Pharmacological evidence for GABAergic regulation of specific behaviors in Drosophila melanogaster

We have identified several GABAergic‐modulated behaviors in Drosophila melanogaster by employing a pharmacological approach to disrupt GABA transporter function in vivo. Systemic treatment of adult female flies with the GABA transport inhibitors DL ‐2,4‐diaminobutyric acid (DABA) or R,S‐nipecotic acid (NipA), resulted in diminished locomotor activity, deficits in geotaxis, and the induction of convulsive behaviors with a secondary loss of the righting reflex. Pharmacological evidence suggested that the observed behavioral phenotypes were specific to disruption of GABA transporter function and GABAergic activity. The effects of GABA reuptake inhibitors on locomotor activity were dose dependent, pharmacologically distinct, and paralleled their known effects in mammalian systems. Recovery of normal locomotor activity and the righting reflex in DABA‐ and NipA‐treated flies was achieved by coadministration of bicuculline (BIC), a GABA receptor antagonist that supresses GABAergic activity in mammals. Recovery of these behaviors was also achieved by coadministration of gabapentin, an anticonvulsant agent that interacts with mammalian GABAergic systems. Finally, behavioral effects were selective because other specific behaviors such as feeding activity and female sexual receptivity were not affected. Related pharmacological analyses performed in vitro on isolated Drosophila synaptic plasma membrane vesicles demonstrated high affinity, saturable uptake mechanisms for [³H]‐GABA; further competitive inhibition studies with DABA and NipA demonstrated their ability to inhibit [³H]‐GABA transport. The existence of experimentally accessible GABA transporters in Drosophila that share conserved pharmacological properties with their mammalian counterparts has resulted in the identification of specific behaviors that are modulated by GABA. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 245–261, 2002; DOI 10.1002/neu.10030     

GABAergic modulation of motor-driven behaviors in juvenile Drosophila and evidence for a nonbehavioral role for GABA transport

We have identified specific GABAergic-modulated behaviors in the juvenile stage of the fruit fly, Drosophila melanogaster via systemic treatment of second instar larvae with the potent GABA transport inhibitor DL-2,4-diaminobutyric acid (DABA). DABA significantly inhibited motor-controlled body wall and mouth hook contractions and impaired rollover activity and contractile responses to touch stimulation. The perturbations in locomotion and rollover activity were reminiscent of corresponding DABA-induced deficits in locomotion and the righting reflex observed in adult flies. The effects were specific to these motor-controlled behaviors, because DABA-treated larvae responded normally in olfaction and phototaxis assays. Recovery of these behaviors was achieved by cotreatment with the vertebrate GABA(A) receptor antagonist picrotoxin. Pharmacological studies performed in vitro with plasma membrane vesicles isolated from second instar larval tissues verified the presence of high-affinity, saturable GABA uptake mechanisms. GABA uptake was also detected in plasma membrane vesicles isolated from behaviorally quiescent stages. Competitive inhibition studies of [3H]-GABA uptake into plasma membrane vesicles from larval and pupal tissues with either unlabeled GABA or the transport inhibitors DABA, nipecotic acid, or valproic acid, revealed differences in affinities. GABAergic-modulation of motor behaviors is thus conserved between the larval and adult stages of Drosophila, as well as in mammals and other vertebrate species. The pharmacological studies reveal shared conservation of GABA transport mechanisms between Drosophila and mammals, and implicate the involvement of GABA and GABA transporters in regulating physiological processes distinct from neurotransmission during behaviorally quiescent stages of development.     

GABAergic modulation of motor‐driven behaviors in juvenile Drosophila and evidence for a nonbehavioral role for GABA transport

