GABA(B) receptors in 5-HT transporter- and 5-HT1A receptor-knock-out mice: further evidence of a transduction pathway shared with 5-HT1A receptors

The functional properties of GABA(B) receptors were examined in the dorsal raphe nucleus (DRN) and the hippocampus of knock-out mice devoid of the 5-HT transporter (5-HTT-/-) or the 5-HT(1A) receptor (5-HT(1A)-/-). Electrophysiological recordings in brain slices showed that the GABA(B) receptor agonist baclofen caused a lower hyperpolarization and neuronal firing inhibition of DRN 5-HT cells in 5-HTT-/- versus 5-HTT+/+ mice. In addition, [(35)S]GTP-gamma-S binding induced by GABA(B) receptor stimulation in the DRN was approximately 40% less in these mutants compared with wild-type mice. In contrast, GABA(B) receptors appeared functionally intact in the hippocampus of 5-HTT-/-, and in both this area and the DRN of 5-HT(1A)-knock-out mice. The unique functional changes of DRN GABA(B) receptors closely resembled those of 5-HT(1A) autoreceptors in 5-HTT-/- mice, further supporting the idea that both receptor types are coupled to a common pool of G-proteins in serotoninergic neurons.     

Assembly of GABA(A) receptors (Review)

GABA(A) receptors are the major inhibitory transmitter receptors in the central nervous system. They are chloride ion channels that can be opened by gamma-aminobutyric acid (GABA) and are the targets of action of a variety of pharmacologically and clinically important drugs. GABA(A) receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to the formation of a large variety of distinct GABA(A) receptor subtypes in the brain. The majority of GABA(A) receptors seems to be composed of two alpha, two beta and one gamma subunit and the occurrence of a defined subunit stoichiometry and arrangement in alphabetagamma receptors strongly indicates that assembly of GABA(A) receptors proceeds via defined pathways. Based on the differential ability of subunits to interact with each other, a variety of studies have been performed to identify amino acid sequences or residues important for assembly. Such residues might be involved in direct protein-protein interactions, or in stabilizing direct contact sites in other regions of the subunit. Several homo-oligomeric or hetero-oligomeric assembly intermediates could be the starting point of GABA(A) receptor assembly but so far no unequivocal assembly mechanism has been identified. Possible mechanisms of assembly of GABA(A) receptors are discussed in the light of recent publications.     

Role of presynaptic 5-HT1A and 5-HT3 receptors in modulation of synaptic GABA transmission in dissociated rat basolateral amygdala neurons

Serotonin (5-HT) is considered to play a significant role in anxiety-related behaviors in animals through actions on the amygdaloid complex. To evaluate this role from the point of neurotransmitter release regulation, nystatin-perforated patch recording was employed on mechanically dissociated basolateral amygdala neurons containing functional synaptic boutons. GABAAergic miniature inhibitory postsynaptic currents (mIPSCs) were pharmacologically separated. In subsets of neurons, 8-OH-DPAT (1 microM), a specific 5-HT1A agonist, continuously inhibited mIPSC frequency without effects on mIPSC amplitude. By comparison, mCPBG (1 microM), a specific 5-HT3 agonist, transiently facilitated mIPSC frequency without effects on mIPSC amplitude. Together these results suggest the presynaptic existence of both 5-HT receptor subtypes. In these neurons, application of 8-OH-DPAT and its subsequent removal still suppressed mCPBG-induced responses on mIPSCs. This suppression was not caused by a reduction of presynaptic 5-HT3 receptor affinities to mCPBG and was completely eliminated by pretreatment with N-ethylmaleimide, a pertussis toxin sensitive GTP-binding protein inhibitor. In the neurons exhibiting presynaptic modulation with mCPBG but not 8-OH-DPAT, such suppression by exposure to 8-OH-DPAT was not observed. In conclusion, activation of presynaptic 5-HT1A receptors inhibited mIPSC frequency and at the same time suppressed, via a G-protein-mediated mechanism, the transient facilitation of mIPSC frequency produced by activation of presynaptic 5-HT3 receptors.     

