RESUMEN
Isoflurane post-conditioning causes an early increase in cardiac progenitor cells; however, during the chronic phase of infarct healing, the number was smaller compared to control, which suggests a positive effect on infarct scar maturity. Myofibroblasts participate in early phase infarct contraction, but their number is small in a mature scar. We investigated whether isoflurane post-conditioning stimulates differentiation of progenitor cells to myofibroblasts and to verify our hypothesis that isoflurane post-conditioning improves maturation of a myocardial scar. Ischemia was induced for 30 min in female rats. From the last 5 min of ischemia until 10 min into reperfusion, the isoflurane group received 1.5% isoflurane, while the control group received only an air-oxygen mixture. Infarct area was analyzed using immunohistochemistry. During the subacute phase of infarct healing, the number of myofibroblasts was greater in isoflurane-treated animals than in the control group. During the chronic phase of infarct healing, post-conditioned animals exhibited fewer myofibroblasts compared to control animals, even those derived from progenitor cells, i.e., α-smooth actin-nestin positive cells. In addition, isoflurane post-conditioning resulted in higher percentage of mature blood vessels compared to control animals. The myocardium of the isoflurane treated animals exhibited more myofibroblasts in granulation tissue compared to control animals. The smaller number of myofibroblasts together with the greater number of mature blood vessels during the chronic phase of healing demonstrated faster healing of the infarct area of isoflurane-treated animals compared to control animals.
Asunto(s)
Isoflurano/farmacología , Infarto del Miocardio/tratamiento farmacológico , Miocardio/patología , Cicatrización de Heridas/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Nestina/metabolismo , Ratas Sprague-DawleyRESUMEN
17ß-Estradiol (E2) crucially affects several processes in the hippocampus of both sexes. E2 acts upon estradiol receptors ERα and ERß, influencing target gene expression and/or modulates intracellular signaling cascades. Another potent modulator of hippocampal function is nucleoside adenosine, the final product of ectonucleotidase cascade, enzymes which hydrolyze extracellular ATP to adenosine. The last and rate-limiting step of the hydrolysis is catalyzed by membrane-bound ecto-5'-nucleotidase (eN). Previous findings obtained on adenosine metabolism in brain suggest that eN may be modulated by ovarian steroids. Therefore, the present study reports that the activity and protein abundance of membrane-bound eN fluctuates across the estrus cycle in the hippocampal synaptosomes of female rats. Further, we analyzed the role of E2 and its intracellular receptors on the expression of eN in ovariectomized females. We found that E2 upregulated eN activity and protein abundance in the hippocampal synaptosomes. Application of nonspecific ER antagonist, ICI 182,780 and selective ERα and ERß agonists, PPT and DPN, respectively, demonstrated the involvement of both receptor subtypes in observed actions. Selective ERα receptor agonist, PPT, induced upregulation of both the protein level and activity of eN, while application of selective ERß receptor agonist, DPN, increased only the activity of eN. In both cases, E2 entered into the intracellular compartment and activated ER(s), which was demonstrated by membrane impermeable E2-BSA conjugate. Together these results imply that E2-induced effects on connectivity and functional properties of the hippocampal synapses may be in part mediated through observed effect on eN.
