RESUMEN
There is increasing evidence that a subset of midbrain dopamine (DA) neurons uses glutamate as a co-transmitter and expresses vesicular glutamate transporter (VGLUT) 2, one of the three vesicular glutamate transporters. In the present study, double in situ hybridization was used to examine tyrosine hydroxylase (TH) and VGLUT2 mRNA expression during the embryonic development of these neurons, and postnatally, in normal rats and rats injected with 6-hydroxydopamine (6-OHDA) at P4 to destroy partially DA neurons. At embryonic days 15 and 16, there was a regional overlap in the labeling of TH and VGLUT2 mRNA in the ventral mesencephalon, which was no longer found at late embryonic stages (E18-E21) and postnatally. In normal pups from P5 to P15, only 1-2% of neurons containing TH mRNA in the ventral tegmental area (VTA) and substantia nigra, pars compacta, also displayed VGLUT2 mRNA. In contrast, after the cerebroventricular administration of 6-OHDA at P4, 26% of surviving DA neurons in the VTA of P15 rats expressed VGLUT2. To search for a colocalization of TH and VGLUT2 protein in axon terminals of these neurons, the nucleus accumbens of normal and 6-OHDA-lesioned P15 rats was examined by electron microscopy after dual immunocytochemical labeling. In normal rats, VGLUT2 protein was found in 28% of TH positive axon terminals in the core of nucleus accumbens. In 6-OHDA-lesioned rats, the total number of TH positive terminals was considerably reduced, and yet the proportion also displaying VGLUT2 immunoreactivity was modestly but significantly increased (37%). These results lead to the suggestion that the glutamatergic phenotype of a VTA DA neurons is highly plastic, repressed toward the end of normal embryonic development, and derepressed postnatally following injury. They also support the hypothesis of co-release of glutamate and DA by mesencephalic neurons in vivo, at least in the developing brain.
Asunto(s)
Dopamina/metabolismo , Ácido Glutámico/metabolismo , Mesencéfalo/metabolismo , Neuronas/metabolismo , Trastornos Parkinsonianos/metabolismo , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Masculino , Mesencéfalo/citología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Vías Nerviosas/metabolismo , Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Núcleo Accumbens/metabolismo , Núcleo Accumbens/patología , Núcleo Accumbens/fisiopatología , Oxidopamina , Trastornos Parkinsonianos/patología , Trastornos Parkinsonianos/fisiopatología , Fenotipo , Terminales Presinápticos/metabolismo , Terminales Presinápticos/patología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancia Negra/metabolismo , Sustancia Negra/patología , Sustancia Negra/fisiopatología , Simpaticolíticos , Tirosina 3-Monooxigenasa/genética , Área Tegmental Ventral/metabolismo , Área Tegmental Ventral/patología , Área Tegmental Ventral/fisiopatología , Proteína 2 de Transporte Vesicular de Glutamato/genéticaRESUMEN
The serotonin1B receptor knockout (5-HT1B KO) mouse is a valuable animal model of addiction to psychostimulants. We previously found selective increases in dopamine (DA) turnover in the nucleus accumbens of these mice, in addition to several changes in their central serotonin system. Here, we searched for further DA adaptations by measuring D1 and D2 receptor as well DA plasma membrane transporter (DAT) sites by ligand binding autoradiography, and G-protein coupling to D1 and D2 receptors by [35S]GTP gamma S autoradiography. Except for a slight increase in the lateral septum, D1 receptor binding did not differ from wild-type in twenty-one other neocortical, limbic or basal ganglia regions examined in the KO. Nor were there changes in D1 agonist-stimulated G-protein coupling in any of these regions, including the lateral septum. Increases in D2 binding sites, presumably involving GABAergic projection neurons, were measured in the nucleus accumbens, olfactory tubercle and ventral tegmental area of the 5-HT1B KO. However, no activation of the efficacy of D2 receptor coupling to G-protein could be measured in these and other brain regions. Binding to DAT was unchanged throughout brain. Because of their implication in cocaine addiction, the functionality of mu-opioid and GABAB receptors was also assessed by [35S]GTP gamma S autoradiography. 5-HT1B KO showed selective decreases in G-protein coupling to mu-opioid receptors in the paraventricular thalamic nucleus, and to GABAB receptors in the basolateral nucleus of amygdala. It is likely that these latter changes underlie some aspects of the addictive behavior of the 5-HT1B KO mouse.
