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We report here that prior social experience modified the behavioral responses of adult crayfish to acute alcohol exposure. Animals housed individually for 1 wk before alcohol exposure were less sensitive to the intoxicating effects of alcohol than animals housed in groups, and these differences are based on changes in the nervous system rather than differences in alcohol uptake. To elucidate the underlying neural mechanisms, we investigated the neurophysiological responses of the lateral giant (LG) interneurons after alcohol exposure. Specifically, we measured the interactions between alcohol and different GABA(A)-receptor antagonists and agonists in reduced crayfish preparations devoid of brain-derived tonic GABAergic inhibition. We found that alcohol significantly increased the postsynaptic potential of the LG neurons, but contrary to our behavioral observations, the results were similar for isolated and communal animals. The GABA(A)-receptor antagonist picrotoxin, however, facilitated LG postsynaptic potentials more strongly in communal crayfish, which altered the neurocellular interactions with alcohol, whereas TPMPA [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid], an antagonist directed against GABA(A)-receptors with ρ subunits, did not produce any effects. Muscimol, an agonist for GABA(A)-receptors, blocked the stimulating effects of alcohol, but this was independent of prior social history. THIP [4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol], an agonist directed against GABA(A)-receptors with δ subunits, which were not previously known to exist in the LG circuit, replicated the suppressing effects of muscimol. Together, our findings provide strong evidence that alcohol interacts with the crayfish GABAergic system, and the interplay between prior social experience and acute alcohol intoxication might be linked to changes in the expression and function of specific GABA(A)-receptor subtypes. NEW & NOTEWORTHY The complex interactions between alcohol and prior social experience are still poorly understood. Our work demonstrates that socially isolated crayfish exhibit lower neurobehavioral sensitivity to acute ethanol compared with communally housed animals, and this socially mediated effect is based on changes in the nervous systems rather than on differences in uptake or metabolism. By combining intracellular neurophysiology and neuropharmacology, we investigated the role of the main inhibitory neurotransmitter GABA, and its receptor subtypes, in shaping this process.

The higher cognitive functions of insects are dependent on their mushroom bodies (MBs), which are particularly large in social insects such as honeybees. MB Kenyon cells (KCs) receive multisensory input and are involved in associative learning and memory. In addition to receiving sensory input via excitatory nicotinic synapses, KCs receive inhibitory GABAergic input from MB feedback neurons. Cultured honeybee KCs exhibit ionotropic GABA receptor currents, but the properties of GABA-mediated inhibition in intact MBs are currently unknown. Here, using whole cell recordings from KCs in acutely isolated honeybee brain, we show that KCs exhibit a tonic current that is inhibited by picrotoxin but not by bicuculline. Bath application of GABA (5 μM) and taurine (1 mM) activate a tonic current in KCs, but l-glutamate (0.1–0.5 mM) has no effect. The tonic current is strongly potentiated by the allosteric GABAAreceptor modulator pentobarbital and is reduced by inhibition of Ca2+channels with Cd2+or nifedipine. Noise analysis of the GABA-evoked current gives a single-channel conductance value for the underlying receptors of 27 ± 3 pS, similar to that of resistant to dieldrin (RDL) receptors. The amount of injected current required to evoke action potential firing in KCs is significantly lower in the presence of picrotoxin. KCs recorded in an intact honeybee head preparation similarly exhibit a tonic GABA receptor conductance that reduces neuronal excitability, a property that is likely to contribute to the sparse coding of sensory information in insect MBs.

Processing of signals within the cerebral cortex requires integration of synaptic inputs and a coordination between excitatory and inhibitory neurotransmission. In addition to the classic form of synaptic inhibition, another important mechanism that can regulate neuronal excitability is tonic inhibition via sustained activation of receptors by ambient levels of inhibitory neurotransmitter, usually GABA. The purpose of this study was to determine whether this occurs in layer II/III pyramidal neurons (PNs) in the prelimbic region of the mouse medial prefrontal cortex (mPFC). We found that these neurons respond to exogenous GABA and to the α4δ-containing GABAA receptor (GABAAR)-selective agonist gaboxadol, consistent with the presence of extrasynaptic GABAAR populations. Spontaneous and miniature synaptic currents were blocked by the GABAAR antagonist gabazine and had fast decay kinetics, consistent with typical synaptic GABAARs. Very few layer II/III neurons showed a baseline current shift in response to gabazine, but almost all showed a current shift (15–25 pA) in response to picrotoxin. In addition to being a noncompetitive antagonist at GABAARs, picrotoxin also blocks homomeric glycine receptors (GlyRs). Application of the GlyR antagonist strychnine caused a modest but consistent shift (∼15 pA) in membrane current, without affecting spontaneous synaptic events, consistent with the tonic activation of GlyRs. Further investigation showed that these neurons respond in a concentration-dependent manner to glycine and taurine. Inhibition of glycine transporter 1 (GlyT1) with sarcosine resulted in an inward current and an increase of the strychnine-sensitive current. Our data demonstrate the existence of functional GlyRs in layer II/III of the mPFC and a role for these receptors in tonic inhibition that can have an important influence on mPFC excitability and signal processing.

Glutamate release at bipolar to ganglion cell synapses activates NMDA and AMPA/kainic acid (KA) ionotropic glutamate receptors. Their relative strength determines the output signals of the retina. We found that this balance is tightly regulated by presynaptic inhibition that preferentially suppresses NMDA receptor (NMDAR) activation. In transient ON-OFF neurons, block of GABA and glycine feedback enhanced total NMDAR charge by 35-fold in the ON response and 9-fold in the OFF compared with a 1.7-fold enhancement of AMPA/KA receptors. Blocking only glycine receptors enhanced the NMDAR excitatory postsynaptic current 10-fold in the ON and 2-fold in the OFF pathway. Blocking GABAA or GABAC receptors (GABACRs or GABAARs) produced small changes in total NMDAR charge. When both GABAARs and GABACRs were blocked, the total NMDAR charge increased ninefold in the ON and fivefold in the OFF pathway. This exposed a strong GABACR feedback to bipolar cells that was suppressed by serial amacrine cell synapses mediated by GABAARs. The results indicate that NMDAR currents are large but latent, held in check by dual GABA and glycine presynaptic inhibition. One example of this controlled NMDAR activation is the cross talk between ON and OFF pathways. Blocking the ON pathway increased NMDAR relative strength in the OFF pathway. Stimulus prolongation similarly increased the NMDAR relative strength in the OFF response. This NMDAR enhancement was produced by a diminution in GABA and glycine feedback. Thus the retinal network recruits NMDAR pathways through presynaptic disinhibition.

Synaptic plasticity rules change during development: while hippocampal synapses can be potentiated by a single action potential pairing protocol in young neurons, mature neurons require burst firing to induce synaptic potentiation. An essential component for spike timing-dependent plasticity is the backpropagating action potential (BAP). BAP along the dendrites can be modulated by morphology and ion channel composition, both of which change during late postnatal development. However, it is unclear whether these dendritic changes can explain the developmental changes in synaptic plasticity induction rules. Here, we show that tonic GABAergic inhibition regulates dendritic action potential backpropagation in adolescent, but not preadolescent, CA1 pyramidal neurons. These developmental changes in tonic inhibition also altered the induction threshold for spike timing-dependent plasticity in adolescent neurons. This GABAergic regulatory effect on backpropagation is restricted to distal regions of apical dendrites (>200 μm) and mediated by α5-containing GABA(A) receptors. Direct dendritic recordings demonstrate α5-mediated tonic GABA(A) currents in adolescent neurons which can modulate BAPs. These developmental modulations in dendritic excitability could not be explained by concurrent changes in dendritic morphology. To explain our data, model simulations propose a distally increasing or localized distal expression of dendritic α5 tonic inhibition in mature neurons. Overall, our results demonstrate that dendritic integration and plasticity in more mature dendrites are significantly altered by tonic α5 inhibition in a dendritic region-specific and developmentally regulated manner. © 2014 the American Physiological Society.

γ-Amino butyric acid (GABA) plays a key role in the regulation of central nervous system by activating synaptic and extrasynaptic GABAA receptors. It is acknowledged that extrasynaptic GABAA receptors located in the soma, dendrites, and axons may be activated tonically by low extracellular GABA concentrations. The activation of these receptors produces a persistent conductance that can hyperpolarize or depolarize nerve cells depending on the Cl− equilibrium potential. In an in vitro preparation of the turtle spinal cord we show that extrasynaptic α5GABAA receptors mediate the tonic state of excitability of primary afferents independently of the phasic primary afferent depolarization mediated by synaptic GABAA receptors. Blockade of α5GABAA receptors with the inverse agonist L-655,708 depressed the dorsal root reflex (DRR) without affecting the phasic increase in excitability of primary afferents. Using RT-PCR and Western blotting, we corroborated the presence of the mRNA and the α5GABAA protein in the dorsal root ganglia of the turtle spinal cord. The receptors were localized in primary afferents in dorsal root, dorsal root ganglia, and peripheral nerve terminals using immunoconfocal microscopy. Considering the implications of the DRR in neurogenic inflammation, α5GABAA receptors may serve as potential pharmacological targets for the treatment of pain.