Asunto(s)
5'-Nucleotidasa/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Estrógenos/farmacología , Animales , Estradiol/análogos & derivados , Antagonistas del Receptor de Estrógeno/farmacología , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/agonistas , Receptor beta de Estrógeno/antagonistas & inhibidores , Ciclo Estral/efectos de los fármacos , Ciclo Estral/fisiología , Femenino , Fulvestrant , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Espacio Intracelular/metabolismo , Nitrilos/farmacología , Fenoles/farmacología , Pirazoles/farmacología , Distribución Aleatoria , Ratas Wistar , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The present study attempted to investigate how chronic cerebral hypoperfusion (CCH) and repeated low-dose progesterone (P) treatment affect gene and protein expression, subcellular distribution of key apoptotic elements within protein kinase B (Akt) and extracellular signal-regulated kinases (Erk) signal transduction pathways, as well as neurodegenerative processes and behavior. The results revealed the absence of Erk activation in CCH in cytosolic and synaptosomal fractions, indicating a lower threshold of Akt activation in brain ischemia, while P increased their levels above control values. CCH induced an increase in caspase 3 (Casp 3) and poly (ADP-ribose) polymerase (PARP) gene and protein expression. However, P restored expression of examined molecules in all observed fractions, except for the levels of Casp 3 in synapses which highlighted its possible non-apoptotic or even protective function. Our study showed the absence of nuclear factor kappa-light-chain-enhancer of activated b cells (NF-κB) response to this type of ischemic condition and its strong activation under the influence of P. Further, the initial increase in the number of apoptotic cells and amount of DNA fragmentation induced by CCH was significantly reduced by P. Finally, P reversed the CCH-induced reduction in locomotor activity, while promoting a substantial decrease in anxiety-related behavior. Our findings support the concept that repeated low-dose post-ischemic P treatment reduces CCH-induced neurodegeneration in the hippocampus. Neuroprotection is initiated through the activation of investigated kinases and regulation of their downstream molecules in subcellular specific manner, indicating that this treatment may be a promising therapy for alleviation of CCH-induced pathologies.
Asunto(s)
Trastornos Cerebrovasculares/tratamiento farmacológico , Trastornos Cerebrovasculares/fisiopatología , Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , Fármacos Neuroprotectores/administración & dosificación , Progesterona/administración & dosificación , Animales , Ansiedad/tratamiento farmacológico , Ansiedad/fisiopatología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Enfermedades de las Arterias Carótidas , Enfermedad Crónica , Modelos Animales de Enfermedad , Trastornos de Somnolencia Excesiva , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Distribución Aleatoria , Ratas WistarRESUMEN
To study time-dependent and gender-specific intracellular and biochemical mechanisms that lead to neurodegeneration due to moderate but persistent reduction of cerebral blood flow, adult male and female Wistar rats were divided into two main groups - controls that underwent sham operation and animals subjected to permanent bilateral occlusion of common carotid arteries. Animals were sacrificed 3, 7 or 90 days following the insult. Expression of several apoptotic proteins in synaptic fractions along with Fluoro-Jade B staining and DNA fragmentation assay were used to estimate the apoptotic processes and potential neurodegeneration in cerebral cortex. Data suggest a time-specific increase of Bax as well as time- and gender-associated downregulation in protein expression of Bcl-2, up-regulation of procaspase 3, accompanied with increased cleavage of procaspase 3 and PARP in synaptic terminals. Furthermore, time- but not gender-specific neurodegeneration was observed. Our findings support the concept of time- and gender-associated response to permanent bilateral occlusion of common carotid arteries, which would enable better understanding of the mechanisms underlying cerebral hypoperfusion.
