Fear conditioning selectively disrupts noradrenergic facilitation of GABAergic inhibition in the basolateral amygdala.

Inappropriate fear memory formation is symptomatic of many psychopathologies, and delineating the neurobiology of non-pathological fear learning may provide critical insight into treating these disorders. Fear memory formation is associated with decreased inhibitory signaling in the basolateral amygdala (BLA), and disrupted noradrenergic signaling may contribute to this decrease. BLA noradrenergic neurotransmission has been implicated in fear memory formation, and distinct adrenoreceptor (AR) subtypes modulate excitatory and inhibitory neurotransmission in this region. For example, α1-ARs promote GABA release from local inhibitory interneurons, while ß3-ARs potentiate neurotransmission at lateral paracapsular (LPC) GABAergic synapses. Conversely, ß1/2-ARs amplify excitatory signaling at glutamatergic synapses in the BLA. As increased BLA excitability promotes fear memory formation, we hypothesized that fear learning shifts the balanced regional effects of noradrenergic signaling toward excitation. To test this hypothesis, we used the fear-potentiated startle paradigm in combination with whole cell patch clamp electrophysiology to examine the effects of AR activation on BLA synaptic transmission following fear conditioning in male Long-Evans rats. We first demonstrated that inhibitory neurotransmission is decreased at both local and LPC synapses following fear conditioning. We next measured noradrenergic facilitation of BLA inhibitory signaling at local and LPC synapses using α1-and ß3-AR agonists (1 µM A61603 and 10 µM BRL37344), and found that the ability of these agents to facilitate inhibitory neurotransmission is disrupted following fear conditioning. Conversely, we found that fear learning does not disrupt noradrenergic modulation of glutamatergic signaling via a ß1/2-AR agonist (1 µM isoproterenol). Taken together, these studies suggest that fear learning increases BLA excitability by selectively disrupting the inhibitory effects of noradrenaline.

Neurobiology of comorbid post-traumatic stress disorder and alcohol-use disorder.

Post-traumatic stress disorder (PTSD) and alcohol-use disorder (AUD) are highly comorbid in humans. Although we have some understanding of the structural and functional brain changes that define each of these disorders, and how those changes contribute to the behavioral symptoms that define them, little is known about the neurobiology of comorbid PTSD and AUD, which may be due in part to a scarcity of adequate animal models for examining this research question. The goal of this review is to summarize the current state-of-the-science on comorbid PTSD and AUD. We summarize epidemiological data documenting the prevalence of this comorbidity, review what is known about the potential neurobiological basis for the frequent co-occurrence of PTSD and AUD and discuss successes and failures of past and current treatment strategies. We also review animal models that aim to examine comorbid PTSD and AUD, highlighting where the models parallel the human condition, and we discuss the strengths and weaknesses of each model. We conclude by discussing key gaps in our knowledge and strategies for addressing them: in particular, we (1) highlight the need for better animal models of the comorbid condition and better clinical trial design, (2) emphasize the need for examination of subpopulation effects and individual differences and (3) urge cross-talk between basic and clinical researchers that is reflected in collaborative work with forward and reverse translational impact.

Behavioral and neurophysiological evidence that lateral paracapsular GABAergic synapses in the basolateral amygdala contribute to the acquisition and extinction of fear learning.