Our laboratory previously reported that gastric activity is controlled by a robust GABAA receptor-mediated inhibition in the medial nucleus of the tractus solitarius (mNTS) ( Herman et al. 2009 ), and that μ-opioid receptor activation inhibits gastric tone by suppression of this GABA signaling ( Herman et al. 2010 ). These data raised two questions: 1) whether any of this inhibition was due to tonic GABAA receptor-mediated conductance in the mNTS; and 2) whether μ-opioid receptor activation suppressed both tonic and phasic GABA signaling. In whole cell recordings from rat mNTS neurons, application of three GABAA receptor antagonists (gabazine, bicuculline, and picrotoxin) produced a persistent reduction in holding current and decrease in population variance or root mean square (RMS) noise, suggesting a blockade of tonic GABA signaling. Application of gabazine at a lower concentration abolished phasic currents, but had no effect on tonic currents or RMS noise. Application of the δ-subunit preferring agonist gaboxadol (THIP) produced a dose-dependent persistent increase in holding current and RMS noise. Pretreatment with tetrodotoxin prevented the action of gabazine, but had no effect on the THIP-induced current. Membrane excitability was unaffected by the selective blockade of phasic inhibition, but was increased by blockade of both phasic and tonic currents. In contrast, activation of tonic currents decreased membrane excitability. Application of the μ-opioid receptor agonist DAMGO produced a persistent reduction in holding current that was not observed following pretreatment with a GABAA receptor antagonist and was not evident in mice lacking the δ-subunit. These data suggest that mNTS neurons possess a robust tonic inhibition that is mediated by GABAA receptors containing the δ-subunit, that determines membrane excitability, and that is partially regulated by μ-opioid receptors.

GABAC receptors (GABACRs) are widely expressed in the mammalian subcortical visual system, particularly in the retina and superior colliculus (SC). GABACRs are composed of specific ρ1–3 subunits the expression of which varies among visual structures. Thus ρ1 subunits are most abundant in retina, and their loss eliminates GABACR expression and function. In the SC, ρ2 subunit expression may be equal to or stronger than ρ1 subunit expression; however, results across studies vary considerably. To more directly assess the expression of GABACR subunits, we characterized inhibition in the SC of wild-type (WT) and GABAC ρ1 Null mice that lack expression of GABAC ρ1 subunits. We used whole cell patch-clamp recordings and evaluated GABACR-mediated modulation of electrically evoked post synaptic currents using either agonists or antagonists in WT mice. In GABAC ρ1 Null stratum griseum superficiale (SGS) cells, inhibitory postsynaptic currents were shorter in duration and their excitatory postsynaptic currents (EPSCs) were longer, indicating that a slow GABACR-mediated inhibitory component was reduced in each case. In contrast to retina, GABACR-mediated currents in the SC were altered but not eliminated in GABAC ρ1 Null mice. In the majority of SC cells in GABAC ρ1 Null mice, GABACR activation could still be induced to alter EPSC peak amplitudes in putative interneurons and in many projection neurons. These results, compared with previously published data, indicate a fundamental difference between retina and SC in the control of GABACR expression and subunit composition.

γ-Aminobutyric acid type A (GABAA) receptor activation inhibits many primary afferent neurons by depolarization and increased membrane conductance. Deterministic (step and sinusoidal) functions are commonly used as stimuli to test such inhibition. We found that when the VS-3 mechanosensory neurons innervating the spider lyriform slit-sense organ were stimulated by randomly varying white-noise mechanical or electrical signals, their responses to GABAA receptor agonists were more complex than the inhibition observed during deterministic stimulation. Instead, there was rapid excitation, then brief inhibition, followed by long-lasting excitation. During the final excitatory phase, VS-3 neuron sensitivity to high-frequency signals increased selectively and their linear information capacity also increased. Using experimental and simulation approaches we found that the excitatory effect could also be achieved by depolarizing the neurons without GABA application and that excitation could override the inhibitory effect produced by increased membrane conductance (shunting). When the VS-3 neurons were exposed to bumetanide, an antagonist of the Cl− transporter NKCC1, the GABA-induced depolarization decreased without any change in firing rate, suggesting that the effects of GABA can be maintained for a long time without additional Cl− influx. Our results show that the VS-3 neuron's response to GABA depends profoundly on the type of signals the neuron is conveying while the transmitter binds to its receptors.

γ-Aminobutyric acid (GABA) is the primary inhibitory transmitter in the mammalian brain. This inhibition is mediated by type A (GABAA) receptors that are pentameric proteins assembled from 14 different subunits. Although inhibitory synaptic transmission has been studied in the amygdala, the subunit composition of receptors present at different synapses is not well understood. In this study we examined the subunit composition of GABAA receptors at synapses in the basolateral and central amygdala. Using receptors expressed in HEK293 cells we first determined the pharmacology of receptors of different subunit compositions. We then used this pharmacological profile to test the properties of receptors present at synapses in the central and basolateral amygdala. These results show that the GABAA receptor subunits are differentially distributed in the amygdala. Our data indicate that in the basolateral amygdala, GABAergic synapses are likely composed of receptors that contain α2βxγ2 subunits. In the central amygdala receptors at the medial input, carrying afferents from the bed nucleus of the stria terminalis contain similar receptors, whereas in the lateral input GABA receptors likely contain γ1 subunits. These inputs arise from the intercalated cells masses, thought to be responsible for mediating extinction of conditioned fear, raising the possibility of new targets for the treatment of anxiety-related disorders.

Previous studies using muscimol inactivations in the frontal eye fields (FEFs) have shown that saccades generated by recall from working memory are eliminated by these lesions, whereas visually guided saccades are relatively spared. In these experiments, we made reversible inactivations in FEFs in alert macaque monkeys and examined the effect on saccades in a choice response task. Our task required monkeys to learn arbitrary pairings between colored stimuli and saccade direction. Following inactivations, the percentage of choice errors increased as a function of the number of alternative (NA) pairings. In contrast, the percentage of dysmetric saccades (saccades that landed in the correct quadrant but were inaccurate) did not vary with NA. Saccade latency increased postlesion but did not increase with NA. We also made simultaneous inactivations in both FEFs. The results following bilateral lesions showed approximately twice as many choice errors. We conclude that the FEFs are involved in the generation of saccades in choice response tasks. The dramatic effect of NA on choice errors, but the lack of an effect of NA on motor errors or response latency, suggests that two types of processing are interrupted by FEF lesions. The first involves the formation of a saccadic intention vector from associate memory inputs, and the second, the execution of the saccade from the intention vector. An alternative interpretation of the first result is that a role of the FEFs may be to suppress incorrect responses. The doubling of choice errors following bilateral FEF lesions suggests that the effect of unilateral lesions is not caused by a general inhibition of the lesioned side by the intact side.

The interstitial nucleus of Cajal (INC) is thought to control torsional and vertical head posture. Unilateral microstimulation of the INC evokes torsional head rotation to positions that are maintained until stimulation offset. Unilateral INC inactivation evokes head position-holding deficits with the head tilted in the opposite direction. However, the underlying muscle synergies for these opposite behavioral effects are unknown. Here, we examined neck muscle activity in head-unrestrained monkeys before and during stimulation (50 muA, 200 ms, 300 Hz) and inactivation (injection of 0.3 mul of 0.05% muscimol) of the same INC sites. Three-dimensional eye and head movements were recorded simultaneously with electromyographic (EMG) activity in six bilateral neck muscles: sternocleidomastoid (SCM), splenius capitis (SP), rectus capitis posterior major (RCPmaj.), occipital capitis inferior (OCI), complexus (COM), and biventer cervicis (BC). INC stimulation evoked a phasic, short-latency ( approximately 5-10 ms) facilitation and later ( approximately 100-200 ms) a more tonic facilitation in the activity of ipsi-SCM, ipsi-SP, ipsi-COM, ipsi-BC, contra-RCPmaj., and contra-OCI. Unilateral INC inactivation led to an increase in the activity of contra-SCM, ipsi-SP, ipsi-RCPmaj., and ipsi-OCI and a decrease in the activity of contra-RCPmaj. and contra-OCI. Thus the influence of INC stimulation and inactivation were opposite on some muscles (i.e., contra-OCI and contra-RCPmaj.), but the comparative influences on other neck muscles were more variable. These results show that the relationship between the neck muscle responses during INC stimulation and inactivation is much more complex than the relationship between the overt behaviors.

