Pre- and postsynaptic localization of the 5-HT7 receptor in rat dorsal spinal cord: immunocytochemical evidence

Several lines of evidence indicate that 5-HT7 receptors are involved in pain control at the level of the spinal cord, although their mechanism of action is poorly understood. To provide a morphological basis for understanding the action of 5-HT on this receptor, we performed an immunocytochemical study of 5-HT7 receptor distribution at the lumbar level. 5-HT7 immunolabelling is localized mainly in the two superficial laminae of the dorsal horn and in small and medium-sized dorsal root ganglion cells, which is consistent with a predominant role in nociception. In addition, moderate labelling is found in the lumbar dorsolateral nucleus (Onuf's nucleus), suggesting involvement in the control of pelvic floor muscles. Electron microscopic examination of the dorsal horn revealed three main localizations: 1) a postsynaptic localization on peptidergic cell bodies in laminae I-III and in numerous dendrites; 2) a presynaptic localization on unmyelinated and thin myelinated peptidergic fibers (two types of axon terminals are observed, large ones, presumably of primary afferent origin, and smaller ones partially from intrinsic cells; this presynaptic labelling represents 60% and 22% of total labelling in laminae I and II, respectively); and 3) 16.9% of labelling in lamina I and 19.8% in lamina II are observed in astrocytes. Labeled astrocytes are either intermingled with neuronal elements or make astrocytic "feet" on blood vessels. In dendrites, the labelling is localized on synaptic differentiations, suggesting that 5-HT may act synaptically on the 5-HT7 receptor. This localization is compared with other 5-HT receptor localizations, and their physiological consequences are discussed.     

Quisqualate-sensitive, chloride-dependent transport of glutamate into rat brain synaptosomes

A chloride-dependent transport process for glutamate has been identified in partially purified rat brain synaptosomes. This process shares many characteristics with the chloride-dependent sequestration process for glutamate in brain sonicates, which was previously thought to represent a quisqualate receptor, such as sensitivity to specific inhibitors and regulation by anions. Increasing the concentrations of chloride led to an increase in the apparent Vmax without affecting the KT. Synaptosomes preincubated with [3H]-L-glutamate exhibit an efflux of the radiolabel, which was stimulated by a substrate for the carrier in the incubating medium, indicating the bidirectional nature of the transport. The chloride-dependent transfer process is restricted to the brain, and regional and developmental profiles clearly distinguish it from the sodium-dependent high-affinity uptake process for glutamate. Nevertheless, the effects of excitotoxic lesions strongly suggest a neuronal localization of the chloride-dependent transport.     

The distribution of [3H]kainic acid binding sites in rat CNS as determined by autoradiography

The distribution of [3H]kainic acid (KA) binding sites in the rat CNS was determined by in vitro autoradiography. KA sites are distributed throughout the CNS gray matter in an anatomically specific pattern with telencephalic structures and the cerebellum accounting for the majority of the binding. These results, together with our previous finding that KA sites are greatly enriched at the synapse, suggest that KA binding sites are associated with select terminal fields, and hence may be involved in neurotransmission in certain CNS pathways.     

The distribution of [H]kainic acid binding sites in rat CNS as determined by autoradiography

The distribution of [³H]kainic acid (KA) binding sites in the rat CNS was determined by in vitro autoradiography. KA sites are distributed throughout the CNS gray matter in an anatomically specific pattern with telencephalic structures and the cerebellum accounting for the majority of the binding. These results, together with our previous finding that KA sites are greatly enriched at the synapse, suggest that KA binding sites are associated with select terminal fields, and hence may be involved in neurotransmission in certain CNS pathways.     

Autoradiographic distribution of serotonin transporters and receptor subtypes in human brain

Several neurochemical in vitro and in vivo imaging studies have been aimed at characterizing the localization of serotonin receptors and transporters in the human brain. In this study, a detailed comparison of the distribution of a number of 5-HT receptor subtypes and the 5-HT transporter was carried out in vitro using human postmortem brain tissue. Anatomically adjacent whole hemisphere sections were incubated with specific radioligands for the 5-HT(1A), 5-HT(1B), 5-HT(2A), 5-HT(4) receptors and the 5-HT transporter. The autoradiograms revealed different laminar and regional distribution patterns in the isocortex, where 5-HT(1A) and 5-HT(4) receptor binding showed highest densities in superficial layers and 5-HT(2A) receptor binding was most abundant in middle layers. In cortical regions, 5-HT transporters were concentrated to several limbic lobe structures (posterior uncus, entorhinal, cingulate, insular and temporal polar regions). 5-HT(1A) receptor densities were also high in limbic cortical regions (hippocampus, posterior entorhinal cortex, and subcallosal area) compared to the isocortex. Subregionally different distribution patterns were observed in the basal ganglia with a trend toward higher levels in ventral striatal (5-HT(1B) receptors) and pallidal (5-HT transporters and 5-HT(1B) receptors) regions. The localization in regions belonging to limbic cortico-striato-pallido-thalamic circuits is in line with the documented role of 5-HT in modulation of mood and emotion, and the suggested involvement of this system in pathophysiology of various psychiatric disorders. The qualitative and quantitative information reported in this study might provide important complements to in vivo neuroimaging studies of the 5-HT system.     

