Mustapha Riad

Cellular and subcellular distribution of the serotonin 5-HT2A receptor in the central nervous system of adult rat.

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.

Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain

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. J. Comp. Neurol. 417:181–194, 2000. ©2000 Wiley‐Liss, Inc.

Acute Treatment with the Antidepressant Fluoxetine Internalizes 5-HT1A Autoreceptors and Reduces the In Vivo Binding of the PET Radioligand [18F]MPPF in the Nucleus Raphe Dorsalis of Rat

Because 5-HT1A receptors located on the soma dendrites of serotonin (5-HT) neurons normally mediate an inhibition of 5-HT firing and release, the desensitization of these autoreceptors is essential for obtaining an enhancement of 5-HT transmission after treatment with 5-HT reuptake inhibitors (SSRIs). We have demonstrated previously, using immunoelectron microscopy with specific 5-HT1A antibodies, that an internalization of 5-HT1A autoreceptors is associated with their desensitization in rats given a single dose of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin. Here, we examined the subcellular distribution of 5-HT1A receptors in dendrites from nucleus raphe dorsalis (NRD) (autoreceptors) and hippocampus (heteroreceptors) after acute treatment with the antidepressant SSRI, fluoxetine (10 mg/kg, i.p.). In parallel experiments, the kinetics of in vivo binding of the 5-HT1A positron emission tomography radioligand 4,2-(methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-fluorobenzamido]ethylpiperazine ([18F]MPPF) was measured in these two brain regions by means of stereotaxically implanted β microprobes. One hour after treatment, there was a 36% decrease in 5-HT1A immunogold labeling of the plasma membrane of NRD dendrites, and a concomitant increase in their cytoplasmic labeling, without any change in hippocampal dendrites. In vivo binding of [18F]MPPF was reduced by 35% in NRD and unchanged in hippocampus. Both effects were blocked by pretreatment with the 5-HT1A receptor antagonist (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide) (1 mg/kg, i.p.). In brain sections of NRD and hippocampus, [18F]MPPF autoradiographic labeling did not differ between fluoxetine- and saline-treated rats. These immunocytochemical results confirmed that internalization of 5-HT1A autoreceptors may account for their desensitization, and the microprobe results suggest that this prerequisite for antidepressant treatment efficacy could be amenable to brain imaging in humans.

Altered Serotonin and Dopamine Metabolism in the CNS of Serotonin 5‐HT1A or 5‐HT1B Receptor Knockout Mice

Abstract: Measurements of serotonin (5‐HT), dopamine (DA), andnoradrenaline, and of 5‐HT and DA metabolites, were obtained by HPLC from 16brain regions and the spinal cord of 5‐HT1A or 5‐HT1Bknockout and wild‐type mice of the 129/Sv strain. In 5‐HT1Aknockouts, 5‐HT concentrations were unchanged throughout, but levels of 5‐HTmetabolites were higher than those of the wild type in dorsal/medial raphenuclei, olfactory bulb, substantia nigra, and locus coeruleus. This was takenas an indication of increased 5‐HT turnover, reflecting an augmented basalactivity of midbrain raphe neurons and consequent increase in theirsomatodendritic and axon terminal release of 5‐HT. It provided a likelyexplanation for the increased anxious‐like behavior observed in5‐HT1A knockout mice. Concomitant increases in DA content and/or DAturnover were interpreted as the result of a disinhibition of DA, whereasincreases in noradrenaline concentration in some territories of projection ofthe locus coeruleus could reflect a diminished activity of its neurons. In5‐HT1B knockouts, 5‐HT concentrations were lower than those of thewild type in nucleus accumbens, locus coeruleus, spinal cord, and probablyalso 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 increasedaggressiveness and the supersensitivity to cocaine reported in5‐HT1B knockout mice. Thus, markedly different alterations in CNSmonoamine metabolism may contribute to the opposite behavioral phenotypes ofthese two knockouts.