Tonic inhibition mediated by extrasynaptic γ-aminobutyric acid type A (GABAA) receptors is a powerful conductance that controls cell excitability. Throughout the CNS, tonic inhibition is expressed at varying degrees across different cell types. Despite a rich history of cortical interneuron diversity, little is known about tonic inhibition in the different classes of cells in the cerebral cortex. We therefore examined the cell-type specificity and functional significance of tonic inhibition in layer 4 of the mouse somatosensory barrel cortex. In situ hybridization and immunocytochemistry showed moderate δ-subunit expression across the barrel structures. Whole cell patch-clamp recordings additionally indicated that significant levels of tonic inhibition can be found across cell types, with differences in the magnitude of inhibition between cell types. To activate tonic currents, we used 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, a superagonist at δ-subunit–containing GABAA receptors) at a concentration that did not affect synaptic decay kinetics. THIP produced greater shifts in baseline holding current in inhibitory cells (low-threshold spiking [LTS], 109 ± 17 pA; fast spiking [FS], 111 ± 15 pA) than in excitatory cells (39 ± 10 pA; P < 0.001). In addition to these differences across cell types, there was also variability within inhibitory cells. FS cells with faster action potentials had larger baseline shifts. Because FS cells are known mediators of feedforward inhibition, we tested whether THIP-induced tonic conductance selectively controls feedforward circuits. THIP application resulted in the abolishment of the inhibitory postsynaptic potential in thalamic-evoked disynaptic responses in a subset of excitatory neurons. These data suggest multiple feedforward circuits can be differentiated by the inhibitory control of the presynaptic inhibitory neuron.

Activity of extrasynaptic GABAA receptors mediating tonic inhibition is thought to play an important role for the excitability of the mammalian cerebral cortex. However, little is known about the cell type–specific expression of tonic inhibition in particular types of cortical interneurons. Here, we used transgenic mice expressing green fluorescent protein (GFP) in somatostatin-positive (SOM) interneurons and investigated tonic inhibition in SOM interneurons versus pyramidal cells in neocortical layers 2/3. In brain slices, pyramidal cells showed a tonic current of 66 ± 19 pA in response to the δ-subunit selective GABAA agonist THIP (1 μM). On the other hand, tonic inhibition was absent in SOM interneurons (8 ± 1 pA) in response to THIP. As opposed to pyramidal cells, SOM interneurons were also insensitive to the δ-subunit preferring neurosteroid allotetrahydrodeoxycorticosterone (THDOC) (100 nM) and to elevated endogenous GABA levels in the slice. Finally, SOM interneurons received only 45% of the phasic charge transfer during GABAA receptor–mediated synaptic activity compared with pyramidal cells. Altogether, our study indicates that SOM interneurons receive relatively weak inhibitory input and cannot be brought under the influence of tonic inhibition.

Cranial visceral afferents enter the brain at the solitary tract nucleus (NTS). GABAergic neurons are scattered throughout the NTS, but their relation to solitary tract (ST) afferent pathways is imprecisely known. We hypothesized that most GABAergic NTS neurons would be connected only indirectly to the ST. We identified GABAergic neurons in brain stem horizontal slices using transgenic mice in which enhanced green fluorescent protein (EGFP) expression was linked to glutamic acid decarboxylase expression (GAD+). Finely graded electrical shocks to ST recruit ST-synchronized synaptic events with all-or-none thresholds and individual waveforms did not change with greater suprathreshold intensities—evidence consistent with initiation by single afferent axons. Most (∼70%) GAD+ neurons received ST-evoked excitatory postsynaptic currents (EPSCs) that had minimally variant latencies (jitter, SD of latency 200 μs including inhibitory postsynaptic currents (IPSCs), indicating indirect connections (polysynaptic). Shocks of suprathreshold intensity delivered adjacent (50–300 μm) to the ST failed to excite non-ST inputs to second-order neurons, suggesting a paucity of axons passing near to ST that connected to these neurons. Despite expectations, we found similar ST synaptic patterns in GAD+ and unlabeled neurons. Generally, ST information that arrived indirectly had small amplitudes (EPSCs and IPSCs) and frequency-dependent failures that reached >50% for IPSCs to bursts of stimuli. This ST afferent pathway organization is strongly use-dependent—a property that may tune signal propagation within and beyond NTS.

γ-Aminobutyric acid type A receptor (GABAA-R) activation leads to depolarization of pyramidal cells during the first postnatal week and produces hyperpolarization from the second week. However, immunohistochemical evidence has suggested that during the second and third postnatal weeks the NKCC1 cotransporter relocates from the soma to the dendrites of CA3 pyramidal cells. We hypothesized that this leads to depolarizing responses in apical dendrites. Here we show that the activation of GABAA-R in the distal dendrites of CA3 pyramidal cells at P15 by restricted application of muscimol or synaptic activation by stimulation of interneurons in stratum radiatum (SR) causes depolarizing postsynaptic potentials (PSPs), which are blocked by NKCC1 cotransporter antagonists. By contrast, activation of proximal GABAA-R by muscimol application or by stimulation of interneurons in s. oriens (SO) leads to hyperpolarizing PSPs. Activation of the dentate gyrus (DG) in the presence of glutamatergic blockers evokes hyperpolarizing responses during the second postnatal week; however, the reversal potential of the DG-evoked inhibitory (I)PSPs is more depolarized than that of IPSPs evoked by activation of SO interneurons. Despite the shift of GABA action from depolarizing to hyperpolarizing, DG-evoked field potentials (f-PSPs) recorded in s. lucidum/radiatum (SL/R) do not change in polarity until the third week. Current source density analysis yielded results consistent with depolarizing actions of GABA in the dendritic compartment. Our data suggest that GABAergic input to apical dendrites of pyramidal cells of CA3 evokes depolarizing PSPs long after synaptic inhibition has become hyperpolarizing in the somata, in the axon initial segments and in basal dendrites.

To elucidate the role of ambient striatal γ-aminobutyric acid (GABA) in the regulation of neuronal activity in the basal ganglia–thalamocortical circuits, we studied the effects of blocking striatal GABAA receptors on the electrical activities of single striatal neurons, on local field potentials (LFPs) in the striatum, and on motor cortical electroencephalograms (EEGs) in two monkeys. Striatal LFPs were recorded with a device that allowed us to simultaneously record field potentials and apply drugs by reverse microdialysis at the same site. Administration of the GABAA-receptor antagonist gabazine (SR95531, 10 and 500 μM) induced large-amplitude LFP fluctuations at the infusion site, occurring every 2–5 s for about 2 h after the start of the 20-min drug administration. These events were prevented by cotreatment with a GABAA-receptor agonist (muscimol, 100 μM) or a combination of ionotropic glutamate receptor antagonists (CNQX and MK-801, each given at 100 μM). Gabazine (10 μM) also increased the firing of single neurons recorded close to the injection site, but in most cases there was no correlation between single-neuron activity and the concomitantly recorded LFP signals from the same striatal region. In contrast, intrastriatal application of gabazine increased the correlation between striatal LFPs and EEG, and resulted in the appearance of recurrent EEG events that were temporally related to the striatal LFP events. These data provide evidence that a GABAergic “tone” in the monkey striatum controls the spontaneous activity of striatal neurons, as well as the level of striatal and cortical synchrony.

Distinguishing between different signals conveyed in a single sensory modality presents a significant problem for sensory processing. The weakly electric fish Apteronotus leptorhynchus use electrosensory information to encode both low-frequency signals associated with environmental and prey signals and high-frequency communication signals between conspecifics. We identify a mechanism whereby the GABABcomponent of a feedback pathway to the electrosensory lobe is recruited to regulate the intrinsic burst dynamics and coding properties of pyramidal cells for these behaviorally relevant input signals. Through recordings in an in vitro slice preparation and a reduced model of pyramidal cells, we show that recruitment of dendritic GABABcurrents can shift the timing of a backpropagating spike and its influence on an intrinsic burst mechanism. This regulation of burst firing alters the coding properties of pyramidal cells by improving the correlation of burst and tonic spikes with respect to low- or high-frequency components of complex stimuli. GABABmodulation of spike backpropagation thus improves the segregation of burst and tonic spikes evoked by simulated sensory input, allowing pyramidal cells to parcel the spike train into coding streams for the low- and high-frequency components. As the feedback pathway is predicted to be activated in circumstances where environmental and communication stimuli coexist, these data reveal a novel means by which inhibitory input can regulate spike backpropagation to improve signal segregation.