The lateral/basolateral amygdala (BLA) is crucial to the acquisition and extinction of Pavlovian fear conditioning, and synaptic plasticity in this region is considered to be a neural correlate of learned fear. We recently reported that activation of BLA ß3-adrenoreceptors (ß3-ARs) selectively enhances lateral paracapsular (LPC) feed-forward GABAergic inhibition onto BLA pyramidal neurons, and that intra-BLA infusion of a ß3-AR agonist reduces measures of unconditioned anxiety-like behavior. Here, we utilized a combination of behavioral and electrophysiological approaches to characterize the role of BLA LPCs in the acquisition of fear and extinction learning in adult male Long-Evans rats. We report that intra-BLA microinjection of ß3-AR agonists (BRL37344 or SR58611A, 1µg/0.5µL/side) prior to training fear conditioning or extinction blocks the expression of these behaviors 24h later. Furthermore,ex vivo low-frequency stimulation of the external capsule (LFS; 1Hz, 15min), which engages LPC synapses, induces LTP of BLA fEPSPs, while application of a ß3-AR agonist (SR58611A, 5µM) induces LTD of fEPSPs when combined with LFS. Interestingly, fEPSP LTP is not observed in recordings from fear conditioned animals, suggesting that fear learning may engage the same mechanisms that induce synaptic plasticity at this input. In support of this, we find that LFS produces LTD of inhibitory postsynaptic currents (iLTD) at LPC GABAergic synapses, and that this effect is also absent following fear conditioning. Taken together, these data provide preliminary evidence that modulation of LPC GABAergic synapses can influence the acquisition and extinction of fear learning and related synaptic plasticity in the BLA.

Adolescent social isolation increases anxiety-like behavior and ethanol intake and impairs fear extinction in adulthood: Possible role of disrupted noradrenergic signaling.

Alcohol use disorder, anxiety disorders, and post-traumatic stress disorder (PTSD) are highly comorbid, and exposure to chronic stress during adolescence may increase the incidence of these conditions in adulthood. Efforts to identify the common stress-related mechanisms driving these disorders have been hampered, in part, by a lack of reliable preclinical models that replicate their comorbid symptomatology. Prior work by us, and others, has shown that adolescent social isolation increases anxiety-like behaviors and voluntary ethanol consumption in adult male Long-Evans rats. Here we examined whether social isolation also produces deficiencies in extinction of conditioned fear, a hallmark symptom of PTSD. Additionally, as disrupted noradrenergic signaling may contribute to alcoholism, we examined the effect of anxiolytic medications that target noradrenergic signaling on ethanol intake following adolescent social isolation. Our results confirm and extend previous findings that adolescent social isolation increases anxiety-like behavior and enhances ethanol intake and preference in adulthood. Additionally, social isolation is associated with a significant deficit in the extinction of conditioned fear and a marked increase in the ability of noradrenergic therapeutics to decrease ethanol intake. These results suggest that adolescent social isolation not only leads to persistent increases in anxiety-like behaviors and ethanol consumption, but also disrupts fear extinction, and as such may be a useful preclinical model of stress-related psychopathology. Our data also suggest that disrupted noradrenergic signaling may contribute to escalated ethanol drinking following social isolation, thus further highlighting the potential utility of noradrenergic therapeutics in treating the deleterious behavioral sequelae associated with early life stress.

Exploring the Neurochemical Basis of Alcohol Addiction-Related Behaviors: Translational Research.

This Editorial presents the position that translational research continues to play a vital role in the field of alcohol addiction research. Using diverse animal models that mimic fundamental features of the disease, tremendous progress has been made in our understanding of alcohol actions in the brain and in identifying key neurobiological adaptations that may contribute to the pathophysiology of alcohol addiction. Current translational research in this field is now focusing on identifying the causal mechanisms that drive the shift from recreational to abusive ethanol drinking behaviors. The relatively recent development and application of optogenetic and chemogenetic techniques is beginning to afford alcohol researchers with the opportunity to identify specific neuronal circuits that govern key elements of the addiction process. These advances are rapidly pointing the way toward novel neural targets for the development of more effective treatments for addictive disorders.

Effect of ß3 adrenoceptor activation in the basolateral amygdala on ethanol seeking behaviors.

