Bath Application Of GABA Research Articles

Paclitaxel Causes Electrophysiological Changes in the Anterior Cingulate Cortex via Modulation of the γ-Aminobutyric Acid-ergic System

Objective: The aim of this study was to elucidate any electrophysiological changes that may contribute to the development of neuropathic pain during treatment with the anticancer drug paclitaxel, particularly in the γ-aminobutyric acid (GABA) system. Materials and Methods: One hundred and eight Sprague-Dawley rats were used (untreated control: 43; vehicle-treated: 21, and paclitaxel-treated: 44). Paclitaxel (8 mg/kg) was administered intraperitoneally on 2 alternate days to induce mechanical allodynia. The rats were sacrificed 7 days after treatment to obtain slices of the anterior cingulate cortex (ACC), a brain region involved in the central processing of pain. Field excitatory postsynaptic potentials (fEPSPs) were recorded in layer II/III of ACC slices, and stimulus-response curves were constructed. The observed effects were pharmacologically characterized by bath application of GABA and appropriate drugs to the slices. Results: The paclitaxel-treated rats developed mechanical allodynia (i.e. reduced withdrawal threshold to mechanical stimuli). Slices from paclitaxel-treated rats produced a significantly higher maximal response (E_max) than those from untreated rats (p < 0.001). Bath application of GABA (0.4 µ<smlcap>M</smlcap>) reversed this effect and returned the excitability to a level similar to control. Pretreatment of the slices with the GABA_B receptor blocker CGP 55845 (50 µ<smlcap>M</smlcap>) increased E_max in slices from untreated rats (p < 0.01) but not from paclitaxel-treated rats. Conclusion: In this study, there was a GABA deficit in paclitaxel-treated rats compared to untreated ones. Such a deficit could contribute to the pathophysiology of paclitaxel-induced neuropathic pain (PINP). Thus, the GABAergic system might be a potential therapeutic target for managing PINP.

PKCɛ mediates substance P inhibition of GABAA receptors-mediated current in rat dorsal root ganglion

The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.

Honeybee Kenyon cells are regulated by a tonic GABA receptor conductance.

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 GABAA receptor modulator pentobarbital and is reduced by inhibition of Ca(2+) channels with Cd(2+) 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.

Control of Breathing in In Vitro Brain Stem Preparation from Goldfish (Carassius auratus; Linnaeus)

In vitro brain stem preparations from goldfish (Carassius auratus) were used to first determine whether this species possesses central chemoreceptors able to modulate respiratory activity. Preparations were superfused with an artificial cerebrospinal fluid (aCSF); fictive breathing was recorded extracellularly by placing a suction electrode on cranial nerve VII. Reducing the level of O2 in the gas mixture used to bubble the aCSF from a hyperoxic level (80% or 98.7% O2) to a relative hypoxic level (20% or 40% O2) increased the frequency of the fictive respiratory burst (P = 0.0002). Reducing the pH of the aCSF from 7.9 to 7.4 by increasing CO2 in the superfusate did not affect fictive breathing. Chloride-mediated neurotransmission (GABA/glycine) is a major modulator of respiratory activity; however, its effect on the neural circuits that regulate breathing in teleosts remains unknown. Bath application of GABA (0.5, 5.0 mM) decreased burst frequency but not amplitude; this effect was dose dependent (drug × concentration: P = 0.01). Superfusion of the preparations with aCSF containing 1.25 μM of bicuculline methochloride and 1.50 μM of strychnine hydrochloride (GABAA and glycine receptor antagonists, respectively) increased burst frequency (P = 0.002) and amplitude (P < 0.001). We conclude that respiratory activity produced by the goldfish brain stem is not responsive to the moderate CO2 levels used in this study; it may contain O2 chemoreceptors, but the relatively small response could also reflect nonspecific effects of hypoxia on the central nervous system. Cl(-)-mediated neurotransmission inhibits fictive breathing; this aspect of respiratory regulation is similar to other groups of vertebrates.