We have identified specific GABAergic‐modulated behaviors in the juvenile stage of the fruit fly, Drosophila melanogaster via systemic treatment of second instar larvae with the potent GABA transport inhibitor DL‐2,4‐diaminobutyric acid (DABA). DABA significantly inhibited motor‐controlled body wall and mouth hook contractions and impaired rollover activity and contractile responses to touch stimulation. The perturbations in locomotion and rollover activity were reminiscent of corresponding DABA‐induced deficits in locomotion and the righting reflex observed in adult flies. The effects were specific to these motor‐controlled behaviors, because DABA‐treated larvae responded normally in olfaction and phototaxis assays. Recovery of these behaviors was achieved by cotreatment with the vertebrate GABA_A receptor antagonist picrotoxin. Pharmacological studies performed in vitro with plasma membrane vesicles isolated from second instar larval tissues verified the presence of high‐affinity, saturable GABA uptake mechanisms. GABA uptake was also detected in plasma membrane vesicles isolated from behaviorally quiescent stages. Competitive inhibition studies of [³H]‐GABA uptake into plasma membrane vesicles from larval and pupal tissues with either unlabeled GABA or the transport inhibitors DABA, nipecotic acid, or valproic acid, revealed differences in affinities. GABAergic‐modulation of motor behaviors is thus conserved between the larval and adult stages of Drosophila, as well as in mammals and other vertebrate species. The pharmacological studies reveal shared conservation of GABA transport mechanisms between Drosophila and mammals, and implicate the involvement of GABA and GABA transporters in regulating physiological processes distinct from neurotransmission during behaviorally quiescent stages of development. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

A new alcohol antagonist: phaclofen

The ability of the GABA(B) receptor antagonist, phaclofen to alter behavioral effects of ethanol was evaluated by loss of righting reflex (sleep time), motor incoordination (bar holding), spontaneous locomotion (open field activity) and hypothermia. Pretreatment with phaclofen significantly decreased the effects of ethanol on motor incoordination, locomotor activity and hypothermia. However, phaclofen had no effect on either pentobarbitalor diazepam-induced motor incoordination. Phaclofen slightly increased the ED50 for loss of the righting reflex but did not alter either the duration of reflex loss produced by ethanol or blood ethanol levels at awakening. Our results suggest phaclofen is rapidly inactivated resulting in difficulty in observing antagonism of long duration ethanol effects. These findings suggest that the GABA(B) system may play a role in mediating several important actions of ethanol.     

The Caenorhabditis elegans snf-11 gene encodes a sodium-dependent GABA transporter required for clearance of synaptic GABA

Sodium-dependent neurotransmitter transporters participate in the clearance and/or recycling of neurotransmitters from synaptic clefts. The snf-11 gene in Caenorhabditis elegans encodes a protein of high similarity to mammalian GABA transporters (GATs). We show here that snf-11 encodes a functional GABA transporter; SNF-11-mediated GABA transport is Na+ and Cl- dependent, has an EC50 value of 168 microM, and is blocked by the GAT1 inhibitor SKF89976A. The SNF-11 protein is expressed in seven GABAergic neurons, several additional neurons in the head and retrovesicular ganglion, and three groups of muscle cells. Therefore, all GABAergic synapses are associated with either presynaptic or postsynaptic (or both) expression of SNF-11. Although a snf-11 null mutation has no obvious effects on GABAergic behaviors, it leads to resistance to inhibitors of acetylcholinesterase. In vivo, a snf-11 null mutation blocks GABA uptake in at least a subset of GABAergic cells; in a cell culture system, all GABA uptake is abolished by the snf-11 mutation. We conclude that GABA transport activity is not essential for normal GABAergic function in C. elegans and that the localization of SNF-11 is consistent with a GABA clearance function rather than recycling.     

Fixation and locomotor activity are impaired by inducing tetanus toxin expression in adult Drosophila brain

Visual fixation and locomotor activity are two important behavioral properties utilized by flies when they approach a landmark. Although previous studies in Drosophila have revealed that the mushroom bodies (Mbs) and the central complex (CC) were regulatory centers for these behaviors, the specific neurons involved still remain largely unknown. We tested visual fixation behavior and locomotor activity of flies in a simple choice assay, Buridan's paradigm, using the GAL4/UAS system to express tetanus toxin light chain (TeTxLC) in adult neurons specifically. Although we explored a variety of mushroom body and central complex-labeling lines, we found that only four GAL4 lines (104y-GAL4, 121y-GAL4, 154y-GAL4 and 210y-GAL4) could produce significant defects in fixation as well as decrease locomotor activity following adult induction of TeTxLC. This suggests a more complex circuit is involved in controlling these behaviors than previously thought. Expression patterns of the GAL4 lines in the central nervous system provide the some clues to which neurons might be involved in this neural circuit.     