Asunto(s)
Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/patología , Circulación Cerebrovascular , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Animales , Apoptosis , Caspasa 3/metabolismo , Corteza Cerebral/fisiopatología , Corteza Cerebral/cirugía , Fragmentación del ADN , Femenino , Fluoresceínas , Masculino , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteolisis , Ratas Wistar , Coloración y Etiquetado , Sinapsis/metabolismo , Factores de Tiempo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Synthetic glucocorticoid dexamethasone (DEX), a highly potent anti-inflammatory and immunosuppressive agent, is widely used in the treatment of brain cancer, as well as for inflammatory and autoimmune diseases. The present study aimed to determine whether low-dose subchronic DEX treatment (100 µg/kg for eight consecutive days) exerts long-term effects on apoptosis in the adult rat prefrontal cortex (PFC) by examining the expression of cell death-promoting molecules [poly(ADP-ribose) polymerase (PARP), p53, procaspase 3, cleaved caspase 3, Bax] and cell-survival molecules (AKT, Bcl-2). The results obtained revealed that body, thymus and adrenal gland weights, as well corticosterone levels, in the serum and PFC were reduced 1 day after the last DEX injection. In the PFC, DEX caused activation of AKT, augmentation of pro-survival Bcl-2 protein and an enhanced Bcl-2/Bax protein ratio, as well Bcl-2 translocation to the mitochondria. An unaltered profile with respect to the protein expression of apoptotic molecules PARP, procaspase 3 and Bax was detected, whereas p53 protein was decreased. Reverse transcriptase -polymerase chain reaction analysis showed a decrease of p53 mRNA levels and no significant difference in Bcl-2 and Bax mRNA expression in DEX-treated rats. Finally, a DNA fragmentation assay and Fluoro-Jade staining demonstrated no considerable changes in apoptosis in the rat PFC. Our findings support the concept that low-dose DEX creates a hypocorticoid state in the brain and also indicate that subchronic DEX treatment activates the pro-survival signalling pathway but does not change apoptotic markers in the rat PFC. This mechanism might be relevant for the DEX-induced apoptosis resistance observed during and after chemotherapy of patients with brain tumours.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Dexametasona/farmacología , Corteza Prefrontal/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Secuencia de Bases , Cartilla de ADN , Relación Dosis-Respuesta a Droga , Masculino , Corteza Prefrontal/metabolismo , Ratas , Ratas WistarRESUMEN
The aim of this study was to examine the rapid non-genomic effect of 17ß-estradiol (E2) on Ca(2+) transport in mitochondria isolated from the nerve terminals (synaptosomes) of caudate nuclei (NC) and brain stems (BS) of ovariectomised female rats. In physiological conditions no effect of E2 on Ca(2+) influx into synaptosomal mitochondria through ruthenium red (RR)-sensitive uniporter was observed. However, in the presence of uncoupling agent carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP) (1µmol/l), pre-treatment with 0.5nmol/l E2 protected mitochondrial membrane potential and consequently increased Ca(2+) influx (2.3-fold in NC and 3.1-fold in BS). At the same time, 0.5nmol/l E2 by increasing the affinity of mitochondrial Na(+)/Ca(2+) exchanger for Na(+) inhibited mitochondrial Ca(2+) efflux in NC and BS by about 40%. Also, the specific binding of physiological E2 concentrations (0.1-10nmol/l) to isolated synaptosomal mitochondria was detected. Using membrane impermeable E2 bound to bovine serum albumin and selective inhibitor of mitochondrial Na(+)/Ca(2+) exchanger, we obtained that E2's action on mitochondrial Ca(2+) efflux at least partially is due to the direct effects on the mitochondrial membrane and/or Na(+)/Ca(2+) exchanger located in inner mitochondrial membrane. Our results implicate E2 as a modulator of Ca(2+) concentration in mitochondrial matrix, and ultimately in the cytosol. Given the vital role of Ca(2+) in regulation of total nerve cells activity, especially energy metabolism, neurotransmission and directing the cells toward survival or cell death, the effects on mitochondrial Ca(2+) transport could be one of the important modes of E2 neuromodulatory action independent of the genome.