Quantitative autoradiography of binding sites for [H]AMPA, a structural analogue of glutamic acid

Binding sites for the potent glutamate agonist [³H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were localized in rat brain frozen sections by quantitative autoradiography. Highest levels of binding were seen in stratum radiatum and stratum oriens of the CA1 hippocampal subfield and in the dorsal subiculum. Substantially less but still high amounts of [³H]AMPA binding occurred in other hippocampal subfields and in rostral forebrain structures. The heterogeneous nature of [³H]AMPA binding is discussed in relation to [³H]glutamate binding visualized by similar methods. From these data it is suggested that [³H]AMPA may label a particular subclass of the glutamate receptor population which exhibits a high affinity for quisqualic acid.     

神经节苷脂(GM-1)对体外培养SH-SY5Y细胞兴奋性氨基酸毒性损伤的作用

目的 探讨神经节苷脂(GM-1)对体外培养SH-SY5Y细胞兴奋性氨基酸毒性损伤的保护作用。方法 采用细胞培养法，以神经母细胞瘤SH-SY5Y细胞系为材料，制备兴奋性氨基酸Glu毒性损伤的离体细胞损伤模型。通过细胞形态学观察、MTT法测定细胞存活率观察不同浓度及不同时间GM-1对上述损伤细胞的保护及治疗作用。结果 GM-1可增强SH-SY5Y细胞的存活，抑制兴奋性氨基酸对细胞的损伤。与损伤组相比GM-1治疗组均好于损伤组，且GM-1高浓度组好于低浓度组。GM-1对损伤SH-SY5Y细胞MTT代谢率的影响显示，相同浓度GM-1作用不同时间相比较12h优于6h，24h与12h无明显差异。结论 GM-1可促进神经细胞生存，对神经细胞具有明显保护作用。     

Excitatory amino acids and synaptic transmission in embryonic rat brainstem motoneurons in organotypic culture

We used brainstem motoneurons recorded in organotypic slice co-cultures maintained for more than 18 days in vitro, together with multibarrel ionophoretic applications of glutamate receptor agonists and bath applications of specific blocking agents, to study the responses of rat brainstem motoneurons to glutamate receptor activation, and the contribution of these receptors to synaptic transmission. Differentiated brainstem motoneurons in vitro are depolarized by glutamate, N-methyl-d-aspartate (NMDA) and dl-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) iontophoresis, and express NMDA, AMPA and also specific kainate receptors, as evidenced by (+/-)2-amino-5-phosphonovaleric acid (APV)- and (-)1-(4-aminophenyl)-3-methyl-carbamoyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzo-diazepine [GYKI 53784 (LY303070)]-resistant depolarizations. Electrical stimulations applied to the dorsal part of the explant trigger excitatory synaptic potentials with latencies distributed in three regularly spaced groups. Excitatory postsynaptic potentials (EPSPs) in the earliest group have a similar latency and time course and correspond to monosynaptic activation. EPSPs in later groups have more scattered latencies and time courses and correspond to polysynaptic activation. Monosynaptic EPSPs are insensitive to the specific NMDA blocker APV, and are completely and reversibly suppressed by the non-competitive AMPA receptor antagonist GYKI 53784 (LY303070). Detailed analysis of the spontaneous excitatory synaptic activity shows that APV decreases the frequency of spontaneous EPSPs without modifying their shape or amplitude. We conclude that excitatory synapses on brainstem motoneurons in vitro are mainly activated through AMPA receptors (AMPA-Rs). NMDA receptors (NMDA-Rs) are present in the membrane, but are located either at extrasynaptic sites or silent synapses, and are not directly involved in synaptic transmission on motoneurons. On the contrary, NMDA receptors contribute to synaptic transmission within the premotor interneuronal network.     