5-HT(2C) receptors localize to dopamine and GABA neurons in the rat mesoaccumbens pathway

The serotonin 5-HT(2C) receptor (5-HT(2C)R) is localized to the limbic-corticostriatal circuit, which plays an integral role in mediating attention, motivation, cognition, and reward processes. The 5-HT(2C)R is linked to modulation of mesoaccumbens dopamine neurotransmission via an activation of γ-aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA). However, we recently demonstrated the expression of the 5-HT(2C)R within dopamine VTA neurons suggesting the possibility of a direct influence of the 5-HT(2C)R upon mesoaccumbens dopamine output. Here, we employed double-label fluorescence immunochemistry with the synthetic enzymes for dopamine (tyrosine hydroxylase; TH) and GABA (glutamic acid decarboxylase isoform 67; GAD-67) and retrograde tract tracing with FluoroGold (FG) to uncover whether dopamine and GABA VTA neurons that possess 5-HT(2C)R innervate the nucleus accumbens (NAc). The highest numbers of FG-labeled cells were detected in the middle versus rostral and caudal levels of the VTA, and included a subset of TH- and GAD-67 immunoreactive cells, of which >50% also contained 5-HT(2C)R immunoreactivity. Thus, we demonstrate for the first time that the 5-HT(2C)R colocalizes in DA and GABA VTA neurons which project to the NAc, describe in detail the distribution of NAc-projecting GABA VTA neurons, and identify the colocalization of TH and GAD-67 in the same NAc-projecting VTA neurons. These data suggest that the 5-HT(2C)R may exert direct influence upon both dopamine and GABA VTA output to the NAc. Further, the indication that a proportion of NAc-projecting VTA neurons synthesize and potentially release both dopamine and GABA adds intriguing complexity to the framework of the VTA and its postulated neuroanatomical roles.     

5-HT(2A) receptors: location and functional analysis in intestines of wild-type and 5-HT(2A) knockout mice

The distribution and function of the 5-hydroxytryptamine (5-HT(2A)) receptor were investigated in the intestines of wild-type (5-HT(2A) +/+) and knockout (5-HT(2A) -/-) mice. In 5-HT(2A) +/+ mice, rats, and guinea pigs, 5-HT(2A) receptor immunoreactivity was found on circular and longitudinal smooth muscle cells, neurons, enterocytes, and Paneth cells. Muscular 5-HT(2A) receptors were concentrated in caveolae; neuronal 5-HT(2A) receptors were found intracellularly and on the plasma membranes of nerve cell bodies and axons. Neuronal 5-HT(2A) immunoreactivity was detected as early as E14 in ganglia, intravillus nerves, and the deep muscle plexus. The 5-HT(2A) -/- colon did not express 5-HT(2A) receptors and did not contract in response to exogenous 5-HT. 5-HT(2A) -/- enterocytes were smaller, Paneth cells fewer, and muscle layers thinner (and showed degeneration) compared with those of 5-HT(2A) +/+ littermates. The 5-HT(2A) receptor may thus be required for the maintenance and/or development of enteric neuroeffectors and other enteric functions, although gastrointestinal and colonic transit times in 5-HT(2A) -/- and +/+ mice did not differ significantly.     

Serotonin 5-HT(3) receptors on mechanosensitive neurons with cardiac afferents

In rats, the mechanosensitive cardiorenal baroreflex influencing renal excretory function might be impaired by serotonin occurring in coronary arteries, e.g., in hypertension. Because the afferent limb of this reflex could be affected, we investigated the responses of nodose ganglion cells (one neuron of reflex) to osmotic, mechanical stress in presence or absence of the serotonin 5-HT(3) receptor agonist phenylbiguanide (PBG). Current-voltage relationships (from -100 to +50 mV) were obtained using cell patch recordings while the cells were exposed to control or hypoosmotic solutions to induce mechanical stress. This protocol was repeated after low doses of PBG (10 microM), angiotensin II (10 nM), or the stretch-activated channel blocker gadolinium (20 microM) were added to the extracellular medium (EM). Hypoosmotic EM induced significant changes in cellular conductance. The full-range current-voltage relationship allowed for the calculation of a mean reversal potential of -13 +/- 1.2 mV with respect to this change in cellular conductance (n = 44). This increase in conductance was impaired after addition of either PBG or gadolinium to the EM,which was statistically evaluated at a voltage of -80 mV, where influences of voltage-gated channels are not likely to interfere with the responses recorded. The serotonin 5-HT(3) receptor antagonist tropisetron (10 nM) prevented the PBG effect on conductance responses. Angiotensin II had no influence. Hence, serotonin might decrease the mechanical sensitivity of afferent cardiac nerves controlling renal sympathetic nerve activity.     

Acetaminophen-induced antinociception via central 5-HT(2A) receptors.

