Presynaptic mu-opioid receptors regulate a late step of the secretory process in rat ventral tegmental area GABAergic neurons

γ-Aminobutyric acid (GABA)-containing interneurons of the ventral tegmental area (VTA) regulate the activity of dopaminergic neurons. These GABAergic interneurons are known to be innervated by synaptic terminals containing enkephalin, an endogenous ligand of μ-opioid receptors. Bath application of μ-opioid receptor agonists inhibits the activity of VTA GABAergic neurons but the mechanism whereby μ-opioid receptors regulate synaptic GABA release from these neurons has not been directly identified. Using cultured VTA neurons we have confirmed that μ-opioid receptor agonists inhibit synaptic GABA release. DAMGO, a selective μ-opioid receptor agonist, had four distinct effects on GABAergic IPSCs: (1) it inhibited the frequency and amplitude of spontaneous IPSCs (sIPSCs), (2) it reduced the amplitude of IPSCs evoked by single action potentials, (3) it inhibited the frequency, but not the amplitude of miniature IPSCs (mIPSCs), and (4) DAMGO inhibited mIPSCs evoked by ionomycin, a Ca²⁺ ionophore. The inhibition of action potential-evoked IPSCs and of spontaneous and ionomycin-evoked mIPSCs by DAMGO was prevented by the K⁺ channel blocker, 4-aminopyridine (4-AP). In conclusion, our work shows that one of the mechanisms through which μ-opioid receptors inhibit GABA release by VTA neurons is through inhibition of the secretory process at the nerve terminal level. In addition, considering that ionomycin stimulates exocytosis through a mechanism that should be insensitive to membrane polarization, our experiments with 4-AP suggest that K⁺ channels are implicated in the inhibition of the efficacy of the secretory process by μ-opioid receptors.     

Presynaptic μ-opioid receptors regulate a late step of the secretory process in rat ventral tegmental area GABAergic neurons

γ-Aminobutyric acid (GABA)-containing interneurons of the ventral tegmental area (VTA) regulate the activity of dopaminergic neurons. These GABAergic interneurons are known to be innervated by synaptic terminals containing enkephalin, an endogenous ligand of μ-opioid receptors. Bath application of μ-opioid receptor agonists inhibits the activity of VTA GABAergic neurons but the mechanism whereby μ-opioid receptors regulate synaptic GABA release from these neurons has not been directly identified. Using cultured VTA neurons we have confirmed that μ-opioid receptor agonists inhibit synaptic GABA release. DAMGO, a selective μ-opioid receptor agonist, had four distinct effects on GABAergic IPSCs: (1) it inhibited the frequency and amplitude of spontaneous IPSCs (sIPSCs), (2) it reduced the amplitude of IPSCs evoked by single action potentials, (3) it inhibited the frequency, but not the amplitude of miniature IPSCs (mIPSCs), and (4) DAMGO inhibited mIPSCs evoked by ionomycin, a Ca²⁺ ionophore. The inhibition of action potential-evoked IPSCs and of spontaneous and ionomycin-evoked mIPSCs by DAMGO was prevented by the K⁺ channel blocker, 4-aminopyridine (4-AP). In conclusion, our work shows that one of the mechanisms through which μ-opioid receptors inhibit GABA release by VTA neurons is through inhibition of the secretory process at the nerve terminal level. In addition, considering that ionomycin stimulates exocytosis through a mechanism that should be insensitive to membrane polarization, our experiments with 4-AP suggest that K⁺ channels are implicated in the inhibition of the efficacy of the secretory process by μ-opioid receptors.     

Presynaptic modulation of the release of GABA by GABAA receptors in pars compacta and by GABAB receptors in pars reticulata of the rat substantia nigra

The effect of GABA agonists and antagonists on K⁺-stimulated [³H]GABA release was studied to assess how presynaptic GABA receptors modulate GABA release. The release was affected in a quite different manner in the pars compacta and in the pars reticulata. Muscimol markedly inhibited the release from the pars compacta but had no effect on the release from the pars reticulata. Baclofen inhibited the release from the pars reticulata without affecting the release from the pars compacta. Bicuculline itself facilitated the release from the pars compacta but inhibited the release from the pars reticulata. Picrotoxin facilitated the release from the pars compacta and had no effect in the pars reticulata. The results suggest that the release of GABA from GABAergic terminals in the substantia nigra of the rat brain is modulated by GABA_A autoreceptors in the pars compacta and by GABA_B receptors in the pars reticulata.     

