos Nus , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologiaRESUMO
Cholinergic activation of the medial septal area (MSA) with carbachol produces thirst, natriuresis and antidiuresis. Hydrogen peroxide (H2O2) injected into the medial septal area (MSA) impairs behavioral and renal responses induced by carbachol at the same site, suggesting the exogenous H2O2 may modulate the responses to cholinergic activation in the MSA. In the present study, we investigated if the accumulation of endogenous H2O2 in the MSA after the injection of the catalase inhibitor 3-amino-1,2,4-triazole (ATZ) also affects cholinergic responses. In addition, the effects of the combination of ATZ with a non-effective dose of H2O2 in the MSA were also tested. Male Holtzman rats (280-320 g) with stainless steel cannulas implanted in the MSA were used. The treatment with ATZ (10 nmol) into the MSA partially reverted the antidiuretic effect of carbachol (10.5 ± 0.7, vs. saline + carbachol: 7.3 ± 0.6 ml/120 min), without changing carbachol-induced water intake (9.5 ± 1.9, vs. saline + carbachol: 10.7 ± 1.6 ml/60 min). The combination of a low dose of ATZ (2.5 nmol) with an ineffective dose of H2O2 (0.5 µmol) into the MSA reduced carbachol-induced thirst (7.5 ± 2.0, vs. saline + carbachol: 14.9 ± 1.2 ml/15 min) and reverted the antidiuresis (8.1 ± 1.1, vs. saline + carbachol: 5.3 ± 0.9 ml/120 min). Sodium and potassium excretion were not modified regardless the treatment. Although exogenous H2O2 injected in the MSA may affect most of the responses to cholinergic activation of the MSA, the antidiuresis is the response clearly modulated by endogenous H2O2.
Assuntos
Antidiuréticos/administração & dosagem , Carbacol/administração & dosagem , Agonistas Colinérgicos/administração & dosagem , Diurese , Peróxido de Hidrogênio/metabolismo , Núcleos Septais/metabolismo , Amitrol (Herbicida)/administração & dosagem , Animais , Comportamento de Ingestão de Líquido/efeitos dos fármacos , Masculino , Ratos Sprague-Dawley , Núcleos Septais/efeitos dos fármacos , Micção/efeitos dos fármacosRESUMO
La catarata es definida por la Organización Mundial de la Salud (OMS) como la opacificación del lente cristalino que generalmente ocurre por el envejecimiento, trauma, o alguna enfermedad sistémica, afectando la capacidad visual de la persona. Esta disminución de la capacidad visual o incluso la ceguera, es un problema de salud pública en adultos y adultos mayores. En el Perú, aproximadamente el 0.6 % de la población tiene ceguera, cuya causa en el 47 % de los casos son por las cataratas. El tratamiento indicado para la catarata es la intervención quirúrgica, la cual consiste en reemplazar el cristalino opacificado o catarata por un lente intraocular. Hay dos formas de realizar esto, mediante la extracción extracapsular del cristalino opacificado, o mediante la facoemulsificación del cristalino, que consiste en un proceso de destrucción mediante ondas vibratorias ultrasónicas. Luego de ello, se realiza la implantación de un nuevo lente intraocular con soporte capsular, el cual reemplaza al cristalino permitiendo que el paciente vuelva tener una visión adecuada. Posterior a la implantación del lente intraocular se realiza la inducción de miosis pupilar para mantener el lente dentro de la bolsa capsular, evitar la captura del lente por el iris y el prolapso del iris por las heridas operatorias. La inducción de miosis luego de una intervención por catarata debe realizarse inmediatamente después de la implantación del lente, por lo que la vía de administración indicada es la inyección intraocular del agente miótico. De este modo, se espera que el efecto miótico se prolongue hasta por 24 horas, mientras que la aplicación tópica de un agente miótico se limita a un efecto que bordea las ocho horas, y sólo se puede realizar en el periodo postoperatorio, por lo que se genera un periodo entre la intervención quirúrgica y la aplicación del agente miótico que expone al paciente a las potenciales complicaciones anteriormente mencionadas. Aunque algunos estudios de serie de casos clínicos indican el uso de pilocarpina 2 %, el cual se encuentra disponible en el Petitorio Farmacológico de EsSalud, este medicamento no ha sido aprobado por la FDA para uso intraocular, sólo para uso tópico, dado que su uso intraocular puede ser tóxico o incrementar el riesgo de infecciones intraoculares. Así, en la actualidad EsSalud no cuenta con un medicamento miótico de uso intraocular autorizado, por lo cual, surge la necesidad de evaluar otras alternativas que pudieran ser de beneficio para dichos pacientes. OBJETIVO: objetivo del presente dictamen fue evaluar la eficacia y seguridad del uso intraocular de carbacol 0.01 % para la inducción de miosis intraoperatoria en las intervenciones quirúrgicas por catarata. Carbacol es um colinérgico o parasimpaticomimético potente, que actúa como agonista del receptor de acetilcolina, inhibiendo la acetilcolinesterasa y estimulando tanto los receptores muscarínicos como nicotínicos, produciendo miosis a través de la constricción del iris y del cuerpo ciliar, y reduciendo la presión intraocular. TECNOLOGÍA SANITARIA DE INTERÉS: CARBACOL. Carbacol, también conocido como carbamilcolina, es un colinérgico o parasimpaticomimético potente, que actúa como agonista del receptor de acetilcolina, inhibiendo la acetilcolinesterasa y estimulando tanto los receptores muscarínicos como nicotínicos, produciendo constricción del iris y del cuerpo ciliar y además reduciendo la presión intraocular. El agente colinérgico fue aprobado por la