Acetaminophen is one of the most widely used analgesic drugs. Although the mechanism of analgesic action of acetaminophen is still not known, the involvement of the central serotonin (5-hydroxytryptamine: 5-HT) system is one possibility. In the present study, we examined the antinociceptive effect of acute and chronic intraperitoneally (i.p.) administered acetaminophen by tail flick latency measurements in the rat. A significantly increased tail flick latency was observed in acute and 15-day acetaminophen-treated rats, but not in 30-day acetaminophen-treated rats, at a dose of 400 mg/kg/day. To investigate the plasticity of receptors at postsynaptic membrane, we conducted a series of experiments by radioligand binding method on frontal cortex and brainstem membrane. The technique involved radioligand binding with [phenyl-4-³H]spiperone and ketanserin for studying 5-HT_2A receptor characteristics. A significant decrease in the maximum number of 5-HT_2A binding sites (B_max) was demonstrated in all treatment groups with acetaminophen 300 and 400 mg/kg on frontal cortex membrane, whereas the value of the dissociation equilibrium constant (K_d) remained unchanged. The down-regulation of 5-HT_2A binding sites in frontal cortex was of a lesser magnitude after 30 days of treatment and the tail flick latency was as in the control animals. These results suggest that down-regulation of 5-HT_2A receptor in response to 5-HT release is a major step in the mechanism underlying analgesia produced by this agent. On the contrary, chronic use of acetaminophen may result in 5-HT depletion, which in turn produces re-adaptation of postsynaptic 5-HT_2A receptors. These data provide further evidence for a central 5-HT-dependent antinociceptive effect of acetaminophen.     

Intracellular trafficking of GABA(A) receptors

Some of the mechanisms that control the intracellular trafficking of GABA(A) receptors have recently been described. Following the synthesis of alpha, beta, and gamma subunits in the endoplasmic reticulum, ternary receptor complexes assemble slowly and are inefficiently inserted into surface membranes of heterologous cells. While beta3, beta4, and gamma2S subunits appear to contain polypeptide sequences that alone are sufficient for surface targeting, these sequences are neither conserved nor essential for surface expression of heteromeric GABA(A) receptors formed from alpha1beta or alpha1betagamma subunits. At the neuronal surface, native GABA(A) receptor clustering and synaptic targeting require a gamma2 subunit and the participation of gephyrin, a clustering protein for glycine receptors. A linker protein, such as the GABA(A) receptor associated protein (GABARAP), may be necessary for the formation of GABA(A) receptor aggregates containing gephyrin. A substantial fraction of surface receptors are sequestered by endocytosis, another process which apparently requires a GABA(A) receptor gamma2 subunit. In heterologous cells, constitutive endocytosis seems to predominate while, in cortical neurons, internalization is evoked when receptors are occupied by GABA(A) agonists. After constitutive endocytosis, receptors are relatively stable and can be rapidly recycled to the cell surface, a process that may be regulated by protein kinase C. On the other hand, a portion of the intracellular GABA(A) receptors derived from ligand-dependent endocytosis is apparently degraded. The clustering of GABA(A) receptors at synapses and at coated pits are two mechanisms that may compete for a pool of diffusable receptors, providing a model for plasticity at inhibitory synapses.     

The dynamin-dependent, arrestin-independent internalization of 5-hydroxytryptamine 2A (5-HT2A) serotonin receptors reveals differential sorting of arrestins and 5-HT2A receptors during endocytosis

5-Hydroxytryptamine 2A (5-HT2A) receptors, a major site of action of clozapine and other atypical antipsychotic medications, are, paradoxically, internalized in vitro and in vivo by antagonists and agonists. The mechanisms responsible for this paradoxical regulation of 5-HT2A receptors are unknown. In this study, the arrestin and dynamin dependences of agonist- and antagonist-mediated internalization were investigated in live cells using green fluorescent protein (GFP)-tagged 5-HT2A receptors (SR2-GFP). Preliminary experiments indicated that GFP tagging of 5-HT2A receptors had no effect on either the binding affinities of several ligands or agonist efficacy. Likewise, both the native receptor and SR2-GFP were internalized via endosomes in vitro. Experiments with a dynamin dominant-negative mutant (dynamin K44A) demonstrated that both agonist- and antagonist-induced internalization were dynamin-dependent. By contrast, both the agonist- and antagonist-induced internalization of SR2-GFP were insensitive to three different arrestin (Arr) dominant-negative mutants (Arr-2 V53D, Arr-2-(319-418), and Arr-3-(284-409)). Interestingly, 5-HT2A receptor activation by agonists, but not antagonists, induced greater Arr-3 than Arr-2 translocation to the plasma membrane. Importantly, the agonist-induced internalization of 5-HT2A receptors was accompanied by differential sorting of Arr-2, Arr-3, and 5-HT2A receptors into distinct plasma membrane and intracellular compartments. The agonist-induced redistribution of Arr-2 and Arr-3 into intracellular vesicles and plasma membrane compartments distinct from those involved in 5-HT2A receptor internalization implies novel roles for Arr-2 and Arr-3 independent of 5-HT2A receptor internalization and desensitization.     