Asunto(s)
Encéfalo/metabolismo , Receptor de Serotonina 5-HT1B/genética , Receptores Dopaminérgicos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neurotransmisores/metabolismo , Serotonina/metabolismo , Animales , Sitios de Unión/fisiología , Unión Competitiva/fisiología , Encéfalo/citología , Química Encefálica/fisiología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Femenino , Masculino , Ratones , Ratones Noqueados , Neurotransmisores/metabolismo , Ensayo de Unión Radioligante , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de GABA-B/metabolismo , Receptores Opioides mu/metabolismoRESUMEN
5-HT(1A) autoreceptors regulate the firing of 5-HT neurons and their release of 5-HT. In previous immuno-electron microscopic studies, we have demonstrated an internalization of 5-HT(1A) autoreceptors in the nucleus raphe dorsalis (NRD) of rats, after the acute administration of a single dose of the specific agonist 8-hydroxy-2-(di-n-propylamine)tetralin (8-OH-DPAT) or of the selective 5-HT reuptake inhibitor, fluoxetine. Twenty-four hours after either treatment, the receptors were back in normal density on the plasma membrane of NRD neurons. Here, we examined the subcellular localization of these receptors and the in vivo binding of the 5-HT(1A) radioligand 4,2-(methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-fluorobenzamido]ethylpiperazine labeled with [(18)F]fluorine ([(18)F]MPPF) after chronic fluoxetine treatment (10 mg/kg daily for 3 weeks, by minipump). Unexpectedly, after such a treatment, there were no more differences between treated and control rats in either the density of plasma membrane labeling of NRD dendrites, or in the in vivo binding of [(18)F]MPPF, as measured with beta-microprobes. This was in keeping with earlier reports of an unchanged density of 5-HT(1A) receptor binding sites after chronic fluoxetine treatment, but quite unexpected from the strong electrophysiological and biochemical evidence for a desensitization of 5-HT(1A) autoreceptors under such conditions. Indeed, when the fluoxetine-treated rats were challenged with a single dose of 8-OH-DPAT, there was no internalization of the 5-HT(1A) autoreceptors, at variance with the controls. Interestingly, several laboratories have reported an uncoupling of 5-HT(1A) autoreceptors from their G protein in the NRD of rats chronically treated with fluoxetine. Therefore, the best explanation for our results is that, after repeated internalization and retargeting, functional 5-HT(1A) autoreceptors are replaced by receptors uncoupled from their G protein on the plasma membrane of NRD 5-HT neurons. Thus, the regulatory function of these autoreceptors may depend on a dynamic balance among their production, activation, internalization and recycling to the plasma membrane in inactivated (desensitized) form.
Asunto(s)
Membrana Celular/efectos de los fármacos , Fluoxetina/farmacología , Neuronas/diagnóstico por imagen , Núcleos del Rafe/citología , Receptor de Serotonina 5-HT1A/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Interacciones Farmacológicas , Fluoxetina/farmacocinética , Hipocampo/ultraestructura , Masculino , Microscopía Inmunoelectrónica , Modelos Neurológicos , Neuronas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , UltrasonografíaRESUMEN
[35S]GTPgammaS autoradiography of slide-mounted tissue sections was used to examine G-protein coupling in the rat spinal cord, as stimulated by dopamine, the D1 receptor agonist SKF 38393, noradrenaline, and noradrenaline in the presence of the alpha adrenoceptor antagonist, phentolamine. Measurements were obtained from the different laminae of spinal grey and from the dorsal, lateral, and ventral columns of white matter, at cervical, thoracic, and lumbar levels. At every level, there was a relatively strong basal incorporation of GTPgammaS in laminae II-III>lamina IV-X of spinal grey, even in presence of DPCPX to block endogenous activation by adenosine A1 receptors. Dopamine, and to a lesser degree SKF 38393, but not the D2 receptor agonist quinpirole, stimulated G-protein coupling in laminae IV-X. Both dopamine and SKF 38393 also induced a weak but significant activation throughout the white matter. In both grey and white matter, the activation by dopamine was markedly reduced in presence of a selective D1 receptor antagonist. Noradrenaline strongly stimulated coupling throughout the spinal grey at all levels, an effect that was uniformly reduced in the presence of phentolamine. With or without phentolamine, there was also significant stimulation by noradrenaline in the white matter. Under the same experimental conditions, alpha 1, alpha 2, and beta adrenergic receptor agonists failed to activate GTPgammaS incorporation in either grey or white matter. However, in the presence of selective alpha 1 or alpha 2 receptor antagonist, significant reductions of noradrenaline-stimulated GTPgammaS incorporation were observed in both grey and white matter. The beta antagonist propanolol reduced GTPgammaS incorporation in grey matter only. Thus, the results confirmed the existence of D1 dopamine receptors and of alpha 1, alpha 2, and beta adrenergic receptors in the grey matter of rat spinal cord. In white matter, they strongly suggested the presence of dopamine D1, and of alpha 1 and alpha 2 adrenergic receptors on glia and/or microvessels, that might be activated by diffuse transmission in vivo.
Asunto(s)
Catecolaminas/farmacología , Proteínas de Unión al GTP/metabolismo , Receptores Adrenérgicos/metabolismo , Receptores Dopaminérgicos/metabolismo , Médula Espinal/metabolismo , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Animales , Dopamina/farmacología , Dopaminérgicos/farmacología , Agonistas de Dopamina/farmacología , Sinergismo Farmacológico , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Masculino , Norepinefrina/farmacología , Fentolamina/farmacología , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Alzheimer's disease (AD) is characterized by increases in amyloid-beta (Abeta) peptides, neurofibrillary tangles, oxidative stress and cholinergic deficits. However, the selectivity of these deficits and their relation with the Abeta pathology or oxidative stress remain unclear. We therefore investigated amyloidosis-related changes in acetylcholine (ACh) and serotonin (5-HT) innervations of hippocampus and parietal cortex by quantitative choline acetyltransferase (ChAT) and 5-HT immunocytochemistry, in 6, 12/14 and 18 month-old transgenic mice carrying familial AD-linked mutations (hAPP(Sw,Ind)). Further, using manganese superoxide dismutase (MnSOD) and nitrotyrosine immunoreactivity as markers, we evaluated the relationship between oxidative stress and the ACh deficit in 18 month-old mice. Thioflavin-positive Abeta plaques were seen in both regions at all ages; they were more numerous in hippocampus and increased in number (>15-fold) and size as a function of age. A majority of plaques exhibited or were surrounded by increased MnSOD immunoreactivity, and dystrophic ACh or 5-HT axons were seen in their immediate vicinity. Counts of immunoreactive axon varicosities revealed significant decreases in ACh innervation, with a sparing of the 5-HT, even in aged mice. First apparent in hippocampus, the loss of ACh terminals was in the order of 20% at 12/14 months, and not significantly greater (26%) at 18 months. In parietal cortex, the ACh denervation was significant at 18 months only, averaging 24% across the different layers. Despite increased perivascular MnSOD immunoreactivity, there was no evidence of dystrophic ACh varicosities or their accentuated loss in the perivascular area. Moreover, there was virtually no sign of tyrosine nitration in ChAT nerve terminals or neuronal cell bodies. These data suggest that aggregated Abeta exerts an early, non-selective and focal neurotoxic effect on both ACh and 5-HT axons, but that a selective, plaque- and oxidative stress-independent diffuse cholinotoxicity, most likely caused by soluble Abeta assemblies, is responsible for the hippocampal and cortical ACh denervation.