The Dual Dopamine-Glutamate Phenotype of Growing Mesencephalic Neurons Regresses in Mature Rat Brain

Coexpression of tyrosine hydroxylase (TH) and vesicular glutamate transporter 2 (VGLUT2) mRNAs in the ventral tegmental area (VTA) and colocalization of these proteins in axon terminals of the nucleus accumbens (nAcb) have recently been demonstrated in immature (15‐day‐old) rat. After neonatal 6‐hydroxydopamine (6‐OHDA) lesion, the proportion of VTA neurons expressing both mRNAs and of nAcb terminals displaying the two proteins was enhanced. To determine the fate of this dual phenotype in adults, double in situ hybridization and dual immunolabeling for TH and VGLUT2 were performed in 90‐day‐old rats subjected or not to the neonatal 6‐OHDA lesion. Very few neurons expressed both mRNAs in the VTA and substantia nigra (SN) of P90 rats, even after neonatal 6‐OHDA. Dually immunolabeled terminals were no longer found in the nAcb of normal P90 rats and were exceedingly rare in the nAcb of 6‐OHDA‐lesioned rats, although they had represented 28% and 37% of all TH terminals at P15. Similarly, 17% of all TH terminals in normal neostriatum and 46% in the dopamine neoinnervation of SN in 6‐OHDA‐lesioned rats were also immunoreactive for VGLUT2 at P15, but none at P90. In these three regions, all dually labeled terminals made synapse, in contradistinction to those immunolabeled for only TH or VGLUT2 at P15. These results suggest a regression of the VGLUT2 phenotype of dopamine neurons with age, following normal development, lesion, or sprouting after injury, and a role for glutamate in the establishment of synapses by these neurons. J. Comp. Neurol. 517:873–891, 2009.

Enhanced glutamatergic phenotype of mesencephalic dopamine neurons after neonatal 6-hydroxydopamine lesion.

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.

Regional changes in density of serotonin transporter in the brain of 5-HT1A and 5-HT1B knockout mice, and of serotonin innervation in the 5-HT1B knockout

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.

Localization of EphA4 in axon terminals and dendritic spines of adult rat hippocampus.

Eph receptors and their ephrin ligands assume various roles during central nervous system development. Several of these proteins are also expressed in the mature brain, and notably in the hippocampus, where EphA4 and ephrins have been shown to influence dendritic spine morphology and long‐term potentiation (LTP). To examine the cellular and subcellular localization of EphA4 in adult rat ventral hippocampus, we used light and electron microscopic immunocytochemistry with a specific polyclonal antibody against EphA4. After immunoperoxidase labeling, EphA4 immunoreactivity was found to be enriched in the neuropil layers of CA1, CA3, and dentate gyrus. In all examined layers of these regions, myelinated axons, small astrocytic leaflets, unmyelinated axons, dendritic spines, and axon terminals were immunolabeled in increasing order of frequency. Neuronal cell bodies and dendritic branches were immunonegative. EphA4‐labeled dendritic spines and axon terminals corresponded to 9–19% and 25–40% of the total number of spines and axon terminals, respectively. Most labeled spines were innervated by unlabeled terminals, but synaptic contacts between two labeled elements were seen. The vast majority of synaptic junctions made by labeled elements was asymmetrical and displayed features of excitatory synapses. Immunogold labeling of EphA4 was located mostly on the plasma membrane of axons, dendritic spines, and axon terminals, supporting its availability for surface interactions with ephrins. The dual preferential labeling of EphA4 on pre‐ or postsynaptic specializations of excitatory synapses in adult rat hippocampus is consistent with roles for this receptor in synaptic plasticity and LTP. J. Comp. Neurol. 501:691–702, 2007.

Pre‐synaptic and post‐synaptic localization of EphA4 and EphB2 in adult mouse forebrain

The ephrin receptors EphA4 and EphB2 have been implicated in synaptogenesis and long‐term potentiation in the cerebral cortex and hippocampus, where they are generally viewed as post‐synaptic receptors. To determine the precise distribution of EphA4 and EphB2 in mature brain synapses, we used subcellular fractionation and electron microscopy to examine the adult mouse forebrain/midbrain. EphA4 and EphB2 were both enriched in microsomes and synaptosomes. In synaptosomes, they were present in the membrane and the synaptic vesicle fractions. While EphA4 was tightly associated with PSD‐95‐enriched post‐synaptic density fractions, EphB2 was easily extracted with detergents. In contrast, both receptors were found in the pre‐synaptic active zone fraction. By electron microscopy, EphA4 was mainly detected in axon terminals, whereas EphB2 was more frequently detected in large dendritic shafts, in the hippocampus and cerebral cortex. However, in the ventrobasal thalamus, EphB2 was detected most frequently in axon terminals and thin dendritic shafts. The localization of EphA4 and EphB2 in multiple compartments of neurons and synaptic junctions suggests that they interact with several distinct scaffolding proteins and play diverse roles at synapses.

Toward brain imaging of serotonin 5-HT1A autoreceptor internalization.

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.