Attenuated Benzodiazepine-Sensitive Tonic GABAA Currents of Supraoptic Magnocellular Neuroendocrine Cells in 24-h Water-Deprived Rats

In supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs), γ-GABA, via activation of GABAA receptors (GABAA Rs), mediates persistent tonic inhibitory currents (Itonic ), as well as conventional inhibitory postsynaptic currents (IPSCs, Iphasic ). In the present study, we examined the functional significance of Itonic in SON MNCs challenged by 24-h water deprivation (24WD). Although the main characteristics of spontaneous IPSCs were similar in 24WD compared to euhydrated (EU) rats, Itonic , measured by bicuculline (BIC)-induced Iholding shifts, was significantly smaller in 24WD compared to EU rats (P < 0.05). Propofol and diazepam prolonged IPSC decay time to a similar extent in both groups but induced less Itonic in 24WD compared to EU rats, suggesting a selective decrease in GABAA receptors mediating Itonic over Iphasic in 24WD rats. THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), a preferential δ subunit agonist, and L-655,708, a GABAA receptor α5 subunit selective imidazobenzodiazepine, caused a significantly smaller inward and outward shift in Iholding , respectively, in 24WD compared to EU rats (P < 0.05 in both cases), suggesting an overall decrease in the α5 subunit-containing GABAA Rs and the δ subunit-containing receptors mediating Itonic in 24WD animals. Consistent with a decrease in 24WD Itonic , bath application of GABA induced significantly less inhibition of the neuronal firing activity in 24WD compared to EU SON MNCs (P < 0.05). Taken together, the results of the present study indicate a selective decrease in GABAA Rs functions mediating Itonic as opposed to those mediating Iphasic in SON MNCs, demonstrating the functional significance of Itonic with respect to increasing neuronal excitability and hormone secretion in 24WD rats.

Effects of propofol on GABAergic and glutamatergic transmission in isolated hippocampal single nerve-synapse preparations

We evaluated the effects of propofol on synaptic transmission using a mechanically dissociated preparation of rat hippocampal CA3 neurons to allow assays of single bouton responses evoked from retained functional native nerve endings. We studied synaptic and extrasynaptic GABAA and glutamate receptor responses in a preparation in which experimental solutions rapidly accessed synaptic terminals. Whole-cell responses were evoked by bath application of GABA and glutamate. Synaptic inhibitory and excitatory postsynaptic currents (IPSC and EPSC) were measured as spontaneous and evoked postsynaptic responses. Evoked currents were elicited by focal electrical stimulation. Propofol (1–100μM) enhanced extrasynaptic GABAA-receptor mediated responses but the increase at clinically relevant concentrations (1μM) were minor. In contrast, 1μM propofol significantly increased both the amplitude and frequency of spontaneous IPSCs (sIPSCs) and increased the amplitudes of evoked IPSCs (eIPSCs) while decreasing failure rates (Rf) and paired-pulse ratios (PPR). Decay times of sIPSCs and eIPSCs were significantly prolonged. Although propofol had no effect on extrasynaptic glutamate responses, only supra-clinical propofol concentrations (≥10µM) increased the spontaneous EPSCs (sEPSCs, amplitudes and frequencies) but suppressed evoked EPSCs (eEPSCs decreased amplitudes with increased Rf and PPR). The decay phases of sEPSCs and eEPSCs were not changed. The propofol-induced changes in sEPSCs and eEPSCs resulted from presynaptic GABAA receptor-mediated depolarization, because these actions were blocked by bicuculline. These results suggest that propofol acts at presynaptic and postsynaptic GABAA receptors within GABAergic synapses, but also increases extrasynaptic GABA responses. Our results expand the locus of propofol actions to GABAergic and glutamatergic synapses.