Administración de Drogas y Alimentos (FDA, por sus siglas en inglés) en el año 2002 (FDA, 2015). METODOLOGÍA: Se realizó una búsqueda sin restricción de idioma hasta mayo del 2018. La formulación de la estrategia de búsqueda incluyó los criterios de elegibilidad, los términos controlados propios de cada base y términos libres. Asimismo, se buscaron otros documentos potencialmente elegibles a través de la revisión del listado de referencias de los documentos seleccionados para lectura a texto completo. Por último, la selección de la evidencia siguió el flujograma mostrado en la subsección de resultados. RESULTADOS: Luego de la búsqueda sistemática realizada, se identificaron dos ensayos clínicos aleatorizados (ECA) Beasley, 1972 y Solomon et al., 1998; y el estudio de serie de casos de Pekel et al., 2014. Si bien estos estudios muestran algunas limitaciones que serán analizadas más adelante, es la evidencia de mayor relevancia en torno al uso de carbacol para la inducción de miosis intraoperatoria en las intervenciones quirúrgicas por catarata. Con respecto a la eficacia de carbacol en la inducción de miosis, el estudio de Beasley, 1972 muestra que el efecto miótico a los dos minutos de la inyección intraocular es significativamente mayor en carbacol, con respecto a placebo (p<0.01) y que el efecto miótico persiste por lo menos por 15 horas. Por otro lado, a la séptima semana postoperatoria, se observó una incidencia significativamente menor de sinequias anteriores periféricas (SAP) en el grupo que recibió carbacol (11 %), en comparación al grupo que recibió a placebo (35 %). Al analizar el impacto sobre la calidad de vida por parte de carbacol mediante el cuestionario modificado de SF-36, el cual mide la percepción del paciente sobre su estado de salud, el estudio de Solomon et al., 1998 muestra que carbacol incrementa la agudeza visual durante el primer día postoperatorio, con respecto a placebo, y muestra una diferencia estadísticamente significativa en el porcentaje de sujetos que pueden descender las escaleras sin ayuda durante la primera semana posterior a la intervención quirúrgica, tanto en un ambiente con luz brillante (p=0.007) o con luz tenue (p=0.037), siendo un potencial factor protector en pacientes con riesgo de presentar caídas acci Con respecto a los eventos adversos, los estudios evaluados no encontraron casos de inflamación intraocular ni de cefalea frontal. El estudio Pekel et al., 2015 muestra que sólo en el primer día postoperatorio hubo un menor volumen macular total (VMD y del grosor macular central (GMC) con respecto al volumen preoperatorio. Mientras que, durante el seguimiento (al primer día, a la primera semana y al primer mes) no se encontraron diferencias estadísticamente significativas en la presencia de edema macular, ni en el calibre de los vasos retinianos (CVR) al comparar los pacientes que recibieron carbacol con los que no lo recibieron. dentales y fractura de caderas. CONCLUSIONES: El presente dictamen preliminar muestra la evidencia disponible hasta mayo 2018 con respecto al uso intraocular de carbacol 0.01 % en comparación con placebo para la inducción de miosis intraoperatoria en pacientes operados de cataratas. No se encontraron guías de práctica clínica, revisiones sistemáticas ni evaluaciones de tecnologías sanitarias que respondan la pregunta PICO de la presente evaluación. Finalmente, se identificaron dos ECA y un estudio de serie de casos como sustento para la elaboración del presente dictamen preliminar. Al evaluar la eficacia de carbacol en la inducción de miosis, el estudio Beasley, 1972 muestra que carbacol genera miosis dentro de los dos minutos de ser aplicado y que su efecto perdura por más de 15 horas. Esto se traduce en una menor incidencia de SAP a la sétima semana postoperatorio, con respecto a placebo. Aunque no se aprecia diferencia en la conservación de la integridad de la cámara vítrea. Con respecto a los eventos adversos, los estudios evaluados no observaron casos de inflamación intraocular ni de cefalea frontal, mientras que el estudio Pekel et al., 2015 muestra que carbacol disminuye el volumen macular total (VMT) y el grosor macular central (GMT) en el primer día postoperatorio, mas no en la primera semana, ni primer mes. Por otro lado, no se ve afectado el calibre de los vasos retinianos (CVR). Estos resultados muestran una cierta protección ante el edema macular en el postoperatorio inmediato y ausencia de secuelas en la morfología macular, sin embargo, se debe tener en cuenta el posible sesgo de medición que conlleva. En resumen, carbacol es un agente miótico que inicia su acción dentro de los dos minutos de su inyección, manteniendo su efecto por más de 15 horas, generando un beneficio en el periodo postoperatorio inmediato en la agudeza visual y en el volumen macular total. Asimismo, los estudios no reportan eventos adversos como edema macular, cefalea, y, por el contrario, refieren una reducción en la incidencia de SAP, mas no hay una diferencia en la preservación de la integridad de la cámara vítrea, al ser comparado con placebo. Con respecto a la calidad de vida en el postoperatorio, se observa un inicio más temprano de la deambulación y autonomía en el uso de las escaleras, lo que podría tener un impacto positivo en el estilo de vida de los pacientes intervenidos. Por lo expuesto, el Instituto de Evaluaciones de Tecnologías en Salud e Investigación - IETSI aprueba el uso de carbacol, según lo establecido en el Anexo N.° 1. La vigencia del presente dictamen preliminar es de dos años a partir de la fecha de publicación.