Asunto(s)
Vías Aferentes/fisiopatología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Corteza Cerebral/fisiopatología , Fibras Colinérgicas/patología , Tirosina/análogos & derivados , Degeneración Walleriana/fisiopatología , Acetilcolina/metabolismo , Vías Aferentes/metabolismo , Vías Aferentes/patología , Enfermedad de Alzheimer/fisiopatología , Precursor de Proteína beta-Amiloide/genética , Animales , Axones/metabolismo , Axones/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/metabolismo , Desnervación , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estrés Oxidativo/fisiología , Terminales Presinápticos/metabolismo , Serotonina/metabolismo , Superóxido Dismutasa/metabolismo , Tirosina/metabolismo , Degeneración Walleriana/metabolismo , Degeneración Walleriana/patologíaRESUMEN
To follow on prior studies of the cerebral cortex, we examined the acetylcholine innervation in the developing hippocampus of rat, by means of light and electron microscopic immunocytochemistry with a highly sensitive antibody against choline acetyltransferease. As in neocortex, the growth of this innervation mostly occurred within the first two weeks after birth. A preliminary ultrastructural survey indicated that a vast majority of these ChAT-immunostained axon varicosities were asynaptic during development as in the adult. In parallel, we quantified the cholinergic innervations of cerebral cortex and hippocampus in transgenic mice overexpressing human beta-amyloid peptide (hAPP(SW,IND)). A selective, widespread, plaque independent cholinergic denervation was thus demonstrated, first in hippocampus and then neocortex, in addition to a non-selective, plaque-dependent, local neurotoxic effect of aggregated beta-amyloid on ACh and 5-HT axons.
Asunto(s)
Acetilcolina/fisiología , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/fisiología , Corteza Cerebral/fisiología , Fibras Colinérgicas/fisiología , Modelos Animales de Enfermedad , Acetilcolina/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/biosíntesis , Péptidos beta-Amiloides/genética , Animales , Corteza Cerebral/química , Corteza Cerebral/ultraestructura , Fibras Colinérgicas/química , Fibras Colinérgicas/ultraestructura , Masculino , Ratones , Ratones Transgénicos , Ratas , Ratas Sprague-DawleyRESUMEN
Enhancing cerebral serotonin (5-hydroxytryptamine, 5-HT) neurotransmission is a common property of antidepressant treatments and the basis for their efficacy. 5-HT1A receptors located on the cell body and dendrites of 5-HT neurons (autoreceptors) play a key role in this regard. Because they normally mediate an inhibition of neuronal firing, their desensitization is a prerequisite to the delayed enhancement of 5-HT neurotransmission upon treatment with monoamine oxidase (MAOI) inhibitors or specific serotonin reuptake inhibitors (SSRI). Using beta-sensitive microprobes in vivo, we measured a significant decrease (-30%) in binding sites for the 5-HT1A PET radioligand [18F]MPPF associated with an equivalent reduction (-34%) in the cell surface density of 5-HT1A receptor immunoreactivity (internalization), in the nucleus raphe dorsalis (autoreceptors), but not hippocampus (heteroreceptors), of rats given a single dose of the specific 5-HT1A receptor agonist, 8-OH-DPAT (0.5 mg/kg, iv). This effect was completely blocked by pretreatment with the selective 5-HT1A antagonist WAY 100635. Having ruled out that this decreased density of [18F]MPPF binding in the nucleus raphe dorsalis of 8-OH-DPAT-treated rats resulted from a local blood flow effect, we obtained autoradiographic evidence indicating that the total amount of specific binding of [18F]MPPF in tissue sections was unaffected by the 8-OH-DPAT treatment in either NRD or hippocampus. It was therefore concluded that the internalization of 5-HT1A autoreceptors accounted for the decreased binding in vivo of [18F]MPPF in the nucleus raphe dorsalis of rats treated with 8-OH-DPAT. Thus, PET imaging might provide a mean to measure 5-HT1A receptor internalization in human brain and thus assess responsiveness to antidepressant treatment.