Asunto(s)
Tronco Encefálico/metabolismo , Calcio/metabolismo , Núcleo Caudado/metabolismo , Estradiol/metabolismo , Mitocondrias/metabolismo , Animales , Tronco Encefálico/efectos de los fármacos , Núcleo Caudado/efectos de los fármacos , Estradiol/farmacología , Femenino , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/efectos de los fármacos , Ovariectomía , Ratas , Ratas Wistar , Sinaptosomas/metabolismoRESUMEN
Ganglioside GM3(Neu5Ac) expression is highly increased in liver 54h following 15% partial hepatectomy in pre-operatively oxygenated rats. GM3(Neu5Gc), GM2, GalNAc-GM1b and gangliosides of the neolacto-series are less affected. GM3(Neu5Ac) is a potent inhibitor of epidermal growth factor signaling. Since GM3(Neu5Ac) growth inhibitory effect depends on its cellular localization, the aim of this study was to detect ganglioside cellular localization during liver regeneration. The experiment was performed using the same rat model which previously showed increased ganglioside expression and more efficient liver regeneration. Frozen sections of liver were analyzed using confocal microscopy after labeling for binding of five ganglioside-specific antibodies, with or without hepatocyte membrane permeabilization. Ganglioside precursors, ceramide (Cer), monohexaosylceramide and lactosylceramide (LacCer) were determined by high-performance thin-layer chromatography. Apoptosis was assessed by fluorescein-dUTP end-labeling of fragmented DNA. Liver of pre-operative oxygenated rats showed high perinuclear labeling of GM3(Neu5Ac) which was absent in post-operative oxygenated and control animals. In the same group, Cer content was lower, monohexaosylceramide and LacCer were absent, and content of apoptotic cells was significantly the lowest, compared to other groups examined (F=20.36, p=0.0001). These findings indicate that ganglioside GM3(Neu5Ac) may be involved in mediation of beneficial effects of pre-operatively oxygenation during the liver regeneration.
Asunto(s)
Gangliósidos/análisis , Inmunohistoquímica/métodos , Hígado/efectos de los fármacos , Oxígeno/farmacología , Animales , Apoptosis/efectos de los fármacos , Ceramidas/análisis , Cromatografía Líquida de Alta Presión , Hepatectomía/métodos , Hígado/metabolismo , Hígado/fisiopatología , Regeneración Hepática/efectos de los fármacos , Microscopía Confocal , Ratas , Ratas WistarRESUMEN
The sympathetic innervation of the rat heart was investigated by retrograde neuronal tracing and multiple label immunohistochemistry. Injections of Fast Blue made into the left ventricular wall labelled sympathetic neurons that were located along the medial border of both the left and right stellate ganglia. Cardiac projecting sympathetic postganglionic neurons could be grouped into one of four neurochemical populations, characterised by their content of calbindin and/or neuropeptide Y (NPY). The subpopulations of neurons contained immunoreactivity to both calbindin and NPY, immunoreactivity to calbindin only, immunoreactivity to NPY only and no immunoreactivity to calbindin or NPY. Sympathetic postganglionic neurons were also labelled in vitro with rhodamine dextran applied to the cut end of a cardiac nerve. The same neurochemical subpopulations of sympathetic neurons were identified by using this technique but in different proportions to those labelled from the left ventricle. Preganglionic terminals that were immunoreactive for another calcium-binding protein, calretinin, preferentially surrounded retrogradely labelled neurons that were immunoreactive for both calbindin and NPY. The separate sympathetic pathways projecting to the rat heart may control different cardiac functions.
Asunto(s)
Ventrículos Cardíacos/inervación , Neuronas/química , Fibras Simpáticas Posganglionares/fisiología , Amidinas/administración & dosificación , Animales , Calbindina 2 , Calbindinas , Femenino , Ganglios Simpáticos/citología , Ganglios Simpáticos/metabolismo , Inmunohistoquímica , Masculino , Neuronas/clasificación , Neuropéptido Y/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína G de Unión al Calcio S100/metabolismo , Coloración y Etiquetado/métodos , Ganglio Estrellado/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The neurochemistry of intracardiac neurons in whole-mount preparations of the intrinsic ganglia was investigated. This technique allowed the study of the morphology of the ganglionated nerve plexus found within the atria as well as of individual neurons. Intracardiac ganglia formed a ring-like plexus around the entry of the pulmonary veins and were interconnected by a series of fine nerve fibres. All intracardiac neurons contained immunoreactivity to PGP-9.5, choline acetyl transferase (ChAT) and neuropeptide Y (NPY). Two smaller subpopulations were immunoreactive to calbindin or nitric oxide synthase. Furthermore, a subpopulation (approximately 6%) of PGP-9.5/ChAT/NPY-immunoreactive cells lacking both calbindin and nitric oxide synthase (NOS) was surrounded by pericellular baskets immunoreactive to ChAT and calbindin. Vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activated peptide (PACAP), substance P and tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibres within the ganglion, but never in neuronal somata. Furthermore, immunoreactivity for NPY was not observed in pericellular baskets surrounding intracardiac neurons, despite being present in all intrinsic neuronal cell bodies. Taken together, the results of this study indicate a moderate level of chemical diversity within the intracardiac neurons of the rat. Such chemical diversity may reflect functional specialisation of neurons in the intracardiac ganglia.