Nicotinic and muscarinic cholinergic receptors coexist on GABAergic nerve endings in the mouse striatum and interact in modulating GABA release

Muscarinic cholinergic receptors (mAChRs) and nicotinic cholinergic receptors (nAChRs) regulating GABA release from striatal nerve endings were studied by monitoring release of previously accumulated [³H]GABA or endogenous GABA from superfused mouse striatal synaptosomes. Oxotremorine inhibited the release of [³H]GABA elicited by depolarization with 4-aminopyridine (4-AP), an effect antagonized by atropine. Agonists at nAChRs, including the α₄β₂^∗ subunit-selective RJR2403, provoked the release of [³H]GABA as well as of the endogenous transmitter; these effects also were prevented by oxotremorine and pilocarpine suggesting coexpression of functional mAChRs and α₄β₂^∗ nAChRs on GABAergic nerve endings. The inhibitory effects of oxotremorine on the release of [³H]GABA evoked by 4-AP or by RJR2403 were: (i) prevented by the M₂/M₄ mAChR antagonist himbacine; (ii) insensitive to the M2 antagonist AFDX116; (iii) blocked by the selective M₄ mAChR antagonists MT3, thus indicating the involvement of receptors of the M₄ subtype. In conclusion, in the corpus striatum, acetylcholine released from cholinergic interneurons can activate α₄β₂^∗ nAChRs mediating release of GABA; this evoked release can be negatively modulated by M₄ mAChRs coexpressed on the same GABAergic terminals.     

The BTB/kelch protein, KRIP6, modulates the interaction of PICK1 with GluR6 kainate receptors

Neuronal proteins of the BTB/kelch and PDZ domain families interact with different regions of the cytoplasmic C-terminal domain of the GluR6 kainate receptor subunit. The BTB/kelch protein KRIP6 binds within a 58 amino acid segment of GluR6 proximal to the plasma membrane. In contrast, PDZ domain proteins, such as PICK1 and PSD95, interact with the last 4 residues of the GluR6 C-terminus. KRIP6 reduces peak currents mediated by recombinant GluR6 receptors and by native kainate receptors in neurons, whereas PICK1 stabilizes kainate receptors at synapses. Thus, protein-protein interactions at the C-terminal domain of GluR6 are important for regulating kainate receptor physiology. Here, we show by co-clustering and co-immunoprecipitation that KRIP6 interacts with PICK1 in heterologous cells. In addition, we demonstrate a novel modulation of GluR6 receptors by PICK1 resulting in increased peak current and relative desensitization of GluR6-mediated currents, phenotypes opposite to those produced by KRIP6. Importantly, these effects cancel out when KRIP6 and PICK1 are co-expressed together with GluR6. KRIP6 and PICK1 strongly co-cluster and co-immunoprecipitate regardless of the presence of GluR6. Immunofluorescence analysis reveals that GluR6 can either join the KRIP6-PICK1 clusters or remain separate; however, co-expression of KRIP6 reduces the fraction of PICK1 that co-immunoprecipitates with GluR6. Taken together, these results indicate that, in addition to a previously demonstrated direct interaction with the GluR6 C-terminal domain, KRIP6 regulates kainate receptors by inhibiting PICK1 modulation via competition or a mutual blocking effect.     

Complex interaction between quisqualate and kainate receptors as revealed by measurement of GABA release from striatal neurons in primary culture