RATIONALE: The interaction between ethanol (EtOH) and anxiety plays an integral role in the development and maintenance of alcoholism. Many medications in pre-clinical or clinical trials for the treatment of alcoholism share anxiolytic properties. However, these drugs typically have untoward side effects, such as sedation or impairment of motor function that may limit their clinical use. We have recently demonstrated that BRL 37344 (BRL), a selective ß3-adrenoceptor (AR) agonist, enhances a discrete population of GABAergic synapses in the basolateral amygdala (BLA) that mediates feed-forward inhibition from lateral paracapsular (LPC) GABAergic interneurons onto BLA pyramidal cells. Behavioral studies revealed that intra-BLA infusion of BRL significantly reduced measures of unconditioned anxiety-like behavior without locomotor depressant effects. OBJECTIVES: The present studies tested the effect of BRL (0.1, 0.5, or 1.0 µg/side) on EtOH self-administration using an intermittent access home cage two-bottle choice procedure and limited access operant responding for EtOH or sucrose. RESULTS: Intra-BLA infusion of BRL did not reduce home cage, intermittent EtOH self-administration. However, using an operant procedure that permits the discrete assessment of appetitive (seeking) and consummatory measures of EtOH self-administration, BRL reduced measures of EtOH and sucrose seeking, but selectively reduced operant responding for EtOH during extinction probe trials. BRL had no effect on consummatory behaviors for EtOH or sucrose. CONCLUSIONS: Together, these data suggest that intra-BLA infusion of BRL significantly reduces motivation to seek EtOH and provide initial evidence that ß3-ARs and LPC GABAergic synapses may represent promising targets for the development of novel pharmacotherapies for the treatment of alcoholism.

Ethanol inhibition of kainate receptor-mediated excitatory neurotransmission in the rat basolateral nucleus of the amygdala.

The neurobiological mechanisms governing alcohol-induced alterations in anxiety-like behaviors are not fully understood. Given that the amygdala is a major emotional center in the brain and regulates the expression of both learned fear and anxiety, neurotransmitter systems within the basolateral amygdala represent likely mechanisms governing the anxiety-related effects of acute ethanol exposure. It is well established that, within the glutamatergic system, N-methyl-d-aspartate (NMDA)-type receptors are particularly sensitive to intoxicating concentrations of ethanol. However, recent evidence suggests that kainate-type glutamate receptors are sensitive to ethanol as well. Therefore, we examined the effect of acute ethanol on kainate receptor (KA-R)-mediated synaptic transmission in the basolateral amygdala (BLA) of Sprague-Dawley rats. Acute ethanol decreased KA-R-mediated excitatory postsynaptic currents (EPSCs) in the BLA in a concentration-dependent manner. Ethanol also inhibited currents evoked by focal application of the kainate receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), and ethanol inhibition of kainate EPSCs was not associated with a change in paired-pulse ratio, suggesting a postsynaptic mechanism of ethanol action. The neurophysiological consequences of this acute sensitivity were tested by measuring ethanol's effects on KA-R-dependent modulation of synaptic plasticity. Acute ethanol, like the GluR5-specific antagonist (R,S)-3-(2-carboxybenzyl)willardiine (UBP 296), robustly diminished ATPA-induced increases in synaptic efficacy. Lastly, to better understand the relationship between KA-R activity and anxiety-like behavior, we bilaterally microinjected ATPA directly into the BLA. We observed an increase in measures of anxiety-like behavior, assessed in the light/dark box, with no change in locomotor activity. This evidence suggests that kainate receptors in the BLA are inhibited by pharmacologically relevant concentrations of ethanol and may contribute to some of the acute anxiolytic effects of this drug.

Distinct mechanisms of ethanol potentiation of local and paracapsular GABAergic synapses in the rat basolateral amygdala.

