Pertussis toxin pretreatment discriminates between pre- and postsynaptic actions of baclofen in rat dorsal raphe nucleus in vitro

doi:10.1016/0304-3940(88)90099-7

Neuroscience Letters

Volume 93, Issues 2–3, 11 November 1988, Pages 300-306

https://doi.org/10.1016/0304-3940(88)90099-7 Get rights and content

Abstract

Intracellular recordings were made from rat dorsal raphe neurons in vitro. Baclofen (30 μM) and 5-carboxamidotryptamine (5-CT, 300 nM to 1 μM) hyperpolarized these neurons by 10 and 13 mV, respectively. Depolarizing synaptic potentials (DSPs) were evoked by single shocks: baclofen reduced the amplitude of the DSP by 81%, but 5-CT reduced it by only 23%. The somatic response to iontophoretically applied glutamate pulses was reduced by 12% by baclofen, and 23% by 5-CT. In slices from rats pretreated with intracerebroventricular pertussis toxin (PTX), the ability of baclofen to reduce the DSP was almost unchanged, although the hyperpolarizing action of baclofen, and both actions of 5-CT were virtually eliminated. We conclude that it is possible to distinguish the pre- and postsynaptic actions of baclofen with PTX, and that the actions of 5-CT are both blocked.

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Cited by (56)