In vivo kinetics of [F-18]MEFWAY: a comparison with [C-11]WAY100635 and [F-18]MPPF in the nonhuman primate

[F‐18]Mefway was developed to provide an F‐18 labeled positron emission tomography (PET) neuroligand with high affinity for the serotonin 5‐HT_1A receptor to improve the in vivo assessment of the 5‐HT_1A system. The goal of this work was to compare the in vivo kinetics of [F‐18]mefway, [F‐18]MPPF, and [C‐11]WAY100635 in the rhesus monkey. Methods: Each of four monkeys were given bolus injections of [F‐18]mefway, [C‐11]WAY100635, and [F‐18]MPPF and scans were acquired with a microPET P4 scanner. Arterial blood was sampled to assay parent compound throughout the time course of the PET experiment. Time activity curves were extracted in the high 5‐HT_1A binding areas of the anterior cingulate cortex (ACG), mesial temporal cortex, raphe nuclei, and insula cortex. Time activity curves were also extracted in the cerebellum, which was used as a reference region. The in vivo kinetics of the radiotracers were compared based on the nondisplaceable distribution volume (V_ND) and binding potential (BP_ND). Results: At 30 min, the fraction of radioactivity in the plasma due to parent compound was 19%, 28%, and 29% and cleared from the arterial plasma at rates of 0.0031, 0.0078, and 0.0069 (min^?1) ([F‐18]mefway, [F‐18]MPPF, [C‐11]WAY100635). The BP_ND in the brain regions were mesial temporal cortex: 7.4 ± 0.6, 3.1 ± 0.4, 7.0 ± 1.2, ACG: 7.2 ± 1.2, 2.1 ± 0.2, 7.9 ± 1.2; raphe nuclei: 3.7 ± 0.6, 1.3 ± 0.3, 3.3 ± 0.7; and insula cortex: 4.2 ± 0.6, 1.2 ± 0.1, 4.7 ± 1.0 for [F‐18]mefway, [F‐18]MPPF, and [C‐11]WAY100635 respectively. Conclusions: In the rhesus monkey, [F‐18]mefway has similar in vivo kinetics to [C‐11]WAY100635 and yields greater than 2‐fold higher BP_ND than [F‐18]MPPF. These properties make [F‐18]mefway a promising radiotracer for 5‐HT_1A assay, providing higher counting statistics and a greater dynamic range in BP_ND. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Distribution of [H]AMPA binding sites in rat brain as determined by quantitative autoradiography

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) is a potent neuroexcitatory compound which acts at the quisqualate class of excitatory amino acid receptors. In this study we describe the pharmacological characteristics and anatomical distribution of [³H]AMPA binding sites in rat brain using quantitative autoradiography. These binding sites exhibit the appropriate pharmacological characteristics and are found in high concentrations in the hippocampus, cerebral cortex (especially layers I–III), induseum griseum, and dorsal lateral septum. Intermediate concentrations are found in the corpus striatum and deeper layers of cerebral cortex. Lower concentrations are found in the diencephalon, midbrain and brainstem. These results demonstrate that [³H]AMPA binding sites are found throughout the CNS and suggest brain regions which may use quisqualate receptors as glutamate neurotransmitter receptors.     

Updated overview of the putative role of the serotoninergic system in obsessive-compulsive disorder

The pathophysiology of obsessive-compulsive disorder (OCD) remains unknown. However, increasing attention has been paid to the putative role of the serotoninergic system, the strongest evidence being based on the widely demonstrated efficacy of serotonin (5HT) reuptake inhibitor antidepressants in the treatment of OCD. The therapeutic effects are correlated with changes in peripheral parameters of 5HT function, which have been found to be altered in OCD, suggesting the possibility of reduced 5HT reuptake capacity. This could reflect a compensatory mechanism presumably due to decreased availability of extracellular 5HT, as evidenced by data derived from direct assessment of central 5HT neurotransmission. The development of new neurochemical probes that explore the sensitivity of various 5HT receptor subtypes has provided precious information. m-Chlorophenylpyperazine (m-CPP), an agonist to 5HT1A, 5HT1D, and 5HT2C receptors, and which also blocks 5HT3 receptors, exacerbates OC symptoms. In contrast, neither MK-212 (6-chloro-2-[1-piperazinyl]-pyrazine), a 5HT1A and 5HT2C receptor agonist, nor ipsapirone or buspirone, which acts as an agonist to 5HT1A receptors, have any effect on OC symptom severity. This suggests the potential implication of the 5HT1D receptor, as shown by the aggravation of OC manifestations in response to sumatriptan, a selective 5HT1D receptor agonist. The 5HT3 plays no specific role, given the lack of influence of the 5HT3 antagonist ondansetron, on OC symptom intensity. Further studies are required to elucidate the pharmacological molecular determinants of the putative 5HT1D receptor dysfunction.     