The effects of non-NMDA receptor agonists were tested on endogenous GABA and [3H]GABA release from highly purified striatal neurons differentiated in primary culture. Kainate (KA), glutamate (Glu) and quisqualate (QA) stimulated [3H]GABA release with EC50S = 85 +/- 20 (n = 6), 6.21 +/- 1.42 (n = 3) and 0.135 +/- 0.035 (n = 3) microM, respectively. KA was the most potent (in term of efficacy) agonist (maximal response at 10 mM: 935 +/- 51% (n = 6) increase over basal release) followed by Glu (at 100 microM: 404 +/- 34% (n = 5) increase) and QA (at 10 microM: 91 +/- 6% (n = 6) increase). Phencyclidine (PCP), which was without effect on QA- and KA-evoked GABA release, inhibited the Glu response by about 50%. QA totally inhibited KA (50 microM)-evoked GABA release with an IC50 = 0.39 +/- 0.11 (n = 4) in a competitive manner (Ki = 0.39 +/- 0.07 microM (n = 3]. Competitive inhibition of the KA response was also observed with the other agonists of the quisqualate receptor, Glu and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), suggesting that Glu, QA and AMPA act as partial agonists at the KA receptor. gamma-D-Glutamylaminomethylsulfonic acid (GAMS) also inhibited (IC50 = 2.1 mM) the KA response competitively. However the inhibition by GAMS and QA was not additive. The response to QA was rapidly inactivated (no response after 3 min stimulation) in contrast to the KA-evoked GABA release which remained maximal for at least 3 min. When neurons were first exposed to concanavalin A (con A), a lectin known to inhibit Glu receptor desensitisation on insect muscles, the QA response remained maximal for at least 6 min. Con A greatly enhanced the maximal responses to QA and AMPA and decreased their apparent affinities. The KA-evoked GABA release (but not the veratridine and NMDA effects) was also augmented (no change in the EC50 value) by con A. It is proposed that QA, AMPA and KA act at the same receptor-channel complex (termed G2 receptor) which is desensitised more rapidly when stimulated by QA or AMPA than when stimulated by KA.     

Presynaptic opioid receptors modulating acetylcholine release in the hippocampus of the rabbit

Summary Inhibition of cardiae adenylate cyclase by adenosine receptor agonists was reinvestigated in a more homogeneous sarcolemmal vesicular preparation than used in a previous study. Microsomal particles obtained by differential centrifugation were further fractionated on a shallow density gradient of Percoll. Two populations of plasma membrane vesicles were partially resolved. Identical peaks were identified for adenylate cyclase activity and [³H]ouabain binding, whereas 5-nucleotidase activity and -adrenoceptor binding displayed an additonal peak at higher density, where angiotensin converting enzyme, a marker for endothelial plasma membranes, was at maximal activity. Significant inhibition by N⁶-cyclohexyladenosine (CHA), as measured in each fractionation step following homogenization, was observed only at the activity peak of adenylate cyclase. Moreover, analysis of the degree and rank order of potency of several adenosine analogs was indicative for interaction with A₁-adenosine receptors. Accordingly, the peak in adenosine receptor binding, using (-)[¹²⁵I]iodo-N⁶-hydroxyphenyl-isopropyladenosine as the radioligand, coincided with CHA-inhibitable adenylate cyclase activity. By contrast, adenylate cyclase was slightly stimulated by CHA in the higher density range, an action suggested to be mediated via A₂-adenosine receptors, which recently have been demonstrated to exist on guinea-pig coronary endothelium. It is concluded that the full extent of adenosine receptor-mediated adenylate cyclase inhibition in the heart is only to be demonstrated if contamination of the sarcolemmal preparation with endothelial membrane components is kept to a minimum.Abbreviations R-PIA (–)N⁶-(R-phenylisopropyl)-adenosine - NECA 5-(N-ethyl-carboxamido)-adenosine - ICYP (–)[¹²⁵I]iodo-cyanopindolol - dATP 2-deoxy-adenosine-5-triphosphate - S-PIA (+)N⁶-(S-phenylisopropyl)-adenosine - HPIA (–)N⁶-(4-hydroxy-phenylisopropyl)-adenosine - CHA N⁶-cyclohexyl-adenosine - Gpp(NH)p guanylyl imidodiphosphate - dAMP 2-deoxy-adenosine-5-monophosphate - HEPES 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid - EDTA (ethylenedinitrilo)-tetraacetic acid - [¹²⁵I]HPIA (–)N⁶-(3-[¹²⁵I]-iodo-4-hydroxyphenylisopropyl)-adenosine     

Influence of AMPA/kainate receptors on extracellular 5-hydroxytryptamine in rat midbrain raphe and forebrain