Converging lines of behavioral and pharmacological evidence suggest that GABAergic synapses in the basolateral amygdala (BLA) may play an integral role in mediating the anxiolytic effects of ethanol (EtOH). Since anxiety is thought to play an important role in the development of, and relapse to, alcoholism, elucidating the mechanisms through which EtOH modulates GABAergic synaptic transmission in the BLA may be fundamental in understanding the etiology of this disease. A recent study in mice has shown that principal cells within the BLA receive inhibitory input from two distinct types of GABAergic interneurons: a loosely distributed population of local interneurons and a dense network of paracapsular (pcs) GABAergic cells clustered along the external capsule border. Here, we sought to confirm the presence of these two populations of GABAergic synapses in the rat BLA and evaluate their ethanol sensitivity. Our results suggest that rat BLA pyramidal cells receive distinct inhibitory input from local and pcs interneurons and that EtOH potentiates both populations of synapses, albeit via distinct mechanisms. EtOH enhancement of local inhibitory postsynaptic currents (IPSCs) was associated with a significant decrease in paired-pulse ratio (PPR) and was significantly potentiated by the GABA(B) receptor antagonist SCH 50911 [(+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid], consistent with a facilitation of GABA release from presynaptic terminals. Conversely, EtOH enhancement of pcs IPSCs did not alter PPR and was not enhanced by SCH 50911 but was inhibited by blockade of noradrenergic receptors. Collectively, these data reveal that EtOH can potentiate GABAergic inhibitory synaptic transmission in the rat BLA through at least two distinct pathways.

Ethanol modulation of GABAergic transmission: the view from the slice.

For almost three decades now, the GABAergic synapse has been the focus of intense study for its putative role in mediating many of the behavioral consequences associated with acute and chronic ethanol exposure. Although it was initially thought that ethanol interacted solely with the postsynaptic GABAA receptors that mediate the majority of fast synaptic inhibition in the mammalian central nervous system (CNS), a number of recent studies have identified novel pre- and postsynaptic mechanisms that may contribute to the acute and long-term effects of ethanol on GABAergic synaptic inhibition. These mechanisms appear to differ in a brain region specific manner and may also be influenced by a variety of endogenous neuromodulatory factors. This article provides a focused review of recent evidence, primarily from in vitro brain slice electrophysiological studies, that offers new insight into the mechanisms through which acute and chronic ethanol exposures modulate the activity of GABAergic synapses. The implications of these new mechanistic insights to our understanding of the behavioral and cognitive effects of ethanol are also discussed.

Growth hormone treatment attenuates age-related changes in hippocampal short-term plasticity and spatial learning.

Downregulation of the growth hormone/insulin-like growth factor-1 (IGF-1)axis is one of the most robust biomarkers of mammalian aging. Reports have suggested that age-related changes in secretion of growth hormone and IGF-1 contribute to the development of some peripheral characteristics of the aged phenotype including decreased bone density and lean body mass. Recent work has focused on the identification of a role for age-related reductions in growth hormone and IGF-1 in the development of cognitive impairments associated with aging. In the current study, we report that aged (30 month-old) Brown Norway x Fisher rats demonstrate impairments in spatial learning compared with adult (10 month-old) animals, and that 4-month treatment with growth hormone (300 microg twice daily) attenuates age-related learning impairments. After 6 months of treatment, we employed an extracellular paired-pulse protocol to investigate age-related changes in hippocampal short-term plasticity, and found that aged rats exhibit significantly increased paired-pulse ratios (PPRs) at an interpulse interval of 50 ms compared with adult rats. Long-term growth hormone administration restored PPRs in aged animals to values comparable to those observed in adult controls. Since the age-related changes observed in PPR may result from decreases in hippocampal inhibitory tone mediated by GABA(A) receptors, we assessed GABA(A) receptor subunit expression by immunoblot analysis. Data revealed significant age-related decreases in GABA(A) receptor alpha-1 subunit expression which were attenuated by growth hormone treatment. However, hippocampal levels of the gamma2 subunit, glutamic acid decarboxylase (GAD)(65), and GAD(67) protein concentrations were not significantly affected by age or growth hormone treatment. In conclusion, we suggest that age-related decreases in growth hormone and IGF-1 contribute to cognitive decline, in part, via alterations in hippocampal short-term plasticity. Changes in plasticity may reflect a shift in the balance of hippocampal inhibitory and excitatory function.