Administration of clozapine to a mother rat potentiates pup ultrasonic vocalization in response to separation and re-separation: contrast with haloperidol

The present study examined how haloperidol (typical) and clozapine (atypical) treatment to mother rats affected their pups’ ultrasonic vocalization (USV) response to maternal separation and re-separation (termed “maternal potentiation”). Clozapine (10 mg/kg, sc) but not haloperidol (0.2 mg/kg, sc) significantly enhanced the maternal potentiation of 40 kHz USVs in pups that were briefly reunited with their dams. This novel paradigm provides an indirect way of assessing the impact of antipsychotic treatment on the quality of maternal care. It may also be useful in examining the impact of antipsychotic treatment on social bonding between infants and mothers.     

Serotonin (5‐HT) regulates neurite outgrowth through 5‐HT1A and 5‐HT7 receptors in cultured hippocampal neurons

Serotonin (5‐HT) production and expression of 5‐HT receptors (5‐HTRs) occur early during prenatal development. Recent evidence suggests that, in addition to its classical role as a neurotransmitter, 5‐HT regulates neuronal connectivity during mammalian development by modulating cell migration and neuronal cytoarchitecture. Given the variety of 5‐HTRs, researchers have had difficulty clarifying the specific role of each receptor subtype in brain development. Signalling mediated by the G‐protein‐coupled 5‐HT_1AR and 5‐HT₇R, however, has been associated with neuronal plasticity. Thus, we hypothesized that 5‐HT promotes neurite outgrowth through 5‐HT_1AR and 5‐HT₇R. The involvement of 5‐HT_1AR and 5‐HT₇R in the morphology of rat hippocampal neurons was evaluated by treating primary cultures at 2 days in vitro with 5‐HT and specific antagonists for 5‐HT_1AR and 5‐HT₇R (WAY‐100635 and SB269970, respectively). The stimulation of hippocampal neurons with 100 nM 5‐HT for 24 hr produced no effect on either the number or the length of primary neurites. Nonetheless, after 5HT₇R was blocked, the addition of 5‐HT increased the number of primary neurites, suggesting that 5HT₇R could inhibit neuritogenesis. In contrast, 5‐HT induced secondary neurite outgrowth, an effect inhibited by 1 μM WAY‐100635 or SB269970. These results suggest that both serotonergic receptors participate in secondary neurite outgrowth. We conclude that 5‐HT_1AR and 5‐HT₇R regulate neuronal morphology in primary hippocampal cultures by promoting secondary neurite outgrowth. © 2014 Wiley Periodicals, Inc.     

5-HT4 receptors in the hippocampus modulate rat locomotor activity

In the present study, the ability of 5-hydroxytryptamine-4 (5-HT4) receptors in the hippocampus to enhance locomotor activity in rats was investigated by local infusion via microdialysis probes. The local infusion of 5-HT bilaterally into the striatum did not alter rat motor activity. The local infusion of 1.0 mM 5-HT into the bilateral hippocampus, but not lower doses, significantly increased motor activity as compared with the baseline values or the control rats. During the day hours (0700-1900, light on), the local infusion of either 5-HT4 agonist, 5-MeOT (100 microM) or mosapride (10 microM), but not in their lower concentrations, into the bilateral hippocampus significantly increased motor activity as compared with the baseline values or the control rats. Almost all increased motor activity was normal forward locomotion. This 5-MeOT-induced hyperlocomotion was completely reversed by the combined infusion of a 5-HT4 antagonist, either GR125487D (100 microM), SB204070 (100 microM) or RS23597-190 (100 microM). During the night hours (1900-0700, light off), the local infusion of either SB204070 (100 microM) or RS23597-190 (100 microM), but not in their lower concentrations, into the bilateral hippocampus significantly decreased rat motor activity and inhibited rat nocturnal hyperactivity. These hypoactivities during the night hours induced by 5-HT4 antagonist were reversed by the combined infusion of a 5-HT4 agonist, 5-MeOT (100 microM). The present study demonstrates that the serotonergic neurons projecting to the hippocampus, but not to the striatum, modulate rat locomotor activity by stimulating 5-HT4 receptors in the hippocampus.     