1. The regulation of 5-hydroxytryptamine (5-HT) release by excitatory amino acid (EAA) receptors was examined by use of microdialysis in the CNS of freely behaving rats. Extracellular 5-HT was measured in the dorsal raphe nucleus (DRN), median raphe nucleus (MRN), nucleus accumbens, hypothalamus, frontal cortex, dorsal and ventral hippocampus.
2. Local infusion of kainate produced increases in extracellular 5-HT in the DRN and MRN. Kainate infusion into forebrain sites had a less potent effect.
3. In further studies of the DRN and nucleus accumbens, kainate-induced increases in extracellular 5-HT were blocked by the EAA receptor antagonists, kynurenate and 6,7-dinitroquinoxaline-2,3-dione (DNQX).
4. The effect of infusing kainate into the DRN or nucleus accumbens was attenuated or abolished by tetrodotoxin (TTX), suggesting that the increase in extracellular 5-HT is dependent on 5-HT neuronal activity. In contrast, ibotenate-induced lesion of intrinsic neurones did not attenuate the effect of infusing kainate into the nucleus accumbens. Thus, the effect of kainate in the nucleus accumbens does not depend on intrinsic neurones.
5. Infusion of α-amino-3-hydroxy-5-methyl-4-isoxazolaproprionate (AMPA) into the DRN and nucleus accumbens induced nonsignificant changes in extracellular 5-HT. Cyclothiazide and diazoxide, which attenuate receptor desensitization, greatly enhanced the effect of AMPA on 5-HT in the DRN, but not in the nucleus accumbens.
6. In conclusion, AMPA/kainate receptors regulate 5-HT in the raphe and in forebrain sites.

     

Role of AMPA and GluR5 kainate receptors in the development and expression of amygdala kindling in the mouse

The role of AMPA and GluR5-containing kainate receptors in the development and expression of amygdala kindling was examined using the selective 2,3-benzodiazepine AMPA receptor antagonist GYKI 52466 [(1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2, 3-benzodiazepine] and the decahydroisoquinoline mixed AMPA receptor and GluR5 kainate receptor antagonist LY293558 {(3S,4aR,6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline- 3-carboxy lic acid)}. Administration of GYKI 52466 (5-40 mg/kg, intraperitoneally) and LY293558 (10-40 mg/kg, intraperitoneally) prior to daily kindling stimulation in mice produced a dose-dependent suppression of the rate of development of behavioral kindled seizure activity and reduced the duration of the stimulation-induced electrographic afterdischarge. In drug-free stimulation sessions after the initial drug-treatment sessions, there was an acceleration in the rate of kindling development compared with the rate during the preceding drug-administration period; the "rebound" rate was also greater than the kindling rate in saline-treated control animals. In fully kindled animals, both GYKI 52466 and LY293558 produced a dose-dependent suppression of evoked seizures (ED(50), 19.3 and 16.7 mg/kg, respectively). Although AMPA receptors appear to be critical to the expression of kindled seizures, since kindling development progressed despite the suppression of behavioral seizure activity, AMPA receptors are less important to the kindling process. LY293558 was modestly less effective at suppressing behavioral seizures during kindling and was not superior to GYKI 52466 in retarding the overall extent of kindling development, indicating that GluR5 kainate receptors do not contribute to epileptogenesis in this model.     

Induction of LTD in the adult hippocampus by the synaptic activation of AMPA/kainate and metabotropic glutamate receptors

It has been suggested that the induction of long-term depression (LTD) may be developmentally regulated since LTD can be readily induced by LFS in slices from young but not adult animals. However, we have recently reported that paired pulse low frequency stimulation (PP-LFS) can reliably induce LTD in the CA1 region of adult hippocampal slices. We now describe the role of glutamate receptors in the induction of LTD in adult hippocampus. The induction of LTD was prevented by the combined application of AMPA/kainate and metabotropic glutamate (mGlu) receptor antagonists (CNQX and LY341495). However, LTD was not blocked by the co-application of NMDA and mGlu receptor antagonists nor by the co-application of NMDA and AMPA/kainate receptor antagonists. Taken together, the above results suggest that activation of either AMPA/kainate or mGlu receptors is sufficient to induce LTD. Therefore, these results suggest that PP-LFS can efficiently activate AMPA/kainate and mGlu receptors in order to induce long-lasting synaptic depression in the CA1 region of the adult hippocampus in vitro.     