Asunto(s)
Ganglios Autónomos/citología , Corazón/inervación , Neuronas/citología , Animales , Axones/metabolismo , Axones/ultraestructura , Biomarcadores , Tronco Encefálico/citología , Tronco Encefálico/metabolismo , Calbindinas , Péptido Relacionado con Gen de Calcitonina/metabolismo , Colina O-Acetiltransferasa/metabolismo , Femenino , Ganglios Autónomos/metabolismo , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Inmunohistoquímica , Masculino , Neuronas/clasificación , Neuronas/metabolismo , Neuropéptido Y/metabolismo , Neuropéptidos/metabolismo , Óxido Nítrico Sintasa/metabolismo , Ganglio Nudoso/citología , Ganglio Nudoso/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Ratas Sprague-Dawley , Proteína G de Unión al Calcio S100/metabolismo , Sustancia P/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
We report an inhibitory effect of an anti-actin monoclonal antibody (mAb) on the human zona pellucida (ZP)-induced acrosome reaction (AR). Motile sperm were incubated with native human ZP for 2 h in medium containing either the anti-actin mAb, an irrelevant control mAb or cytochalasins B or D (40 micromol/l). Sperm bound to the ZP were recovered and the AR was determined by fluorescein-labelled Pisum Sativum agglutinin. Anti-mouse immunoglobulin G (mIgG) Dynabeads, immunofluorescence and immunogold were used to detect the location of the anti-actin mAb in sperm. The anti-actin mAb significantly inhibited the ZP-induced AR (equivalent to cytochalasins), the ionophore A23187-induced AR and hyperactivation of sperm in medium. After incubation with anti-actin mAb, anti-mIgG beads bound to the head of >50% of sperm recovered after binding to the ZP and 10% of sperm remaining in the medium. The proportion of sperm that bound anti-mIgG beads after recovery from binding to the ZP in the presence of the anti-actin mAb was significantly correlated with the ZP-induced AR in the absence of the antibody. Immunofluorescence and immunogold demonstrated entry of the anti-actin mAb into sperm. This study suggests that the sperm plasma membrane becomes permeable to the anti-actin mAb during capacitation and initiation of the AR.
Asunto(s)
Reacción Acrosómica/fisiología , Actinas/metabolismo , Anticuerpos Monoclonales/metabolismo , Motilidad Espermática/fisiología , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Actinas/inmunología , Animales , Calcimicina/farmacología , Membrana Celular/química , Membrana Celular/metabolismo , Citocalasina B/farmacología , Citocalasina D/farmacología , Femenino , Humanos , Ionóforos/farmacología , Masculino , Ratones , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Espermatozoides/efectos de los fármacos , Espermatozoides/ultraestructura , Estadística como Asunto , Zona Pelúcida/ultraestructuraRESUMEN
The amounts of neurokinin 1 (NK(1)) receptor immunolabelling on the membranes of myenteric cell bodies at appositions with tachykinin-immunoreactive nerve terminals, other nerve terminals, and glial cells were compared at the ultrastructural level using pre-embedding, double-label immunocytochemistry. NK(1) receptor immunoreactivity was revealed using silver-intensified, 1 nm gold, and tachykinin-immunoreactive nerve terminals were revealed using diaminobenzidine. The density of NK(1) receptor immunolabelling (silver particles per length of cell membrane) on the membrane at appositions with tachykinin-immunoreactive nerve terminals was not significantly different from that at appositions with other (nonimmunoreactive) nerve terminals or with glial cells. Synaptic specializations ("active zones") were present at a small proportion of the appositions between NK(1) receptor-immunoreactive cell bodies and tachykinin-immunoreactive or other nerve terminals. The density of NK(1) receptor immunolabelling at synaptic specializations was lower than that at regions of appositions where no synaptic specializations were present. The presence of NK(1) receptor on the cell surface in areas not directly apposed to tachykinin-containing nerve terminals suggests that tachykinins that diffuse away from their site of release may still exert an action via NK(1) receptors. Although NK(1) receptors do not appear to be targetted to particular sites on the surfaces of myenteric nerve cell bodies and proximal dendrites, they are reduced in density at regions of the membrane-forming synaptic specializations.