In rat olfactory bulb slices, external tufted (ET) cells spontaneously generate spike bursts. Although ET cell bursting is intrinsically generated, its strength and precise timing may be regulated by synaptic input. We tested this hypothesis by analyzing whether the burst properties are modulated by activation of ionotropic γ-aminobutyric acid (GABA) and glutamate receptors. Blocking GABAA receptors increased—whereas blocking ionotropic glutamate receptors decreased—the number of spikes/burst without changing the interburst frequency. The GABAA agonist (isoguvacine, 10 μM) completely inhibited bursting or reduced the number of spikes/burst, suggesting a shunting effect. These findings indicate that the properties of ET cell spontaneous bursting are differentially controlled by GABAergic and glutamatergic fast synaptic transmission. We suggest that ET cell excitatory and inhibitory inputs may be encoded as a change in the pattern of spike bursting in ET cells, which together with mitral/tufted cells constitute the output circuit of the olfactory bulb.

Autoinhibitory serotonin 1A receptors (5-HT1A) in dorsal raphé nucleus (DRN) have been implicated in chronic depression and in actions of selective serotonin reuptake inhibitors (SSRI). Due to experimental limitations, it was never studied at single-cell level whether changes in 5-HT1Areceptor functionality occur in depression and during SSRI treatment. Here we address this question in a social stress paradigm in rats that mimics anhedonia, a core symptom of depression. We used whole cell patch-clamp recordings of 5-HT- and baclophen-induced G-protein-coupled inwardly rectifying potassium (GIRK) currents as a measure of 5-HT1A- and GABABreceptor functionality. 5-HT1A- and GABABreceptor-mediated GIRK-currents were not affected in socially stressed rats, suggesting that there was no abnormal (auto)inhibition in the DRN on social stress. However, chronic fluoxetine treatment of socially stressed rats restored anticipatory behavior and reduced the responsiveness of 5-HT1Areceptor-mediated GIRK currents. Because GABABreceptor-induced GIRK responses were also suppressed, fluoxetine does not appear to desensitize 5-HT1Areceptors but rather one of the downstream components shared with GABABreceptors. This fluoxetine effect on GIRK currents was also present in healthy animals and was independent of the animal's “depressed” state. Thus our data show that symptoms of depression after social stress are not paralleled by changes in 5-HT1Areceptor signaling in DRN neurons, but SSRI treatment can alleviate these behavioral symptoms while acting strongly on the 5-HT1Areceptor signaling pathway.

The pedunculopontine tegmental (PPT) GABAergic system plays a crucial role in the regulation of rapid eye movement (REM) sleep. I recently reported that the activation of PPT GABAB receptors suppressed REM sleep by inhibiting REM-on cells. One of the important mechanisms for GABAB receptor activation–mediated physiological action is the inhibition of the intracellular cAMP-dependent protein kinase A (cAMP-PKA) signaling pathway. Accordingly, I hypothesized that the PPT GABAB receptor activation–mediated REM sleep suppression effect could be mediated through inhibition of cAMP-PKA activation. To test this hypothesis, a GABAB receptor selective agonist, baclofen hydrochloride (baclofen), cAMP-PKA activator, Sp-adenosine 3′,5′-cyclic monophosphothioate triethylamine (SpCAMPS), and vehicle control were microinjected into the PPT in selected combinations to determine effects on sleep-waking states of chronically instrumented, freely moving rats. Microinjection of SpCAMPS (1.5 nmol) induced REM sleep within a short latency (12.1 ± 3.6 min) compared with vehicle control microinjection (60.0 ± 6.5 min). On the contrary, microinjection of baclofen (1.5 nmol) suppressed REM sleep by delaying its appearance for ∼183 min; however, the suppression of REM sleep by baclofen was prevented by a subsequent microinjection of SpCAMPS. These results provide evidence that the activation of cAMP-PKA within the PPT can successfully block the GABAB receptor activation–mediated REM sleep suppression effect. These findings suggest that the PPT GABAB receptor activation–mediated REM sleep regulating mechanism involves inactivation of cAMP-PKA signaling in the freely moving rat.

The function and the origin of replay of motor activity during sleep are currently unknown. Spontaneous activity patterns in the nucleus robustus of the arcopallium (RA) and in HVC (high vocal center) of the sleeping songbird resemble premotor patterns in these areas observed during singing. We test the hypothesis that the nucleus interface of the nidopallium (NIf) has an important role for initiating and shaping these sleep-related activity patterns. In head-fixed, sleeping zebra finches we find that injections of the GABAA-agonist muscimol into NIf lead to transient abolishment of premotor-like bursting activity in HVC neurons. Using antidromic activation of NIf neurons by electrical stimulation in HVC, we are able to distinguish a class of HVC-projecting NIf neurons from a second class of NIf neurons. Paired extracellular recordings in NIf and HVC show that NIf neurons provide a strong bursting drive to HVC. In contrast to HVC neurons, whose bursting activity waxes and wanes in burst epochs, individual NIf projection neurons are nearly continuously bursting and tend to burst only once on the timescale of song syllables. Two types of HVC projection neurons—premotor and striatal projecting—respond differently to the NIf drive, in agreement with notions of HVC relaying premotor signals to RA and an anticipatory copy thereof to areas of a basal ganglia pathway.

Down syndrome (DS) is the most common nonheritable cause of mental retardation. DS is the result of the presence of an extra chromosome 21 and its phenotype may be a consequence of overexpressed genes from that chromosome. One such gene is Kcnj6/Girk2, which encodes the G-protein-coupled inward rectifying potassium channel subunit 2 (GIRK2). We have recently shown that the DS mouse model, Ts65Dn, overexpresses GIRK2 throughout the brain and in particular the hippocampus. Here we report that this overexpression leads to a significant increase (∼2-fold) in GABAB-mediated GIRK current in primary cultured hippocampal neurons. The dose response curves for peak and steady-state GIRK current density is significantly shifted left toward lower concentrations of baclofen in Ts65Dn neurons compared with diploid controls, consistent with increased functional expression of GIRK channels. Stationary fluctuation analysis of baclofen-induced GIRK current from Ts65Dn neurons indicated no significant change in single-channel conductance compared with diploid. However, significant increases in GIRK channel density was found in Ts65Dn neurons. In normalized baclofen-induced GIRK current and GIRK current kinetics no difference was found between diploid and Ts65Dn neurons, which suggests unimpaired mechanisms of interaction between GIRK channel and GABAB receptor. These results indicate that increased expression of GIRK2 containing channels have functional consequences that likely affect the balance between excitatory and inhibitory neuronal transmission.

Suppression of reactions to one noxious stimulus by a spatially distant noxious stimulus is termed heterotopic antinociception. In lightly anesthetized rats, a noxious visceral stimulus, colorectal distension (CRD), suppressed motor withdrawals but not blood pressure or heart rate changes evoked by noxious hindpaw heat. Microinjection of muscimol, a GABAAreceptor agonist, into raphe magnus (RM) reduced CRD-evoked suppression of withdrawals, evidence that RM neurons contribute to this heterotopic antinociception. To understand how brain stem neurons contribute to heterotopic antinociception, RM neurons were recorded during CRD-elicited suppression of hindpaw withdrawals. Although subsets of RM neurons that were excited (on cells) or inhibited (off cells) by noxious cutaneous stimulation were either excited or inhibited by CRD, on cells were inhibited and off cells excited by an intracerebroventricularly administered opioid, evidence that the nociception-facilitating and -inhibiting functions of on and off cells, respectively, are predicted by the cellular response to noxious cutaneous stimulation alone and not by the response to CRD. When recorded during CRD-elicited antinociception, RM cell discharge resembled the pattern observed in response to CRD stimulation alone. However, when hindpaw withdrawal suppression was incomplete, RM cell discharge resembled the pattern observed in response to heat alone. We propose that on cells excited by CRD facilitate responses to CRD itself, which in turn augments excitation of off cells that then act to suppress cutaneous nociception. RM cells may thereby contribute to the dominance of quiet recuperative reactions evoked by potentially life-threatening visceral stimuli over transient somatomotor activity elicited by less-injurious noxious cutaneous stimuli.

A whole cell patch-clamp study was carried out in slices obtained from young rat brain to elucidate the roles of somatostatin in the modulation of synaptic transmission onto cholinergic neurons in the basal forebrain (BF), a region that contains cholinergic and GABAergic corticopetal neurons and somatostatin (SS)-containing local circuit neurons. Cholinergic neurons within the BF were identified by in vivo prelabeling with Cy3 IgG. Because in many cases SS is contained in GABAergic neurons in the CNS, we investigated whether exogenously applied SS can influence GABAergic transmission onto cholinergic neurons. Bath application of somatostatin (1 μM) reduced the amplitude of the evoked GABAergic inhibitory presynaptic currents (IPSCs) in cholinergic neurons. SS also reduced the frequency of miniature IPSCs (mIPSCs) without affecting their amplitude distribution. SS-induced effect on the mIPSC frequency was significantly larger in the solution containing 7.2 mM Ca2+ than in the standard (2.4 mM Ca2+) external solution. Similar effects were observed in the case of non-NMDA glutamatergic excitatory postsynaptic currents (EPSCs). SS inhibited the amplitude of evoked EPSCs and reduced the frequency of miniature EPSCs dependent on the external Ca2+ concentration with no effect on their amplitude distribution. Pharmacological analyses using SS-receptor subtype–specific drugs suggest that SS-induced action of the IPSCs is mediated mostly by the sst2 subtype, whereas sst subtypes mediating SS-induced inhibition of EPSCs are mainly sst1 or sst4. These findings suggest that SS presynaptically inhibits both GABA and glutamate release onto BF cholinergic neurons in a Ca2+-dependent way, and that SS-induced effect on IPSCs and EPSCs are mediated by different sst subtypes.