Blockade of the alpha3alpha4 N-cholinoreceptors and GluR1 AMPA receptors eliminates clonic-tonic nicotinic and kainate seizures

Monoammonium N-alkyl derivative of decylamine (IEM-1678), which blocks alpha3beta4 N-cholinoreceptors (but does not block GluR1 AMPA receptors), in doses of 1.0 - 3.0 mg/kg produces a 4-fold decrease in the frequency and lethality of nicotinic clonic-tonic seizures. However, even in the maximum dose of 3 mg/kg, IEM-1678 only slightly decreases kainate clonic-tonic seizures. Bis-ammonium compound IEM-1460 (containing adamantyl radical), which blocks both GluR1 AMPA receptors and alpha3beta4 N-cholinoreceptors, in a range of doses 0.1 - 3 mg/kg produces a 5- to 8-fold decrease in the frequency and virtually completely eliminates lethality of both clonic-tonic nicotinic and kainate seizures. Hence, the complete elimination of generalized kainate and nicotinic seizures requires combined blockade GluR1 AMPA and alpha3beta4 N-cholinoreceptors.     

Presynaptic muscarinic and alpha-adrenoceptor-mediated regulation of GABA release from myenteric neurones of the guinea-pig small intestine.

The effects of cholinomimetic and sympathomimetic drugs on the release of [3H]-gamma-aminobutyric acid ([3H]-GABA) evoked by high K+ from the isolated small intestine of the guinea-pig were investigated, in the presence of tetrodotoxin. Acetylcholine and oxotremorine, at concentrations ranging from 10(-9) to 10(-6) M inhibited the evoked release of [3H]-GABA in a concentration-dependent manner, while nicotine was without effect. Scopolamine and pirenzepine inhibited the effect of oxotremorine, while hexamethonium had no effect. The IC50 values for scopolamine and pirenzepine of the oxotremorine (3 X 10(-8) M)-induced inhibition were 1.02 X 10(-9) M and 9.78 X 10(-10) M, respectively. Noradrenaline, but not isoprenaline inhibited the evoked release of [3H]-GABA. Clonidine (10(-10)-10(-6) M) reduced the evoked release of [3H]-GABA in a concentration-dependent manner, but phenylephrine had no effect. The inhibitory effect of clonidine was antagonized by yohimbine but not by prazosin. These findings provide evidence for the localization of M1-muscarinic and alpha 2-adrenoceptors on GABAergic nerve terminals and their involvement in the presynaptic control of the release of GABA from the guinea-pig small intestine.     

NMDA and AMPA/kainate receptors are involved in the anticonvulsant activity of riluzole in DBA/2 mice

The anticonvulsant activity of riluzole against sound-induced seizures was studied in the DBA/2 mouse model. Riluzole (0.1-4 mg kg(-1), intraperitoneal (i.p.)) produced dose-dependent effects with ED(50) values for the suppression of tonic, clonic and wild running phases of 0.72, 1.38 and 2.71 mg kg(-1), respectively. Riluzole also protected DBA/2 mice from seizures induced by an intracerebroventricular (i.c.v.) injection of N-methyl-D-aspartate (NMDA) with ED(50) values of 3.03 and 5.0 mg kg(-1) for tonus and clonus, respectively. Pretreatment with glycine, an agonist to the glycine/NMDA receptors, shifted the dose-response effect of riluzole to the right (ED(50)=6.53 against tonus and 9.34 mg kg(-1) vs. clonus). Similarly, D-serine, an agonist at the glycine site, shifted the ED(50) of riluzole against the tonic component of audiogenic seizures from 0.72 to 1.97, and that against clonus from 1.38 to 2.77 mg kg(-1). Riluzole was also potent to prevent seizures induced by administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), an AMPA/kainate receptor agonist (ED(50)=1.80 and 3.35 mg kg(-1), against tonus and clonus, respectively). Pretreatment with aniracetam, a positive allosteric modulator of AMPA/kainate receptors, shifted the dose-response curve of riluzole to the right (ED(50)=1.78 against tonus and 2.58 mg kg(-1) vs. clonus). The data indicate that riluzole is an effective anticonvulsant drug in the genetic model of seizure-prone DBA/2 mice. Our findings suggest that the anticonvulsant properties of riluzole depend upon its interaction with neurotransmission mediated by both the glycine/NMDA and the AMPA/kainate receptor complex.     