Assuntos
Humanos , Carbacol/administração & dosagem , Extração de Catarata/métodos , Miose/induzido quimicamente , Análise Custo-Eficiência , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do Tratamento , Cuidados Intraoperatórios/métodosRESUMO
INTRODUCCIÓN: El presente dictamen preliminar expone la evaluación de tecnología de la eficacia y seguridad del uso intraocular de carbacol 0.01% para la inducción de miosis intraoperatoria en pacientes operados de cataratas. La catarata es definida por la Organización Mundial de la Salud (OMS) como la opacificación del lente cristalino que generalmente ocurre por el envejecimiento, trauma, o alguna enfermedad sistémica, afectando la capacidad visual de la persona. Esta disminución de la capacidad visual o incluso la ceguera, es un problema de salud pública en adultos y adultos mayores. En el Perú, aproximadamente el 0.6 % de la población tiene ceguera, cuya causa en el 47 % de los casos son por las cataratas. El tratamiento indicado para la catarata es la intervención quirúrgica, la cual consiste en reemplazar el cristalino opacificado o catarata por un lente intraocular. Hay dos formas de realizar esto, mediante la extracción extracapsular del cristalino opacificado, o mediante la facoemulsificación del cristalino, que consiste en un proceso de destrucción mediante ondas vibratorias ultrasónicas. Luego de ello, se realiza la implantación de un nuevo lente intraocular con soporte capsular, el cual reemplaza al cristalino permitiendo que el paciente vuelva tener una visión adecuada. Posterior a la implantación del lente intraocular se realiza la inducción de miosis pupilar para mantener el lente dentro de la bolsa capsular, evitar la captura del lente por el iris y el prolapso del iris por las heridas operatorias. TECNOLOGÍA SANITARIA DE INTERÉS: Carbacol, también conocido como carbamilcolina, es un colinérgico o parasimpaticomimético potente, que actúa como agonista del receptor de acetilcolina, inhibiendo la acetilcolinesterasa y estimulando tanto los receptores muscarínicos como nicotínicos, produciendo constricción del iris y del cuerpo ciliar y además reduciendo la presión intraocular. METODOLOGÍA: Se realizó una búsqueda sin restricción de idioma hasta mayo del 2018. La formulación de la estrategia de búsqueda incluyó los criterios de elegibilidad, los términos controlados propios de cada base y términos libres. Asimismo, se buscaron otros documentos potencialmente elegibles a través de la revisión del listado de referencias de los documentos seleccionados para lectura a texto completo. Por último, la selección de la evidencia siguió el flujograma mostrado en la subsección de resultados. RESULTADOS: Luego de la búsqueda sistemática realizada, se identificaron dos ensayos clínicos aleatorizados (ECA) Beasley, 1972 y Solomon et al., 1998; y el estudio de serie de casos de Pekel et al., 2014. Si bien estos estudios muestran algunas limitaciones que serán analizadas más adelante, es la evidencia de mayor relevancia en torno al uso de carbacol para la inducción de miosis intraoperatoria en las intervenciones quirúrgicas por catarata. CONCLUSIONES: El presente dictamen preliminar muestra la evidencia disponible hasta mayo 2018 con respecto al uso intraocular de carbacol 0.01 % en comparación con placebo para la inducción de miosis intraoperatoria en pacientes operados de cataratas. No se encontraron guías de práctica clínica, revisiones sistemáticas ni evaluaciones de tecnologías sanitarias que respondan la pregunta PICO de la presente evaluación. Finalmente, se identificaron dos ECA y un estudio de serie de casos como sustento para la elaboración del presente dictamen preliminar. Al evaluar la eficacia de carbacol en la inducción de miosis, el estudio Beasley, 1972 muestra que carbacol genera miosis dentro de los dos minutos de ser aplicado y que su efecto perdura por más de 15 horas. Esto se traduce en una menor incidencia de SAP a la sétima semana postoperatorio, con respecto a placebo. Aunque no se aprecia diferencia en la conservación de la integridad de la cámara vítrea. Con respecto a los eventos adversos, los estudios evaluados no observaron casos de inflamación intraocular ni de cefalea frontal, mientras que el estudio Pekel et al., 2015 muestra que carbacol disminuye el volumen macular total (VMT) y el grosor macular central (GMT) en el primer día postoperatorio, mas no en la primera semana, ni primer mes. Por otro lado, no se ve afectado el calibre de los vasos retinianos (CVR). Estos resultados muestran una cierta protección ante el edema macular en el postoperatorio inmediato y ausencia de secuelas en la morfología macular, sin embargo, se debe tener en cuenta el posible sesgo de medición que conlleva. El Instituto de Evaluaciones de Tecnologías en Salud e Investigación IETSI aprueba el uso de carbacol. La vigencia del presente dictamen preliminar es de dos años a partir de la fecha de publicación.