Ethanol modulation of excitatory and inhibitory synaptic transmission in rat and monkey dentate granule neurons.

BACKGROUND: The physiological mechanisms underlying the behavioral and cognitive effects of ethanol are not fully understood. However, there is now compelling evidence that ethanol acts, at least in part, by modulating the function of a small group of proteins that mediate excitatory and inhibitory synaptic transmission. For example, intoxicating concentrations of ethanol have been shown to enhance GABAergic synaptic inhibition and depress glutamatergic excitatory neurotransmission in a number of brain regions. Because all of these electrophysiological studies have been performed in rodent brain slice or neuronal culture preparations, direct evidence that ethanol exerts similar effects on synaptic transmission in the primate central nervous system is lacking. METHODS: We have therefore developed methods to perform patch-clamp electrophysiological recordings from neurons in acutely prepared monkey (Macaca fascicularis) hippocampal slices. We have used these methods to compare the acute effects of ethanol on excitatory and inhibitory synaptic transmission in rat and monkey dentate granule neurons. RESULTS: Under our recording conditions, ethanol significantly potentiated gamma-aminobutyric acid type A inhibitory postsynaptic currents in both rat and monkey neurons. In addition, ethanol significantly inhibited NMDA, but not AMPA, excitatory postsynaptic currents in dentate granule neurons from both species. Notably, no significant differences were observed in any of the pharmacological properties of inhibitory or excitatory synaptic responses recorded from rat and monkey neurons. CONCLUSIONS: These data suggest that the differences in the behavioral effects of ethanol that have been observed between rats and higher-order mammals, such as monkeys and humans, may not reflect differences in the sensitivity of some of the major synaptic sites of ethanol action. Moreover, our results provide empirical evidence for the use of rodent brain slice preparations in elucidating synaptic mechanisms of ethanol action in the primate central nervous system.

Ethanol antagonizes kainate receptor-mediated inhibition of evoked GABA(A) inhibitory postsynaptic currents in the rat hippocampal CA1 region.

Many studies have demonstrated that ethanol reduces glutamatergic synaptic transmission primarily by inhibiting the N-methyl-D-aspartate subtype of glutamate receptor. In contrast, the other two subtypes of ionotropic glutamate receptor (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and kainate) have generally been shown to be insensitive to intoxicating concentrations of ethanol. However, we have previously identified a population of kainate receptors that mediate slow excitatory postsynaptic currents in the rat hippocampal CA3 pyramidal cell region that is potently inhibited by low concentrations of ethanol. In this study, we examined the effect of ethanol on kainate receptor-mediated inhibition of evoked GABA(A) inhibitory postsynaptic currents (IPSCs) in the rat hippocampal CA1 pyramidal cell region. Under our recording conditions, bath application of 1 microM kainate significantly inhibited GABA(A) IPSCs. This inhibition seemed to be mediated by the activation of somatodendritic kainate receptors on GABAergic interneurons and the subsequent activation of metabotropic GABA(B) receptors, because the kainate inhibition was largely blocked by pretreating slices with a GABA(B) receptor antagonist. Ethanol pretreatment significantly antagonized the inhibitory effect of kainate on GABA(A) IPSCs, at concentrations as low as 20 mM. In contrast, ethanol did not block the direct inhibitory effect of a GABA(B) receptor agonist on GABA(A) IPSCs. The results of this study suggest that modest concentrations of ethanol may antagonize presynaptic, as well as postsynaptic, kainate receptor function in the rat hippocampus.

Glycine and gamma-aminobutyric acid(A) receptor function is enhanced by inhaled drugs of abuse.