Remote control of behavior through genetically targeted photostimulation of neurons

Optically gated ion channels were expressed in circumscribed groups of neurons in the Drosophila CNS so that broad illumination of flies evoked action potentials only in genetically designated target cells. Flies harboring the "phototriggers" in different sets of neurons responded to laser light with behaviors specific to the sites of phototrigger expression. Photostimulation of neurons in the giant fiber system elicited the characteristic escape behaviors of jumping, wing beating, and flight; photostimulation of dopaminergic neurons caused changes in locomotor activity and locomotor patterns. These responses reflected the direct optical activation of central neuronal targets rather than confounding visual input, as they persisted unabated in carriers of a mutation that eliminates phototransduction. Encodable phototriggers provide noninvasive control interfaces for studying the connectivity and dynamics of neural circuits, for assigning behavioral content to neurons and their activity patterns, and, potentially, for restoring information corrupted by injury or disease.     

Casein kinase I epsilon does not rescue double-time function in Drosophila despite evolutionarily conserved roles in the circadian clock

Double-time (dbt) is a casein kinase gene involved in cell survival, proliferation, and circadian rhythms in the fruit fly, Drosophila melanogaster. Genetic and biochemical studies have shown that dbt and its mammalian ortholog casein kinase I epsilon (hckI epsilon) regulate the circadian phosphorylation of period (per), thus controlling per subcellular localization and stability. Mutations in these kinases can shorten the circadian period in both mammals and Drosophila. Since similar activities in circadian clock have been described for these kinases, we investigated whether the expression of mammalian casein kinase I can replace the activity of dbt in flies. Global expression of the full-length dbt rescued lethality of the null mutant dbt revVIII and rescued flies showed normal locomotor activity rhythms. Global expression of dbt also restored the locomotor activity rhythm of the arrhythmic genotype, dbt ar/dbt revVIII. In contrast, global expression of hckI epsilon or hckI alpha did not rescue lethality or locomotor activity of dbt mutants. Furthermore dbt overexpression in wild-type clock cells had only a small effect on period length, whereas hckI epsilon expression in clock cells greatly lengthened period to ~30.5 hours and increased the number of arrhythmic flies. These results indicate that hckI epsilon cannot replace the activity of dbt in flies despite the high degree of similarity in primary sequence and kinase function. Moreover, expression of hck Iepsilon in flies appears to interfere with dbt activity. Thus, caution should be used in interpreting assays that measure activity of mammalian casein kinase mutants in Drosophila, or that employ vertebrate CKI in studies of dPER phosphorylations.     

The Rhythmic GABAergic System

GABA is the major inhibitory neurotransmitter in the mammalian brain, and has been implicated in the regulation of a variety of behavioral functions, including biological rhythms. The focus of this minireview is the rhythmic variation of the central GABAergic system, comprising fluctuations of GABA levels and turnover, GABA receptor affinity and postsynaptic activity on the chloride ionophore in rodent's brain. Neurochemical rhythms correlated with diurnal and circadian changes in several behaviors associated with the GABA_A receptor, e.g., anxiolysis-related behavior. GABA is considered to be the principal neurotransmitter of the mammalian circadian system, being present in the suprachiasmatic nuclei and the intergeniculate leaflet. Pharmacological manipulations of GABA_A receptors phase shift circadian rhythms and alter circadian responses to light. Administration of putative modulators of GABA function, like melatonin or neuroactive steroids, affects the timing of biological rhythms. Therefore, not only does the GABAergic system exhibit strong diurnal and circadian variations, but it also serves as one of the key modulators of the circadian apparatus.     

A role for the Drosophila fragile X-related gene in circadian output

Mutations that abolish expression of an X-linked gene, FMR1, result in the pathogenesis of fragile X syndrome, the most common form of inherited mental retardation. To understand the normal function of the FMR1 protein, we have produced fly strains bearing deletions in a Drosophila homolog of FMR1 (dfmr1). Since fragile X patients show a number of abnormal behaviors including sleep problems, we investigated whether a loss-of-function mutation of dfmr1 affect circadian behavior. Here we show that under constant darkness (DD), a lack of dfmr1 expression causes arrhythmic locomotor activity, but in light:dark cycles, their behavioral rhythms appear normal. In addition, the clock-controlled eclosion rhythm is normal in DFMR1-deficient flies. These results suggest that DFMR1 plays a critical role in the circadian output pathway regulating locomotor activity in Drosophila.     