Assuntos
Humanos , Carbacol/administração & dosagem , Extração de Catarata , Miose/induzido quimicamente , Avaliação da Tecnologia Biomédica , Análise Custo-EficiênciaRESUMO
BACKGROUND: It is actually known that acetylcholine works as a signaling molecule in non-neuronal cells and tissues, in addition to its neuronal function as neurotransmitter. It can act on two types of receptors nicotinic and muscarinic receptors (mAChRs). The latter belong to the G protein coupled receptor family and there are five subtypes genetically cloned. Their activation triggers classical and non-classical intracellular signals that could be linked to the proliferation of normal and/or transformed cells. The M3 subtype was identified in different types of tumors and its stimulation with agonists triggers cell proliferation, migration, invasion and metastasis. RESULTS: Our laboratory has extensively investigated the expression and function of mAChRs in breast tumors from animal and human origins. We found a profuse expression of mAChRs in breast tumors, but opposite to this, an absence of these receptors in normal breast cells and tissues. The stimulation of mAChRs with the cholinergic agonist carbachol for 20 h increased tumor cell death. Moreover, the combination of subthreshold concentrations of the agonist with paclitaxel potentiates cell death. The usage of low dose chemotherapy with short drug free intervals was named metronomic therapy and it has emerged as a novel regimen for cancer treatment with very low incidence of side effects. CONCLUSION: Our work and that of others indicate that mAChRs that are over-expressed in different types of tumor cells could be a useful target for metronomic therapy in cancer treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Paclitaxel/farmacologia , Receptores Muscarínicos/metabolismo , Administração Metronômica , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carbacol/administração & dosagem , Agonistas Colinérgicos/administração & dosagem , Feminino , Humanos , Paclitaxel/administração & dosagem , Receptores Muscarínicos/genéticaRESUMO
In this work, we investigated the spasmolytic effect of caulerpine, a bisindole alkaloid isolated from marine algae of the Caulerpa genus, on guinea pig ileum. Our findings indicated that caulerpine inhibited phasic contractions induced by carbachol (IC50 = 7.0 ± 1.9 × 10â»5 M), histamine (IC50 = 1.3 ± 0.3 × 10â»4 M) and serotonin (IC50 = 8.0 ± 1.4 × 10â»5 M) in a non-selective manner. Furthermore, caulerpine concentration-dependently inhibited serotonin-induced cumulative contractions (pD'2 = 4.48 ± 0.08), shifting the curves to the right with Emax reduction and slope of 2.44 ± 0.21, suggesting a noncompetitive antagonism pseudo-irreversible. The alkaloid also relaxed the ileum pre-contracted by KCl (EC50 = 9.0 ± 0.9 × 10â»5 M) and carbachol (EC50 = 4.6 ± 0.7 × 10â»5 M) in a concentration-dependent manner. This effect was probably due to inhibition of Ca²âº influx through voltage-gated calcium channels (CaV), since caulerpine slightly inhibited the CaCl2-induced contractions in depolarizing medium without Ca²âº, shifting the curves to the right and with Emax reduction. According to these results, the spasmolytic effect of caulerpine on guinea pig ileum seems to involve inhibition of Ca²âº influx through CaV. However, other mechanisms are not discarded.
Assuntos
Cálcio/metabolismo , Caulerpa/química , Indóis/farmacologia , Parassimpatolíticos/farmacologia , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Carbacol/administração & dosagem , Carbacol/farmacologia , Relação Dose-Resposta a Droga , Feminino , Cobaias , Histamina/administração & dosagem , Histamina/farmacologia , Íleo/efeitos dos fármacos , Íleo/metabolismo , Indóis/administração & dosagem , Indóis/isolamento & purificação , Concentração Inibidora 50 , Masculino , Contração Muscular/efeitos dos fármacos , Parassimpatolíticos/administração & dosagem , Parassimpatolíticos/isolamento & purificação , Serotonina/administração & dosagem , Serotonina/farmacologiaRESUMO
The paraventricular nucleus of the hypothalamus (PVN) is an important area of the brain involved in the control of cardiovascular system and fluid-electrolyte balance. In the present study we evaluated the effects of hypothalamic disconnection (HD) caudal to PVN in the pressor and dipsogenic responses induced by intracerebroventricular (icv) injections of angiotensin II (ANG II) or carbachol (cholinergic agonist). Male Holtzman rats (280-320 g) with a stainless steel cannula implanted into the lateral ventricle and submitted to sham or HD surgery were used. HD (2 or 15 days) reduced the pressor responses to ANG II (50 ng/1µl) icv (8±3 and 11±3 mm Hg, respectively, vs. sham: 23±3 and 21±2 mm Hg) or carbachol (4 nmol/1 µl) icv (8±2 and 21±3 mm Hg, respectively, vs. sham: 33±3 and 33±3 mm Hg), without changing baseline arterial pressure. Acutely (2-4 days), HD also reduced water intake to icv ANG II (3.3±2.2 vs. sham: 14.2±3.0 ml/60 min) or carbachol (4.4±1.8 vs. sham: 11.4±1.6 ml/60 min); however, chronically (15-17 days), HD produced no change on ANG II- and carbachol-induced water intake, in spite of the increased daily water intake and urinary volume. The results suggest that medial projections caudal to PVN are important for pressor and dipsogenic responses to central angiotensinergic and cholinergic activation.
Assuntos
Angiotensina II/administração & dosagem , Carbacol/administração & dosagem , Sistema Cardiovascular/efeitos dos fármacos , Agonistas Colinérgicos/administração & dosagem , Ingestão de Líquidos/fisiologia , Núcleo Hipotalâmico Paraventricular/fisiologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Fenômenos Fisiológicos Cardiovasculares , Injeções Intraventriculares , Masculino , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
Clinical and experimental data have shown that the preoptic area of the hypothalamus (POA) is involved in the generation and maintenance of NREM sleep. However, the activity of specific populations of POA neurons during REM sleep, NREM sleep and different waking conditions is still not firmly established. Consequently, we performed a quantitative, regionally-specific analysis of the Fos immunoreactivity of neurons in the POA of the cat during NREM sleep and REM sleep induced by microinjections of carbachol into the nucleus pontis oralis (REMc), as well as during quiet and alert wakefulness. We observed that while the total number of Fos immunoreactive neurons in the POA did not change as a function of these behavioral states, state-specific differences in neuronal activity were detected in restricted regions of the POA. An increase in the number of Fos+ neurons was observed in the rostral tip of the suprachiasmatic nucleus (SCN) during NREM (83.4+/-25.6) compared to quiet wakefulness (5.1+/-1.3, p<0.05) but not with the other behavioral states. In the median preoptic nucleus (MnPN), the number of Fos immunoreactive neurons was greater during NREM sleep (39.5+/-6.1) compared with quiet wakefulness (13.5+/-1.4, p<0.05) and REMc (16.2+/-2.0, p<0.05). State-specific Fos immunoreactive neurons were not observed in the ventro-lateral preoptic nucleus (VLPO). Finally, there was no significant increase in the number of Fos+ neurons during REMc in any of the subregions of the POA. In conclusion, within the POA, a selective neuronal activation during NREM sleep was found only in the MnPN. In addition, our data suggest a potential role of the SCN in NREM sleep. Finally, based on the distribution of Fos+ neurons in the entire POA, we conclude that the neuronal network involved in the regulation of NREM sleep is dispersed and intermingled with waking-related neurons.
