Immunohistochemical studies of the localization of neurons containing the enzyme that synthesizes dopamine, GABA, or gamma-hydroxybutyrate in the rat substantia nigra and striatum

gamma-Hydroxybutyrate (GHB) is an endogenous metabolite of gamma-aminobutyric acid (GABA), which is synthesized in the neuronal compartment of the central nervous system. This substance possesses several properties that support its role as a neurotransmitter/neuromodulator in brain. In particular, it is synthesized by a specific pathway that transforms GABA into succinic semialdehyde via GABA-T activity; then succinic semialdehyde is converted into GHB by a specific succinic semialdehyde reductase (SSR). The last enzyme is considered as a marker for neurons that synthesize GHB. This compound binds in brain to receptors whose distribution, ontogenesis, kinetics, and pharmacology are specific. Endogenous GHB, but also GHB exogenously administered to rats, participate in the regulation of dopaminergic activity of the nigrostriatal pathway. To investigate the distribution of GHB neurons in this pathway and the anatomic relationships between dopaminergic and GHB neurons, immunocytochemical identification of dopamine, GABA, and GHB neurons was carried out in the substantia nigra and striatum of the rat. The following markers for these neurons were used: anti-tyrosine hydroxylase (TH) antibodies for dopamine neurons, anti-glutamate decarboxylase (GAD) antibodies for GABA neurons, and anti-succinic semialdehyde reductase (SSR) antibodies for GHB neurons. GABA neurons were studied because GAD and SSR co-exist frequently in the same neuron, and GABA alone also exerts its own regulatory effects on dopaminergic neurons. This study reveals the co-existence of GAD/SSR and GAD/SSR/TH in numerous neurons of the substantia nigra. However, some neurons appear to be only GAD or SSR positive. In the striatum, TH-positive terminals surround many GHB neurons. GAD innervation is abundant in close contact with unlabeled neurons in the caudate-putamen, whereas distinct SSR-positive punctuates are also present. The existence of SSR-reactive synapses and neurons was confirmed in the striatum at the electron microscopic level. On the basis of these results, a clear anatomo-functional relationship between GHB and dopamine networks cannot be defined; however, we propose the modulation by GHB of striatal intrinsic neurons that could then interfere with the presynaptic control of dopaminergic activity.     

Anti-aggressive effects of GHB in OF.1 strain mice: involvement of dopamine D2 receptors

Numerous studies indicate that gamma-hydroxybutyric acid (GHB) influences the endogenous dopamine system. Both GHB and most dopaminergic D(2) receptor antagonists are effective anti-aggressive agents in animal models. The present study aimed to investigate the effects of GHB on agonistic behaviour and to implicate D(2) dopamine receptor on these behaviours. For this purpose, the effects of GHB (80, 120 and 160 mg/kg, IP) and tiapride (60 mg/kg) administered alone or in combination were examined on agonistic behaviour elicited by 'isolation' in male mice. Individually housed mice were exposed to anosmic "standard opponents" 30 min after drug administration, and the encounters were videotaped and evaluated using an ethologically based analysis. The administration of 80 and 120 mg/kg of GHB reduced threat without impairing motor activity, but the administration of 160 mg/kg of GHB or the co-administration of GHB+tiapride (a selective D(2) receptor antagonist) significantly reduced threat and attack but concomitantly increased immobility. The co-administration of GHB+tiapride had different effects to those observed by the administration of these drugs separately. It is concluded that the anti-aggressive effect of GHB appears to be mediated, at least in part, by D(2) dopamine receptors. This anti-dopaminergic activity is an indirect effect, probably induced by the activation of GHB receptors of low affinity, and in this way, this compound would reduce levels of dopamine without blockading of D(2) postsynaptic dopamine receptors.     

Naloxone pretreatment alters the local cerebral metabolic effect of γ-hydroxybutyrate in rats

The effect of naloxone on γ-hydroxybutyrate (GHB)-induced cerebral metabolic depression was studied in rats with the 2-[¹⁴C]deoxyglucose method. Naloxone pretreatment statistically significantly antagonized the cerebral metabolic effect of GHB in 10 of 38 structures examined. These results are consistent with previous data showing reversal of the dopaminergic, electroencephalographic, and behavioral effects of GHB by naloxone and suggest that some of the neuropharmacological effects of GHB are mediated by the endogenous opiate system.     