Asunto(s)
Autorreceptores/metabolismo , Encéfalo/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Autorradiografía , Sitios de Unión , Encéfalo/ultraestructura , Membrana Celular/metabolismo , Citoplasma/metabolismo , Líquido Extracelular/metabolismo , Cinética , Ligandos , Masculino , Microdiálisis , Microscopía Inmunoelectrónica , Concentración Osmolar , Piperazinas/farmacología , Piridinas/farmacología , Núcleos del Rafe/metabolismo , Ratas , Ratas Sprague-Dawley , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Fracciones Subcelulares/metabolismo , Distribución TisularRESUMEN
As visualized by light and electron microscopic immunocytochemistry, the distribution of the neuronal serotonin-2A (5-HT(2A)) receptor is mainly intracellular throughout adult rat brain. This localization is particularly striking in the pyramidal cells of cerebral cortex, the dendrites of which are intensely immunoreactive, but without any labeling of their spines. In view of recent yeast two-hybrid and biochemical results suggesting an association of 5-HT(2A) receptors with the cytoskeletal microtubule-associated protein MAP1A, the respective subcellular distributions of the receptors and of MAP1A were compared by quantitative electron microscopic immunocytochemistry in dendrites of adult rat frontoparietal cortex. Counts of silver-intensified immunogold particles revealed a higher density of 5-HT(2A) receptors in smaller rather than larger dendrites, and an apportionment between pre-defined compartments representing the plasma membrane and the cytoplasm that was proportional to the relative surface area of these compartments. MAP1A immunoreactivity also predominated in smaller versus larger dendrites, but with a slightly lower proportion of labeling in the plasma membrane versus cytoplasmic compartment. The co-localization of 5-HT(2A) receptors and MAP1A protein in the same dendrites could be demonstrated in double immunolabeling experiments. These results confirmed the predominantly somato-dendritic, intracellular localization of 5-HT(2A) receptors in cerebral cortex, showed their higher concentration in distal as opposed to proximal dendrites, and suggested their potential association to the cytoskeleton in cortical neurons in vivo. Such a distribution of 5-HT(2A) receptors reinforces our earlier hypothesis that 5-HT(2A) receptors participate in intraneuronal signaling processes involving the cytoskeleton, and raises the possibility that their activation could be dependent upon that of another co-localized, plasma membrane-bound, 5-HT receptor.
Asunto(s)
Dendritas/química , Proteínas Asociadas a Microtúbulos/análisis , Neocórtex/química , Receptores de Serotonina/análisis , Animales , Anticuerpos Monoclonales/análisis , Inmunohistoquímica , Masculino , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/inmunología , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT2A , Receptores de Serotonina/inmunología , Distribución TisularRESUMEN
The firing of central serotonin (5-hydroxytryptamine, 5-HT) neurons and their capacity to release 5-HT are subjected to a receptor-mediated auto-control via 5-HT(1A) and 5-HT(1B) receptors respectively located on the somata/dendrites (5-HT(1A) autoreceptors) and preterminal axon arborizations (5-HT(1B) autoreceptors) of these neurons. To further characterize mutual adaptations of these two receptor subtypes in the absence of one of them, activation of G-protein coupling by agonist was measured and compared to wild-type (WT) in 5-HT(1A) and 5-HT(1B) homozygous knockout (KO) mice. As expected, in WT, the non-selective 5-HT(1A/1B) receptor agonist 5-carboxyamidotryptamine (5-CT) stimulated guanosine 5'-O-(gamma-[(35)S]thio)triphosphate ([(35)S]GTP(gamma)S) incorporation in many brain regions endowed with one and/or the other receptor. In the respective KOs, no stimulation was measured in regions known to express only or mainly the deleted receptor. In the 5-HT(1A) KOs, the amplitude of G-protein activation in regions endowed with 5-HT(1B) receptors was unchanged by comparison to WT. In the 5-HT(1B) KOs, the magnitude of the 5-CT stimulation was the same as WT in all regions containing 5-HT(1A) receptors, except in the amygdala, where it was significantly lower, even if this region was one of the most strongly activated in the WT. A similar result was obtained in the amygdala of 5-HT(1B) KOs after activation by the selective 5-HT(1A) receptor agonist R-(+)8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). Under these conditions, however, there was in addition a significant lowering of the stimulated (but not basal) [(35)S]GTP(gamma)S incorporation by comparison to WT in all regions endowed with 5-HT(1A) receptors, including the dorsal raphe nucleus. Thus, eventhough agonist radioligand binding to either 5-HT(1A) or 5-HT(1B) receptors is unchanged in the reciprocal KOs, it appears that a compensatory decrease in the efficiency of G-protein coupling to 5-HT(1A) receptors has developed in the 5-HT(1B) mutant. This could represent the first indication of a cross-talk between these two 5-HT receptor subtypes, at least in brain regions where they are co localized in the same neurons.
Asunto(s)
Encéfalo/fisiología , Proteínas de Unión al GTP/metabolismo , Neuronas/fisiología , Receptores de Serotonina/fisiología , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Serotonina/análogos & derivados , Animales , Autorradiografía , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Ratones , Ratones Noqueados , Neuronas/efectos de los fármacos , Receptor de Serotonina 5-HT1B , Receptores de Serotonina/deficiencia , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/genética , Receptores de Serotonina 5-HT1 , Serotonina/farmacologíaRESUMEN
A recently developed method for determining the length of cholinergic axons and number of cholinergic axon varicosities (terminals) in brain sections immunostained for choline acetyltransferase was used to estimate the areal and laminar densities of the cholinergic innervation in rat frontal (motor), parietal (somatosensory) and occipital (visual) cortex at different postnatal ages. This cortical innervation showed an early beginning, a few immunostained fibers being already present in the cortical subplate at birth. In the first two postnatal weeks, it developed rapidly along three parameters: a progressive increase in the number of varicosities per unit length of axon, and a lengthening and branching of the axons. Between postnatal days 4 and 16, the number of varicosities increased steadily from two to four per 10 microm of cholinergic axon. The mean densities of cholinergic axons increased from 1.4 to 9.6, 1.7 to 9.3 and 0.7 to 7.2 m/mm(3), and the corresponding densities of varicosities from 0.4 to 3.9, 0.4 to 3.5, and 0.2 to 2.6x10(6)/mm(3) in the frontal, parietal and occipital areas, respectively. The rate of growth was maximal during these first two weeks, after which the laminar pattern characteristic of each area appeared to be established. Adult values were almost reached by postnatal day 16 in the parietal cortex, but maturation proceeded further in the frontal and particularly in the occipital cortex. These quantitative data on the ingrowth and maturation of the cholinergic innervation in postnatal rat cerebral cortex substantiate a role for acetylcholine in the development of this brain region and emphasize the striking growth capacity of individual cholinergic neurons.