Inhalable solvents possess significant abuse liability and produce many of the neurobehavioral effects typically associated with central nervous system-depressant agents, including motor incoordination, anxiolysis, and the elicitation of signs of physical dependence on withdrawal. We tested the hypothesis that the commonly abused solvents toluene, 1,1,1-trichloroethane (TCE), and trichloroethylene (TCY) affect ligand-gated ion channel activity, as do other classes of central nervous system-depressive agents. TCE and toluene, like ethanol, reversibly enhanced gamma-aminobutyric acid (GABA)(A) receptor-mediated synaptic currents in rat hippocampal slices. All three inhalants significantly and reversibly enhanced neurotransmitter-activated currents at alpha1beta1 GABA(A) and alpha1 glycine receptors expressed in Xenopus oocytes. We previously identified specific amino acids of glycine and GABA(A) receptor subunits mediating alcohol and volatile anesthetic enhancement of receptor function. Toluene, TCE, and TCY were tested on several glycine receptor mutants, some of which were insensitive to ethanol and/or enflurane. Toluene and TCY enhancement of glycine receptor function was seen in all these mutants. However, the potentiating effects of TCE were abolished in three mutants and enhanced in two, a pattern more akin to that seen with enflurane than ethanol. These data suggest that inhaled drugs of abuse affect ligand-gated ion channels, and that the molecular sites of action of these compounds may overlap with those of ethanol and the volatile anesthetics.

Ethanol inhibition of synaptically evoked kainate responses in rat hippocampal CA3 pyramidal neurons.

Many studies have demonstrated that intoxicating concentrations of ethanol (10-100 mM) can selectively inhibit the component of glutamatergic synaptic transmission mediated by N-methyl-D-aspartate (NMDA) receptors while having little or no effect on excitatory synaptic transmission mediated by non-NMDA receptors [i.e., alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and/or kainate (KA) receptors]. However, until the recent development of highly selective AMPA receptor antagonists, it was not possible to assess the relative contribution of AMPA and KA receptors to non-NMDA receptor-mediated synaptic transmission or to determine whether these glutamate receptor subtypes differed in their sensitivity to ethanol. In the present experiments, we used the highly selective AMPA receptor antagonist LY 303070 to pharmacologically isolate KA receptor-mediated excitatory postsynaptic currents (EPSCs) in rat hippocampal CA3 pyramidal neurons and tested their sensitivity to ethanol. Concentrations of ethanol as low as 20 mM significantly and reversibly depressed KA EPSCs. Ethanol also inhibited KA currents evoked by direct pressure application of KA in the presence of LY 303070, suggesting that this inhibition was mediated by a postsynaptic action. In contrast, ethanol had no effect on AMPA EPSCs in these cells, even at the highest concentration tested (80 mM). Ethanol significantly inhibited NMDA EPSCs in these neurons, but these responses were less sensitive to ethanol than KA EPSCs. These results suggest that in addition to its well-described depressant effect on NMDA receptor-mediated synaptic transmission, ethanol has an even greater inhibitory effect on glutamatergic synaptic transmission mediated by KA receptors in rat hippocampal CA3 pyramidal neurons.

Synaptic potentials mediated via alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in rat hippocampal interneurons.

Exogenous application of acetylcholine elicits inward currents in hippocampal interneurons that are mediated via alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors, but synaptic responses mediated via such receptors have never been reported in mammalian brain. In the present study, EPSCs were evoked in hippocampal interneurons in rat brain slices by electrical stimulation and were recorded by using whole-cell voltage-clamp techniques. Nicotinic EPSCs were isolated pharmacologically, using antagonists to block other known types of ligand-gated ion channels, and then were tested with either alpha-bungarotoxin or methyllycaconitine, which are selective antagonists for nicotinic acetylcholine receptors that contain the alpha7 receptor subunit. Each antagonist proved highly effective at reducing the remaining synaptic current. Evoked alpha7-mediated nicotinic EPSCs also were desensitized by superfusion with 1 microM nicotine, had extrapolated reversal potentials near 0 mV, and showed strong inward rectification at positive potentials. In several interneurons, methyllycaconitine-sensitive spontaneous EPSCs also were observed that exhibited a biphasic decay rate very similar to that of the alpha7-mediated evoked response. These studies provide the first demonstration of a functional cholinergic synapse in the mammalian brain, in which the primary postsynaptic receptors are alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors.