Gamma-hydroxybutyrate in the treatment of schizophrenia

Gamma-Hydroxybutyrate (GHB) inhibits firing of dopaminergic neurons and is thus potentially useful in the treatment of schizophrenia. GHB was administered to 10 schizophrenics concurrently with low-dose fluphenazine in a 6-week double-blind crossover study. No antipsychotic efficacy of GHB was noted. GHB had little if any effect on plasma prolactin levels after a single administration and caused few side effects. Trials with higher doses of GHB may be warranted.     

Gamma-hydroxybutyrate and ethanol depress spontaneous excitatory postsynaptic currents in dopaminergic neurons of the substantia nigra

Gamma-hydroxybutyrate (GHB) has been shown to have therapeutical properties in various psychiatric disorders, especially in alcohol abuse, and to mimic different actions of ethanol at the cellular and system level. Using whole-cell patch-clamp recordings on brain slices of 21- to 25-day-old rats, the present study investigated the effects of GHB and ethanol on spontaneous excitatory postsynaptic currents (sEPSCs) in dopaminergic neurons of the substantia nigra pars compacta (SNc). sEPSCs are an index of glutamate release from the excitatory input to dopamine cells, which play a key role in different reward-related behaviors. We found that GHB and ethanol depressed both the frequency and the amplitude of sEPSCs. These effects were GABA(B)-independent and the GHB-induced depression was blocked by the GHB receptor antagonist 6,7,8,9-tetrahydro-5[H]benzocyclohepte-5-ol-4-ylidene acetic acid (NCS-382), pointing to a specific effect of this drug. The effects of ethanol were not affected by NCS-382. This study indicates that GHB and ethanol share the effect of reducing the efficacy of excitatory glutamatergic neurotransmission in the SNc by acting through different mechanisms.     

Restless legs syndrome in narcolepsy: a side effect of sodium oxybate?

Gamma-hydroxybutyrate (GHB) has re-emerged as a major treatment for narcolepsy. As dopaminergic transmission is clearly involved in the pathophysiology of restless legs syndrome (RLS), and GHB reduces dopamine release, one may hypothesize that RLS may occur in narcolepsy in the presence of GHB. We report a case of narcolepsy with a severe occurrence of typical RLS with GHB, symptoms never previously experienced by the subject and reversible after withdrawal.     

Low doses of gamma-hydroxybutyric acid stimulate the firing rate of dopaminergic neurons in unanesthetized rats.

In unanesthetized rats the intravenous (i.v.) administration of gamma-hydroxybutyric acid (GHB) at the doses of 50-400 mg/kg produced a dose-related stimulation (10-56%) of the firing rate of dopaminergic (DA) neurons in the pars compacta of the substantia nigra. Doses of 1000 and 1500 mg/kg inhibited the firing rate almost completely. In unanesthetized rats the intraperitoneal injection of GHB at the dose of 750 mg/kg produced a brief initial stimulation (23%) followed by a modest reduction in the firing rate (29%). On the other hand, in chloral hydrate-anesthetized rats the i.v. administration of GHB at cumulative doses of up to 200 mg/kg failed to modify the firing rate of DA neurons, while a cumulative dose of 400 mg/kg suppressed neuronal firing. The results indicate that sub-anesthetic doses of GHB stimulate the firing rate of DA neurons in unanesthetized rats.     

Lack of effects of GHB precursors GBL and 1,4-BD following i.c.v. administration in rats

Gamma-hydroxybutyrate (GHB) is used therapeutically and recreationally worldwide. Since the scheduling of GHB by the USA and the United Nations in 2000-2001, the recreational use of GHB precursors has reportedly increased. The aim of this study was to examine if potency differences of GHB and GHB-like compounds are due to their blood-brain barrier permeability. The effects of peripheral and central administration of GHB, GHB precursors gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD), and the gamma-aminobutyric acid (GABA)(B) receptor agonist baclofen on schedule-controlled responding were examined in rats. GHB and baclofen were 276- and 253-fold more potent, respectively, after intracerebroventricular (i.c.v.) administration than after intraperitoneal (i.p.) administration, whereas GBL and 1,4-BD, up to a dose of 1780 microg were without effect after i.c.v. administration. These data suggest that GBL and 1,4-BD are not metabolically converted to GHB in the brain, that enhanced brain penetration cannot account for potency differences between compounds, and that baclofen, like GHB, can readily cross the blood-brain barrier.     