Asunto(s)
Acetilcolina/análisis , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Fibras Colinérgicas/química , Factores de Edad , Animales , Colina O-Acetiltransferasa/análisis , Fibras Colinérgicas/enzimología , Lóbulo Frontal/citología , Lóbulo Frontal/crecimiento & desarrollo , Inmunohistoquímica , Masculino , Vías Nerviosas , Lóbulo Occipital/citología , Lóbulo Occipital/crecimiento & desarrollo , Lóbulo Parietal/citología , Lóbulo Parietal/crecimiento & desarrollo , Ratas , Ratas Sprague-DawleyRESUMEN
Serotonin-1A (5-HT(1A)) receptors in the CNS are a major target for psychotropic drugs. In nucleus raphe dorsalis (NRD) and hippocampus (CA3), the selective 5-HT(1A) agonist (+)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT) reduces the firing activity of serotoninergic (5-HT) and pyramidal neurons, respectively. When located on 5-HT (autoreceptors), but not on non-5-HT (heteroreceptors) neurons, 5-HT(1A) receptors are known to be subject to desensitization. Using quantitative electron microscopy after pre-embedding immunogold labeling with specific antibodies, we examined the subcellular distribution of these receptors after acute administration of 8-OH-DPAT (0.5 mg/kg, i.v.). Silver-intensified immunogold particles associated with the plasma membrane or the cytoplasm were counted in somata and dendrites within the NRD, 15 min, 1 hr and 24 hr after 8-OH-DPAT injection, and in hippocampal dendrites 1 hr after the same treatment. Significant decrease in the density of membrane labeling and concomitant increase of cytoplasmic labeling were demonstrated in the NRD, 15 min and 1 hr after 8-OH-DPAT administration, with a return to baseline level at 24 hr. Internalization was blocked by previous administration of the 5-HT(1A) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide (WAY 100635), which, by itself, was without apparent effect. In hippocampus (CA3), there were no apparent changes in the distribution of the receptor after 8-OH-DPAT administration. These findings are in line with earlier results showing a desensitization of 5-HT(1A) autoreceptors but not heteroreceptors after treatment with 5-HT(1A) receptor agonist. They suggest that this desensitization is the result of autoreceptor internalization.
Asunto(s)
Hipocampo/metabolismo , Núcleos del Rafe/metabolismo , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/administración & dosificación , 8-Hidroxi-2-(di-n-propilamino)tetralin/administración & dosificación , Animales , Autorreceptores/efectos de los fármacos , Autorreceptores/metabolismo , Compartimento Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Citoplasma/metabolismo , Citoplasma/ultraestructura , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Dendritas/ultraestructura , Hipocampo/efectos de los fármacos , Hipocampo/ultraestructura , Inmunohistoquímica , Inyecciones Intravenosas , Masculino , Microscopía Electrónica , Piperazinas/administración & dosificación , Piridinas/administración & dosificación , Núcleos del Rafe/efectos de los fármacos , Núcleos del Rafe/ultraestructura , Ratas , Ratas Sprague-Dawley , Receptores de Serotonina 5-HT1 , Antagonistas de la Serotonina/administración & dosificaciónRESUMEN
5-HT1A knockout (KO) mice display an anxious-like phenotype, whereas 5-HT1B KOs are over-aggressive. To identify serotoninergic correlates of these altered behaviors, autoradiographic measurements of 5-HT1A and 5-HT1B serotonin (5-HT) receptors and transporter (5-HTT) were obtained using the radioligands [3H]8-OH-DPAT, [125I]cyanopindolol and [3H]citalopram, respectively. By comparison to wild-type, density of 5-HT1B receptors was unchanged throughout brain in 5-HT1A KOs, and that of 5-HT1A receptors in 5-HT1B KOs. In contrast, decreases in density of 5-HTT binding were measured in several brain regions of both genotypes. Moreover, 5-HTT binding density was significantly increased in the amygdalo-hippocampal nucleus and ventral hippocampus of the 5-HT1B KOs. Measurements of 5-HT axon length and number of axon varicosities by quantitative 5-HT immunocytochemistry revealed proportional increases in the density of 5-HT innervation in these two regions of 5-HT1B KOs, whereas none of the decreases in 5-HTT binding sites were associated with any such changes. Several conclusions could be drawn from these results: (i) 5-HT1B receptors do not adapt in 5-HT1A KOs, nor do 5-HT1A receptors in 5-HT1B KOs. (ii) 5-HTT is down-regulated in several brain regions of 5-HT1A and 5-HT1B KO mice. (iii) This down-regulation could contribute to the anxious-like phenotype of the 5-HT1A KOs, by reducing 5-HT clearance in several territories of 5-HT innervation. (iv) The 5-HT hyperinnervation in the amygdalo-hippocampal nucleus and ventral hippocampus of 5-HT1B KOs could play a role in their increased aggressiveness, and might also explain their better performance in some cognitive tests. (v) These increases in density of 5-HT innervation provide the first evidence for a negative control of 5-HT neuron growth mediated by 5-HT1B receptors.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Neuronas/metabolismo , Pindolol/análogos & derivados , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/metabolismo , Animales , Autorradiografía , Conducta Animal/fisiología , Proteínas Portadoras/genética , Citalopram/metabolismo , Femenino , Inmunohistoquímica , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Neuronas/citología , Pindolol/metabolismo , Ensayo de Unión Radioligante , Receptor de Serotonina 5-HT1B , Receptores de Serotonina/genética , Receptores de Serotonina 5-HT1 , Antagonistas de la Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Agonistas de Receptores de Serotonina/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/metabolismoRESUMEN
