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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 ß-amyloid peptide (hAPPSW,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 ß-amyloid on ACh and 5-HT axons. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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]GTPγS 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 μ-opioid and GABAB receptors was also assessed by [35S]GTPγS autoradiography. 5-HT1B KO showed selective decreases in G-protein coupling to μ-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. [Copyright &y& Elsevier]

Abstract: 5-HT1A 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-HT1A 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-HT1A radioligand 4,2-(methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-fluorobenzamido]ethylpiperazine labeled with [18F]fluorine ([18F]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 [18F]MPPF, as measured with β-microprobes. This was in keeping with earlier reports of an unchanged density of 5-HT1A receptor binding sites after chronic fluoxetine treatment, but quite unexpected from the strong electrophysiological and biochemical evidence for a desensitization of 5-HT1A 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-HT1A autoreceptors, at variance with the controls. Interestingly, several laboratories have reported an uncoupling of 5-HT1A 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-HT1A 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. [Copyright &y& Elsevier]

Abstract: [35S]GTPγS 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 GTPγS 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 GTPγS 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 GTPγS incorporation were observed in both grey and white matter. The beta antagonist propanolol reduced GTPγS 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. [Copyright &y& Elsevier]

Alzheimer’s disease (AD) is characterized by increases in amyloid-β (Aβ) peptides, neurofibrillary tangles, oxidative stress and cholinergic deficits. However, the selectivity of these deficits and their relation with the Aβ 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 (hAPPSw,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 Aβ 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 Aβ 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 Aβ assemblies, is responsible for the hippocampal and cortical ACh denervation. [Copyright &y& Elsevier]

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 β-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. [Copyright &y& Elsevier]

As visualized by light and electron microscopic immunocytochemistry, the distribution of the neuronal serotonin-2A (5-HT2A) 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-HT2A 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-HT2A 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-HT2A 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-HT2A 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-HT2A receptors reinforces our earlier hypothesis that 5-HT2A 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. [Copyright &y& Elsevier]

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-HT1A and 5-HT1B receptors respectively located on the somata/dendrites (5-HT1A autoreceptors) and preterminal axon arborizations (5-HT1B 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-HT1A and 5-HT1B homozygous knockout (KO) mice. As expected, in WT, the non-selective 5-HT1A/1B receptor agonist 5-carboxyamidotryptamine (5-CT) stimulated guanosine 5′-O-(γ-[35S]thio)triphosphate ([35S]GTPγS) 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-HT1A KOs, the amplitude of G-protein activation in regions endowed with 5-HT1B receptors was unchanged by comparison to WT. In the 5-HT1B KOs, the magnitude of the 5-CT stimulation was the same as WT in all regions containing 5-HT1A 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-HT1B KOs after activation by the selective 5-HT1A 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) [35S]GTPγS incorporation by comparison to WT in all regions endowed with 5-HT1A receptors, including the dorsal raphe nucleus. Thus, eventhough agonist radioligand binding to either 5-HT1A or 5-HT1B receptors is unchanged in the reciprocal KOs, it appears that a compensatory decrease in the efficiency of G-protein coupling to 5-HT1A receptors has developed in the 5-HT1B 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. [Copyright &y& Elsevier]

5-HT[sub 1A] knockout (KO) mice display an anxious-like phenotype, whereas 5-HT[sub 1B] KOs are over-aggressive. To identify serotoninergic correlates of these altered behaviors, autoradiographic measurements of 5-HT[sub 1A] and 5-HT[sub 1B] serotonin (5-HT) receptors and transporter (5-HTT) were obtained using the radioligands [[sup 3]H]8-OH-DPAT, [[sup 125]I]cyanopindolol and [[sup 3]H]citalopram, respectively. By comparison to wild-type, density of 5-HT[sub 1B] receptors was unchanged throughout brain in 5-HT[sub 1A] KOs, and that of 5-HT[sub 1A] receptors in 5-HT[sub 1B] 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-HT[sub 1B] 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-HT[sub 1B] 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-HT[sub 1B] receptors do not adapt in 5-HT[sub 1A] KOs, nor do 5-HT[sub 1A] receptors in 5-HT[sub 1B] KOs. (ii) 5-HTT is down-regulated in several brain regions of 5-HT[sub 1A] and 5-HT[sub 1B] KO mice. (iii) This down-regulation could contribute to the anxious-like phenotype of the 5-HT[sub 1A] 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-HT[sub 1B] 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-HT[sub 1B] receptors. [ABSTRACT FROM AUTHOR]

Abstract 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 γ-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. [ABSTRACT FROM AUTHOR]