Neurons within the intact respiratory network produce bursts of action potentials that cause inspiration or expiration. Within inspiratory bursts, activity is synchronized on a shorter timescale to generate clusters of action potentials that occur in a set frequency range and are called synchronous oscillations. We investigated how GABA and glycine modulate synchronous oscillations and respiratory rhythm during postnatal development. We recorded inspiratory activity from hypoglossal nerves using the in vitro rhythmically active mouse medullary slice preparation from P0–P11 mice. Average oscillation frequency increased with postnatal development, from 17 ± 12 Hz in P0–P6 mice ( n = 15) to 38 ± 7 Hz in P7–P11 mice ( n = 37) ( P < 0.0001). Bath application of GABAA and GlyR antagonists significantly reduced oscillation power in neonates (P0–P6) and juveniles (P7–P10) and increased peak integrated activity in both age groups. To test whether elevating slice excitability is sufficient to reduce oscillation power, Substance P was bath applied alone. Substance P, although increasing peak integrated activity, had no significant effect on oscillation power. Prolonging the time course of GABAergic synaptic currents with zolpidem decreased the median oscillation frequency in P9–P10 mouse slices. These data demonstrate that oscillation frequency increases with postnatal development and that both GABAergic and glycinergic transmission contribute to synchronization of activity. Further, the time course of synaptic GABAergic currents is a determinant of oscillation frequency.

The superior colliculus (SC) provides signals for the generation of saccades via a direct pathway to the brain stem burst generator (BG). In addition, it sends saccade-related activity to the BG indirectly through the cerebellum via a relay in the nucleus reticularis tegmenti pontis (NRTP). Lesions of the oculomotor vermis, lobules VIc and VII, and inactivation of the caudal fastigial nucleus, the cerebellar output nucleus to which it projects, produce saccade dysmetria but have little effect on saccade peak velocity and duration. We expected similar deficits from inactivation of the NRTP. Instead, injections as small as 80 nl into the NRTP first slowed ipsiversive saccades and then gradually reduced their amplitudes. Postinjection saccades had slower peak velocities and longer durations than preinjection saccades with similar amplitudes. Contraversive saccades retained their normal kinematics. When the gains of ipsiversive saccades to 10° target steps had fallen to their lowest values (0.28 ± 0.19; mean ± SD; n = 10 experiments), the gains of contraversive saccades to 10° target steps had decreased very little (0.82 ± 0.11). Eventually, ipsiversive saccades did not exceed 5°, even to 20° target steps. Moreover, these small remaining saccades apparently were made with considerable difficulty because their latencies increased substantially. When ipsiversive saccade gain was at its lowest, the gain and kinematics of vertical saccades to 10° target steps exhibited inconsistent changes. We argue that our injections did not compromise the direct SC pathway. Therefore these data suggest that the cerebellar saccade pathway does not simply modulate BG activity but is required for horizontal saccades to occur at all.

Functionally, γ-aminobutyric acid receptor (GABAR)–mediated inhibition can be classified as phasic (synaptic) and tonic (extrasynaptic). The GABARs underlying tonic inhibition assemble from subunits different from those responsible for phasic inhibition. We wanted to assess the excitability of hippocampal pyramidal cell (PC) networks following a selective impairment of tonic inhibition. This is difficult to accomplish by pharmacological means. Because the GABAR α5 subunits mostly mediate the tonic inhibition in CA1 and CA3 PCs, we quantified changes in tonic inhibition and examined network excitability in slices of adult gabra5−/− mice. In gabra5−/− CA1 and CA3 PCs tonic inhibitory currents were 60 and 53%, respectively, of those recorded in wild type (WT), with no alterations in phasic inhibition. The amount of tonic inhibition recorded in slices was significantly affected by the method of slice storage (interface or submerged chamber). Field recordings in gabra5−/− CA3 pyramidal layer showed an increased network excitability that was decreased by the GABAR agonist muscimol at a concentration that restored the tonic inhibition of gabra5−/− PCs to the WT level without altering phasic inhibition. Through a battery of pharmacological experiments, we have identified δ subunit–containing GABARs as the mediators of the residual tonic inhibition in gabra5−/− PCs. Our study is consistent with an important role of tonic inhibition in the control of hippocampal network excitability and highlights selective enhancers of tonic inhibition as promising therapeutic approaches for diseases involving network hyperexcitability.

Thalamocortical synapses may be able to undergo activity-dependent long-term changes in efficacy, such as long-term potentiation. Indeed, studies conducted in vivo have found that theta-burst stimulation (TBS) of the thalamus induces a long-term enhancement (LTE) of field potential responses evoked in the neocortex of adult rodents. Because the thalamus and neocortex form a complex interconnected network that is highly active in vivo, it is possible that a change in thalamic excitability would be reflected in the neocortex. To test this possibility, we recorded from barrel neocortex and applied TBS to the thalamic radiation while the somatosensory thalamus was inactivated with muscimol. Thalamocortical LTE was absent when the thalamus was inactivated, suggesting that changes in thalamic excitability are involved. Single-unit recordings from thalamocortical cells revealed that TBS causes a significant reduction in the spontaneous firing rate of thalamocortical cells. Reducing the spontaneous firing of thalamocortical cells directly enhances the efficacy of the thalamocortical pathway because it relieves the tonic depression of the thalamocortical connection caused by thalamocortical activity. Because these changes in thalamic excitability are triggered by corticothalamic activity, this may be a useful top-down mechanism to regulate afferent sensory input to the neocortex during behavior as a function of experience.

In the paraventricular nucleus of the hypothalamus (PVN), experimental stress paradigms that suppress γ-aminobutyric acid (GABA) inputs to parvocellular neuroendocrine cells (PNCs) also increase the expression of brain-derived neurotrophic factor (BDNF). In the adult CNS, BDNF regulates the efficacy of GABAergic transmission, but its contributions to functional changes at inhibitory synapses in the PVN have not been investigated. Analysis of quantal transmission revealed a decrease in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in response to BDNF with no accompanying changes in their amplitude. These effects were completely blocked by prior inclusion of the TrKB receptor antagonist K252a in the patch pipette. Inclusion of a dynamin inhibitory peptide in the patch pipette also blocked the effects of BDNF, consistent with an all-or-none removal of clusters of postsynaptic GABAA receptors. Finally, to confirm a decrease in the availability of postsynaptic GABAA receptors, we tested the effects of BDNF on focal application of the GABAA agonist muscimol. Postsynaptic responses to muscimol were reduced after BDNF. Collectively, these data indicate that BDNF remodels functional synaptic contacts putatively by reducing the surface expression of postsynaptic GABAA receptors.

Domestic Mongolian gerbils, a model of inherited epilepsy, begin having spontaneous seizures at ∼1.5 mo of age, making it possible to evaluate them during epileptic and pre-epileptic stages. Previous studies have shown that GABA binding is reduced in the substantia nigra pars reticulata (SNr) of both epileptic and pre-epileptic gerbils compared with controls, suggesting that reduced expression of GABAA receptors in SNr might be epileptogenic in this model. To test this hypothesis, we measured the expression of the GABAA receptor α1 subunit, the dominant α subunit expressed in the SNr, and evaluated GABAA receptor–mediated postsynaptic currents in SNr neurons. GABAA α1 subunit mRNA levels in substantia nigra–rich tissue from pre-epileptic animals were similar to controls, and immunocytochemistry for the α1 subunit showed similar strong expression in the SNr in both groups. Western analysis confirmed that expression of the α1 subunit protein was similar in substantia nigra–rich tissue from pre-epileptic and control gerbils. The frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) and the frequency of miniature (m)IPSCs in SNr neurons of pre-epileptic gerbil were similar to those of controls. The amplitude of mIPSCs in the pre-epileptics was significantly larger than controls. Zolpidem, an α1 subunit–specific modulator of the GABAA receptor, was equally efficacious in prolonging the decay time of mIPSCs in both groups. Hence, contrary to the predictions of the hypothesis, mRNA and protein expression levels of the major GABAA receptor α subunit were normal, and neurons of the SNr in pre-epileptic gerbils displayed normal or enhanced IPSC frequencies and amplitudes. Therefore reduced expression of GABAA receptors in SNr is not likely to be an epileptogenic mechanism in this model.