Measurements of serotonin (5-HT), dopamine (DA), and noradrenaline, and of 5-HT and DA metabolites, were obtained by HPLC from 16 brain regions and the spinal cord of 5-HT(1A) or 5-HT(1B) knockout and wild-type mice of the 129/Sv strain. In 5-HT(1A) knockouts, 5-HT concentrations were unchanged throughout, but levels of 5-HT metabolites were higher than those of the wild type in dorsal/medial raphe nuclei, olfactory bulb, substantia nigra, and locus coeruleus. This was taken as an indication of increased 5-HT turnover, reflecting an augmented basal activity of midbrain raphe neurons and consequent increase in their somatodendritic and axon terminal release of 5-HT. It provided a likely explanation for the increased anxious-like behavior observed in 5-HT(1A) knockout mice. Concomitant increases in DA content and/or DA turnover were interpreted as the result of a disinhibition of DA, whereas increases in noradrenaline concentration in some territories of projection of the locus coeruleus could reflect a diminished activity of its neurons. In 5-HT(1B) knockouts, 5-HT concentrations were lower than those of the wild type in nucleus accumbens, locus coeruleus, spinal cord, and probably also several other territories of 5-HT innervation. A decrease in DA, associated with increased DA turnover, was measured in nucleus accumbens. These changes in 5-HT and DA metabolism were consistent with the increased aggressiveness and the supersensitivity to cocaine reported in 5-HT(1B) knockout mice. Thus, markedly different alterations in CNS monoamine metabolism may contribute to the opposite behavioral phenotypes of these two knockouts.
Asunto(s)
Sistema Nervioso Central/metabolismo , Dopamina/análogos & derivados , Dopamina/metabolismo , Receptores de Serotonina/genética , Serotonina/metabolismo , Ácido 3,4-Dihidroxifenilacético/análisis , Animales , Autorradiografía , Ganglios Basales/química , Ganglios Basales/metabolismo , Sistema Nervioso Central/química , Cerebelo/química , Cerebelo/metabolismo , Corteza Cerebral/química , Corteza Cerebral/metabolismo , Cromatografía Líquida de Alta Presión , Dopamina/análisis , Femenino , Ácido Homovanílico/análisis , Ácido Hidroxiindolacético/análisis , Hidroxitriptofol/análisis , Hipotálamo/química , Hipotálamo/metabolismo , Masculino , Mesencéfalo/química , Mesencéfalo/metabolismo , Ratones , Ratones Noqueados , Norepinefrina/análisis , Bulbo Olfatorio/química , Bulbo Olfatorio/metabolismo , Especificidad de Órganos , Receptor de Serotonina 5-HT1B , Receptores de Serotonina/deficiencia , Receptores de Serotonina 5-HT1 , Serotonina/análisis , Médula Espinal/química , Médula Espinal/metabolismoRESUMEN
A method for determining the length of acetylcholine (ACh) axons and number of ACh axon varicosities (terminals) in brain sections immunostained for choline acetyltransferase (ChAT) was used to estimate the areal and laminar densities of this innervation in the frontal (motor), parietal (somatosensory), and occipital (visual) cortex of adult rat. The number of ACh varicosities per length of axon (4 per 10 microm) appeared constant in the different layers and areas. The mean density of ACh axons was the highest in the frontal cortex (13.0 m/mm(3) vs. 9.9 and 11.0 m/mm(3) in the somatosensory and visual cortex, respectively), as was the mean density of ACh varicosities (5.4 x 10(6)/mm(3) vs. 3.8 and 4.6 x 10(6)/mm(3)). In all three areas, layer I displayed the highest laminar densities of ACh axons and varicosities (e.g., 13.5 m/mm(3) and 5.4 x 10(6)/mm(3) in frontal cortex). The lowest were those of layer IV in the parietal cortex (7.3 m/mm(3) and 2.9 x 10(6)/mm(3)). The lengths of ACh axons under a 1 mm(2) surface of cortex were 26.7, 19.7, and 15.3 m in the frontal, parietal, and occipital areas, respectively, for corresponding numbers of 11.1, 7.7, and 6.4 x 10(6) ACh varicosities. In the parietal cortex, this meant a total of 1.2 x 10(6) synaptic ACh varicosities under a 1 mm(2) surface, 48% of which in layer V alone, according to previous electron microscopic estimates of synaptic incidence. In keeping with the notion that the synaptic component of ACh transmission in cerebral cortex is preponderant in layer V, these quantitative data suggest a role for this innervation in the processing of cortical output as well as input. Extrapolation of particular features of this system in terms of total axon length and number of varicosities in whole cortex, length of axons and number of varicosities per cortically projecting neuron, and concentration of ACh per axon varicosity, should also help in arriving at a better definition of its roles and functional properties in cerebral cortex.