Assuntos
Neurônios/metabolismo , Área Pré-Óptica/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Sono/fisiologia , Análise de Variância , Animais , Carbacol/administração & dosagem , Carbacol/farmacologia , Gatos , Imuno-Histoquímica , Masculino , Microinjeções , Fotomicrografia , Ponte/efeitos dos fármacos , Sono/efeitos dos fármacos , Núcleo Supraquiasmático/metabolismoRESUMO
Tonic immobility (TI) is an inborn defensive behavior characterized by a temporary state of profound and reversible motor inhibition elicited by some forms of physical restraint. It is known that endogenous antinociceptive systems are activated during the emission of defensive behaviors including TI. The nucleus raphe magnus (NRM) is related to the modulation of nociceptive and behavioral responses. In the present study, we investigated the role of the cholinergic system of the NRM in the modulation of TI and nociception in guinea pigs. Microinjection of the cholinergic agonist carbachol (0.5 microg/0.2 microl) into the NRM promoted a reduction in the duration of TI episodes and nociception, the latter measured by the vocalization test in guinea pigs. The effect of microinjection of carbachol on TI reduction and antinociception was blocked by the previous microinjection of the cholinergic antagonist atropine (0.5 microg/0.2 microl and 1 microg/0.2 microl, respectively), demonstrating the participation of muscarinic receptors in the modulation of these responses. Microinjection of atropine per se did not interfere with the duration of TI episodes. In summary, the present results demonstrate that cholinergic stimulation of the NRM promoted analgesia and a reduction in the duration of TI in guinea pigs. These data indicate that the NRM possibly contributes to the modulation of defensive and nociceptive behavioral responses, probably by modulating the activity of neurons in the ventral and dorsal horn of the spinal cord, respectively.
Assuntos
Fibras Colinérgicas/fisiologia , Resposta de Imobilidade Tônica/fisiologia , Limiar da Dor/fisiologia , Núcleos da Rafe/metabolismo , Animais , Carbacol/administração & dosagem , Agonistas Colinérgicos/administração & dosagem , Fibras Colinérgicas/efeitos dos fármacos , Cobaias , Masculino , Microinjeções , Atividade Motora/fisiologia , Núcleos da Rafe/efeitos dos fármacos , Vocalização Animal/efeitos dos fármacos , Vocalização Animal/fisiologiaRESUMO
Several studies have demonstrated the involvement of the central nucleus of the amygdala (CEA) in the modulation of defensive behavior and in antinociceptive regulation. In a previous study, we demonstrated the existence of a cholinergic-opioidergic interaction in the CEA, modulating the defensive response of tonic immobility in guinea pigs. In the present study, we investigated a similar interaction in the CEA, but now involved in the regulation of the nociceptive response. Microinjection of carbachol (2.7 nmol) and morphine (2.2 nmol) into the CEA promoted antinociception up to 45 min after microinjection in guinea pigs as determined by a decrease in the vocalization index in the vocalization test. This test consists of the application of a peripheral noxious stimulus (electric shock into the subcutaneous region of the thigh) that provokes the emission of a vocalization response by the animal. Furthermore, the present results demonstrated that the antinociceptive effect of carbachol (2.7 nmol; N = 10) was blocked by previous administration of atropine (0.7 nmol; N = 7) or naloxone (1.3 nmol; N = 7) into the same site. In addition, the decrease in the vocalization index induced by the microinjection of morphine (2.2 nmol; N = 9) into the CEA was prevented by pretreatment with naloxone (1.3 nmol; N = 11). All sites of injection were confirmed by histology. These results indicate the involvement of the cholinergic and opioidergic systems of the CEA in the modulation of antinociception in guinea pigs. In addition, the present study suggests that cholinergic transmission may activate the release of endorphins/enkephalins from interneurons of the CEA, resulting in antinociception.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Analgésicos/farmacologia , Medição da Dor/métodos , Receptores Colinérgicos/efeitos dos fármacos , Receptores Opioides/efeitos dos fármacos , Vocalização Animal , Animais , Atropina/administração & dosagem , Atropina/farmacologia , Carbacol/administração & dosagem , Carbacol/farmacologia , Estimulação Elétrica/métodos , Cobaias , Masculino , Microinjeções , Morfina/administração & dosagem , Morfina/farmacologia , Naloxona/administração & dosagem , Naloxona/farmacologiaRESUMO
Tonic immobility (TI), also known as death feigning or animal hypnosis, is a reversible state of motor inhibition that is triggered by postural inversion and/or movement restraining maneuvers but also by repetitive stimulation and pressure on body parts. Our previous studies demonstrated that cholinergic stimulation of the central amygdala (CEA) decreases the duration of TI in guinea pigs. Some reports have demonstrated that electrical or chemical stimulation of the CEA promotes antinociception. Evidence suggests that the CEA performs part of its functions by means of a connection with the ventrolateral periaqueductal gray (vlPAG). In the current study, we investigated the participation of a possible functional and anatomical CEA-vlPAG connection in guinea pigs in the regulation of the TI response and antinociception. Our results showed that the functional CEA-vlPAG connection is essential for the participation of the CEA in the modulation of TI and of antinociception. The reversible exclusion of the vlPAG by means of microinjection of 2% lidocaine blocked the inhibitory effect on TI duration and the antinociceptive effect, as determined by a decrease of the vocalization index (VI) obtained with the administration of carbachol (2.7 nmol/0.2 microl) into the CEA. On the other hand, the exclusion of the CEA by lidocaine did not block the antinociception or the increase in TI induced by microinjection of CCh into the vlPAG. Finally, microinjection of the retrograde neurotracer Fast Blue into the CEA or into the vlPAG demonstrated the existence of a reciprocal anatomical connection between the CEA and vlPAG.