Chloride currents in cones modify feedback from horizontal cells to cones in goldfish retina

In neuronal systems, excitation and inhibition must be well balanced to ensure reliable information transfer. The cone/horizontal cell (HC) interaction in the retina is an example of this. Because natural scenes encompass an enormous intensity range both in temporal and spatial domains, the balance between excitation and inhibition in the outer retina needs to be adaptable. How this is achieved is unknown. Using electrophysiological techniques in the isolated retina of the goldfish, it was found that opening Ca(2+)-dependent Cl(-) channels in recorded cones reduced the size of feedback responses measured in both cones and HCs. Furthermore, we show that cones express Cl(-) channels that are gated by GABA released from HCs. Similar to activation of I(Cl(Ca)), opening of these GABA-gated Cl(-) channels reduced the size of light-induced feedback responses both in cones and HCs. Conversely, application of picrotoxin, a blocker of GABA(A) and GABA(C) receptors, had the opposite effect. In addition, reducing GABA release from HCs by blocking GABA transporters also led to an increase in the size of feedback. Because the independent manipulation of Ca(2+)-dependent Cl(-) currents in individual cones yielded results comparable to bath-applied GABA, it was concluded that activation of either Cl(-) current by itself is sufficient to reduce the size of HC feedback. However, additional effects of GABA on outer retinal processing cannot be excluded. These results can be accounted for by an ephaptic feedback model in which a cone Cl(-) current shunts the current flow in the synaptic cleft. The Ca(2+)-dependent Cl(-) current might be essential to set the initial balance between the feedforward and the feedback signals active in the cone HC synapse. It prevents that strong feedback from HCs to cones flood the cone with Ca(2)(+). Modulation of the feedback strength by GABA might play a role during light/dark adaptation, adjusting the amount of negative feedback to the signal to noise ratio of the cone output.

Dynamic Neuronal Excitability

Event Abstract Back to Event Dynamic Neuronal Excitability Christoph Kirst1, 2*, Julian Ammer3, 4, Felix Felmy3, 4, Andreas Herz3, 4 and Martin Stemmler3, 4 1 Max Planck Institute for Dynamics and Self-Organization, Germany 2 Bernstein Center for Computational Neuroscience Göttingen, Germany 3 Ludwigs Maximilians University Munich, Biology II, Germany 4 Bernstein Center for Computational Neuroscience Munich, Germany Neurons generally show two types of excitability [1,2]: Type I neurons support arbitrary long inter-spike-intervals, while type II neurons start firing with a non-zero frequency upon current injection. Here we show that a transition from type I to type II can be dynamically controlled in a large number of conductance-based neuron models (including Wang-Buszaki, Morris-Lecar, Connor- Stevens, Erisir et al.), e.g. by an increase in leak conductance. We mathematically prove that the bifurcation structure of this transition is organized by a degenerate Bogdanov-Takens-cusp bifurcation point of co-dimension 3 [3] which implies a switch from type I to type II for the spiking dynamics, a transition from integration to resonance near spike threshold, as well as a region of bistability of resting and regular spiking dynamics. We confirm these predictions experimentally for different neurons using dynamic patch clamp recordings to artificially change the leak conductance. Interestingly, the neuronal excitability type can also be switched dynamically via activation of shunting synapses, which we mimicked experimentally by bath application of GABA. These results imply that inhibitory cells can dynamically control the neuronal excitability type of postsynaptic neurons and as a consequence their synchronization properties. In particular, we show that inhibition can separately synchronize several coexisting sub-populations of excitatory neurons. Moreover, the maximal amount of synchrony in the network can be efficiently regulated by dynamically forcing the neurons into the region of bistability. In conclusion, inhibition-induced dynamic neuronal excitability switching provides a mechanism for flexible and activity controlled dynamic formation of synchronized neuronal cell assemblies.