Baclofen and gamma-hydroxybutyrate differentially altered behavior, EEG activity and sleep in rats
2015, Neuroscience
Citation Excerpt :
There is evidence that median raphe nucleus (MRN) may serve to desynchronize hippocampal EEG and thus to block theta rhythm (Vertes, 1981). Bac, through GABAB receptors, might suppress the firing of serotonergic neurons in the raphe nucleus (Colmers and Williams, 1988; Innis et al., 1988) and promote theta rhythms generation. Indeed, Bac infused into the serotonin-containing MRN, promoted theta rhythm in anaesthetized rats (Varga et al., 2002; Li et al., 2005).
Objectives: Animal and human studies have shown that sleep may have an impact on functional recovery after brain damage. Baclofen (Bac) and gamma-hydroxybutyrate (GHB) have been shown to induce physiological sleep in humans, however, their effects in rodents are unclear. The aim of this study is to characterize sleep and electroencelphalogram (EEG) after Bac and GHB administration in rats. We hypothesized that both drugs would induce physiological sleep.
Methods: Adult male Sprague-Dawley rats were implanted with EEG/electromyogram (EMG) electrodes for sleep recordings. Bac (10 or 20 mg/kg), GHB (150 or 300 mg/kg) or saline were injected 1 h after light and dark onset to evaluate time of day effect of the drugs. Vigilance states and EEG spectra were quantified.
Results: Bac and GHB induced a non-physiological state characterized by atypical behavior and an abnormal EEG pattern. After termination of this state, Bac was found to increase the duration of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep (∼90 and 10 min, respectively), reduce sleep fragmentation and affect NREM sleep episode frequency and duration (p < 0.05). GHB had no major effect on vigilance states. Bac drastically increased EEG power density in NREM sleep in the frequencies 1.5–6.5 and 9.5–21.5 Hz compared to saline (p < 0.05), while GHB enhanced power in the 1–5-Hz frequency band and reduced it in the 7–9-Hz band. Slow-wave activity in NREM sleep was enhanced 1.5–3-fold during the first 1–2 h following termination of the non-physiological state. The magnitude of drug effects was stronger during the dark phase.
Conclusion: While both Bac and GHB induced a non-physiological resting state, only Bac facilitated and consolidated sleep, and promoted EEG delta oscillations thereafter. Hence, Bac can be considered a sleep-promoting drug and its effects on functional recovery after stroke can be evaluated both in humans and rats.
γ-Hydroxybutyric acid induces actions via the GABA<inf>B</inf> receptor in arousal and motor control-related nuclei: Implications for therapeutic actions in behavioral state disorders
2013, Neuroscience
Citation Excerpt :
Stimulation of the GIRK in LDT cells has been shown to be sensitive to the bee venom toxin, tertiapin (Kohlmeier and Leonard, 2006; Kohlmeier and Kristiansen, 2010). Within the DR, others have shown that baclofen actions at GABAB receptors are linked to a GIRK conductance similar to that linked to activation of serotonergic autoreceptors located on DR neurons (Colmers and Williams, 1988). In the present study, the GHB-activated conductance was tertiapin-sensitive within cells of both nuclei.
γ-Hydroxybutyric acid (GHB) is used as an effective therapeutic for reducing the hypersomnolence and cataplexy (loss of motor control) of the sleeping disorder, narcolepsy, with an immediate pharmacologic behavioral action of inducing a natural sleep-like state. Despite its clinical use, few studies have examined the cellular actions of this drug on behavioral state-related neurons. Therefore, we monitored GHB-induced responses using calcium imaging within the laterodorsal tegmentum (LDT) and the dorsal raphe (DR), two pontine nuclei important in state and motor control. In addition, we recorded GHB-induced membrane responses using whole cell, patch clamp electrophysiology of immunohistochemically-identified principal neurons within these nuclei.
GHB induced GABA_B receptor-mediated rises in calcium in neurons of the LDT and the DR. However, the pattern and amplitude of calcium rises differed greatly between these two nuclei. GHB induced GABA_B receptor antagonist-sensitive outward currents/hyperpolarizations in immunohistochemically-identified cholinergic LDT and serotonergic DR neurons. However, GHB had this action in a greater proportion of DR cells than LDT neurons. Further, larger inhibitory currents were induced in DR cells when compared to the amplitude of GHB-induced current in LDT-responding cells. Finally, NCS-382 and HOCPCA, a reported antagonist and agonist specific to activity at the putative GHB receptor, respectively, with no demonstrated binding at the GABA_B receptor, failed to block GHB-induced effects or elicit any discernible electrophysiological action when applied alone, indicating a lack of involvement of a GHB receptor in mediating GHB actions.
