RESUMEN
Both estrous cycle and sex affect the numbers and types of neuronal and glial profiles containing the classical estrogen receptors α and ß, and synaptic levels in the rodent dorsal hippocampus. Here, we examined whether the membrane estrogen receptor, G-protein-coupled estrogen receptor 1 (GPER1), is anatomically positioned in the dorsal hippocampus of mice to regulate synaptic plasticity. By light microscopy, GPER1-immunoreactivity (IR) was most noticeable in the pyramidal cell layer and interspersed interneurons, especially those in the hilus of the dentate gyrus. Diffuse GPER1-IR was found in all lamina but was most dense in stratum lucidum of CA3. Ultrastructural analysis revealed discrete extranuclear GPER1-IR affiliated with the plasma membrane and endoplasmic reticulum of neuronal perikarya and dendritic shafts, synaptic specializations in dendritic spines, and clusters of vesicles in axon terminals. Moreover, GPER1-IR was found in unmyelinated axons and glial profiles. Overall, the types and amounts of GPER1-labeled profiles were similar between males and females; however, in females elevated estrogen levels generally increased axonal labeling. Some estradiol-induced changes observed in previous studies were replicated by the GPER agonist G1: G1 increased PSD95-IR in strata oriens, lucidum, and radiatum of CA3 in ovariectomized mice 6 h after administration. In contrast, estradiol but not G1 increased Akt phosphorylation levels. Instead, GPER1 actions in the synapse may be due to interactions with synaptic scaffolding proteins, such as SAP97. These results suggest that although estrogen's actions via GPER1 may converge on the same synaptic elements, different pathways are used to achieve these actions.
Asunto(s)
Hipocampo/fisiología , Hipocampo/ultraestructura , Plasticidad Neuronal/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Sinapsis/fisiología , Sinapsis/ultraestructura , Animales , Homólogo 1 de la Proteína Discs Large , Homólogo 4 de la Proteína Discs Large , Ciclo Estral/fisiología , Femenino , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Hipocampo/efectos de los fármacos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Estrógenos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Presinapticos/metabolismo , Receptores Presinapticos/ultraestructura , Caracteres Sexuales , Sinapsis/efectos de los fármacosRESUMEN
The amyloid precursor protein (APP) and its mammalian homologs, APLP1, APLP2, have been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a cell surface-localized pool. In the brain, all APPs are restricted to neurons; however, their precise localization at the plasma membrane remained enigmatic. Employing a variety of subcellular fractionation steps, we isolated two synaptic vesicle (SV) pools from rat and mouse brain, a pool consisting of synaptic vesicles only and a pool comprising SV docked to the presynaptic plasma membrane. Immunopurification of these two pools using a monoclonal antibody directed against the 12 membrane span synaptic vesicle protein2 (SV2) demonstrated unambiguously that APP, APLP1 and APLP2 are constituents of the active zone of murine brain but essentially absent from free synaptic vesicles. The specificity of immunodetection was confirmed by analyzing the respective knock-out animals. The fractionation experiments further revealed that APP is accumulated in the fraction containing docked synaptic vesicles. These data present novel insights into the subsynaptic localization of APPs and are a prerequisite for unraveling the physiological role of all mature APP proteins in synaptic physiology.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Receptores Presinapticos/metabolismo , Animales , Western Blotting , Femenino , Inmunoprecipitación , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Microscopía Electrónica , Simulación del Acoplamiento Molecular , Proteínas del Tejido Nervioso/metabolismo , Neurotransmisores/metabolismo , Ratas , Ratas Wistar , Receptores Presinapticos/ultraestructura , Fracciones Subcelulares/metabolismo , Sinapsis/ultraestructuraRESUMEN
Adolescent experiences of social deprivation result in profound and enduring perturbations in adult behavior, including impaired sensorimotor gating. The behavioral deficits induced by adolescent social isolation in rats can be ameliorated by antipsychotic drugs blocking dopamine D2 receptors in the prefrontal cortex (PFC) or by chronic administration of a cannabinoid CB1 receptor antagonist. The patterning and abundance of D2 receptors in the PFC evolves concurrently with CB1 receptors through the period of adolescence. This evidence suggests that mature expression and/or surface distribution of D2 and CB1 receptors may be influenced by the adolescent social environment. We tested this hypothesis using electron microscopic immunolabeling to compare the distribution of CB1 and D2 receptors in the PFC of adult male Sprague-Dawley rats that were isolated or socially reared throughout the adolescent transition period. Prepulse inhibition (PPI) of acoustic startle was assessed as a measure of sensorimotor gating. Social isolation reduced PPI and selectively decreased dendritic D2 immunogold labeling in the PFC. However, the decrease was only evident in dendrites that were not contacted by axon terminals containing CB1. There was no apparent change in the expression of CB1 or D2 receptors in presynaptic terminals. The D2 deficit therefore may be tempered by local CB1-mediated retrograde signaling. This suggests a biological mechanism whereby the adolescent social environment can persistently influence cortical dopaminergic activity and resultant behavior.