γ-Hydroxybutyric acid-induced psychosis and seizures

Disulfiram and γ-hydroxybutyric acid (GHB) are used to treat alcohol dependence and may both increase dopamine brain levels and modulate GABAergic transmission. We describe a patient affected by bipolar disorder (on valproate as mood-stabilizing treatment) and alcohol dependence who developed a disulfiram-induced hypomanic episode and in whom the switch from disulfiram to GHB induced recurrent convulsive seizures, not responsive to treatment with diazepam, and psychosis. Seizures and psychiatric symptoms ceased after GHB discontinuation. We outline the deregulation of the neurotransmitter systems (GABAergic and dopaminergic networks) that are involved in these drug-drug interactions and that might be responsible for both psychosis and generalized tonic-clonic seizures resistant to standard treatments.     

gamma-Hydroxybutyric acid is not a GABA-mimetic agent in the spinal cord.

gamma-Hydroxybutyric acid (GHB), a pharmacologically active central nervous system constituent, has been postulated to function as a gamma-aminobutyric acid (GABA) agonist. This hypothesis was tested directly on GABAergic synapses in isolated, superfused frog spinal cord. Addition of GHB to the superfusate produced effects on primary afferent terminals that were distinctly different from the effects of GABA. Thus, although both compounds depressed dorsal root potentials, GHB hyperpolarized terminals while GABA depolarized the same structures. The GABA responses were antagonized by bicuculline and picrotoxin, but these alkaloids did not change GHB's actions. In addition, GHB altered neither high-affinity uptake by cord slices, nor potassium-evoked release of tritiated GABA from them. GHB did not directly release GABA from spinal slices preloaded with [3H]GABA. These observations suggest that the central nervous system actions of GHB are not dependent upon its ability to activate GABAergic synapses or to modify GABAergic mechanisms.     

Gamma hydroxybutyrate in the monkey. I. Electroencephalographic, behavioral, and pharmacokinetic studies.

Gamma hydroxybutyrate (GHB) was administered to adult and prepubescent rhesus monkeys intravenously in varying dosages while an electroencephalogram (EEG) was recorded from scalp electrodes and the body core temperature was monitored. Blood and cerebrospinal fluid samples were assayed for gamma hydroxybutyrate. GHB produced a trancelike stupor in all the monkeys, associated with marked EEG changes and hypothermia. There was a striking age specificity in that prepubescent rhesus monkeys responded to a lower threshold dosage, had a higher incidence of myoclonic jerking, and showed characteristic EEG changes not seen in the adult animals. The EEG-behavioral changes paralleled the hypothermia. There was good correlation between the serum levels of GHB and the EEG-behavioral effects. These studies suggest that the GHB-treated monkey may have utility as a petit mal seizure model.     

Gamma hydroxybutyrate in the monkey. II. Effect of chronic oral anticonvulsant drugs.

Gamma hydroxybutyrate (GHB) was administered intravenously to monkeys that had been pretreated orally for 2 weeks with various anticonvulsant drugs or with L-DOPA at different dosage levels. Continuous electroencephalographic (EEG) monitoring was performed during and after GHB administration. Bloood was assayed for GHB and for the anticonvulsant drug the animal was receiving. The EEG and behavioral changes produced by GHB were improved by ethosuximide and phenobarbital, made worse by phenytoin, and unchanged by L-DOPA.     

Gamma-butyrolactone (GBL) disruption of passive avoidance learning in the day-old chick appears to be due to its effect on GABAB not gamma-hydroxybutyric [corrected] acid (GHB) receptors

Gamma-butyrolactone (GBL) is a prodrug to gamma-hydroxybutyric acid (GHB) and metabolises to GHB when ingested. Discrimination stimulus studies report generalisation of effects of GHB to GBL. While amnesia is one of the most commonly reported symptoms of GHB's ingestion in human users, as yet few studies have examined this effect. Although an endogenous GHB specific receptor is present in the brain, several studies have indicated that the clinical effects of exogenous doses of GBL/GHB are due to its action on GABA(B) receptors rather than on the GHB receptor. In this series of studies, New Hampshire x White leghorn cockerels were trained using a modified version of the passive avoidance learning task. Subcutaneous injections of GBL induced a memory deficit by 10 min post-training, which persisted for at least 24 h. No effect on memory was seen with administration of the specific GHB agonist NCS-356 (gamma-p-chlorophenyl-trans-4-hydroxycrotonate). The GBL-induced memory deficit appeared similar to the deficit produced by baclofen, where the antagonist facilitated learning. Additionally, GBL-induced memory deficit was ameliorated by application of a GABA(B) antagonist. The results support the hypothesis that GBL exerts its influence on memory via the GABA(B) receptor rather than by the specific GHB receptor.     