BDNF Val66met and 5‐HTTLPR polymorphisms predict a human in vivo marker for brain serotonin levels

Brain‐derived neurotrophic factor (BDNF) has been implicated in multiple aspects of brain function including regulation of serotonin signaling. The BDNF val66met polymorphism (rs6265) has been linked to aspects of serotonin signaling in humans but its effects are not well understood. To address this, we evaluated whether BDNF val66met was predictive of a putative marker of brain serotonin levels, serotonin 4 receptor (5‐HT₄) binding assessed with [¹¹C]SB207145 positron emission tomography, which has also been associated with the serotonin‐transporter‐linked polymorphic region (5‐HTTLPR) polymorphism. We applied a linear latent variable model (LVM) using regional 5‐HT₄ binding values (neocortex, amygdala, caudate, hippocampus, and putamen) from 68 healthy humans, allowing us to explicitly model brain‐wide and region‐specific genotype effects on 5‐HT₄ binding. Our data supported an LVM wherein BDNF val66met significantly predicted a LV reflecting [¹¹C]SB207145 binding across regions (P = 0.005). BDNF val66met met‐carriers showed 2–9% higher binding relative to val/val homozygotes. In contrast, 5‐HTTLPR did not predict the LV but S‐carriers showed 7% lower neocortical binding relative to LL homozygotes (P = 7.3 × 10^?6). We observed no evidence for genetic interaction. Our findings indicate that BDNF val66met significantly predicts a common regulator of brain [¹¹C]SB207145 binding, which we hypothesize reflects brain serotonin levels. In contrast, our data indicate that 5‐HTTLPR specifically affects 5‐HT₄ binding in the neocortex. These findings implicate serotonin signaling as an important molecular mediator underlying the effects of BDNF val66met and 5‐HTTLPR on behavior and related risk for neuropsychiatric illness in humans. Hum Brain Mapp, 36:313–323, 2015. © 2014 Wiley Periodicals, Inc .     

Distribution and neurochemical characterization of neurons expressing GIRK channels in the rat brain

G-protein inwardly rectifying potassium (GIRK) channels mediate the synaptic actions of numerous neurotransmitters in the mammalian brain and play an important role in the regulation of neuronal excitability in most brain regions through activation of various G-protein-coupled receptors such as the serotonin 5-HT(1A) receptor. In this report we describe the localization of GIRK1, GIRK2, and GIRK3 subunits and 5-HT(1A) receptor in the rat brain, as assessed by immunohistochemistry and in situ hybridization. We also analyze the co-expression of GIRK subunits with the 5-HT(1A) receptor and cell markers of glutamatergic, gamma-aminobutyric acid (GABA)ergic, cholinergic, and serotonergic neurons in different brain areas by double-label in situ hybridization. The three GIRK subunits are widely distributed throughout the brain, with an overlapping expression in cerebral cortex, hippocampus, paraventricular nucleus, supraoptic nucleus, thalamic nuclei, pontine nuclei, and granular layer of the cerebellum. Double-labeling experiments show that GIRK subunits are present in most of the 5-HT(1A) receptor-expressing cells in hippocampus, cerebral cortex, septum, and dorsal raphe nucleus. Similarly, GIRK mRNA subunits are found in glutamatergic and GABAergic neurons in hippocampus, cerebral cortex, and thalamus, in cholinergic cells in the nucleus of vertical limb of the diagonal band, and in serotonergic cells in the dorsal raphe nucleus. These results provide a deeper knowledge of the distribution of GIRK channels in different cell subtypes in the rat brain and might help to elucidate their physiological roles and to evaluate their potential involvement in human diseases.     

Synaptic coexistence of AMPA and NMDA receptors in the rat hippocampus: A postembedding immunogold study

Synaptic distributions of N-methyl-d -aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptor subunits, NMDAR1 and GluR2, respectively, were examined by electron microscopy with the high spatial resolution of postembedding immunogold localization. We provide direct evidence for colocalization at individual axodendritic asymmetric synapses within the CA1 subfield of rat hippocampus. AMPA/ NMDA receptor colocalization was found both in γ-aminobutyric acid (GABA)ergic dendrites and non-GABAergic dendritic shafts, as well as dendritic spines. Some asymmetric synapses were found to contain only NMDAR1 or GluR2; however, most immunopositive synapses contained both subunits. Many NMDAR1 and/ or GluR2 immunopositive profiles received GABAergic innervation at an adjacent synapse, providing a substrate for GABAergic modulation of both GluR classes. These data suggest that excitatory neuronal transmission in CA1 neurons may generally involve activation of both NMDA and AMPA receptor subunits at a single synapse, however, they also offer ultrastructural evidence for NMDAR1-only synapses that might represent silent synapses. J. Neurosci. Res. 54:444–449, 1998. © 1998 Wiley-Liss, Inc.