Asunto(s)
Corteza Prefrontal/metabolismo , Receptor Cannabinoide CB1/fisiología , Receptores de Dopamina D2/fisiología , Aislamiento Social , Estimulación Acústica , Animales , Interpretación Estadística de Datos , Dendritas/metabolismo , Dendritas/fisiología , Dendritas/ultraestructura , Técnicas para Inmunoenzimas , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Neuronas/metabolismo , Neuronas/ultraestructura , Corteza Prefrontal/fisiología , Corteza Prefrontal/ultraestructura , Terminales Presinápticos/metabolismo , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Receptores Presinapticos/fisiología , Receptores Presinapticos/ultraestructura , Reflejo de Sobresalto , Filtrado Sensorial/efectos de los fármacos , Filtrado Sensorial/fisiologíaRESUMEN
Synapses of hippocampal neurons play important roles in learning and memory processes and are involved in aberrant hippocampal function in temporal lobe epilepsy. Major neuronal types in the hippocampus as well as their input and output synapses are well known, but it has remained an open question to what extent conventional electron microscopy (EM) has provided us with the real appearance of synaptic fine structure under in vivo conditions. There is reason to assume that conventional aldehyde fixation and dehydration lead to protein denaturation and tissue shrinkage, likely associated with the occurrence of artifacts. However, realistic fine-structural data of synapses are required for our understanding of the transmission process and for its simulation. Here, we used high-pressure freezing and cryosubstitution of hippocampal tissue that was not subjected to aldehyde fixation and dehydration in ethanol to monitor the fine structure of an identified synapse in the hippocampal CA3 region, that is, the synapse between granule cell axons, the mossy fibers, and the proximal dendrites of CA3 pyramidal neurons. Our results showed that high-pressure freezing nicely preserved ultrastructural detail of this particular synapse and allowed us to study rapid structural changes associated with synaptic plasticity.
Asunto(s)
Fibras Nerviosas/ultraestructura , Sinapsis/ultraestructura , Fijación del Tejido/métodos , Aldehídos , Animales , Artefactos , Agua Corporal/metabolismo , Región CA3 Hipocampal/patología , Desecación , Epilepsia del Lóbulo Temporal/patología , Etanol , Congelación , Indicadores y Reactivos , Potenciación a Largo Plazo , Ratones , Fibras Nerviosas/patología , Perfusión , Desnaturalización Proteica , Receptores Presinapticos/ultraestructura , Sinapsis/patología , Transmisión Sináptica/fisiología , Técnicas de Cultivo de TejidosRESUMEN
We employed optical detection of the lipophylic dye FM1-43 and focal recordings of quantal release to investigate how synapsin affects vesicle cycling at the neuromuscular junction of synapsin knockout (Syn KO) Drosophila. Loading the dye employing high K+ stimulation, which presumably involves the recycling pool of vesicles in exo/endocytosis, stained the periphery of wild type (WT) boutons, while in Syn KO the dye was redistributed towards the center of the bouton. When endocytosis was promoted by cyclosporin A pretreatment, the dye uptake was significantly enhanced in WT boutons, and the entire boutons were stained, suggesting staining of the reserve vesicle pool. In Syn KO boutons, the same loading paradigm produced fainter staining and significantly faster destaining. When the axon was stimulated electrically, a distinct difference in dye loading patterns was observed in WT boutons at different stimulation frequencies: a low stimulation frequency (3 Hz) produced a ring-shaped staining pattern, while at a higher frequency (10 Hz) the dye was redistributed towards the center of the bouton and the fluorescence intensity was significantly increased. This difference in staining patterns was essentially disrupted in Syn KO boutons, although synapsin did not affect the rate of quantal release. Stimulation of the nerve in the presence of bafilomycin, the blocker of the transmitter uptake, produced significantly stronger depression in Syn KO boutons. These results, taken together, suggest that synapsin maintains the reserve pool of vesicles and segregation between the recycling and reserve pools, and that it mediates mobilization of the reserve pool during intense stimulation.
Asunto(s)
Drosophila/fisiología , Terminales Presinápticos/fisiología , Receptores Presinapticos/fisiología , Sinapsinas/fisiología , Vesículas Sinápticas/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Estimulación Eléctrica , Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Larva , Macrólidos/farmacología , Microscopía Confocal , Terminales Presinápticos/ultraestructura , Receptores Presinapticos/ultraestructura , Sinapsinas/genética , Vesículas Sinápticas/ultraestructuraRESUMEN
Diacylglycerol (DAG) is a prominent endogenous modulator of synaptic transmission. Recent studies proposed two apparently incompatible pathways, via protein kinase C (PKC) and via Munc13. Here we show how these two pathways converge. First, we confirm that DAG analogs indeed continue to potentiate transmission after PKC inhibition (the Munc13 pathway), but only in neurons that previously experienced DAG analogs, before PKC inhibition started. Second, we identify an essential PKC pathway by expressing a PKC-insensitive Munc18-1 mutant in munc18-1 null mutant neurons. This mutant supported basic transmission, but not DAG-induced potentiation and vesicle redistribution. Moreover, synaptic depression was increased, but not Ca2+-independent release evoked by hypertonic solutions. These data show that activation of both PKC-dependent and -independent pathways (via Munc13) are required for DAG-induced potentiation. Munc18-1 is an essential downstream target in the PKC pathway. This pathway is of general importance for presynaptic plasticity.