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Imobilização/fisiologia , Substância Cinzenta Periaquedutal/fisiologia , Amidinas/metabolismo , Tonsila do Cerebelo/fisiologia , Anestésicos Locais/administração & dosagem , Anestésicos Locais/farmacologia , Animais , Mapeamento Encefálico , Carbacol/administração & dosagem , Agonistas Colinérgicos/administração & dosagem , Corantes Fluorescentes/metabolismo , Cobaias , Lidocaína/administração & dosagem , Lidocaína/farmacologia , Masculino , Microinjeções , Inibição Neural/fisiologia , Vias Neurais/fisiologia , Medição da Dor , Substância Cinzenta Periaquedutal/anatomia & histologia , Estimulação Física , Tempo de Reação , Fatores de Tempo , Vocalização AnimalRESUMO
The present work studied the effects of dopaminergic and muscarinic receptor agonists and antagonists on rat locomotor activity and catalepsy. Results showed that carbachol at the highest dose used (10 mg/kg, p.o.) decreased and pimozide at the dose used abolished locomotor activity. Atropine at a low dose (1 mg/kg, p.o.) increased and at a high dose decreased this parameter. Mazindol at a high dose also increased locomotor activity. A significant and dose-dependent increase in the time on the bar was observed in animals treated with carbachol or pimozide as compared to controls. The increase observed with pimozide was greater than 60 s. Effects of carbachol on locomotor activity were observed already after the first drug exposure, but the increased time on bar produced by this drug in the test of catalepsy was observed only after repeated exposure (7th day). The effect of the highest dose (10 mg/kg, p.o.) of atropine (decreased activity) as related to the lowest one was evident at the 7th day, but the increased locomotor activity seen at the low dose was detected already at the first day. There was a predominance of the effect of pimozide on the open field as well as on catalepsy after its association with each one of the three doses of carbachol. The association of atropine and mazindol did not seem to alter locomotor activity and catalepsy as related to each drug alone. Our results indicate that interactions between dopaminergic and cholinergic systems play an important role on behavior and motor functions.
Assuntos
Comportamento Animal/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Administração Oral , Animais , Atropina/farmacologia , Carbacol/administração & dosagem , Carbacol/farmacologia , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Feminino , Locomoção/efeitos dos fármacos , Mazindol/farmacologia , Pimozida/farmacologia , Ratos , Ratos WistarRESUMO
As espécies reativas de oxigênio geradas in vivo têm sido implicadas em vários mecanismos como apoptose, crescimento e diferenciação celular, câncer e inflamação. O estresse oxidativo é o produto do desbalanço entre os agentes pro-oxidantes e antioxidantes celulares, causando danos por meio da peroxidação lipídica, oxidação de proteínas e de DNA. Neste trabalho, avaliou-se o efeito do estresse oxidativo em glândulas submandibulares de rato e em células acinares dispersas tratadas com isoproterenol (ISO), cicloheximida (CHX), carbacol (CA) e propanolol (PROP). Observou-se aumento dos níveis de MDA em homogenados de glândulas de ratos tratados com ISO e CHX e em células acinares dispersas incubadas com ISO e CHX...
Assuntos
Animais , Ratos , 3,4-Metilenodioxianfetamina , Carbacol/administração & dosagem , Isoproterenol/administração & dosagem , Estresse Oxidativo , Propranolol/administração & dosagem , Antioxidantes , Western Blotting , Técnicas de Cultura de Células , Meios de Cultura , Peroxidação de Lipídeos , Interpretação Estatística de Dados , Superóxido Dismutase , Transdução de SinaisRESUMO
A 33 de 48 pacientes con hipoacusia sensorioneural, cuya principal molestia era el tinitus, se les colocó en la caja timpánica mediante una aguja de punción lumbar Nº 26, punta de lápiz, a través del cuadrante anterior de la membrana timpánica solución de pilocarpina al 1 por ciento y a los otros 15 pacientes solución isopto de carbachol al 2 por ciento. Ambos grupos fueron seleccionados al azar sin considerar edad, sexo o daño topográfico de la vía acústica. La evaluación de su efecto se hace audiométricamente, después de 30 minutos de la inyección intratimpánica, equiparando el tinnitus antes y después del procedimiento, con el mínimo de intensidad de sonido puro o de banda angosta, que más se asemeje al ruido del paciente. Los resultados fueron positivos, desde la anulación completa a diferentes grados de atenuación del tinnitus, suficiente para comprobarlo con la equiparación audiométrica. Estos casos positivos alcanzan a más de la mitad de ellos. Los resultados serían mejores si se hubieran descartado los casos de hipoacusia por exposición a ruido enviados por las mutuales de seguros por accidentes del trabajo y enfermedades profesionales o los casos de presbiacusia. Estos resultados serían buenos si no fuera por la corta duración del efecto de las drogas que no es mayor que 12 a 72 horas del procedimiento. Luego reaparece el tinnitus en su intensidad original. Los resultados mejores fueron con el uso del carbachol frente a los de la pilocarpina. La sustitución de pilocarpina por carbachol se basó en el razonamiento de que el deterioro de las vías eferentes colinérgicas inhibitorias, que se intentan estimular en el oído interno vía ventana redonda, se dañan primero que las vías aferentes. El uso de la pilocarpina presupone un buen funcionamiento colinérgico, ya que su acción es agonista indirecta anulando las enzimas colinoesterasas