Asunto(s)
Axones , Corteza Cerebral/anatomía & histología , Fibras Colinérgicas , Acetilcolina/análisis , Animales , Axones/química , Axones/ultraestructura , Corteza Cerebral/química , Corteza Cerebral/ultraestructura , Colina O-Acetiltransferasa/análisis , Fibras Colinérgicas/química , Inmunohistoquímica , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The autosomal recessive mutation dystonia musculorum (dt(J)/dt(J)) causes degenerative alterations of peripheral and central sensory pathways that lead to ataxia. To investigate possible changes in the central serotonin system of these mice, HPLC measurements of 5-hydroxytryptophan, 5-hydroxy-tryptamine (serotonin; 5-HT), and 5-HT metabolites were obtained from 22 brain regions and the spinal cord of wild type and dt(J)/dt(J) mutant mice. Also, 5-HT transporters were quantified by [(3)H]citalopram autoradiography in 72 brain regions, subregions, and nuclei, and the 5-HT innervation visualized by immunocytochemistry throughout the brain and spinal cord. In all brain regions measured for indoleamine content, there were no significant differences between the two genotypes. In the spinal cord, an increased tissue concentration of 5-HT (+34%), 5-hydroxyindole-3-acetic acid (+33%), 5-hydroxytryptophol (+21%), and 5-hydroxytryptophan (+45%) in dt(J)/dt(J) actually corresponded to the same total amount of each of these indoleamines in the entire spinal cord, when taking into account its reduced size in the mutants. Quantification of the binding to 5-HT transporters showed increases in the medial geniculate nucleus (+14%), medial (+24%) and lateral (+18%) hypothalamus, interpeduncular (+13%), vestibular (+22%), and deep cerebellar nuclei (+37%) of dt(J)/dt mice, and decreases in the ventral tegmental area (-13%), median and linear raphe nuclei (-20%), as well as in the solitary complex (-35%). There were no apparent differences in the distribution of 5-HT-immunostained fibers in these and other regions of brain and in the spinal cord of dt(J)/dt(J) compared to wild type mice. The bulk of these results indicates a relative sparing of the central 5-HT system in the dt(J)/dt(J) mice, even though alterations in 5-HT transporters could justify attempts at improving the sensorimotor dysfunction by administration of serotoninergic agents in these mice.
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Trastornos Distónicos/metabolismo , Trastornos Distónicos/patología , Serotonina/metabolismo , 5-Hidroxitriptófano/metabolismo , Animales , Autorradiografía , Encéfalo/metabolismo , Encéfalo/patología , Química Encefálica , Cromatografía Líquida de Alta Presión , Citalopram/metabolismo , Ácido Hidroxiindolacético/metabolismo , Hidroxitriptofol/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Mutantes Neurológicos , Especificidad de Órganos , Médula Espinal/química , Médula Espinal/metabolismo , Médula Espinal/patología , TritioRESUMEN
The 5-HT1A and 5-HT1B receptors of serotonin play important roles as auto- and heteroreceptors controlling the release of serotonin itself and of other neurotransmitters/modulators in the central nervous system (CNS). To determine the precise localization of these receptors, we examined their respective cellular and subcellular distributions in the nucleus raphe dorsalis and hippocampal formation (5-HT1A) and in the globus pallidus and substantia nigra (5-HT1B), using light and electron microscopic immunocytochemistry with specific antibodies. Both immunogold and immunoperoxidase preembedding labelings were achieved. In the nucleus raphe dorsalis, 5-HT1A immunoreactivity was found exclusively on neuronal cell bodies and dendrites, and mostly along extrasynaptic portions of their plasma membrane. After immunogold labeling, the density of membrane-associated 5-HT1A receptors could be estimated to be at least 30-40 times that in the cytoplasm. In the hippocampal formation, the somata as well as dendrites of pyramidal and granule cells displayed 5-HT1A immunoreactivity, which was also prominent on the dendritic spines of pyramidal cells. In both substantia nigra and globus pallidus, 5-HT1B receptors were preferentially associated with the membrane of fine, unmyelinated, preterminal axons, and were not found on axon terminals. A selective localization to the cytoplasm of endothelial cells of microvessels was also observed. Because the 5-HT1A receptors are somatodendritic, they are ideally situated to mediate serotonin effects on neuronal firing, both as auto- and as heteroreceptors. The localization of 5-HT1B receptors to the membrane of preterminal axons suggests that they control transmitter release from nonserotonin as well as serotonin neurons by mediating serotonin effects on axonal conduction. The fact that these two receptor subtypes predominate at extrasynaptic and nonsynaptic sites provides further evidence for diffuse serotonin transmission in the CNS.