Binding of beta-carbolines at 5-HT(2) serotonin receptors

A series of ring-substituted (i.e., methoxy and bromo) 3,4-dihydro- and 1,2,3,4-tetrahydro-β-carbolines was examined at 5-HT_2A and 5-HT_2C serotonin receptors. Whereas most of the methoxy-substituted derivatives typically displayed affinities similar to their unsubstituted parents, certain (particularly 8-substituted) bromo derivatives displayed enhanced affinity. A binding profile was obtained for selected β-carbolines.     

Subunit specificity and interaction domain between GABA(A) receptor-associated protein (GABARAP) and GABA(A) receptors

GABARAP (GABA(A) receptor-associated protein) interacts with both microtubules and GABA(A) receptors in vitro and in vivo and is capable of modulating receptor channel kinetics. In this study, we use the intracellular loop of 15 GABA(A) receptor subunits to show that the interaction between GABARAP and GABA(A) receptor is specific for the gamma subunits. Pharmacological characterization of proteins purified by GABARAP affinity column indicates that native GABA(A) receptors interact with GABARAP. Quantitative yeast two-hybrid assays were used to identify the interaction domain in the gamma2 subunit for GABARAP binding, and to identify the interaction domain in GABARAP for GABA(A) receptor binding. A peptide corresponding to the GABARAP interaction domain in the gamma2 subunit was used to inhibit the interaction between GABARAP and the gamma2 subunit. In addition, the ability of GABARAP to promote cluster formation of recombinant receptors expressed in QT-6 fibroblasts was inhibited by a membrane-permeable form of this peptide in a time-dependent manner. The establishment of a model for GABARAP-induced clustering of GABA(A) receptors in living cells and the identification of subunit specificity and interaction domains in the interaction between GABARAP and GABA(A) receptors is a step in dissecting the function of GABARAP in GABA(A) receptor clustering and/or targeting.     

Serotonin 5-HT(2C) receptors regulate anxiety-like behavior

Central serotonin (5-hydroxytryptamine, 5-HT) systems have been implicated in the pathophysiology and treatment of anxiety disorders, which are among the world's most prevalent psychiatric conditions. Here, we report that the 5-HT(2C) receptor (5-HT(2C)R) subtype is critically involved in regulating behaviors characteristic of anxiety using male 5-HT(2C)R knockout (KO) mice. Specific neural substrates underlying the 5-HT(2C)R KO anxiolytic phenotype were investigated, and we report that 5-HT(2C)R KO mice display a selective blunting of extended amygdala corticotropin-releasing hormone neuronal activation in response to anxiety stimuli. These findings illustrate a mechanism through which 5-HT(2C)Rs affect anxiety-related behavior and provide insight into the neural circuitry mediating the complex psychological process of anxiety.     

5-HT(3) receptors mediate inflammation-induced unmasking of functional tachykinin responses in vitro.

Exogenously applied tachykinins produce no measurable electrophysiological responses in the somata of vagal afferent neurons [nodose ganglion neurons (NGNs)] isolated from naive guinea pigs. By contrast, after in vitro antigen challenge of nodose ganglia from guinea pigs immunized with chick ovalbumin, approximately 60% (53 of 89) of NGNs were depolarized an average of 13 +/- 1.2 mV by substance P (SP; 100 nM; n = 53). Receptor antagonists and enzyme inhibitors were utilized to screen a number of mast cell-derived mediators for their role in the uncovering or "unmasking" of functional tachykinin receptors after antigen challenge. Two chemically distinct 5-hydroxytryptamine-3-receptor antagonists significantly reduced the percentage of NGNs displaying depolarizing SP responses. Treatment with Y-25130 (1 or 10 microM) or tropisetron (1 microM) 15 min before and during antigen challenge reduced the percentage of SP-responsive neurons to approximately 20 and approximately 15% respectively. These results suggest that activation of 5-hydroxytryptamine-3 receptors plays an integral role in the unmasking of functional tachykinin receptors after specific antigen challenge of nodose ganglia. The mediator(s) underlying tachykinin-receptor unmasking in the remainder of the NGNs has yet to be characterized. However, it does not appear to be histamine, prostanoids, or peptidoleukotrienes.     