Baclofen antagonises intravenous self-administration of gamma-hydroxybutyric acid in mice.

gamma-Hydroxybutyric acid (GHB) is a widely used recreational drug known to exert positive reinforcing effects in animals and humans. The GABA(B) receptor agonist baclofen has been proved to possess antimotivational effect and to inhibit alcohol, cocaine, heroin and nicotine intake. In the present study we evaluated the effect of baclofen on i.v. self-administration of GHB in drug-naive mice under a fixed-ratio (FR-1) schedule of reinforcement and nose-poking-like response as operandum. Results show that baclofen was able to completely prevent GHB seeking behaviour, decreasing the rate of responding to basal values, without showing any reinforcing properties when made contingent on nose-poking response. Our findings demonstrate that baclofen antagonises GHB i.v. self-administration, supporting an important role for the GABA(B) receptor in reward-related mechanisms underlying addictive behaviour.     

Recreational use of GHB is associated with alterations of resting state functional connectivity of the central executive and default mode networks

Gamma‐hydroxybutyrate acid (GHB) is a recreational drug with a high addictive potential. Severe side effects such as GHB‐induced coma are common and linked to increased emergency room attendances. Task‐based functional‐imaging studies have revealed an association between the regular use of GHB and multiple GHB‐induced comas, and altered neurocognitive function. However the effects of multiple GHB‐induced comas and regular GHB‐use on intrinsic brain connectivity during rest remain unknown. The study population consisted of 23 GHB‐users with ≥4 GHB‐induced comas (GHB‐Coma), 22 GHB‐users who never experienced a GHB‐induced coma (GHB‐NoComa) and 24 polydrug users who never used GHB (No‐GHB). Resting‐state scans were collected to assess resting‐state functional‐connectivity within and between the default mode network (DMN), the bilateral central executive network (CEN) and the salience network (SN). The GHB‐NoComa group showed decreased rsFC of the right CEN with a region in the anterior cingulate cortex (p_FWE = 0.048) and decreased rsFC between the right CEN and the DMN (p_FWE = 0.048) when compared with the No‐GHB group. These results suggest that regular GHB‐use is associated with decreased rsFC within the right CEN and between the right CEN and the DMN. The presence of multiple GHB‐induced comas is not associated with (additional) alterations in rsFC.     

Possible long-term effects of γ-hydroxybutyric acid (GHB) due to neurotoxicity and overdose

In several countries, including the Netherlands, the use of GHB seems to be rising. GHB is regarded by recreational users as an innocent drug without any side effects. Recently, the number of patients in treatment due to GHB addiction sharply increased. In addition, various studies report incidents following risky GHB use or GHB overdosing. Other sedative drugs, like ketamine and alcohol have been shown to result in unintended neurotoxic harm at the level of memory and cognitive function. As outlined in the present review, GHB and ketamine have a common mode of action, which suggests that GHB may also lead to similar neurotoxicity as ketamine. GHB overdosing, as well as binge drinking (and high ketamine doses), induce profound coma which is probably neurotoxic for the brain especially in the maturing brain of young adults. It is therefore advocated to investigate possible long-term neurotoxic effects in recreational GHB users e.g. by studying the residual effects on cognition and memory.     

Gamma-hydroxybutyrate--a neurotransmitter, medicine, and drug

Gamma-hydroxybutyrate (GHB) is a short fatty acid and physiologic neurotransmitter. Initially, it was synthesized as a GABA agonist and used as a narcotic agent, because it rapidly induces sleep without major cardiovascular or respiratory side effects. Recently, a specific GHB receptor was identified, but while the clinical use of GHB as an anaesthetic was reduced due to putative pro-convulsive effects, it now is used to treat alcohol withdrawal and sleep disorders. Furthermore, GHB was postulated to be a regulator of energy metabolism, and tissue-protective effects were demonstrated in different animal models. Besides its clinical use, GHB (also called "liquid ecstasy") is increasingly consumed in the disco scene because of its mild sedative and euphoric effects. Intoxication from GHB is common with GHB users. For this reason and because GHB is not easy to detect, it is important to be aware of the symptoms of GHB intoxication. Moreover, some recent case reports document the danger of GHB dependence.     