Effects of ethanol on recombinant glycine receptors expressed in mammalian cell lines.

We examined the effects of acute ethanol exposure on recombinant human glycine receptors transiently transfected into HEK 293 cells and stably transfected into Ltk- fibroblast-like cells. In our study of the effects of ethanol, we used the whole-cell patch-clamp configuration. Relatively low concentrations of ethanol (25 mM and 50 mM) did not affect glycine-gated currents in any of the cell lines studied. Higher concentrations of ethanol (100 mM and 200 mM) significantly potentiated glycine responses only in stably transfected Ltk- cells expressing alpha1 and alpha2 subunits and in HEK 293 cells transiently expressing alpha2 subunits. Cells stably expressing alpha1 versus alpha2 glycine receptors were modulated equally by ethanol. Both glycine alpha1 and glycine alpha1beta receptors transiently expressed in HEK 293 cells were insensitive to all concentrations of ethanol tested; however, there was a trend toward potentiation at 100 and 200 mM ethanol concentrations. A population of cells (41-87%) that was sensitive to the potentiating effects of 100 and 200 mM ethanol (defined as more than 10% potentiation) was identified in both cell lines tested. In these sensitive cells, ethanol (100 and 200 mM) produced significant potentiation, independent of the cell line and the glycine receptor subunit tested. Together with published results from studies with Xenopus oocytes, these data indicate that the sensitivity of recombinant glycine receptors to ethanol depends upon the expression system.

Colchicine is a competitive antagonist at human recombinant gamma-aminobutyric acidA receptors.

Colchicine is an alkaloid that is used clinically in the treatment of arthritic gout. This potent microtubule disrupting agent has also been used extensively as an experimental tool in studies characterizing the role of the cytoskeleton in a variety of cellular processes. Colchicine has also been used as a selective neurotoxin and in animal models of Alzheimer's disease and epilepsy. Although the mechanism(s) mediating the neurotoxic actions of colchicine have not been established, most studies have attributed these effects to its microtubule depolymerizing actions. Here we report another central nervous system action of colchicine, competitive antagonism of gamma-aminobutyric acid (GABA)A receptor function. By use of a rapid drug perfusion system, colchicine (10-1000 microM) significantly inhibited GABA currents recorded from L(tk-) cells stably transfected with human alpha 1 beta 2 gamma 2L GABAA receptor subunits. The inhibition was rapid and reversible, with 100 microM colchicine shifting the GABA EC50 from 2.5 to 5.1 microM with no effect on currents evoked by saturating concentrations of GABA. Colchicine also significantly inhibited binding of the competitive GABAA receptor antagonist [3H]SR-95531. Other microtubule disrupting agents (10 microM vinblastine, 10 micrograms/ml nocodazole, 1 microM taxol) had no acute effects on GABA currents, nor did the inactive analog gamma-lumicolchicine (100 microM). Moreover, pretreating cells with colchicine, vinblastine, nocodazole or taxol for 1 to 4 hr did not occlude the acute inhibitory action of colchicine. We conclude that, in addition to its well characterized effects on microtubule assembly, colchicine can also inhibit GABAA receptor function through a direct interaction with the receptor/ion channel complex.

Roles of platelet-derived growth factor in the developing and mature nervous systems.