Taken together, our data support the conclusion that GHB may be exerting its actions on state and motor control, in part, via an acutely mediated strong inhibition of serotonergic DR neurons and a more modest inhibitory action on a smaller proportion of LDT cholinergic neurons. Given the roles played by these nuclei, these actions are consistent with acute pharmacologic effects of GHB: hypotonia and promotion of sleep, including presence of REM, a sub-state of sleep. Differences in GHB-mediated calcium suggest differential regulation of calcium-dependent processes, which may also contribute to functioning of the LDT and DR in state and motor control and the therapeutic pharmacologic actions of GHB, which develop following chronic administration. These findings add to knowledge of cellular actions of GHB and it is hoped that, combined with findings from other studies examining GHB neurotransmission, these data can contribute to development of highly targeted therapeutics at the GABA_B receptor for management of human disorders presenting with alterations in motor and arousal control.
GABA <inf>B</inf> receptors. Physiological functions and mechanisms of diversity
2010, Advances in Pharmacology
Citation Excerpt :
Differences in native GABAB responses were first observed between pre- and postsynaptic receptors (Bowery, 1993; Nicoll, 2004). Electrophysiological studies in the neocortex and hippocampus showed different sensitivities of pre- and postsynaptic GABAB receptors to the antagonist phaclofen and to pertussis toxin, which suggested the existence of distinct receptors (Colmers & Williams, 1988; Deisz et al., 1993; Dutar & Nicoll, 1988; Harrison, 1990). However, studies in other brain regions or hippocampal cultures using more potent GABAB receptor antagonists did not detect any pharmacological difference between pre- and postsynaptic receptors (Seabrook et al., 1990; Thompson & Gahwiler, 1992; Yoon & Rothman, 1991).
GABA_B receptors are the G-protein-coupled receptors (GPCRs) for γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the central nervous system. GABA_B receptors are implicated in the etiology of a variety of psychiatric disorders and are considered attractive drug targets. With the cloning of GABA_B receptor subunits 13 years ago, substantial progress was made in the understanding of the molecular structure, physiology, and pharmacology of these receptors. However, it remained puzzling that native studies demonstrated a heterogeneity of GABA_B responses that contrasted with a very limited diversity of cloned GABA_B receptor subunits. Until recently, the only firmly established molecular diversity consisted of two GABA_B1 subunit isoforms, GABA_B1a and GABA_B1b, which assemble with GABA_B2 subunits to generate heterodimeric GABA_B(1a,2) and GABA_B(1b,2) receptors. Using genetic, ultrastructural, biochemical, and electrophysiological approaches, it has been possible to identify functional properties that segregate with these two receptors. Moreover, receptor modifications and factors that can alter the receptor response have been identified. Most importantly, recent data reveal the existence of a family of auxiliary GABA_B receptor subunits that assemble as tetramers with the C-terminal domain of GABA_B2 subunits and drastically alter pharmacology and kinetics of the receptor response. The data are most consistent with native GABA_B receptors minimally forming dimeric assemblies of units composed of GABA_B1, GABA_B2, and a tetramer of auxiliary subunits. This represents a substantial departure from current structural concepts for GPCRs.
GABAergic modulation of 5-HT<inf>7</inf> receptor-mediated effects on 5-HT efflux in the guinea-pig dorsal raphe nucleus
2004, Neuropharmacology
5-HT₇ receptor mRNA and protein are localised in the dorsal raphe nucleus (DRN) on non-serotonergic neurones. The effect of 5-HT₇ receptor antagonism on 5-HT efflux was measured from guinea-pig DRN slices, using the technique of fast cyclic voltammetry.
The 5-HT₇ receptor antagonist, SB-269970-A, significantly inhibited 5-HT efflux. The GABA_A receptor agonist, muscimol, significantly inhibited 5-HT efflux, to a similar degree as SB-269970-A. In contrast, the GABA_A receptor antagonist, bicuculline, significantly increased 5-HT efflux and attenuated the muscimol-induced inhibition. The muscimol and SB-269970-A effects were not additive and in the presence of bicuculline the SB-269970-A-induced inhibition of 5-HT efflux was attenuated.
These data suggest that 5-HT₇ receptor antagonist-induced inhibition of 5-HT efflux occurs indirectly via activation of GABA_A receptors. That is, 5-HT₇ receptors may be located on GABA interneurones and when activated decrease GABA release and hence decrease the inhibitory tone on 5-HT neurones, increasing 5-HT efflux in the DRN. Therefore, in the presence of GABAergic tone 5-HT₇ receptor antagonists would decrease 5-HT release from the DRN.
Dopamine-induced synaptic depression in the parabrachial nucleus is independent of CTX- and PTX-sensitive G-proteins, PKA and PLC signalling pathways
2004, Brain Research
Citation Excerpt :