Long-term depression (LTD) of the parallel fiber-Purkinje cell synapse in the cerebellum is a cellular model system that has been suggested to underlie certain forms of motor learning. Induction of cerebellar LTD requires a postsynaptic kinase limb involving activation of mGluR1, protein kinase Cα (PKCα), and phosphorylation of ser-880 on the AMPA receptor subunit GluR2. Several lines of evidence have also implicated a complementary phosphatase limb in which N-methyl-d-aspartate (NMDA) receptor-mediated Ca2+ influx activates neuronal nitric oxide synthase (nNOS), the ultimate consequences of which are mediated by nitric oxide (NO), cGMP, and inhibition of postsynaptic protein phosphatases. However, the cellular localization of an NMDA/NO cascade has been complicated by the fact that neither functional NMDA receptors nor nNOS are expressed in Purkinje cells. This has lead to a proposal in which NMDA receptors activate nNOS in parallel fibers. Here, we confirm that pharmacological blockade of NMDA receptor or NO signaling blocks induction of LTD. However, no evidence was found for functional NMDA receptors in parallel fiber terminals: blockade of NMDA receptors did not alter either presynaptic Ca2+ transients or the frequency of miniature excitatory postsynaptic currents. NMDA receptor blockade did abolish a slow depolarization evoked by burst stimulation of parallel fiber-stellate cell synapses. The application of NMDA evoked a Ca2+ transient in stellate cell terminals but not in parallel fiber terminals. These results are consistent with the hypothesis that an NMDA receptor/NO cascade involved in cerebellar LTD is localized to interneurons rather than parallel fibers.

Neurogenesis in the dentate gyrus begins before birth but then continues into adulthood. Consequently, many newborn granule cells must integrate into a preexisting hippocampal network. Little is known about the timing of this process or the characteristics of the first established synapses. We used mice that transiently express enhanced green fluorescent protein in newborn granule cells to examine their synaptic input. Although newborn granule cells had functional glutamate receptors, evoked and spontaneous synaptic currents were exclusively GABAergic with immature characteristics including slow rise and decay phases and depolarized reversal potentials. Synaptic currents in newborn granule cells were relatively insensitive to the GABAA receptor modulator zolpidem compared with neighboring mature granule cells. Consistent with the kinetics and pharmacology, newborn granule cells isolated by fluorescent cell sorting lacked the α1 GABAA receptor subunit. Our results indicate that newborn granule cells initially receive only GABAergic synapses even in the adult.

We investigated the mechanisms of presynaptic inhibition of GABAergic neurotransmission by group III metabotropic glutamate receptors (mGluRs) and GABAB receptors, in dopamine (DA) neurons of the substantia nigra pars compacta (SNc). Both the group III mGluRs agonist l-(+)-2-amino-4-phosphonobutyric acid (AP4, 100 μM) and the GABAB receptor agonist baclofen (10 μM) reversibly depressed the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) to 48.5 ± 2.7 and 79.3 ± 1.6% (means ± SE) of control, respectively. On the contrary, the frequency of action potential-independent miniature IPSCs (mIPSCs), recorded in tetrodotoxin (TTX, 1 μM) and cadmium (100 μM) were insensitive to AP4 but were reduced by baclofen to 49.7 ± 8.6% of control. When the contribution of voltage-dependent calcium channels (VDCCs) to synaptic transmission was boosted with external barium (1 mM), AP4 became effective in reducing TTX-resistant mIPSCs to 65.4 ± 3.9% of control, thus confirming a mechanism of presynaptic inhibition involving modulation of VDCCs. Differently from AP4, baclofen inhibited to 58.5 ± 6.7% of control the frequency mIPSCs recorded in TTX and the calcium ionophore ionomycin (2 μM), which promotes Ca2+-dependent, but VDCC-independent, transmitter release. Moreover, in the presence of α-latrotoxin (0.3 nM), to promote a Ca2+-independent vesicular release of GABA, baclofen reduced mIPSC frequency to 48.1 ± 3.2% of control, while AP4 was ineffective. These results indicate that group III mGluRs depress GABA release to DA neurons of the SNc through inhibition of presynaptic VDCCs, while presynaptic GABAB receptors directly impair transmitter exocytosis.

Neurons in the external and internal segment of the globus pallidus (GPe and GPi, respectively) receive substantial GABAergic inputs from the striatum and through axon collaterals of neighboring pallidal neurons. The effects of GABA on pallidal activity depend on the synaptic localization of GABA receptors and the distribution and activity of GABA transporters (GATs). To explore the contribution of GABA receptors and transporters to pallidal function, we recorded the activity of single neurons in GPe or GPi before, during, and after local microinjections of GABAergic compounds in awake rhesus monkeys. Activation of GABAA or GABAB receptors with muscimol or baclofen, respectively, inhibited pallidal activity. These effects were reversed by concomitant infusion of the respective GABA receptor antagonists, gabazine and CGP-55845. Given alone, the antagonists were without consistent effect. Application of the selective GAT-1 inhibitor, SKF-89976A, and the semiselective GAT-3 blocker, SNAP-5114, decreased pallidal activity. Both GAT inhibitors increased GABA levels in the pallidum, as measured by microdialysis. Electron microscopic observations revealed that these transporters are located on glial processes and unmyelinated axonal segments, but rarely on terminals. Our results indicate that activation of GABAA and GABAB receptors inhibits neuronal activity in both segments of the pallidum. GAT-1 and GAT-3 are involved in the modulation of endogenous GABA levels and may be important in regulating the extrasynaptic levels of GABA. Together with previous evidence that a considerable proportion of pallidal GABA receptors are located outside the synaptic cleft, our experiments strongly support the importance of extrasynaptic GABAergic transmission in the primate pallidum.

The cerebellar interposed nuclei (IN) are critical components of a neural network that controls the expression of classically conditioned eyeblinks. The IN receive 2 major inputs: the massive, γ-aminobutyric acid (GABA)–mediated input from the Purkinje cells of the cerebellar cortex and the relatively weaker, glutamate-mediated input from collaterals of mossy and climbing fiber cerebellar afferent systems. To elucidate the role of IN glutamate neurotransmission in conditioned response (CR) expression, effects of blocking fast glutamatergic neurotransmission in the IN with γ-d-glutamylglycine (DGG) on the expression of conditioned eyeblinks and on cerebellar nuclear neuronal activity were examined. Surprisingly, blocking fast glutamate receptors in the IN did not abolish CRs. DGG decreased CR incidence and slightly increased CR latency. In contrast, identical amounts of DGG applied to the cerebellar cortex abolished CRs. Similar to the behavioral effects, DGG had unexpectedly mild effects on IN neurons. At the population level, the baseline firing frequency of IN cells was not affected. After DGG injections, the incidence of excitatory modulation of cell activity in the interstimulus interval decreased but was not abolished. A combined block of fast glutamate and GABAA neurotransmission using a mixture of DGG and picrotoxin dramatically reduced CR incidence, increased the firing frequency of all cell types, and virtually abolished all modulation of neuronal activity. These results indicate that fast glutamate neurotransmission in the IN plays only an accessory role both in the expression of behavioral CRs and in the generation of associated neuronal activity in the IN.

One way we keep track of our movements is by monitoring corollary discharges or internal copies of movement commands. This study tested a hypothesis that the pathway from superior colliculus (SC) to mediodorsal thalamus (MD) to frontal eye field (FEF) carries a corollary discharge about saccades made into the contralateral visual field. We inactivated the MD relay node with muscimol in monkeys and measured corollary discharge deficits using a double-step task: two sequential saccades were made to the locations of briefly flashed targets. To make second saccades correctly, monkeys had to internally monitor their first saccades; therefore deficits in the corollary discharge representation of first saccades should disrupt second saccades. We found, first, that monkeys seemed to misjudge the amplitudes of their first saccades; this was revealed by systematic shifts in second saccade end points. Thus corollary discharge accuracy was impaired. Second, monkeys were less able to detect trial-by-trial variations in their first saccades; this was revealed by reduced compensatory changes in second saccade angles. Thus corollary discharge precision also was impaired. Both deficits occurred only when first saccades went into the contralateral visual field. Single-saccade generation was unaffected. Additional deficits occurred in reaction time and overall performance, but these were bilateral. We conclude that the SC-MD-FEF pathway conveys a corollary discharge used for coordinating sequential saccades and possibly for stabilizing vision across saccades. This pathway is the first elucidated in what may be a multilevel chain of corollary discharge circuits extending from the extraocular motoneurons up into cerebral cortex.