Intrinsic activation of GABAA receptors suppresses epileptiform activity in the cerebral cortex of immature mice

Activation of ionotropic γ-aminobutyric acid type A (GABA(A) ) receptors induces in immature neocortical neurons a membrane depolarization that may contribute to the higher epilepsy susceptibility in newborns. To elucidate whether depolarizing GABAergic responses enhance or attenuate epileptiform activity in the immature neocortex, we investigated the effect of agonists, antagonists, and positive modulators of GABA(A) receptors on epileptiform activity. We performed in vitro field potential recordings on isolated whole neocortex preparations and whole cell recordings of identified pyramidal neurons in 400-μm slices of immature (postnatal day 1-7) mice. Epileptiform activity was induced by low Mg²(+) solutions with or without 50-100 μm 4-aminopyridine. Bath application of GABA (3-100 μm, in the presence of tiagabine) attenuated epileptiform activity. The GABA transporter isoform 1 (GAT-1) inhibitor tiagabine (30 μm) and the GAT-2/3 specific inhibitor SNAP 5114 (40 μm) reduced the frequency of epileptiform activity. The benzodiazepines midazolam (0.2 μm) and zolpidem (0.5 μm) as well as the barbiturate phenobarbital (30 μm) slightly attenuated epileptiform activity. Continuous bath application of the GABAergic antagonist gabazine (SR-95531, 2-3 μm) or picrotoxin (15 μm) induced epileptiform discharges. These results demonstrate, that (1) the activation or positive modulation of GABA(A) receptors attenuates epileptiform activity, (2) GABA(A) antagonists mediate a disinhibition, and (3) GABA uptake contributes to the regulation of extracellular GABA in immature neocortex. We conclude from these findings that a constant inhibition via GABA(A) receptors is required to suppress epileptiform activity already in the immature neocortex.

Spread of excitation across modality borders in spinal dorsal horn of neuropathic rats

Under physiological conditions, nociceptive information is mainly processed in superficial laminae of the spinal dorsal horn, whereas non-nociceptive information is processed in deeper laminae. Neuropathic pain patients often suffer from touch-evoked pain (allodynia), suggesting that modality borders are disrupted in their nervous system. We studied whether excitation evoked in deep dorsal horn neurons either via stimulation of primary afferent Aβ-fibres, by direct electrical stimulation or via glutamate microinjection leads to activation of neurons in the superficial dorsal horn. We used Ca 2+-imaging in transversal spinal cord slices of neuropathic and control animals to monitor spread of excitation from the deep to the superficial spinal dorsal horn. In neuropathic but not control animals, a spread of excitation occurred from the deep to the superficial dorsal horn. The spread of excitation was synaptically mediated as it was blocked by the AMPA receptor antagonist CNQX. In contrast, block of NMDA receptors was ineffective. In control animals, the violation of modality borders could be reproduced by bath application of GABA A and glycine receptor antagonists. Furthermore, we could show that neuropathic animals were more prone to synchronous network activity than control animals. Thus, following peripheral nerve injury, excitation generated in dorsal horn areas which process non-nociceptive information can invade superficial dorsal horn areas which normally receive nociceptive input. This may be a spinal mechanism of touch-evoked pain.

GABAB receptor‐mediated modulation of hypocretin/orexin neurones in mouse hypothalamus

Hypocretin/orexin (Hcrt) is a critical neurotransmitter for the maintenance of wakefulness and has been implicated in several other functions, including energy metabolism and reward. Using whole-cell patch-clamp recordings from transgenic mice in which enhanced green fluorescent protein was linked to the Hcrt promoter, we investigated GABAergic control of the Hcrt neurones in hypothalamic slices. Bath application of GABA or muscimol caused an early hyperpolarization mediated by Cl(-) and a late depolarization mediated by the efflux of bicarbonate. These GABA(A) receptor-mediated responses were blocked by picrotoxin and bicuculline. Under the GABA(A) blockade condition, GABA produced consistent hyperpolarization, decreased firing rate and input resistance. The selective GABA(B) agonist (R)-baclofen caused a similar response with an EC(50) of 7.1 mum. The effects of (R)-baclofen were blocked by the GABA(B) antagonist CGP 52432 but persisted in the presence of tetrodotoxin, suggesting direct postsynaptic effects. The existence of GABA(B) modulation was supported by GABA(B(1)) subunit immunoreactivity on Hcrt cells colabelled with antisera to the Hcrt-2 peptide. Furthermore, GABA(B) receptor activation inhibited the presynaptic release of both glutamate and GABA. (R)-Baclofen depressed the amplitude of evoked excitatory postsynaptic currents (EPSCs) and inhibitory synaptic currents (IPSCs), and also decreased the frequency of both spontaneous and miniature EPSCs and IPSCs with a modest effect on their amplitudes. These data suggest that GABA(B) receptors modulate Hcrt neuronal activity via both pre- and postsynaptic mechanisms, which may underlie the promotion of non-rapid eye movement sleep and have implications for the use of GABA(B) agonists in the treatment of substance addiction through direct interaction with the Hcrt system.