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Encéfalo/metabolismo , Receptores de Serotonina/metabolismo , Animales , Axones/metabolismo , Dendritas/metabolismo , Inmunohistoquímica , Masculino , Microscopía Electrónica , Terminaciones Nerviosas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT1B , Receptores de Serotonina 5-HT1 , Fracciones Subcelulares/metabolismoRESUMEN
Light and electron microscope immunocytochemistry with a monoclonal antibody against the N-terminal domain of the human protein was used to determine the cellular and subcellular localization of serotonin 5-HT2A receptors in the central nervous system of adult rat. Following immunoperoxidase or silver-intensified immunogold labeling, neuronal, somatodendritic, and/or axonal immunoreactivity was detected in numerous brain regions, including all those in which ligand binding sites and 5-HT2A mRNA had previously been reported. The distribution of 5-HT2A-immunolabeled soma/dendrites was characterized in cerebral cortex, olfactory system, septum, hippocampal formation, basal ganglia, amygdala, diencephalon, cerebellum, brainstem, and spinal cord. Labeled axons were visible in every myelinated tract known to arise from immunoreactive cell body groups. In immunopositive soma/dendrites as well as axons, the 5-HT2A receptor appeared mainly cytoplasmic rather than membrane bound. Even though the dendritic labeling was generally stronger than the somatic, it did not extend to dendritic spines in such regions as the cerebral and piriform cortex, the neostriatum, or the molecular layer of the cerebellum. Similarly, there were no labeled axon terminals in numerous regions known to be strongly innervated by the immunoreactive somata and their axons (e.g., molecular layer of piriform cortex). It was concluded that the 5-HT2A receptor is mostly intracellular and transported in dendrites and axons, but does not reach into dendritic spines or axon terminals. Because it has previously been shown that this serotonin receptor is transported retrogradely as well as anterogradely, activates intracellular transduction pathways and intervenes in the regulation of the expression of many genes, it is suggested that one of its main functions is to participate in retrograde signaling systems activated by serotonin.
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Química Encefálica , Ratas Sprague-Dawley/fisiología , Receptores de Serotonina/análisis , Células 3T3 , Factores de Edad , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Axones/química , Axones/ultraestructura , Western Blotting , Sistema Nervioso Central/química , Sistema Nervioso Central/citología , Dendritas/química , Dendritas/ultraestructura , Humanos , Masculino , Ratones , Microscopía Inmunoelectrónica , Neuronas/química , Neuronas/ultraestructura , Ratas , Receptor de Serotonina 5-HT2A , Receptores de Serotonina/inmunología , Fracciones Subcelulares/química , TransfecciónRESUMEN
Numerous features of its primary structure demonstrate that the orphan transporter Rxt1 belongs to the Na+/Cl--dependent neurotransmitter plasma membrane transporter superfamily, which includes the dopamine, norepinephrine, serotonin and gamma-aminobutyric acid (GABA) transporters. Initial immunocytochemical investigations with affinity-purified antibodies have established that Rxt1 is localized, almost exclusively, in axon terminals of glutamatergic neurons and subsets of GABAergic neurons in the CNS. Further studies were carried out to determine its subcellular distribution. In a first series of experiments, PC-12 cells were transfected with plasmids encoding either the dopamine transporter or Rxt1. Immunofluorescence experiments showed that the dopamine transporter was expressed in these cells, and, as expected, addressed to their plasma membrane. Surprisingly, this was never the case with Rxt1, which was targeted to the same subcellular compartment as synaptophysin, a vesicular protein. In a second set of experiments, subcellular fractionation of rat striatum showed that Rxt1, but not the dopamine transporter, was relatively abundant in the purified synaptic vesicle fraction. Finally, electron microscopic immunocytochemistry with anti-Rxt1 antibodies showed peroxidase as well as pre- and post-embedding immunogold labelling confined to the intracellular compartment in various brain regions. Moreover, quantitative analysis of post-embedding experiments demonstrated that the immunogold particles corresponding to Rxt1 immunoreactivity were mostly localized to small synaptic vesicles. These data indicate that, in contrast with the other members of the Na+/Cl--dependent neurotransmitter transporter superfamily, which are targeted to the plasma membrane, Rxt1 is distributed as a vesicular protein in the CNS.
Asunto(s)
Proteínas Portadoras/análisis , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso/análisis , Vesículas Sinápticas/química , Animales , Western Blotting , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Microscopía Electrónica , Proteínas del Tejido Nervioso/metabolismo , Células PC12 , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/química , TransfecciónRESUMEN
Transgenic mice expressing human light neurofilament protein (NF-L) display early perikaryal accumulations of disarrayed neurofilaments in layers II/III of the parietal cortex and in the ventrobasal complex of thalamus. This cytoskeletal abnormality, reflected by strong NF-L immunoreactivity, is transient in the developing cortex but persists until old age in the thalamus. To investigate whether it leads to neuronal death, the unbiased cell counting method of the dissector was applied to the parietal cortex and the thalamus of normal and transgenic mice at various postnatal (P10, P20, P90) and advanced ages (14-18 months). Similar data were also obtained from the primary visual cortex free of NF-L accumulation. Compared with normal, the total number of neurons in the parietal (but not occipital) cortex of transgenic mice showed little change during the postnatal period, but decreased markedly with old age, particularly in layers II/III. Severe neuronal loss was also documented in the thalamic ventrobasal complex of aged transgenic mice. The delayed neuronal death in the parietal cortex, occurring long after recovery from the NF-L accumulations, was suggestive of a combination of deleterious factors, including the early overproduction of neurofilament protein and subsequent loss of afferent input from the affected somatosensory thalamic nuclei. Furthermore, strong accumulation of lipofuscin in the neurons of aged transgenic mice suggested that oxidative stress partakes in the mechanisms through which NF-L overproduction compromises neuronal viability.