Increased methamphetamine-induced locomotor activity and behavioral sensitization in histamine-deficient mice

We have recently suggested that the brain histamine has an inhibitory role on the behavioral effects of methamphetamine by pharmacological studies. In this study, we used the histidine decarboxylase gene knockout mice and measured the spontaneous locomotor activity, the changes of locomotion by single and repeated administrations of methamphetamine, and the contents of brain monoamines and amino acids at 1 h after a single administration of methamphetamine. In the histidine decarboxylase gene knockout mice, spontaneous locomotor activity during the dark period was significantly lower than in the wild-type mice. Interestingly, methamphetamine-induced locomotor hyperactivity and behavioral sensitization were facilitated more in the histidine decarboxylase gene knockout mice. In the neurochemical study, noradrenaline and O-phosphoserine were decreased in the midbrain of the saline-treated histidine decarboxylase gene knockout mice. On the other hand, single administration of methamphetamine decreased GABA content of the midbrain of the wild-type mice, but did not alter that of histidine decarboxylase gene knockout mice. These results suggest that the histamine neuron system plays a role as an awakening amine in concert with the noradrenaline neuron system, whereas it has an inhibitory role on the behavioral effects of methamphetamine through the interaction with the GABAergic neuron system.     

Amino Acid Neurotransmitters in the CNS: Properties of Diaminobutyric Acid Transport

Uptake of L-2,4-diaminobutyric acid (DABA), a positively charged analogue of gamma-aminobutyric acid (GABA), by a synaptosomal fraction isolated from rat brain occurred with a Km of 54 +/- 12 microM and a Vmax of 1.3 +/- 0.2 nmol/min/mg protein. The transport of DABA was inhibited competitively by GABA whereas that of GABA was affected in the same manner by addition of DABA. The maximal accumulation of DABA ([DABA]i/[DABA]c) was observed to increase as the second power of the transmembrane electrical potential ([K+]i/[K+]e) and the first power of the sodium ion concentration gradient. These findings indicate that DABA is transported on the GABA carrier with a net charge of +2, where one charge is provided by the cotransported Na+ and the second is contributed by the amino acid itself. Since uptake of GABA, an electroneutral molecule, is accompanied by transfer of two sodium ions, the results obtained with DABA suggest that one of the sodium binding sites on the GABA transporter is in proximity to the amino acid binding site.     

Drosophila pacemaker neurons require g protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms

Intercellular signaling is important for accurate circadian rhythms. In Drosophila, the small ventral lateral neurons (s-LN(v)s) are the dominant pacemaker neurons and set the pace of most other clock neurons in constant darkness. Here we show that two distinct G protein signaling pathways are required in LN(v)s for 24?hr rhythms. Reducing signaling in LN(v)s via the G alpha subunit Gs, which signals via cAMP, or via the G alpha subunit Go, which we show signals via Phospholipase 21c, lengthens the period of behavioral rhythms. In contrast, constitutive Gs or Go signaling makes most flies arrhythmic. Using dissociated LN(v)s in culture, we found that Go and the metabotropic GABA(B)-R3 receptor are required for the inhibitory effects of GABA on LN(v)s and that reduced GABA(B)-R3 expression in?vivo lengthens period. Although no clock neurons produce GABA, hyperexciting GABAergic neurons disrupts behavioral rhythms and s-LN(v) molecular clocks. Therefore, s-LN(v)s require GABAergic inputs for 24?hr rhythms.     