Sleep is differently modulated by basal forebrain GABA(A) and GABA(B) receptors

There is evidence that GABA plays a major role in sleep regulation. GABA(A) receptor agonists and different compounds interacting with the GABA(A) receptor complex, such as barbiturates and benzodiazepines, can interfere with the sleep/wake cycle. On the other hand, there is very little information about the possible role of GABA(B) receptors in sleep modulation. The nucleus basalis of Meynert (NBM), a cholinergic area in the basal forebrain, plays a pivotal role in the modulation of sleep and wakefulness, and both GABA(A) and GABA(B) receptors have been described within the NBM. This study used unilateral infusions in the NBM to determine the effects of 3-hydroxy-5-aminomethylisoxazole hydrobromide (muscimol hydrobromide, a GABA(A) receptor subtype agonist) and beta-(aminomethyl)-4-chlorobenzenepropanoic acid (baclofen, a GABA(B) receptor subtype agonist) on sleep parameters in freely moving rats by means of polygraphic recordings. Muscimol (0.5 nmol) and baclofen (0.7 nmol) induced an increase in slow-wave sleep and an inhibition of wakefulness. Muscimol, but not baclofen, also caused a decrease in desynchronized sleep parameters. The results reported here indicate that 1) the NBM activation of both GABA(A) and GABA(B) receptors influences the sleep/wake cycle, and 2) GABA(A) but not GABA(B) receptors are important for desynchronized sleep modulation, suggesting that the two GABAergic receptors play different roles in sleep modulation.     

Hypothalamic 5-HT functional regulation through 5-HT1A and 5-HT2C receptors during pancreatic regeneration

5-HT receptors are predominantly located in the brain and are involved in pancreatic function and cell proliferation through sympathetic nervous system. The objective of this study was to investigate the role of hypothalamic 5-HT, 5-HT1A and 5-HT2C receptor binding and gene expression in rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content, 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content was quantified by HPLC. 5-HT1A receptor assay was done by using specific agonist [3H]8-OH DPAT. 5-HT2C receptor assay was done by using specific antagonist [3H]mesulergine. The expression of 5-HT1A and 5-HT2C receptor gene was analyzed by RT-PCR. 5-HT content was higher in the hypothalamus of 72 h pancreatectomised rats. 5-HT1A and 5-HT2C receptors were down-regulated in the hypothalamus. RT-PCR analysis revealed decreased 5-HT1A and 5-HT2C receptor mRNA expression. The 5-HT1A and 5-HT2C receptors gene expression in the 7 days pancreatectomised rats reversed to near sham level. This study is the first to identify 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus during pancreatic regeneration in rats. Our results suggest the hypothalamic serotonergic receptor functional regulation during pancreatic regeneration.     

The alteration of 5-HT(2A) and 5-HT(2C) receptors is involved in neuronal apoptosis of goldfish cerebellum following traumatic experience

5-HT receptor changes remain controversial in posttraumatic stress disorder (PTSD) models. This study looks at the relationship between traumatic injuries and the alterations in 5-HT(2A) and 5-HT(2C) receptors in the goldfish brain. The effect of treatment with doxepin and fluoxetine, known to be selective serotonin reuptake inhibitor (SSRI) antidepressants, on 5-HT receptor expression in goldfish with fin ablation was also investigated. We demonstrated that fin ablation induced anxiety-like behavioural alterations and significant up-regulation of c-fos expression in goldfish cerebellum. The behavioural alterations correlated well with an increased expression of 5-HT(2A) receptors in the cerebellum of the fish with traumatic injury. An increase in the number of apoptotic cells and a higher caspase-8 protein level was present in the brains of goldfish with fin ablation compared to the control. Our findings suggest that neuronal apoptosis occured in the cerebellum as a result of fin ablation and may be related to the alterations of 5-HT(2A) and 5-HT(2C) levels and that the beneficial clinical effects of doxepin/fluoxetine treatment are due to the down-regulation of 5-HT(2A) and up-regulation of 5-HT(2C) receptors in the brain.