We verified that the PTX used in this study was active by examining the effects of baclofen in the PTX-treated slices. We took advantage of the finding that baclofen's postsynaptic, but not its presynaptic effects, are sensitive to PTX treatment [9]. Compared to control slices, PTX incubation (500 ng/ml for 20 h) abolished baclofen-induced outward current (not shown) as well as the change in slope resistance obtained by a slow voltage ramp protocol (−120 to −40 mV over 20 s; Fig. 3).
We have previously reported that dopamine (DA) depresses non-NMDA receptor-mediated glutamatergic transmission in the rat parabrachial nucleus (PBN), an interface between brainstem and forebrain that is implicated in autonomic regulation. This work examined cellular signalling pathways that might underlie this DA-induced synaptic depression. Direct activation of adenylyl cyclase with 10 μM forskolin increased the evoked EPSC but did not occlude DA-induced EPSC depression. Similarly, a preferential protein kinase A inhibitor, H-7 (10 μM), did not block DA's synaptic effects. Incubation of slices with cholera toxin (CTX; 1 μg/ml) or pertussis toxin (PTX; 0.5 μg/ml) for 20 h, procedures used to irreversibly activate or disable the G_s and G_i proteins, respectively, did not change DA's effects. The putative phospholipase C inhibitor, U-73122 (10 μM) and its inactive analogue U-73343 (10 μM) did not alter DA-induced reduction in the EPSCs. Alterations in signalling molecules downstream of phospholipase C including depleting internal calcium stores by thapsigargin and cyclopiazonic acid and blocking protein kinase C with chelerythrine, had no effect on DA-induced synaptic depression. Furthermore, DA's depression of the non-NMDA response was not blocked by APV, an NMDA receptor antagonist. Finally, DA depressed evoked, pharmacologically isolated NMDA receptor-mediated synaptic responses while increasing NMDA-induced inward currents in the PBN. These results indicate that DA-induced synaptic effects in the PBN are not through the activation of cholera or pertussis toxin sensitive G proteins. Furthermore, it does not employ the adenylyl cyclase–cAMP–PKA cascade, the phospholipase C signalling pathway and NMDA receptor-coupled mechanisms to depress excitatory synaptic transmission in the PBN.
Control of serotonergic neurons in the dorsal raphe nucleus by the lateral hypothalamus
2002, Brain Research
Citation Excerpt :
GABAB receptors are located pre- and postsynaptically. Pre- and postsynaptic actions of baclofen in different brain areas have been discriminated [1,18,21,31,42]. Since baclofen preferentially activates presynaptic GABAB receptors [1,19,20,30] its local application likely disinhibited lateral hypothalamic neurons, thus mimicking the effect of bicuculline.
Anatomical evidence indicates the presence of projections from the lateral hypothalamus to serotonergic (5-hydroxytryptamine, 5-HT) neurons of the dorsal raphe nucleus (DR). Using dual probe microdialysis and extracellular recordings in the DR, we show that the application of GABAergic agents in the lateral hypothalamus modulates the activity of 5-HT neurons in the DR. GABA and bicuculline or baclofen, applied in the lateral hypothalamus significantly reduced and increased, respectively, the 5-HT output in the DR. Likewise, the intrahypothalamic application of GABA and bicuculline reduced (14/20 neurons) and increased (8/12 neurons), respectively, the firing rate of 5-HT neurons in the DR. A smaller percentage of neurons, however, were excited by GABA (3/20) and inhibited by bicuculline (1/12). Application of tetrodotoxin in the lateral hypothalamus suppressed the local 5-HT output and reduced that in the DR. The 5-HT output in the DR increased transiently soon after darkness. The hypothalamic application of GABA attenuated and that of bicuculline potentiated this spontaneous change with an efficacy similar to that seen in light conditions. These results indicate that the lateral hypothalamus is involved in the control of 5-HT activity in the DR, possibly through excitatory (major) and inhibitory (minor) inputs.

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^∗: Present address: Department of Pharmacology, University of Alberta, 9–12 MSB, Edmonton, Alta., T6G 2R7, Canada.

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Pertussis toxin pretreatment discriminates between pre- and postsynaptic actions of baclofen in rat dorsal raphe nucleus in vitro

Abstract

Brain Res.

Trends Neurosci.

Brain Res.

Neurosci. Lett.

A G protein couples serotonin and GABAB receptors to the same channel in hippocampus

Science

(−)Baclofen decreases neurotransmitter release in the mammalian CNS by an action at a novel GABA receptor

Nature (Lond.)

Inhibition of calcium spikes and transmitter release by γ-aminobutyric acid in the guinea pig myenteric plexus

Br. J. Pharmacol.

Differential modulation of three separate K-conductances in hippocampal CAl neurons by serotonin

Nature (Lond.)

Calcium currents and their inhibition by (−)-baclofen in rat sensory neurones: modulation by guanine nucleotides

J. Physiol. (Lond.)

A G protein couples serotonin and GABA_B receptors to the same channel in hippocampus