Sequence and chromosomal assignment of a human novel cDNA: similarity to gamma-aminobutyric acid transporter

Gamma-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the mammalian brain. Although initially thought to be confined to the central nervous system, GABAergic activity has also been described in other tissues throughout the body. In the present study, we report the cloning and localization of human GABA transporter cDNA and document its expression in various human tissues. A human liver cDNA library was initially screened by a 32P-labeled murine brain GABA transporter 3 (GAT-3) cDNA probe, and full-length cDNA was cloned by employing Marathon-Ready human kidney cDNA. The human GABA transporter cDNA encoded a 569 amino acid hydrophobic protein with 12 transmembrane domains (TMs). Search of published sequences revealed high homology with rat GAT-2, murine GAT-3 cDNA, human solute carrier family 6 member 13 (SLC6A13), and a human peripheral betaine/GABA transporter. Northern blot analyses demonstrated that the human GABA transporter is expressed strongly in the kidney and to a lesser extent in the liver and brain. The sequence was well matched with human chromosome 12p13.3, suggesting the human GABA transporter contains 14 exons. The above findings confirm the existence of and further characterize a specific GABA transporter in human tissues.     

Electrophysiological correlates of rest and activity in Drosophila melanogaster

Extended periods of rest in Drosophila melanogaster resemble mammalian sleep states in that they are characterized by heightened arousal thresholds and specific alterations in gene expression. Defined as inactivity periods spanning 5 or more min, amounts of this sleep-like state are, as in mammals, sensitive to prior amounts of waking activity, time of day, and pharmacological intervention. Clearly recognizable changes in the pattern and amount of brain electrical activity accompany changes in motor activity and arousal thresholds originally used to identify mammalian sleeping behavior. Electroencephalograms (EEGs) and/or local field potentials (LFPs) are now widely used to quantify sleep state amounts and define types of sleep. Thus, slow-wave sleep (SWS) is characterized by EEG spindles and large-amplitude delta-frequency (0-3.5 Hz) waves. Rapid-eye movement (REM) sleep is characterized by irregular gamma-frequency cortical EEG patterns and rhythmic theta-frequency (5-9 Hz) hippocampal EEG activity. It is unknown whether rest and activity in Drosophila are associated with distinct electrophysiological correlates. To address this issue, we monitored motor activity levels and recorded LFPs in the medial brain between the mushroom bodies, structures implicated in the modulation of locomotor activity, of Drosophila. The results indicate that LFPs can be reliably recorded from the brains of awake, moving fruit flies, that targeted genetic manipulations can be used to localize sources of LFP activity, and that brain electrical activity of Drosophila is reliably correlated with activity state.     

GABA transporters inDrosophila melanogaster: molecular cloning, behavior, and physiology

Molecular cloning of GABA transporter-homologous cDNAs from aDrosophila melanogaster headspecific library was accomplished using a conserved oligomer from a highly conserved domain within the mammalian GABA transporters. Partial DNA sequencing of these cDNAs demonstrated homology with the mammalian transporters, indicating these are ancient, evolutionarily conserved molecules. Although theDrosophila cDNAs had distinct restriction enzyme patterns, they recognized the same locus inDrosophila genomic DNA, suggesting that the multiple isoforms might arise via alternative splicing. Antibodies specific for the mammalian GABA transporters GAT1, GAT2 and GAT3 recognized non-overlapping and developmentally distinct patterns of expression inDrosophila neuronal tissues. Treatment of larval instars with nipecotic acid, a generalized GABA reuptake inhibitor, revealed specific, dose-dependent alterations in behavior consistent with the presence of multiple transporter molecules with differing affinities for this drug. Synaptic current recordings revealed that nipecotic acid treated larvae have an increase in latency jitter of evoked quantal release, resulting in a broader average excitatory junctional current which was manifested in a broader EJP. These results imply that alterations in the development of the CNS occur if GABAergic neurotransmission is protentiated during development. The data suggest that, as in mammals, there are multiple GABA transporters inDrosophila whose expression is differentially regulated.     