The effects of gamma-hydroxybutyrate on sleep.

Sodium gamma-hydroxybutyrate (GHB) is a remarkably safe and nontoxic hypnotic agent which is reported to be free of addicting properties. It is also a normal metabolite of the mammalian nervous system. We examined its effects on the sleep-EEG of eight patients with histories of impaired sleep, as a prelude to a more detailed study of its clinical potential. Sleep induced with GHB was indistinguishable subjectively from natural sleep as well as by behavioral and electroencephalographic criteria. Unlike most synthetic hypnotics, GHB increased delta sleep and did not suppress REM sleep. It shortened the REM sleep latency and shifted REM sleep into the first third of the night. On one occasion it induced a sleep onset REM period which was experienced as an attack of sleep paralysis. Withdrawal was simple; there was no REM sleep rebound and sleep patterns immediately returned to their pre-drug form. Its major clinical drawback was its short duration of action: its hypnotic effect lasting only 2 to 3 hr. We suggest that GHB may serve as the prototype for a new class of hypnotic compounds derived from natural sources and capable of activating the neurological mechanisms of normal human sleep.     

Specific gamma-hydroxybutyrate-binding sites but loss of pharmacological effects of gamma-hydroxybutyrate in GABA(B)(1)-deficient mice

gamma-Hydroxybutyrate (GHB), a metabolite of gamma-aminobutyric acid (GABA), is proposed to function as a neurotransmitter or neuromodulator. gamma-Hydroxybutyrate and its prodrug, gamma-butyrolactone (GBL), recently received increased public attention as they emerged as popular drugs of abuse. The actions of GHB/GBL are believed to be mediated by GABAB and/or specific GHB receptors, the latter corresponding to high-affinity [3H]GHB-binding sites coupled to G-proteins. To investigate the contribution of GABAB receptors to GHB actions we studied the effects of GHB in GABAB(1)-/- mice, which lack functional GABAB receptors. Autoradiography reveals a similar spatial distribution of [3H]GHB-binding sites in brains of GABAB(1)-/- and wild-type mice. The maximal number of binding sites and the KD values for the putative GHB antagonist [3H]6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene acetic acid (NCS-382) appear unchanged in GABAB(1)-/- compared with wild-type mice, demonstrating that GHB- are distinct from GABAB-binding sites. In the presence of the GABAB receptor positive modulator 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol GHB induced functional GTPgamma[35S] responses in brain membrane preparations from wild-type but not GABAB(1)-/- mice. The GTPgamma[35S] responses in wild-type mice were blocked by the GABAB antagonist [3-[[1-(S)-(3,4dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]-cyclohexylmethyl phosphinic acid hydrochloride (CGP54626) but not by NCS-382. Altogether, these findings suggest that the GHB-induced GTPgamma[35S] responses are mediated by GABAB receptors. Following GHB or GBL application, GABAB(1)-/- mice showed neither the hypolocomotion, hypothermia, increase in striatal dopamine synthesis nor electroencephalogram delta-wave induction seen in wild-type mice. It, therefore, appears that all studied GHB effects are GABAB receptor dependent. The molecular nature and the signalling properties of the specific [3H]GHB-binding sites remain elusive.     

Gamma hydroxybutyrate in the monkey. III. Effect of intravenous anticonvulsant drugs.

Monkeys were treated intravenously with various anticonvulsant drugs before and after the intravenous administration of gamma hydroxybutyrate (GHB). Continuous electroencephalographic (EEG) and temperature monitoring was performed throughout all experiments. The GHB-induced EEG changes were abolished by ethosuximide and clonazepam, marginally improved by diazepam, and unaffected by phenobarbital. The GHB-induced myoclonic jerks were abolished by ethosuximide, significantly improved by diazepam, and worsened by clonazepam. Phenobarbital was effective in diminishing the frequency of GHB-induced myoclonic jerks only when given prior to administration of GHB. The GHB-induced stupor was improved only by ethosuximide. The GHB model of petit mal seizures is quite specific for drugs used in this disorder. GHB may play a role in the pathogenesis of absence seizures in children.