Interaction between facilitation and presynaptic inhibition at the crayfish neuromuscular junction

Action potential-mediated calcium (Ca) entry into excitor nerve terminal boutons during presynaptic inhibition and the effects of co-activation of the inhibitor on the kinetics of muscle contraction were studied at crayfish claw opener muscle. Inhibition reduced postsynaptic excitatory junction potentials (EJPs) below the threshold to initiate contraction. Upon cessation of inhibition, EJP amplitudes immediately increased several-fold due to the build-up of presynaptic facilitation during inhibition. Consequently, muscle contraction was initiated more rapidly after a period of inhibitor-excitor coactivation. Presynaptic inhibition reduced Ca entry into presynaptic excitor terminal boutons (range 0-50%, mean +/-s.e.m.=20+/-1%, N=122 terminals; 12 preparations) and reduced the EJP amplitude (range 30-70%, mean +/- s.e.m.=51+/-2%, N=27 cells). The decline in the EJP was proportional to the reduction of Ca influx raised to the power of 2.8. Since presynaptic inhibition reduces the number of Ca channels opened by an action potential, our data suggest cooperativity between Ca channel microdomains to initiate vesicle fusion at this synapse. The amount of inhibition of Ca influx into an excitor bouton was not correlated with either the distance to the closest inhibitor bouton or the main excitor branch, although slightly more inhibition was seen for excitor boutons on tertiary versus secondary branches. Unlike inhibitor axon stimulation, bath application of GABA caused inhibition of Ca influx that steadily increased from proximal to distal terminal boutons on a branch. We propose a model where presynaptic inhibition causes localized shunting of an actively propagated action potential in the vicinity of release sites, which can recover its amplitude outside the shunted region.

Prenatal nicotine exposure increases the strength of GABAA receptor‐mediated inhibition of respiratory rhythm in neonatal rats

Infants born to mothers that smoke while pregnant have a relatively high incidence of central respiratory control abnormalities. Recent studies have shown that prenatal nicotine exposure increases GABA release and the frequency of GABAergic currents, leading to an up-regulation of GABA(A) receptors in central neurones. Activation of GABA(A) receptors inhibits ventilatory activity, with intense activation causing apnoea. These observations lead us to hypothesize that prenatal nicotine exposure alters GABAergic control of respiratory motor pattern in the early neonatal period. Osmotic minipumps were implanted in pregnant Sprague-Dawley rats on the fifth day of gestation, and filled with nicotine (6 mg kg(-1) day(-1), 2.5 microl h(-1)) or physiological saline (2.5 microl h(-1)). Brainstem-spinal cord preparations from 1- to 3-day-old neonates were studied under in vitro conditions. Electrical activity was recorded from the fourth cervical ventral root (C4 VR), which contains the axons of phrenic motoneurones. Bath application of GABA(A) receptor agonists muscimol (250 microM) or pentobarbital sodium (60 microM) to the brainstem led to consistent, reversible and significant reductions in C4 VR burst frequency. In saline-exposed animals, frequency (bursts min(-1)) fell from 6.8 +/- 0.4 to a nadir of 2.8 +/- 0.5 with muscimol, and from 6.5 +/- 0.3 to a nadir of 2.9 +/- 0.3 for pentobarbital; in nicotine-exposed animals, frequency fell from 6.3 +/- 0.4 to 1.0 +/- 0.4 with muscimol and from 6.4 +/- 0.2 to 1.7 +/- 0.4 with pentobarbital (P < 0.05 in all cases). The decrease in C4 VR frequency was significantly greater in nicotine-exposed compared to saline-exposed preparations with both muscimol and pentobarbital (P < 0.001 for both). There were no changes in the amplitude of C4 VR bursts under any condition. The GABA(A) receptor antagonist bicuculline methiodide (8 microM) did not change C4 VR frequency or amplitude in either group, although it was effective in reversing the effects of muscimol. These experiments demonstrate that prenatal nicotine exposure alters the GABAergic regulation of respiratory rhythm in a reduced preparation. The results may lead to a better understanding of the perturbed breathing pattern observed in neonates that are exposed to nicotine in utero.