Asunto(s)
Diglicéridos/fisiología , Plasticidad Neuronal/fisiología , Proteína Quinasa C/fisiología , Receptores Presinapticos/fisiología , Transducción de Señal/fisiología , Animales , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Células Cromafines/metabolismo , Diglicéridos/metabolismo , Electrofisiología , Inhibidores Enzimáticos/farmacología , Femenino , Hipocampo/citología , Hipocampo/efectos de los fármacos , Cinética , Lentivirus/genética , Ratones , Ratones Noqueados , Microscopía Electrónica , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Mutación/fisiología , Neuronas/citología , Neuronas/metabolismo , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Ésteres del Forbol/farmacología , Fosforilación , Embarazo , Proteína Quinasa C/antagonistas & inhibidores , Receptores Presinapticos/ultraestructuraRESUMEN
Synapses may undergo long-term increases or decreases in synaptic strength dependent on critical differences in the timing between pre-and postsynaptic activity. Such spike-timing-dependent plasticity (STDP) follows rules that govern how patterns of neural activity induce changes in synaptic strength. Synaptic plasticity in the dorsal cochlear nucleus (DCN) follows Hebbian and anti-Hebbian patterns in a cell-specific manner. Here we show that these opposing responses to synaptic activity result from differential expression of two signaling pathways. Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling underlies Hebbian postsynaptic LTP in principal cells. By contrast, in interneurons, a temporally precise anti-Hebbian synaptic spike-timing rule results from the combined effects of postsynaptic CaMKII-dependent LTP and endocannabinoid-dependent presynaptic LTD. Cell specificity in the circuit arises from selective targeting of presynaptic CB1 receptors in different axonal terminals. Hence, pre- and postsynaptic sites of expression determine both the sign and timing requirements of long-term plasticity in interneurons.
Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Plasticidad Neuronal/fisiología , Receptores Presinapticos/fisiología , Transducción de Señal/fisiología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Moduladores de Receptores de Cannabinoides/fisiología , Núcleo Coclear/citología , Núcleo Coclear/fisiología , Núcleo Coclear/ultraestructura , Electrofisiología , Substitución por Congelación , Técnicas In Vitro , Aprendizaje/fisiología , Potenciación a Largo Plazo/fisiología , Ratones , Ratones Endogámicos ICR , Microscopía Electrónica , Fibras Nerviosas/fisiología , Receptor Cannabinoide CB1/fisiología , Receptores Presinapticos/ultraestructura , Transmisión Sináptica/fisiologíaRESUMEN
Analyses of the effect of ryanodine in vertebrate brain slices have led to the conclusion that presynaptic ryanodine receptors (RYRs) may have several functions in synaptic release, including causing large-amplitude miniature postsynaptic currents (mPSCs) by promoting concerted multivesicular release. However, the role of RYRs in synaptic release is controversial. To better understand the role of RYRs in synaptic release, we analyzed the effect of RYR mutation on mPSCs and evoked postsynaptic currents (ePSCs) at the Caenorhabditis elegans neuromuscular junction (NMJ). Amplitudes of mPSCs varied greatly at the C. elegans NMJ. Loss-of-function mutations of the RYR gene unc-68 (uncoordinated 68) essentially abolished large-amplitude mPSCs. The amplitude of ePSCs was also greatly suppressed. These defects were completely rescued by expressing wild-type UNC-68 specifically in neurons but not in muscle cells, suggesting that RYRs acted presynaptically. A combination of removing extracellular Ca2+ and UNC-68 function eliminated mPSCs, suggesting that influx and RYR-mediated release are likely the exclusive sources of Ca2+ for synaptic release. Large-amplitude mPSCs did not appear to be caused by multivesicular release, as has been suggested to occur at vertebrate central synapses, because the rise time of mPSCs was constant regardless of the amplitude but distinctive from that of ePSCs, and because large-amplitude mPSCs persisted under conditions that inhibit synchronized synaptic release, including elimination of extracellular Ca2+, and mutations of syntaxin and SNAP25 (soluble N-ethylmaleimide-sensitive factor attachment protein 25). These observations suggest that RYRs are essential to normal quantal size and are potential regulators of quantal size.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Unión Neuromuscular/fisiología , Receptores Presinapticos/fisiología , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Transmisión Sináptica/fisiología , Acetilcolina/fisiología , Animales , Caenorhabditis elegans , Calcio/metabolismo , Exocitosis/fisiología , Microscopía Electrónica , Mutación , Unión Neuromuscular/ultraestructura , Receptores Presinapticos/ultraestructura , Rianodina/farmacología , Transmisión Sináptica/efectos de los fármacos , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/ultraestructura , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Although the localization and role of kainate receptors in the CNS remain poorly known, complex, and rather unusual, pre-synaptic auto- and heteroreceptor functions have been disclosed in various brain regions. Basal ganglia nuclei, including the globus pallidus, are enriched in GluR6/7 immunoreactivity. Using electron microscopic immunocytochemistry for GluR6/7 combined with post-embedding immunogold labeling for GABA, we demonstrate that GluR6/7 immunoreactivity is enriched in a large subpopulation of small unmyelinated, presumably pre-terminal, axons as well as GABAergic and putative glutamatergic axon terminals in the internal and external segments of the globus pallidus in monkey. Our findings suggest that kainate receptors are located to subserve pre-synaptic modulation of inhibitory and excitatory transmission in the primate globus pallidus.