褪黑素受体与GABAA受体在褪黑素延长小鼠睡眠时间中的作用

目的：研究褪黑素受体和GABAA受体在褪黑素延长小鼠睡眠时间中的作用。方法：以翻正反射消失为睡眠开始的指标,至翻正反射恢复作为睡眠时间。观察不同受体激动剂或拮抗剂对褪黑素催眠作用的影响。结果：褪黑素3型受体拮抗剂盐酸哌唑嗪对褪黑素延长小鼠睡眠时间的作用无明显影响。GABA受体内源性激动剂GABA能明显增强褪黑素延长小鼠睡眠时间的作用,而GABAA受体上的印防己毒素结合位点的配基,即氯离子通道阻断剂印防己毒素能明显拮抗褪黑素的催眠作用,GABAA受体上的GABA结合位点的拮抗剂荷包牡丹碱则对褪黑素延长小鼠睡眠作用无明显影响。结论：褪黑素延长小鼠睡眠时间的作用与褪黑素3型受体无关,而与GABAA受体关系密切,其作用主要由印防己毒素结合位点介导。     

GABA Transporter-1 Deficiency Confers Schizophrenia-Like Behavioral Phenotypes

The mechanism underlying the pathogenesis of schizophrenia remains poorly understood. The hyper-dopamine and hypo-NMDA receptor hypotheses have been the most enduring ideas. Recently, emerging evidence implicates alterations of the major inhibitory system, GABAergic neurotransmission in the schizophrenic patients. However, the pathophysiological role of GABAergic system in schizophrenia still remains dubious. In this study, we took advantage of GABA transporter 1 (GAT1) knockout (KO) mouse, a unique animal model with elevated ambient GABA, to study the schizophrenia-related behavioral abnormalities. We found that GAT1 KO mice displayed multiple behavioral abnormalities related to schizophrenic positive, negative and cognitive symptoms. Moreover, GAT1 deficiency did not change the striatal dopamine levels, but significantly enhanced the tonic GABA currents in prefrontal cortex. The GABA_A receptor antagonist picrotoxin could effectively ameliorate several behavioral defects of GAT1 KO mice. These results identified a novel function of GAT1, and indicated that the elevated ambient GABA contributed critically to the pathogenesis of schizophrenia. Furthermore, several commonly used antipsychotic drugs were effective in treating the locomotor hyperactivity in GAT1 KO mice, suggesting the utility of GAT1 KO mice as an alternative animal model for studying schizophrenia pathogenesis and developing new antipsychotic drugs.     

EVIDENCE FOR THE HETEROGENEITY OF L-2,4-DIAMINOBUTYRIC ACID UPTAKE IN RAT CORTEX

The uptake of L-[³H]DABA by rat cerebral cortex slices was studied. Analysis of the kinetic data obtained provides evidence that DABA entry is mediated by both high and low affinity carriers. When cortical slices were incubated in the presence of equimolar [³H]DABA and [¹⁴C]GABA the ratio of entry of the two radionuclides was found to depend upon the loading concentration. The specificity of the uptake of 1 μM and 1 mM-L-DABA was examined: GABA and DABA were relatively potent inhibitors of 1 μM-DABA uptake whereas an equal concentration of histidine did not produce significant inhibition. In contrast, DABA and histidine were markedly more potent as inhibitors of 1 mM-DABA uptake than was GABA. It is concluded from these experiments that L-DABA is transported into cortical slices by a carrier which has high affinities for both DABA and GABA and by a second lower affinity carrier which prefers DABA as a substrate to GABA. On the basis of a comparison of the effects of inhibitors on [³H]DABA and [³H]GABA uptake it is estimated that approx 26% of DABA uptake at 1 μM does not occur by the high affinity carrier whereas at 1 mM-DABA this proportion rises to 62–67%.     

Evidence for a gamma-hydroxybutyrate (GHB) uptake by rat brain synaptic vesicles

gamma-Hydroxybutyrate (GHB) is an endogenous metabolite of mammalian brain which is derived from GABA. Much evidence favours its role as an endogenous neuromodulator, synthesized, stored and released at particular synapses expressing specific receptors. One key step for GHB involvement in neurotransmission is its uptake by a specific population of synaptic vesicles. We demonstrate that this specific uptake exists in a crude synaptic vesicle pool obtained from rat brain. The kinetic parameters and the pharmacology of this transport are in favour of an active vesicular uptake system for GHB via the vesicular inhibitory amino acid transporter. This result supports the idea that GABA and GHB accumulate together and are coliberated in some GABAergic synapses of the rat brain, where GHB acts as a modulatory factor for the activity of these synapses following stimulation of specific receptors.