Asunto(s)
Íleon/metabolismo , Plexo Mientérico/metabolismo , Neuronas/metabolismo , Receptores de Neuroquinina-1/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Femenino , Cobayas , Íleon/inervación , Inmunohistoquímica , Masculino , Plexo Mientérico/citología , Neuroglía/metabolismo , Neuroglía/ultraestructura , Neuronas/ultraestructura , Receptores de Neuroquinina-1/ultraestructura , Sustancia P/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Taquicininas/metabolismoRESUMEN
Individual autonomic postganglionic neurons are surrounded by pericellular baskets of preganglionic terminals that are easily identifiable with the light microscope. It has been assumed that the target cell of a pericellular basket of preganglionic terminals is the neuron at the centre of the basket. This assumption has enabled the connectivity of preganglionic neurons to be determined at the light microscopic level. However, if the preganglionic terminals in a pericellular basket make synapses with the dendrites of nearby, but functionally different, postganglionic neurons, then the conclusions of light microscopic studies are far less certain. We have used a serial section ultrastructural study to determine the target of the preganglionic pericellular basket in a situation where the apparent target cell is surrounded by neurons of dissimilar function. In the rat superior cervical ganglion, postganglionic neurons projecting to the iris were identified, using retrograde tracers, as single neurons (i.e., not in clusters). We have used immunohistochemistry to show that iris-projecting neurons are surrounded by preganglionic nerve terminals containing calcitonin gene-related peptide (CGRP). We have demonstrated that the pericellular basket of CGRP-immunoreactive preganglionic terminals provides inputs only to the soma at the centre of the basket and not to the dendrites of surrounding neurons. This suggests that, in autonomic ganglia, light microscopic identification of the preganglionic terminal baskets is likely to be a reliable method for identifying the targets of subclasses of preganglionic neurons.