Midbrain dopaminergic (DA) neurons in vivo exhibit two major firing patterns: single-spike firing and burst firing. The firing pattern expressed is dependent on both the intrinsic properties of the neurons and their excitatory and inhibitory synaptic inputs. Experimental data suggest that the activation of N-methyl-d-aspartate (NMDA) and GABAA receptors is a crucial contributor to the initiation and suppression of burst firing, respectively, and that blocking Ca2+-activated potassium SK channels can facilitate burst firing. A multi-compartmental model of a DA neuron with a branching structure was developed and calibrated based on in vitro experimental data to explore the effects of different levels of activation of NMDA and GABAA receptors as well as the modulation of the SK current on the firing activity. The simulated tonic activation of GABAA receptors was calibrated by taking into account the difference in the electrotonic properties in vivo versus in vitro. Although NMDA-evoked currents are required for burst generation in the model, currents evoked by GABAA-receptor activation can also regulate the firing pattern. For example, the model predicts that increasing the level of NMDA receptor activation can produce excessive depolarization that prevents burst firing, but a concurrent increase in the activation of GABAA receptors can restore burst firing. Another prediction of the model is that blocking the SK channel current in vivo will facilitate bursting, but not as robustly as blocking the GABAA receptors.

The effect of γ-aminobutyric acid (GABA) on neuronal firing rate in rat suprachiasmatic nucleus (SCN) slices was examined using continuous recording methods. GABA inhibited neuronal discharge during both the subjective day and the subjective night in a concentration-dependent manner characterized by two apparent affinity states. The GABAA receptor agonist muscimol caused potent inhibition regardless of circadian time; repeated applications of the agonist did not reverse the direction of effect. The GABAA receptor antagonists bicuculline and picrotoxin increased excitability when applied during either subjective day or subjective night. A significant increase in GABAA receptor– mediated inhibition, as well as endogenous GABAergic tone, was observed on the second day after slice preparation. The GABAB receptor agonist baclofen inhibited cell firing during subjective day and night, but the GABAB antagonist phaclofen had no significant effect. These data provide additional strong support for a predominantly inhibitory role of GABA in the rat SCN, regardless of the time of application in relation to the circadian rhythm, and demonstrate an important level of plasticity of this system in vitro.

The principal cells of the chick tangential nucleus are vestibular nucleus neurons participating in the vestibular reflexes. In 16-day embryos, the application of glutamate receptor antagonists abolished the postsynaptic responses generated on vestibular-nerve stimulation, but spontaneous synaptic activity was largely unaffected. Here, spontaneous synaptic activity was characterized in principal cells from brain slices at E16 using whole cell voltage-clamp recordings. With KCl electrodes, the frequency of spontaneous inward currents was 3.1 Hz at –60 mV, and the reversal potential was +4 mV. Cs-gluconate pipette solution allowed the discrimination of glycine/GABAA versus glutamate receptor-mediated events according to their different reversal potentials. The ratio for spontaneous excitatory to inhibitory events was about 1:4. Seventy-four percent of the outward events were GABAA, whereas 26% were glycine receptor-mediated events. Both pre- and postsynaptic GABAB receptor effects were shown, with presynaptic GABAB receptors inhibiting 40% of spontaneous excitatory postsynaptic currents (sEPSCs) and 53% of spontaneous inhibitory postsynaptic currents (sIPSCs). With TTX, the frequency decreased ∼50% for EPSCs and 23% for IPSCs. These data indicate that the spontaneous synaptic activity recorded in the principal cells at E16 is primarily inhibitory, action potential-independent, and based on the activation of GABAA receptors that can be modulated by presynaptic GABAB receptors.

The dorsolateral pontine nucleus (DLPN) is a major component of the cortico-ponto-cerebellar pathway that carries signals essential for smooth pursuit. This pathway also carries visual signals that could play a role in visually guided motor learning in the vestibular ocular reflex (VOR). However, there have been no previous studies that tested this possibility directly. The aim of this study was to determine the potential role of the DLPN in short-term VOR gain adaptation produced by viewing a scene through lenses placed in front of both eyes. In control experiments, adaptation of VOR gain was achieved by sinusoidal rotation (0.2 Hz, 30°/s) for 2 h while the monkey viewed a stationary visual surround through either magnifying (×2) or minifying (×0.5) lenses. This led to increases (23–32%) or decreases (22–48%) of VOR gain as measured in complete darkness (VORd). We used injections of muscimol, a potent GABAA agonist (0.5 μl; 2%), to reversibly inactivate the DLPN, unilaterally, in three monkeys. After DLPN inactivation, initial acceleration of ipsilateral smooth-pursuit was reduced by 35–68%, and steady-state gain was reduced by 32–61%. Despite these significant deficits ( P < 0.01) in ipsilesional smooth pursuit, the VOR during lens viewing was similar to that measured in preinjection control experiments. Similarly, after 2 h of adaptation, VORd gain was not significantly different ( P > 0.61) from control adaptation values for either ipsi- or contralesional directions of head rotation. This was the case even though a stable ipsilesional smooth pursuit deficit persisted throughout the full adaptation period. Our results suggest that visual error signals for short-term adaptation of the VOR are derived from sources other than the DLPN perhaps including other basilar pontine nuclei and the accessory optic system.

Trains of brief iontophoretic glutamate pulses were delivered onto the apical dendrites of CA1 pyramidal cells at variable frequencies (3–100 Hz) to examine how the activation of a G protein–activated, inwardly rectifying K+ (GIRK) conductance alters the postsynaptic processing of repetitive excitatory input. Application of the GIRK channel agonist baclofen (20 μM) reduced the amplitude of individual glutamate-evoked postsynaptic potentials (GPSPs) and attenuated summation of GPSPs so that higher stimulus intensities were required to fire the cell. Notably, GIRK channel activation not only decreased GPSPs, but also suppressed the subsequent afterhyperpolarization (AHP), which arises from a transient deactivation of the hyperpolarization-activated cation current ( I h). Voltage-clamp recordings ruled out a direct modulatory action of baclofen on I h. GIRK channel activation alone accounts for AHP suppression, firstly because, with smaller GPSP amplitudes, fewer I h channels are deactivated, resulting in a diminished AHP, and secondly because, owing to its progressive increase in the hyperpolarizing direction, the GIRK conductance shunts a large portion of the remaining AHP. We provide experimental evidence that the suppression of the I h-dependent AHP by GIRK channel activation bears particular significance on the processing of repetitive excitatory inputs at frequencies at which the deactivation kinetics of I h exert a prominent depressing effect. In functional terms, activation of GIRK current not only produces a time-independent mitigation of incoming excitatory input, which results directly from the opening of an instantaneous K+ conductance, but might also cause a time-dependent redistribution of synaptic weight within a stimulus train, which we link to an interplay with the deactivation of I h.

It has been suggested that the GABAAreceptor-mediated depolarization in immature neurons depends on a high intracellular Cl−concentration maintained by Na-K-Cl cotransporter isoform1 (NKCC1). We previously found that activation of the GABAAreceptor led to stimulation of NKCC1. This stimulation could be a result of GABAAreceptor-mediated Cl−efflux. However, a loss of intracellular Cl−is associated with cell shrinkage, membrane depolarization, as well as a rise of intracellular Ca2+concentration ([Ca2+]i). To determine which cellular mechanism is underlying NKCC1 stimulation, we investigated changes of intracellular Cl−content, [Ca2+]i, cell volume, and NKCC1 activity following GABAAreceptor activation. The basal levels of intracellular36Cl were 0.70 ± 0.04 μmol/mg protein. The intracellular36Cl content decreased to 0.53 ± 0.03 μmol/mg protein in response to 30 μM muscimol ( P < 0.05). The loss of intracellular36Cl was blocked by 10 μM bicuculline. Muscimol triggered a rise in [Ca2+]i, but did not cause cell shrinkage. In contrast, 10–50 mM [Cl−]oor hypertonic HEPES–MEM resulted in reversible cell shrinkage ( P < 0.05). Moreover, the GABA-mediated stimulation of NKCC1 activity was not abolished either by removal of extracellular Ca2+or BAPTA-AM. An increase in phosphorylation of NKCC1 was detected under both 10 mM [Cl−]oand muscimol conditions. These results suggest that a GABA-mediated loss of intracellular Cl−, but not a subsequent rise in [Ca2+]ior shrinkage, leads to stimulation of NKCC1. This stimulation may be an important positive feedback mechanism to maintain intracellular Cl−level and GABA function in immature neurons.