Asunto(s)
Globo Pálido/química , Ácido Glutámico/análisis , Terminales Presinápticos/química , Receptores de Ácido Kaínico/análisis , Ácido gamma-Aminobutírico/análisis , Animales , Globo Pálido/ultraestructura , Macaca mulatta , Terminales Presinápticos/ultraestructura , Receptores de Ácido Kaínico/ultraestructura , Receptores Presinapticos/análisis , Receptores Presinapticos/ultraestructura , Receptor de Ácido Kaínico GluK2 , Receptor Kainato GluK3RESUMEN
We compared somatic and dendritic inhibition in paired recordings from two classes of anatomically identified interneurons and granule cells of the dentate gyrus. Inhibitory postsynaptic current (IPSC) amplitude and decay were remarkably similar at somatic and dendritic synapses. Slower IPSC rise times and longer latencies at dendritic synapses were consistent with their distal location, without requiring differences in postsynaptic gamma-aminobutyric acid type A (GABA(A)) receptor properties. In contrast, higher transmission failure rate and greater paired-pulse depression at dendritic synapses suggest that somatic and dendritic inhibition differ in presynaptic properties. Cholinergic input has been suggested to modulate hippocampal rhythmicity as well as episodic memory function. We therefore tested the effects of acetylcholine (ACh) on paired IPSCs and on spontaneous synaptic activity in interneurons and granule cells. We found no effect of ACh on paired IPSCs; however, spontaneous IPSCs recorded in granule cells were enhanced in amplitude and frequency. ACh potentiated spontaneous excitatory postsynaptic potentials (sEPSPs) and induced spiking in both types of interneuron, and preferentially increased sEPSP frequency in dendritic interneurons. Our findings suggest that patterns of activity in the two classes of interneurons, coupled with differences in their presynaptic properties, are likely to determine the roles of somatic and dendritic inhibition in network function.
Asunto(s)
Dendritas/fisiología , Giro Dentado/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Receptores Presinapticos/fisiología , Sinapsis/fisiología , Acetilcolina/farmacología , Potenciales de Acción/fisiología , Algoritmos , Animales , Dendritas/ultraestructura , Giro Dentado/citología , Giro Dentado/ultraestructura , Electrofisiología , Técnicas In Vitro , Interneuronas/fisiología , Interneuronas/ultraestructura , Masculino , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores Presinapticos/ultraestructura , Sinapsis/ultraestructura , Transmisión Sináptica/fisiologíaRESUMEN
1. When buccal neuron B2 of Aplysia californica is co-cultured with sensory neurons (SNs), slow peptidergic synapses are formed. When B2 is co-cultured with neurons B3 or B6, fast cholinergic synapses are formed. 2. Patch pipettes were used to voltage clamp pre- and postsynaptic neurons and to load the caged Ca2+ chelator o-nitrophenyl EGTA (NPE) and the Ca2+ indicator BTC into presynaptic neurons. The relationships between presynaptic [Ca2+]i and postsynaptic responses were compared between peptidergic and cholinergic synapses formed by cell B2. 3. Using variable intensity flashes, Ca2+ stoichiometries of peptide and acetylcholine (ACh) release were approximately 2 and 3, respectively. The difference did not reach statistical significance. 4. ACh quanta summate linearly postsynaptically. We also found a linear dose-response curve for peptide action, indicating a linear relationship between submaximal peptide concentration and response of the SN. 5. The minimum intracellular calcium concentrations ([Ca2+]i) for triggering peptidergic and cholinergic transmission were estimated to be about 5 and 10 microM, respectively. 6. By comparing normal postsynaptic responses to those evoked by photolysis of NPE, we estimate [Ca2+]i at the release trigger site elicited by a single action potential (AP) to be at least 10 microM for peptidergic synapses and probably higher for cholinergic synapses. 7. Cholinergic release is brief (half-width approximately 200 ms), even in response to a prolonged rise in [Ca2+]i, while some peptidergic release appears to persist for as long as [Ca2+]i remains elevated (for up to 10 s). This may reflect differences in sizes of reserve pools, or in replenishment rates of immediately releasable pools of vesicles. 8. Electron microscopy revealed that most synaptic contacts had at least one morphologically docked dense core vesicle that presumably contained peptide; these were often located within conventional active zones. 9. Both cholinergic and peptidergic vesicles are docked within active zones, but cholinergic vesicles may be located closer to Ca2+ channels than are peptidergic vesicles.