Assuntos
Humanos , Masculino , Feminino , Pilocarpina/administração & dosagem , Zumbido/tratamento farmacológico , Carbacol/administração & dosagem , Audiometria , Vertigem/epidemiologia , Nistagmo Patológico/epidemiologia , Orelha MédiaRESUMO
Pharmacological, neurochemical, and behavioral techniques were used to characterize DA-ACh interaction within the lateral hypothalamus (LH) in the context of locomotion, feeding behavior, and reinforcement. In Experiment 1, the muscarinic agonist carbachol injected in the LH increased locomotor activity in proportion to dose. In Experiment 2, the same doses of carbachol proportionately increased exctracellular DA in the nucleus accumbens (Nac) as monitored by brain microdialysis. Dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) also increased. In Experiment 3, LH infusion by reverse microdialysis of the D(2) receptor blocker sulpiride released ACh in the LH in a dose-response manner. This suggested that sulpiride disinhibits ACh release via D(2) receptors in the LH and thereby facilitates behavior. Confirming this in Experiment 4, local LH atropine 5 min before sulpiride suppressed the locomotor response to sulpiride for about 20 min. These results suggest that sulpiride acts in the LH by disinhibiting a hypothalamic locomotor mechanism that is cholinergically driven and connected with the mesoaccumbens dopamine pathway. Given prior results that local sulpiride in the LH can induce hyperphagia and reward, this system may be involved in searching for food and rewarding feeding behavior. In conclusion, DA acts in the LH via D(2) receptors to inhibit cholinergic neurons or terminals that are part of an approach system for eating.
Assuntos
Acetilcolina/fisiologia , Dopamina/fisiologia , Região Hipotalâmica Lateral/fisiologia , Locomoção/fisiologia , Animais , Atropina/administração & dosagem , Carbacol/administração & dosagem , Carbacol/farmacologia , Agonistas Colinérgicos/administração & dosagem , Agonistas Colinérgicos/farmacologia , Antagonistas de Dopamina/administração & dosagem , Antagonistas de Dopamina/farmacologia , Interações Medicamentosas , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologia , Região Hipotalâmica Lateral/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Masculino , Microinjeções , Ratos , Ratos Sprague-Dawley , Reforço Psicológico , Sulpirida/administração & dosagem , Sulpirida/farmacologiaRESUMO
Tonic immobility (TI) is thought to represent the terminal reaction in the chain of antipredatory responses involved in maintaining survival. TI is an inhibitory behavioral response in which the animal presents a significant decrease in body activity and responsiveness to the environment induced by some form of physical restraint. This response is induced in the laboratory by inversion of the animal and brief postural contention of its movements. In nature, the TI response may be triggered by some threatening or predatory stimulus, indicating the physical contact between response occurs when there is physical contact between prey and predator. In this case, the physical inactivity of the prey may prevent the continuation of the attack. The neural substrate of this response is not well known, and the objective of the present study was to investigate the effect of cholinergic stimulation of amygdala regions on TI modulation in guinea pigs. Microinjection of carbachol (0.5 microg/0.2 microL) into the central (CEA), basolateral (BLA), and lateral posterior (LPA) nuclei of the amygdala promoted a reduction in the duration of TI episodes. Pretreatment with atropine (0.5 microg/0.2 microL) showed that the action of carbachol is mediated by muscarinic receptors.
Assuntos
Tonsila do Cerebelo/fisiologia , Comportamento Animal/fisiologia , Tonsila do Cerebelo/anatomia & histologia , Animais , Atropina/administração & dosagem , Atropina/farmacologia , Comportamento Animal/efeitos dos fármacos , Carbacol/administração & dosagem , Carbacol/farmacologia , Cobaias , Microinjeções , Agonistas Muscarínicos/administração & dosagem , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/administração & dosagem , Antagonistas Muscarínicos/farmacologia , Sistema Nervoso Parassimpático/efeitos dos fármacos , Sistema Nervoso Parassimpático/fisiologia , Receptores Muscarínicos/fisiologiaRESUMO
Tonic immobility is an inborn defensive behavior characterized by a temporary state of profound and reversible motor inhibition elicited by some forms of physical restraint. The periaqueductal gray matter (PAG) contains neural circuits involved in descending pain modulation, as well as in the modulation of TI. We have reported previously that the cholinergic stimulation of the ventrolateral PAG increases the duration of TI in guinea pigs. In the present study, we attempted to characterize further the modulation of TI by pharmacological alteration of the neurochemistry of the ventrolateral PAG circuitry. We observed that both cholinergic (carbachol, 5.4 nmol/0.2 microl) and opioidergic stimulations (morphine, 4.48 nmol/0.2 microl) of the ventrolateral PAG increase the duration of TI and that these effects can be reversed by pre-treatment with naloxone (2.74 nmol/0.2 microl). Our results also showed that microinjection of the GABAergic agonist muscimol (1, 0.5, and 0.26 nmol/0.2 microl) decreased the duration of TI episodes, while microinjection of the GABAergic antagonist bicuculline (1 nmol/microl) increased it. Moreover, we observed that preadministration of muscimol (0.13 nmol/0.2 microl) at a dose that had no effect per se at this site antagonized the potentiating effect of morphine. Our results suggest that this modulation of TI from the ventrolateral PAG circuitry is accomplished by a complex interaction of cholinergic, opioidergic, and GABAergic mechanisms, similar to that proposed for descending antinociceptive circuits.