AMPA/kainate receptors in the ventromedial hypothalamus mediate the effects of glutamate on estrus termination in the rat

Infusions of glutamate or its selective receptor agonists to the VMH of ovariectomized (OVX) female rats primed with estradiol benzoate (EB) and progesterone (P) inhibit both appetitive and consummatory aspects of sexual behavior whereas selective glutamate receptor antagonists facilitate these measures in females primed with EB alone. Because vaginocervical stimulation (VCS) activates glutamate neurons in the VMH, and induces a faster termination of estros behavior, the present study examined the effects of the AMPA/kainate receptor antagonist DNQX on the induction of estrus termination by manual VCS. Ovx, sexually-experienced rats were primed with EB and P and subsequently received either 1 or 50 distributed VCSs, over the course of an hour, 12 h before a test with sexually vigorous males. Half of the females in each stimulus group received bilateral infusions of 1 μl/side of either DNQX (19.8 mmol/μl) or saline aimed at the VMH immediately prior to VCS or sham stimulation. Saline-infused females given VCS had lower lordosis quotients compared to females given sham stimulation. In contrast, females infused with DNQX prior to VCS displayed more appetitive behaviors and higher lordosis quotients and magnitudes compared to females infused with saline. These data indicate that activation of AMPA/kainate receptors in the VMH by increased glutamate transmission induced by VCS mediates estrus termination.     

Influence of LY 300164, an antagonist of AMPA/kainate receptors, on the anticonvulsant activity of clonazepam

LY 300164 [7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo(4,5H)-2,3-benzodiazepine], an antagonist of AMPA/kainate receptors, at 5 mg/kg exerted a significant anticonvulsant effect, as regards seizure and afterdischarge durations in amygdala-kindled seizures in rats. At lower doses, LY 300164 did not exert anticonvulsant activity. Clonazepam alone (0.003–0.1 mg/kg) significantly diminished seizure severity, seizure and afterdischarge durations. Coadministration of LY 300164 (2 mg/kg) with clonazepam (0.001 mg/kg) resulted in the significant anticonvulsant activity. Seizure severity score, seizure and afterdischarge durations were reduced from 5 to 4, from 32.6 s to 12.3 s, and 42.7 s to 23.2 s. LY 300164 (2 mg/kg), clonazepam (0.001–0.1 mg/kg) and the combination of clonazepam (0.001 mg/kg) with LY 300164 (2 mg/kg) did not affect long-term memory evaluated in the passive avoidance task in rats. LY 300164 (at the subprotective dose of 2 mg/kg) significantly potentiated the anticonvulsant action of clonazepam against maximal electroshock but not against pentylenetetrazol-induced convulsions in mice. The results indicate that blockade of glutamate-mediated events at AMPA/kainate receptors may differently affect the protection offered by clonazepam, which seems dependent upon the model of experimental seizures.     

Functional role of the endocannabinoid system and AMPA/kainate receptors in 5-HT2A receptor-mediated wet dog shakes

These experiments sought to determine the influence of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors and the endocannabinoid system in the functional expression of the serotonin (5-HT) type 2A receptor-mediated wet dog shake response. Male Long-Evans rats were pretreated with either 1 mg/kg i.p. of the 5-HT(2A/2C) receptor antagonist ketanserin; 1, 10 or 30 mg/kg i.p. of the AMPA/kainate antagonist 6,7-dinitroquinnoxaline-2,3-dione (DNQX); 1, 5 or 10 mg/kg i.p. of the endocannabinoid uptake inhibitor AM404; or 1, 5 or 10 mg/kg i.p. of the cannabinoid CB(1) receptor antagonist AM 251 prior to injection of the 5-HT(2A/2C) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI, 1 mg/kg i.p.). Results demonstrated that 10 mg/kg of AM404 significantly reduced the expression of DOI-induced wet dog shakes, but lower doses were ineffective. Administration of AM251 did not induce wet dog shakes behavior when administered alone, but significantly potentiated DOI-induced wet dog shaking behavior at a dose of 10 mg/kg. Pretreatment with DNQX significantly reduced the expression of DOI-induced wet dog shakes at all doses tested. These data suggest that AMPA/kainate receptors play a role in the mediation of 5-HT(2A) receptor activity, whereas the endocannabinoid system may act as a regulatory buffer system during periods of elevated activity, but not under basal conditions.     