Asunto(s)
Acetilcolina/metabolismo , Calcio/fisiología , Neuronas/metabolismo , Neuropéptidos/metabolismo , Receptores Presinapticos/metabolismo , Potenciales de Acción/efectos de los fármacos , Algoritmos , Animales , Aplysia , Calibración , Células Cultivadas , Quelantes/farmacología , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Microscopía Electrónica , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Receptores Presinapticos/ultraestructura , Transmisión Sináptica , Rayos UltravioletaRESUMEN
We have used the styryl dye FM 2-10 to monitor changes in synaptic activity mediated by nicotinic acetylcholine receptors (AChRs) on cultured hippocampal neurons. We show that both 100 microM ACh and nicotine at 20 microM causes a significant increase in staining intensities of presynaptic boutons in the presence of 0.5 microM tetrodotoxin (TTX). This effect of nicotine is blocked by d-tubocurarine. Interestingly, nicotine also had a long-lasting effect on high potassium-induced staining of boutons. These results suggest that nicotine can have significant and sustained effects on synaptic efficacy in cultured hippocampal neurons.
Asunto(s)
Hipocampo/metabolismo , Receptores Nicotínicos/metabolismo , Receptores Presinapticos/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Colorantes , Colorantes Fluorescentes , Hipocampo/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Sistema Nervioso Parasimpático/fisiología , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Ratas , Receptores Nicotínicos/ultraestructura , Receptores Presinapticos/ultraestructura , Tetrodotoxina/farmacologíaRESUMEN
Substance P (SP) is a peptide that is present in unmyelinated primary afferents to the dorsal horn and is released in response to painful or noxious stimuli. Opiates active at the mu-opiate receptor (MOR) produce antinociception, in part, through modulation of responses to SP. MOR ligands may either inhibit the release of SP or reduce the excitatory responses of second-order neurons to SP. We examined potential functional sites for interactions between SP and MOR with dual electron microscopic immmunocytochemical localization of the SP receptor (NK1) and MOR in rat trigeminal dorsal horn. We also examined the relationship between SP-containing profiles and NK1-bearing profiles. We found that 56% of SP-immunoreactive terminals contact NK1 dendrites, whereas 34% of NK1-immunoreactive dendrites receive SP afferents. This result indicates that there is not a significant mismatch between sites of SP release and available NK1 receptors, although receptive neurons may contain receptors at sites distant from the peptide release site. With regard to opioid receptors, we found that many MOR-immunoreactive dendrites also contain NK1 (32%), whereas a smaller proportion of NK1-immunoreactive dendrites contain MOR (17%). Few NK1 dendrites (2%) were contacted by MOR-immunoreactive afferents. These results provide the first direct evidence that MORs are on the same neurons as NK1 receptors, suggesting that MOR ligands directly modulate SP-induced nociceptive responses primarily at postsynaptic sites, rather than through inhibition of SP release from primary afferents. This colocalization of NK1 and MORs has significant implications for the development of pain therapies targeted at these nociceptive neurons.
Asunto(s)
Células del Asta Posterior/metabolismo , Células del Asta Posterior/ultraestructura , Receptores de Neuroquinina-1/metabolismo , Receptores de Neuroquinina-1/ultraestructura , Receptores Opioides mu/metabolismo , Receptores Opioides mu/ultraestructura , Sustancia P/metabolismo , Nervio Trigémino/metabolismo , Nervio Trigémino/ultraestructura , Animales , Dendritas/metabolismo , Dendritas/ultraestructura , Inmunohistoquímica , Masculino , Microscopía Electrónica , Células del Asta Posterior/citología , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Receptores Presinapticos/metabolismo , Receptores Presinapticos/ultraestructura , Sinapsis/metabolismo , Sinapsis/ultraestructura , Nervio Trigémino/citologíaRESUMEN
The effects of corticosteroid (CS) treatment (prednisolone continuously administered subcutaneously at a flow rate of 2.5 microl/h, daily dose 5.6 mg/kg, for 3 wk) on neuromuscular junction (NMJ) morphology and neuromuscular transmission in rat diaphragm muscle (Dimus) were compared with weight-matched (Sham) and ad libitum fed control (Ctl) groups. Fibers were classified on the basis of myosin heavy chain (MHC) isoform expression. CS treatment caused significant atrophy of fibers expressing MHC2X (type IIx), either alone or with MHC2B (type IIx/b). Fibers expressing MHCslow (type I) and MHC2A (type IIa) were unaffected by CS. The planar areas of nerve terminals and motor endplates at type IIx/b fibers were smaller in CS-treated Dimus compared with Sham and Ctl. However, CS-induced atrophy of type IIx/b fibers exceeded changes in NMJ morphology. Thus, when normalized for fiber diameter, NMJs were relatively larger in the CS-treated group compared with Ctl. Neuromuscular transmission failure, assessed in vitro by comparing force loss during repetitive (40 Hz) nerve vs. direct muscle stimulation, was less in CS-treated Dimus. These results indicate that alterations in NMJ morphology after CS treatment are dependent on fiber type and may contribute to improved neuromuscular transmission.