The hypothalamus influences a number of autonomic functions. The activity of hypothalamic neurons is modulated in part by release of the inhibitory neurotransmitter GABA onto these neurons. GABAA receptors are formed from a number of distinct subunits, designated α, β, γ, δ, ε, and θ, many of which have multiple isoforms. Little data exist, however, on the functional characteristics of the GABAA receptors present on hypothalamic neurons. To gain insight into which GABAA receptor subunits are functionally expressed in the hypothalamus, we used an array of pharmacologic assessments. Whole cell recordings were made from thin hypothalamic slices obtained from 1- to 14-day-old rats. GABAA receptor-mediated currents were detected in all neurons tested and had an average EC50 of 20 ± 1.6 μM. Hypothalamic GABAA receptors were modulated by diazepam (EC50 = 0.060 μM), zolpidem (EC50 = 0.19 μM), loreclezole (EC50 = 4.4 μM), methyl-6,7-dimethoxy-4-ethyl-β-carboline (EC50= 7.7 μM), and 5α-pregnan-3α-hydroxy-20-one (3α-OH-DHP). Conversely, these receptors were inhibited by Zn2+ (IC50 = 70.5 μM), dehydroepiandrosterone sulfate (IC50 = 16.7 μM), and picrotoxin (IC50 = 2.6 μM). The α4/6-selective antagonist furosemide (10–1,000 μM) was ineffective in all hypothalamic neurons tested. The results of our pharmacological analysis suggest that hypothalamic neurons express functional GABAA receptor subtypes that incorporate α1 and/or α2 subunits, β2 and/or β3 subunits, and the γ2 subunit. Our results suggest receptors expressing α3–α6, β1, γ1, and δ, if present, represent a minor component of functional hypothalamic GABAA receptors.

There is general agreement that saccades are guided to their targets by means of a motor error signal, which is produced by a local feedback circuit that calculates the difference between desired saccadic amplitude and an internal copy of actual saccadic amplitude. Although the superior colliculus (SC) is thought to provide the desired saccadic amplitude signal, it is unclear whether the SC resides in the feedback loop. To test this possibility, we injected muscimol into the brain stem region containing omnipause neurons (OPNs) to slow saccades and then determined whether the firing of neurons at different sites in the SC was altered. In 14 experiments, we produced saccadic slowing while simultaneously recording the activity of a single SC neuron. Eleven of the 14 neurons were saccade-related burst neurons (SRBNs), which discharged their most vigorous burst for saccades with an optimal amplitude and direction (optimal vector). The optimal directions for the 11 SRBNs ranged from nearly horizontal to nearly vertical, with optimal amplitudes between 4 and 17°. Although muscimol injections into the OPN region produced little change in the optimal vector, they did increase mean saccade duration by 25 to 192.8% and decrease mean saccade peak velocity by 20.5 to 69.8%. For optimal vector saccades, both the acceleration and deceleration phases increased in duration. However, during 10 of 14 experiments, the duration of deceleration increased as fast as or faster than that of acceleration as saccade duration increased, indicating that most of the increase in duration occurred during the deceleration phase. SRBNs in the SC changed their burst duration and firing rate concomitantly with changes in saccadic duration and velocity, respectively. All SRBNs showed a robust increase in burst duration as saccadic duration increased. Five of 11 SRBNs also exhibited a decrease in burst peak firing rate as saccadic velocity decreased. On average across the neurons, the number of spikes in the burst was constant. There was no consistent change in the discharge of the three SC neurons that did not exhibit bursts with saccades. Our data show that the SC receives feedback from downstream saccade-related neurons about the ongoing saccades. However, the changes in SC firing produced in our study do not suggest that the feedback is involved with producing motor error. Instead, the feedback seems to be involved with regulating the duration of the discharge of SRBNs so that the desired saccadic amplitude signal remains present throughout the saccade.

We investigated the effects of linear perspective and binocular disparity, as monocular and binocular depth cues, respectively, on the response of surface-orientation–selective (SOS) neurons in the caudal part of the lateral bank of the intraparietal sulcus (area CIP). During the single-unit recording, monkeys were required to perform the delayed-matching-to-sample (successive same/different discrimination) of discriminating surface orientation in stereoscopic computer graphics. Of 211 visually responsive neurons, 66 were intensively tested using the solid-figure stereogram (SFS) of a square plate with both disparity and perspective cues (D+P condition), and 62 of these were identified as SOS neurons for responding selectively to the orientation of stimuli. All these neurons were further tested using a solid figure with perspective cues alone (P-only condition), and 58% (36/62) of these showed selective response to the orientation of the stimuli. Of the 62 SOS neurons, 35 neurons were also tested using SFS with disparity cues alone (D-only condition) in addition to the D+P and P-only conditions. We classified these 35 neurons into four groups by comparing the response selectivity under the P-only and D-only conditions. More than one-half of these (19/35) were sensitive to both perspective and disparity cues (DP neurons), and nearly one-third (11/35) of these were sensitive to disparity cues alone (D neurons), but a few (2/35) were sensitive to perspective cues alone (P neurons). The remaining (3/35) neurons exhibited orientation selectivity only when both cues were present. In DP neurons, the preferred orientation under the D+P condition was correlated to those under the D-only and P-only conditions, and the response magnitude under the D+P condition was greater than those under the D-only and P-only conditions, suggesting the integration of both cues for the perception of surface orientation. However, in these neurons, the orientation tuning sharpness under the D+P and D-only conditions was higher than that under the P-only condition, suggesting the dominance of disparity cues. After the single-unit recording experiments, muscimol was microinjected into the recording site to temporarily inactivate its function. In all three effective cases out of six microinjection experiments, discrimination of a three-dimensional (3D) surface orientation was impaired when disparity cues alone were present. In only one effective case, when a relatively large amount of muscimol was microinjected, discrimination of a 3D surface orientation was impaired even when both disparity and perspective cues were present. These results suggest that linear perspective is an important cue for representations of a 3D surface of SOS neurons in area CIP, although it is less effective than binocular disparity, and that both of these depth cues may be integrated in area CIP for the perception of surface orientation in depth.

In primates, dorsolateral areas of the prefrontal cortex (PFC) play a major role in visuospatial working memory. To examine the functional organization of the PFC for representing visuospatial working memory, we produced reversible local inactivation, with the local injection of muscimol (5 μg, 1 μl), at various sites ( n = 100) in the dorsolateral PFC of monkeys and observed the behavioral consequences in an oculomotor delayed-response task that required memory-guided saccades for locations throughout both visual fields. At 82 sites, the local injection of muscimol induced deficits in memory-guided saccades to a few specific, usually contralateral, target locations that varied with the location of the injection site. Such deficits depended on the delay length, and longer delays were associated with larger deficits in memory-guided saccades. The injection sites and affected spatial locations of the target showed a gross topographical relationship. No deficits appeared for a control task in which the subject was required to make a visually guided saccade to a visible target. These findings suggest that a specific site in the dorsolateral PFC is responsible for the working memory process for a specific visuospatial coordinate to guide goal-directed behavior. Further, memoranda for specific visuospatial coordinates appear to be represented in a topographical memory mapwithin the dorsolateral PFC to represent visuospatial working memory processes.

The subthalamic nucleus (STN) influences the output of the basal ganglia, thereby interfering with motor behavior. The main inputs to the STN are GABAergic. We characterized the GABAA receptors expressed in the STN and investigated the response of subthalamic neurons to the activation of GABAA receptors. Cell-attached and whole cell recordings were made from rat brain slices using the patch-clamp technique. The newly identified ε subunit confers atypical pharmacological properties on recombinant receptors, which are insensitive to barbiturates and benzodiazepines. We tested the hypothesis that native subthalamic GABAAreceptors contain ε proteins. Applications of increasing concentrations of muscimol, a selective GABAAagonist, induced Cl− and HCO[Formula: see text]currents with an EC50 of 5 μM. Currents induced by muscimol were fully blocked by the GABAAreceptor antagonists, bicuculline and picrotoxin. They were strongly potentiated by the barbiturate, pentobarbital (+190%), and by the benzodiazepines, diazepam (+197%) and flunitrazepam (+199%). Spontaneous inhibitory postsynaptic currents were also significantly enhanced by flunitrazepam. Furthermore, immunohistological experiments with an ε subunit-specific antibody showed that the ε protein was not expressed within the STN. Native subthalamic GABAA receptors did not, therefore, display pharmacological or structural properties consistent with receptors comprising ε. Burst firing is a hallmark of Parkinson's disease. Half of the subthalamic neurons have the intrinsic capacity of switching from regular-firing to burst-firing mode when hyperpolarized by current injection. This raises the possibility that activation of GABAA receptors might trigger the switch. Statistical analysis of spiking activity established that 90% of intact neurons in vitro were in single-spike firing mode, whereas 10% were in burst-firing mode. Muscimol reversibly stopped recurrent electrical activity in all intact neurons. In neurons held in whole cell configuration, membrane potential hyperpolarized by −10 mV whilst input resistance decreased by 50%, indicating powerful membrane shunting. Muscimol never induced burst firing, even in neurons that exhibited the capacity of switching from regular- to burst-firing mode. These molecular and functional data indicate that native subthalamic GABAA receptors do not contain the ε protein and activation of GABAA receptors induces membrane shunting, which is essential for firing inhibition but prevents switching to burst-firing. They suggest that the STN, like many other parts of the brain, has the physiological and structural features of the widely expressed GABAA receptors consisting of αβγ subunits.