Presynaptic cross-talk of beta-adrenoreceptor and 5-hydroxytryptamine receptor signalling in the modulation of glutamate release from cerebrocortical nerve terminals

1. The presynaptic interactions between facilitatory beta-adrenoreceptors and inhibitory 5-hydroxytryptamine (5-HT) receptors modulating glutamate release from cerebrocortical nerve terminals were examined. 2. 4-aminopyridine (4-AP, 1 mM)-evoked glutamate release was facilitated by the membrane permeant cyclic-3',5'-adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (8-Br-cAMP), used to directly activate cAMP-dependent protein kinase (PKA). 3. The beta-adrenoreceptor agonist, isoprenaline (ISO), effected a concentration-dependent potentiation of 4-AP-evoked glutamate release which was abolished by the beta-adrenoreceptor antagonist, propranolol, and the PKA inhibitor, Rp-cyclic-3',5'-adenosine-monophosphothioate (Rp-cAMPS). 4. 5-HT receptor activation by 100 microM 5-HT produced an inhibition of 4-AP-evoked glutamate release in nerve terminals. The inhibitory effect of 5-HT could be mimicked by the selective 5-HT(1A) receptor agonist, 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) and antagonized by 1-(2-methoxyphenyl)-4-(4-phthalimidobutyl)piperazine (NAN-190). 5. When 5-HT (or 8-OH-DPAT) was used in conjunction with ISO or 8-Br-cAMP, the beta-adrenoreceptor- and PKA-mediated potentiation of glutamate release was abrogated. 6. The inhibitory crosstalk of 5-HT(1A) receptors to beta-adrenoceptor-mediated facilitation of glutamate release was abolished in the presence of NAN-190. 7. Examination of voltage-dependent Ca(2+) influx revealed that, while ISO and 5-HT alone caused a respective potentiation and diminution of the 4-AP-evoked increase in [Ca(2+)](c), the co-presence of 5-HT abolished the ISO mediated potentiation of Ca(2+) influx. 8. Together, these results suggest that beta-adrenoreceptors and 5-HT(1A) receptors coexist on the cerebrocortical nerve terminals and that the cross-talk between the two receptor signalling pathways occurs at a locus downstream from cAMP production, possibly at the level of voltage-dependent Ca(2+) influx.     

Presynaptic serotonin receptors regulate the release of 3H-serotonin in hypothalamic slices of the rabbit

Summary Hypothalamic slices of the rabbit brain were incubated with 10^–7 M of ³H-serotonin (³H-5HT). After the incubation and an initial washout period, a nearly constant basal efflux of tritium was detected. This basal efflux was not significantly altered by Ca²⁺-free solution or by the 5HT-antagonist metitepin (10^–5 M), but was augmented by chlorimipramine (10^–5 M) and by unlabelled 5HT (10^–6 M); the acceleration caused by unlabelled 5HT was absent in presence of chlorimipramine (10^–5 M). Both electrical stimulation (4 Hz, 50 mA, 2 min) and high K⁺ (50 mM) induced an overflow of ³H. This overflow was nearly abolished in Ca²⁺-free solution. In presence of chlorimipramine (10^–5 M) both the tritium overflow evoked by electrical stimulation and that evoked by high K⁺ were augmented by metitepin (10^–5 M) and decreased in a concentration dependent manner by unlabelled serotonin (10^–8–10^–6 M); the latter effect was antagonized by metitepin (10^–6 M and 10^–5 M). These experiments suggest that in rabbit hypothalamic slices, the release of ³H-5HT is controlled by a negative feedback mechanism acting via presynaptic serotonin receptors.