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Corticoesteroides/farmacología , Diafragma/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Animales , Diafragma/inervación , Glucocorticoides/farmacología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Masculino , Microscopía Confocal , Placa Motora/efectos de los fármacos , Placa Motora/ultraestructura , Neuronas Motoras/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/ultraestructura , Unión Neuromuscular/ultraestructura , Prednisolona/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Presinapticos/efectos de los fármacos , Receptores Presinapticos/ultraestructura , Transmisión Sináptica/efectos de los fármacos , Hormonas Tiroideas/sangreRESUMEN
The purpose of this study was to determine whether presynaptic cholinergic receptors are present in sympathetic nerves in human dental pulp. Pulp was incubated with [3H]noradrenaline (0.6 mumol/l) for 30 min and then superfused with Krebs' solution at 1.0 ml/min. Electrical stimulation (100 sec, 5 Hz) increased the overflow of [3H]noradrenaline into the superfusate. Carbachol (10 and 100 mumol/l), an agonist of muscarinic receptors, decreased the stimulation-induced (SI) overflow of 3H, an effect blocked by atropine but not hexamethonium. Carbachol, atropine and hexamethonium had no effect on the resting overflow. Nicotine (10 mumol/l) increased the resting overflow and inhibited the SI overflow, although the inhibition was variable. Cytisine, another agonist of nicotinic receptors, also increased the resting overflow, but did not affect the SI overflow. To ascertain whether the actions of nicotine and electrical stimulation were influenced by the release of nitric oxide (NO), the effects of an NO donor and two NO-synthase inhibitors were examined. With the exception of one of the NO-synthase inhibitors (L-NAME), the agents were without effect on the overflow of 3H in the absence or presence of nicotine. It was concluded that sympathetic nerves in human dental pulp possess (a) presynaptic muscarinic receptors that inhibit the SI release of noradrenaline, and (b) nicotinic receptors that evoke the release of noradrenaline and that inhibit the SI release of the transmitter. The results do not point to a significant role for NO in the effects of stimulation or nicotine on the overflow of 3H.
Asunto(s)
Pulpa Dental/inervación , Receptores Muscarínicos/ultraestructura , Receptores Presinapticos/ultraestructura , Sistema Nervioso Simpático/ultraestructura , Adolescente , Adulto , Alcaloides/farmacología , Atropina/farmacología , Azocinas , Carbacol/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Femenino , Hexametonio/farmacología , Humanos , Soluciones Isotónicas , Masculino , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , NG-Nitroarginina Metil Éster/farmacología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Norepinefrina/farmacología , Quinolizinas , Radiofármacos , Receptores Muscarínicos/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/ultraestructura , Receptores Presinapticos/antagonistas & inhibidores , Receptores Presinapticos/efectos de los fármacos , Sistema Nervioso Simpático/efectos de los fármacos , Simpatomiméticos/farmacología , TritioRESUMEN
Alpha-adrenergic receptor, subtype 2A (alpha2A-AR), activation is one of the primary modes of action for norepinephrine (NE) in the rat hippocampal formation. In this study, alpha2A-AR immunoreactivity (alpha2A-AR-I) was localized by light and electron microscopy in the rat hippocampus and dentate gyrus by using a previously characterized antibody to the rat alpha2A-AR. By light microscopy, dense alpha2A-AR-I was observed in the pyramidal and granule cell layers. Diffuse and slightly granular alpha2A-AR-I was found in the neuropil in all other laminae, notably stratum lacunosum-moleculare. Ultrastructurally, alpha2A-AR-I was found in neuronal cytoplasm associated with large multivesicular-like organelles and with clusters adjacent to endoplasmic reticula and/or plasmalemma. The distribution of alpha2A-AR-I in the strata oriens, radiatum, and lacunosum-moleculare of hippocampal CA1 and CA3 regions and in the molecular layer of the dentate gyrus was remarkably similar (n > 2,000 profiles examined): alpha2A-AR-I was found in axons and terminals (approximately 40%), glia (approximately 30%), dendritic spines (approximately 25%), and dendritic shafts (approximately 5%). This mixed pre- and postsynaptic distribution was not seen in the stratum lucidum of the CA3 region and the dentate hilar region, where most alpha2A-AR-I was found in axons (approximately 60%) and glia (approximately 30%). Alpha-2A-AR-labeled axons were small and unmyelinated; labeled terminals usually formed asymmetric synapses on unlabeled spines; and labeled dendritic spines were morphologically similar to pyramidal or granule cells. Dual labeling studies demonstrated that some axons contained alpha2A-AR-I and tyrosine hydroxylase (TH), the catecholaminergic synthesizing enzyme, and that some TH-labeled terminals were in close proximity to alpha2A-AR-labeled spines and glia. These studies demonstrate that hippocampal alpha2A-AR-I is localized (1) presynaptically in both noncatecholaminergic and catecholaminergic terminals, (2) postsynaptically in the dendritic spines of pyramidal and granule cells near catecholaminergic terminals, and (3) in some glial processes. These results suggest several sites for NE to exert its effects on hippocampal alpha2A-ARs.