Assuntos
Carbacol/farmacologia , Reação de Fuga/efeitos dos fármacos , Morfina/farmacologia , Substância Cinzenta Periaquedutal/fisiologia , Animais , Bicuculina/administração & dosagem , Bicuculina/análogos & derivados , Bicuculina/farmacologia , Mapeamento Encefálico , Carbacol/administração & dosagem , Reação de Fuga/fisiologia , Cobaias , Masculino , Microinjeções , Morfina/administração & dosagem , Muscimol/administração & dosagem , Muscimol/farmacologia , Naloxona/administração & dosagem , Naloxona/farmacologia , Vias Neurais/fisiologia , Postura , Restrição FísicaRESUMO
In this study we investigated: (a) the effects of intracerebroventricular (i.c.v.) injections of moxonidine (an alpha2-adrenergic and imidazoline receptor agonist) on the ingestion of water and NaCl induced by 24 h of water deprivation; (b) the effects of i.c.v. injection of moxonidine on central angiotensin II (ANG II)- and carbachol-induced water intake; (c) the effects of the pre-treatment with i.c.v. idazoxan (an alpha2-adrenergic and imidazoline receptor antagonist) and RX 821002 (a selective alpha2-adrenergic antagonist) on the antidipsogenic action of central moxonidine. Male Holtzman rats had stainless steel cannulas implanted in the lateral cerebral ventricle. Intracerebroventricular injection of moxonidine (5 and 20 nmol/1 microl) reduced the ingestion of 1.5% NaCl solution (4.1 +/- 1.1 and 2.9 +/- 2.5 ml/2 h, respectively vs. control = 7.4 +/- 2.1 ml/2 h) and water intake (2.0 +/- 0.6 and 0.3 +/- 0.2 ml/h, respectively vs. control = 13.0 +/- 1.4 ml/h) induced by water deprivation. Intracerebroventricular moxonidine (5 nmol/1 microl) also reduced i.c.v. ANG II-induced water intake (2.8 +/- 0.9 vs. control = 7.9 +/- 1.7 ml/1 h) and i.c.v. moxonidine (10 and 20 nmol/1 microl) reduced i.c.v. carbachol-induced water intake (4.3 +/- 1.7 and 2.1 +/- 0.9, respectively vs. control = 9.2 +/- 1.0 ml/1 h). The pre-treatment with i.c.v. idazoxan (40 to 320 nmol/1 microl) abolished the inhibitory effect of i.c.v. moxonidine on carbachol-induced water intake. Intracerebroventricular idazoxan (320 nmol/1 microl) partially reduced the inhibitory effect of moxonidine on water deprivation-induced water intake and produced only a tendency to reduce the antidipsogenic effect of moxonidine on ANG II-induced water intake. RX 821002 (80 and 160 nmol/1 microl) completely abolished the antidipsogenic action of moxonidine on ANG II-induced water intake. The results show that central injections of moxonidine strongly inhibit water and NaCl ingestion. They also suggest the involvement of central alpha2-adrenergic receptors in the antidipsogenic action of moxonidine.
Assuntos
Anti-Hipertensivos/administração & dosagem , Ingestão de Líquidos/efeitos dos fármacos , Imidazóis/administração & dosagem , Cloreto de Sódio na Dieta/administração & dosagem , Antagonistas Adrenérgicos alfa/administração & dosagem , Antagonistas Adrenérgicos alfa/farmacologia , Angiotensina II/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Carbacol/administração & dosagem , Carbacol/farmacologia , Agonistas Colinérgicos/administração & dosagem , Agonistas Colinérgicos/farmacologia , Idazoxano/administração & dosagem , Idazoxano/análogos & derivados , Idazoxano/farmacologia , Imidazóis/farmacologia , Injeções Intraventriculares , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Vasoconstritores/farmacologiaRESUMO
The serotonergic Retzius neurons of the leech midbody ganglia respond in a complex manner to pressure pulses of acetylcholine (ACh) applied onto their soma with a fast depolarization followed by a slower hyperpolarization and an additional delayed long-lasting depolarization. The delayed depolarization is the subject of the present study. The delayed depolarization could be elicited by long (> 1 s) ACh pressure pulses or by short pulses (10 ms) of carbachol, nicotine and DMPP, but not by muscarinic agonists. It was inhibited by bath application of nicotine (10-100 mumol l-1), strychnine (100 mumol l-1) and atropine (10-100 mumol l-1). Nicotinic antagonists that blocked the fast depolarization and the slow hyperpolarization (100 mumol l-1 mecamylamine and d-tubocurarine) did not affect the delayed depolarization induced by carbachol. Partial replacement of the extracellular Na+ by glucamine caused a decrease in the amplitude of the response and a shift of its reversal potential to more negative values. Carbachol pulses applied to Retzius neurons of the ganglia innervating the reproductive segments elicited delayed depolarizations of much smaller amplitude than the ones recorded in Retzius neurons from standard segments. The delayed depolarization could be elicited by the application of short agonist pulses onto different loci over the surface of the ganglion, at a distance from the soma. Isolated cultured Retzius neurons did not exhibit the delayed depolarization although they readily expressed the earlier phases of the complex cholinergic response. Carbachol pulses applied to the soma of other neurons in the leech ganglion produced a variety of specific responses. The results suggest that the delayed depolarization was produced by the activation of a cationic conductance mediated by receptors with a pharmacological profile similar to that of the alpha 9 nicotinic receptors and was not a byproduct of the early phases of the cholinergic response. The response seemed to be initiated in the extensive neuropilar processes of the Retzius cell, enabling a persistent excitatory signal.