Asunto(s)
Fibras Autónomas Posganglionares/química , Fibras Autónomas Preganglionares/química , Iris/citología , Neuronas/química , Ganglio Cervical Superior/química , Animales , Fibras Autónomas Posganglionares/citología , Fibras Autónomas Preganglionares/citología , Péptido Relacionado con Gen de Calcitonina/análisis , Femenino , Masculino , Microscopía/métodos , Microscopía Electrónica , Microscopía Fluorescente , Vías Nerviosas/fisiología , Ratas , Ratas Sprague-Dawley , Ganglio Cervical Superior/citologíaRESUMEN
Neurons performing the same function can be identified immunohistochemically because they often share the same neurochemistry. The distribution of calcium-binding proteins, like calbindin, has been used previously to identify functional subpopulations of neurons in many parts of the nervous system. In this study we have investigated the distribution of calbindin D28K-immunoreactivity in subpopulations of sympathetic preganglionic neurons in the intermediolateral nucleus of the rat spinal cord. The majority of calbindin D28K-immunoreactive preganglionic neurons also had co-localised nitric oxide synthase, although a population of preganglionic neurons in the mid- to low thoracic intermediolateral nucleus expressed only calbindin D28K-immunoreactivity. Retrograde-tracing studies showed that calbindin D28K-immunoreactive neurons projected to the superior cervical and stellate ganglia, with smaller numbers of cells projecting to the lumbar sympathetic chain and superior mesenteric ganglia. Very few calbindin D28K-immunoreactive neurons projected to the inferior mesenteric ganglion, and none projected to the adrenal medulla. The distribution of calbindin D28K-immunoreactive terminals and postganglionic neurons in the superior cervical and stellate ganglia was also investigated. Many postganglionic neurons were calbindin D28K-immunoreactive, and most of these lacked neuropeptide Y-immunoreactivity. Calbindin D28K-immunoreactive nerve terminals were common and formed dense pericellular baskets around many postganglionic neurons, including some of those that were calbindin D28K-immunoreactive, but only rarely formed pericellular baskets around neuropeptide Y-immunoreactive neurons. The function of some of the classes of postganglionic neurons that were the target of calbindin D28K-immunoreactive preganglionic terminals was determined by combining immunohistochemistry with retrograde-tracer injections into a range of peripheral tissues. Calbindin D28K-immunoreactive nerve terminals, with co-localised nitric oxide synthase-immunoreactivity, surrounded secretomotor neurons projecting to the submandibular salivary gland and pilomotor neurons projecting to skin, but did not surround neurons projecting to brown fat or vasomotor neurons projecting to the skin, muscle, or salivary glands.
Asunto(s)
Fibras Autónomas Preganglionares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Estilbamidinas , Fibras Simpáticas Posganglionares/metabolismo , Tejido Adiposo Pardo/inervación , Animales , Calbindina 1 , Calbindinas , Recuento de Células , Femenino , Colorantes Fluorescentes , Inmunohistoquímica , Masculino , Terminaciones Nerviosas/metabolismo , Neuropéptido Y/metabolismo , Óxido Nítrico/metabolismo , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Ganglio Estrellado/citología , Ganglio Estrellado/metabolismo , Ganglio Cervical Superior/citología , Ganglio Cervical Superior/metabolismo , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/fisiologíaRESUMEN
The distribution of immunoreactivity to the neurokinin3 receptor (NK3R) was examined in segments C7, T11-12, L1-2, and L4-6 of the rat spinal cord. NK3R immunoreactivity was visualized by using two antisera generated against sequences of amino acids contained in the C-terminal region of the NK3R. NK3R-immunoreactive cells were numerous in the substantia gelatinosa of all spinal segments examined as well as the dorsal commissural nucleus of spinal segments L1-2. Isolated, immunoreactive cells were scattered throughout other regions of the spinal cord. The relationship of NK3R-immunoreactivity with neurons was demonstrated by colocalization with microtubule associated protein 2-immunoreactivity in individual cells. Within neurons, NK3R-immunoreactivity was associated predominately with the plasma membrane of cell bodies and dendrites. Within the substantia gelatinosa, 86% of nitric oxide synthase (NOS)-immunoreactive neurons were also NK3R-immunoreactive. Although NOS-immunoreactive neurons were found throughout all other regions of the spinal cord in the segments examined, these were not NK3R-immunoreactive. When preganglionic sympathetic neurons in spinal segments T11-12 and L1-2 were visualized by intraperitoneal injection of Fluorogold, less than 1% of the Fluorogold-labeled neurons were also immunoreactive for NK3R. The large number of NK3R-immunoreactive neurons in the substantia gelatinosa suggests that some effects of tachykinins on somatosensation may be mediated by NK3R.