Asunto(s)
Astrocitos/metabolismo , Hipocampo/metabolismo , Receptores Adrenérgicos alfa 2/biosíntesis , Receptores Presinapticos/metabolismo , Sinapsis/fisiología , Animales , Astrocitos/enzimología , Astrocitos/ultraestructura , Dendritas/enzimología , Dendritas/metabolismo , Dendritas/ultraestructura , Giro Dentado/citología , Giro Dentado/metabolismo , Giro Dentado/ultraestructura , Hipocampo/citología , Hipocampo/ultraestructura , Inmunohistoquímica , Masculino , Microscopía Electrónica , Terminaciones Nerviosas/enzimología , Terminaciones Nerviosas/metabolismo , Terminaciones Nerviosas/ultraestructura , Células Piramidales/enzimología , Células Piramidales/metabolismo , Células Piramidales/ultraestructura , Ratas , Ratas Sprague-Dawley , Receptores Presinapticos/ultraestructura , Sinapsis/ultraestructura , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The ins and outs of the synaptic vesicle cycle are being examined in increasing detail with diverse investigative tools in a variety of cell types, particularly those with large granules. The cycle begins with the opening of a fusion pore that connects the vesicle lumen to the extracellular fluid. Sensitive electrophysiological techniques reveal the often-stuttering behavior of single pores in non-neuronal cells, through which small molecules trickle until the fusion pore expands and the remaining contents erupt from the vesicle. The granule membranes are then retrieved by multiple processes that appear to act in parallel and that are distinguished from each other kinetically and ultrastructurally. Following endocytosis, synaptic vesicles are then shuttled back into the vesicle pool, where they briefly mix with other vesicles, become immobilized, and remain gelled with their neighbors, even while moving en masse again to the presynaptic membrane as a prelude for another round of exocytosis.
Asunto(s)
Vesículas Sinápticas/fisiología , Animales , Endocitosis/fisiología , Humanos , Receptores Presinapticos/fisiología , Receptores Presinapticos/ultraestructura , Vesículas Sinápticas/ultraestructuraRESUMEN
1. Intracellular recordings were obtained from 21 amacrine cells and 12 ganglion cells in the isolated, superfused retina-eyecup of the rabbit. Cells were subsequently labeled with horseradish peroxidase (HRP) or N-(2-aminoethyl)-biotinamide hydrochloride (Neurobiotin) for morphologic identification. 2. Initial experiments performed on three amacrine cells and three ganglion cells showed that 1 microM tetrodotoxin (TTX) abolished all spiking. This included both large-amplitude and small-amplitude spikes recorded in many amacrine cells, indicating that they are mediated by voltage-gated sodium channels. 3. The center-receptive-field size of 18 amacrine cells and 9 ganglion cells was measured with the use of a 50-microns-wide/6.0-mm-long rectangular slit of light that was displaced along its minor axis (parallel to the visual streak) in steps as small as 3 microns. The retina was then bathed in 1 microM TTX, or individual cells were injected with 50 mM QX-314, a quatemary lidocaine derivative, to abolish all spiking, and the center-receptive field of each cell was then remeasured. 4. Although TTX blocked spiking in all ganglion cells (dendritic diameters ranging from 302 to 969 microns), it produced no significant change in the size of their center-receptive fields. This finding argues that passive, electrotonic spread of synaptic inputs to ganglion cell dendritic arbors is adequate for efficient propagation from terminal branches to the soma; active propagation via voltage-gated sodium channels plays no apparent role. 5. In contrast, TTX and QX-314 had variable effect on the receptive fields of amacrine cells, which was related to the size of their dendritic arbors. Whereas TTX had no significant effect on the receptive-field size of amacrine cells whose dendritic arbors were < 525 microns across, the center-receptive fields of larger amacrine cells were reduced, on average, by 40%; QX-314 produced a very similar average reduction of 39%. Moreover, for these larger cells, there was a direct relationship between the magnitude of the reduction in receptive-field size produced by TTX or QX-314 and the size of a cell's dendritic arbor. This relationship was true whether the change in receptive-field size was measured in absolute terms or as percent reduction from control values. 6. Interestingly, TTX and QX-314 also significantly reduced the amplitude of slow potentials recorded in amacrine cells by an average of 22 and 24%, respectively. However, the amplitude of slow potentials recorded in ganglion cells were relatively uneffected by TTX. 7. These findings are consistent with the idea that, for amacrine cells with dendritic arbors spanning > 525 microns, active propagation of synaptic signals mediated by voltage-gated sodium channels is necessary for efficient movement of information across a cell's dendritic arbor and thus plays a major role in shaping their receptive fields. Although the TTX effects may also reflect an indirect contribution from altered synaptic input derived from presynaptic spiking neurons, the strong similarity between the effects of TTX and QX-314 argues that any such contribution was minor. For smaller amacrine cells, passive, electrotonic spread of signals appears adequate for efficient propagation within their limited dendritic arbors.