In spite of its association by history and name to platelets, platelet-derived growth factor (PDGF) exerts important actions in a myriad of tissues, including the nervous system. PDGF and PDGF receptors are widely expressed in neuronal and glial cells of many regions of both the central and peripheral nervous systems. In this topical review, the roles played by PDGF in the development and maintenance of the nervous system are discussed. We also discuss the modulatory effects of PDGF on synaptic transmission, its role in neoplastic and non-neoplastic conditions of the central nervous system, and the neuroprotective effects of this growth factor.

Elevation of basal protein kinase C activity increases ethanol sensitivity of GABA(A) receptors in rat hippocampal CA1 pyramidal neurons.

The ability of ethanol to enhance GABA(A) receptor function remains controversial; conflicting observations have been made even in the same brain region, and when using apparently similar methodologies. In this study we characterized a single protocol variable, the initial incubation temperature of brain slices, that had dramatic effects on the ethanol sensitivity of GABA(A) inhibitory postsynaptic currents (IPSCs) recorded from rat hippocampal CA1 pyramidal neurons. Incubation of hippocampal slices at relatively low temperatures (11-15 degrees C) immediately after slice preparation significantly affected a number of physiological and biochemical parameters. Such slices showed a decrease in extracellular inhibitory postsynaptic potential amplitude, a significant increase in the ethanol sensitivity of GABA(A) IPSCs in CA1 pyramidal neurons, no change in pentobarbital or flunitrazepam potentiation of IPSCs, and an increase in basal protein kinase C (PKC) activity relative to slices incubated at 31-33 degrees C. In addition, the increase in ethanol sensitivity of GABA(A) IPSCs was blocked by chelerythrine, a selective inhibitor of PKC. These results suggest that differences in hippocampal slice incubation protocols may have contributed to the disparate results of previous investigations of ethanol modulation of GABA(A) receptor-mediated synaptic transmission in the rat hippocampus. In addition, these findings provide further evidence that PKC activity positively modulates the interaction between ethanol and GABA(A) receptors in the mammalian brain.

Differential ethanol sensitivity of subpopulations of GABAA synapses onto rat hippocampal CA1 pyramidal neurons.

The actions of ethanol on gamma-aminobutyric acid-A (GABAA) receptor-mediated synaptic transmission in rat hippocampal CA1 neurons remain controversial. Recent studies have reported that intoxicating concentrations of ethanol (10-100 mM) can potentiate, inhibit, or have no effect on GABAA receptor-mediated synaptic responses in this brain region. The essential determinants of ethanol sensitivity have not been defined; however, GABAA receptor subunit composition, as well as posttranslational modifications of these receptors, have been suggested as important factors in conferring ethanol sensitivity to the GABAA receptor complex. Multiple types of GABAA receptor-mediated synaptic responses have been described within individual hippocampal CA1 neurons. These responses have been shown to differ in some of their physiological and pharmacological properties. In the present study we tested hypothesis that some of the disparate findings concerning the effects of ethanol may have resulted from differences in the ethanol sensitivity of GABAA receptor-mediated synapses on single CA1 pyramidal cells. Electrical stimulation adjacent to the stratum pyramidale (proximal) and within the stratum lacunosum-moleculare (distal) activated nonoverlapping populations of GABAA receptors on rat hippocampal CA1 neurons. Proximal inhibitory postsynaptic currents (IPSCs) decayed with a single time constant and were significantly potentiated by ethanol at all concentrations tested (40, 80, and 160 mM). Distal IPSCs had slower decay rates that were often described better by the sum of two exponentials and were significantly less sensitive to ethanol at all concentrations tested. Three other allosteric modulators of GABAA receptor function with well-defined GABAA receptor subunit requirements, pentobarbital, flunitrazepam, and zolpidem, potentiated proximal and distal GABAA IPSCs to the same extent. These results demonstrate that the ethanol sensitivity of GABAA receptors can differ, not only between brain regions but within single neurons. These findings offer a possible explanation for the conflicting results of previous studies on ethanol modulation of GABAA receptor-mediated synaptic transmission in rat hippocampal CA1 neurons.