Asunto(s)
Neuronas/citología , Neuronas/metabolismo , Receptores de Neuroquinina-3/análisis , Médula Espinal/metabolismo , Estilbamidinas , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Colorantes Fluorescentes , Proteína Ácida Fibrilar de la Glía/análisis , Sueros Inmunes , Masculino , Proteínas Asociadas a Microtúbulos/análisis , Óxido Nítrico Sintasa/análisis , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Sustancia Gelatinosa/citología , Sustancia Gelatinosa/metabolismoRESUMEN
The distribution of immunoreactivity to the receptor for substance P, the neurokinin 1 (NK1) receptor, was examined in preganglionic sympathetic neurons of the rat by using immunohistochemistry and retrograde neuronal tracing. About one-third of all sympathetic preganglionic neurons were NK1 receptor immunoreactive, and most of the NK1 receptor-immunoreactive neurons were also nitric oxide synthase immunoreactive. The proportions of sympathetic preganglionic neurons projecting to the superior and inferior mesenteric ganglia, adrenal gland, and lumbar sympathetic chain which were NK1 receptor-immunoreactive were determined. Most (89%) of the preganglionic neurons projecting to the adrenal glands were NK1 receptor immunoreactive. Few (17%) of the preganglionic neurons projecting to the L5 sympathetic chain ganglion were immunoreactive for the receptor, while preganglionic neurons projecting to the prevertebral ganglia were NK1 receptor immunoreactive at intermediate frequencies (61-64%). Thus, substance P acting on NK1 receptors is likely to be important in the preganglionic pathways to the adrenal medulla and viscera via the prevertebral ganglia, but is unlikely to be important in pathways to the lumbar sympathetic chain. The co-localisation of the NK1 receptor with the enzyme nitric oxide synthase was also examined. The majority of NK1 receptor-immunoreactive neurons were also nitric oxide synthase immunoreactive. Thus NK1 receptors occur on preganglionic neurons over many spinal segments and in a range of preganglionic pathways, as well as in a range of combinations with nitric oxide synthase. The heterogeneity of preganglionic neurons showing NK1 receptor immunoreactivity may reflect the involvement of NK1-mediated transmission in a variety of functional pathways, most notably the preganglionic projections to the adrenal medulla and to the viscera.
Asunto(s)
Fibras Adrenérgicas/metabolismo , Fibras Autónomas Preganglionares/metabolismo , Receptores de Neuroquinina-1/metabolismo , Estilbamidinas , Glándulas Suprarrenales/inervación , Animales , Recuento de Células , Colorantes Fluorescentes , Ganglios Simpáticos/citología , Ganglios Simpáticos/metabolismo , Inmunohistoquímica , Masculino , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Médula Espinal/metabolismo , Sustancia P/fisiologíaRESUMEN
The distribution and targets of calretinin-immunoreactive preganglionic nerve terminals in the superior cervical ganglion of the rat were examined using immunohistochemistry and retrograde neuronal tracing. Calretinin-immunoreactive nerve terminals were found throughout the ganglion, forming distinct pericellular baskets around a sub-population of postganglionic neurons. The targets of postganglionic neurons surrounded by calretinin-immunoreactive nerve terminals were determined after injection of tracer into the submandibular salivary gland, the extra-orbital lacrimal gland, the thyroid gland, the anterior chamber of the eye or the skin of the forehead. Only when tracer was injected into the submandibular gland were neurons labelled that were surrounded by calretinin-immunoreactive nerve terminals. When immunohistochemistry using antisera to neuropeptide Y was combined with retrograde tracing, only submandibular gland projecting neurons lacking neuropeptide Y were surrounded by calretinin-immunoreactive terminals. When retrograde neuronal tracer was injected into the superior cervical ganglion, a proportion of retrogradely-labelled neurons in the upper thoracic spinal cord showed relatively weak calretinin-immunoreactivity. All calretinin-immunoreactive terminals in the superior cervical ganglion disappeared following section of the sympathetic chain distal to the superior cervical ganglion. Thus, calretinin is present in a population of preganglionic neurons projecting exclusively to neuropeptide Y non-immunoreactive (presumably secretomotor) neurons innervating the submandibular salivary gland of the rat.