Asunto(s)
Retina/fisiología , Células Ganglionares de la Retina/fisiología , Campos Visuales/fisiología , Anestésicos Locales/farmacología , Animales , Tamaño de la Célula/efectos de los fármacos , Dendritas/efectos de los fármacos , Dendritas/ultraestructura , Potenciales Evocados Visuales/fisiología , Activación del Canal Iónico/fisiología , Lidocaína/análogos & derivados , Lidocaína/farmacología , Microelectrodos , Estimulación Luminosa , Conejos , Receptores Presinapticos/efectos de los fármacos , Receptores Presinapticos/ultraestructura , Retina/citología , Retina/efectos de los fármacos , Células Ganglionares de la Retina/efectos de los fármacos , Canales de Sodio/fisiología , Tetrodotoxina/farmacologíaRESUMEN
The endogenous opioid peptide, enkephalin, and epincphrine are distributed in varicose processes throughout the nucleus locus coeruleus (LC) in the dorsolateral tegmentum of the rat brain. In this brain region, micro-opioid and alpha-2-adrenergic receptors have been shown to share the same potassium channel suggesting that they may be co-localized in the same terminal or that they may be present in terminals that innervate the same target neuron. However, this has not been demonstrated at the ultrastructural level. Thus, the present study combined the immunocytochemical localization of the opioid peptide leucine5-enkephalin (ENK) and the epinephrine synthesizing enzyme, phenylethanolamine-N-methyltransferase (PNMT) in the same section of tissue within the LC at the electron microscopic level. At the light microscopic level, both ENK and PNMT varicose processes were dense and overlapped the region known to contain the noradrenergic cell bodies and dendrites of the LC. However, the morphological features of the two immunolabeled fiber types appeared different in 30-microns thick coronal sections. PNMT-labeled process were thin, beaded and ramified within the coronal plane. ENK-immunoreactive fibers, however, were more punctate in appearance and processes joining these puncta were not often evident in the frontal plane examined. Varicose fibers immunolabeled for either ENK or PNMT were confirmed to be axons and axon terminals by electron microscopy. Both types contained small clear as well as large dense core vesicles and formed heterogeneous types of synaptic specializations with postsynaptic targets. A common feature encountered in dually labeled tissue sections was convergence of the separately labeled axon terminals on common targets. Another common feature was the apposition of PNMT-labeled axon terminals with ENK-immunoreactive axon terminals that formed synaptic contacts in the plane of section examined. Although numerous ENK and PNMT-labeled axon terminals were identified in similar regions of the neuropil, few terminals were found to contain both labels. These findings indicate that the opioid peptide ENK and epinephrine may elicit concerted actions on common noradrenergic neurons in the LC via separate sets of afferents.
Asunto(s)
Encefalina Leucina/fisiología , Epinefrina/fisiología , Locus Coeruleus/fisiología , Locus Coeruleus/ultraestructura , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Receptores Presinapticos/fisiología , Receptores Presinapticos/ultraestructura , Animales , Encefalina Leucina/metabolismo , Epinefrina/metabolismo , Inmunohistoquímica , Locus Coeruleus/metabolismo , Masculino , Microscopía Electrónica , Vías Nerviosas/metabolismo , Vías Nerviosas/fisiología , Vías Nerviosas/ultraestructura , Feniletanolamina N-Metiltransferasa/metabolismo , Terminales Presinápticos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Presinapticos/metabolismoRESUMEN
Activation of alpha 2-adrenergic receptors (alpha 2AR) in the cerebral cortex has been shown to modulate visually guided delayed response tasks as well as anxiety and depression. We used an antiserum directed specifically against the A subtype of alpha 2AR (alpha 2AAR) to determine the cell types and subcellular sites for noradrenergic reception mediated by this receptor in the adult and the developing rat visual cortices. Light microscopic examination of adult tissue revealed numerous labeled perikarya in layers II-VI, many of which appeared distinctly pyramidal. A few perikarya in layer I also were immunoreactive. In all layers, alpha 2AAR immunoreactivity (alpha 2AAR-ir) was present within proximal dendrites and fine processes. In neonatal tissue, there was an intense, distinct band of immunoreactivity spanning the layer composed of tightly packed immature cell bodies, i.e., the cortical plate. The band dissipated as this tier differentiated postnatally into the supragranular layers. Electron microscopy showed that the supragranular layers, which contain the highest density of noradrenergic fibers, also contain the highest areal density of labeled postsynaptic junctions beyond 2 weeks of age. Throughout the ages, the majority of immunoreactivity occurred at sites which, in single ultrathin sections, appeared to be nonjunctional sites of axons, dendrites, and in glial processes. Our observations indicate that (1) both pyramidal and nonpyramidal neurons are receptive to norepinephrine via alpha 2AAR, (2) alpha 2AAR synthesis is robust prior to synaptogenesis, and (3) alpha 2AAR operates both pre- and postsynaptically.