Functional synaptic projections onto subplate neurons in neonatal rat somatosensory cortex.

Subplate neurons (SPn) play an important role in the formation of thalamocortical connections during early development and show glutamatergic and GABAergic spontaneous synaptic activity. We characterized these synaptic inputs by performing whole-cell recordings from SPn in somatosensory cortical slices of postnatal day 0-3 rats. At -70 mV, electrical stimulation of the thalamocortical afferents elicited in 68% of the SPn a monosynaptic CNQX-sensitive postsynaptic current (PSC). These fast PSCs were mediated by AMPA receptors, because they were prolonged by cyclothiazide and blocked by GYKI 52466. On membrane depolarization, thalamocortical stimulation elicited in 50% of the cells an additional slow monosynaptic component mediated by NMDA receptors. Stimulation of the cortical plate evoked in 72% of SPn a monosynaptic AMPA receptor-mediated PSC with an additional NMDA component at depolarized membrane potentials and in 40% of the investigated cells polysynaptic responses, depending on GABA(A) and NMDA receptors. Stimulation of the subplate elicited in 67% of SPn a monosynaptic dual-component PSC mediated by AMPA and NMDA receptors activated at -70 mV and in 12% of SPn a monosynaptic single-component PSC mediated by AMPA receptors with an additional NMDA component activated at depolarized membrane potentials. A monosynaptic GABAergic response could be observed in 68% of SPn after stimulation of the subplate. In gramicidin-perforated patch recordings, bath application of GABA caused membrane depolarization to -40 mV and elicited action potentials. These results demonstrate that SPn receive distinct functional synaptic inputs arising from the thalamus, cortical plate, and subplate, indicating that SPn are capable of integrating and processing information from cortical and subcortical regions.

Functional Synaptic Projections onto Subplate Neurons in Neonatal Rat Somatosensory Cortex

Subplate neurons (SPn) play an important role in the formation of thalamocortical connections during early development and show glutamatergic and GABAergic spontaneous synaptic activity. We characterized these synaptic inputs by performing whole-cell recordings from SPn in somatosensory cortical slices of postnatal day 0–3 rats. At –70 mV, electrical stimulation of the thalamocortical afferents elicited in 68% of the SPn a monosynaptic CNQX-sensitive postsynaptic current (PSC). These fast PSCs were mediated by AMPA receptors, because they were prolonged by cyclothiazide and blocked by GYKI 52466. On membrane depolarization, thalamocortical stimulation elicited in 50% of the cells an additional slow monosynaptic component mediated by NMDA receptors. Stimulation of the cortical plate evoked in 72% of SPn a monosynaptic AMPA receptor-mediated PSC with an additional NMDA component at depolarized membrane potentials and in 40% of the investigated cells polysynaptic responses, depending on GABA_A and NMDA receptors. Stimulation of the subplate elicited in 67% of SPn a monosynaptic dual-component PSC mediated by AMPA and NMDA receptors activated at –70 mV and in 12% of SPn a monosynaptic single-component PSC mediated by AMPA receptors with an additional NMDA component activated at depolarized membrane potentials. A monosynaptic GABAergic response could be observed in 68% of SPn after stimulation of the subplate. In gramicidin-perforated patch recordings, bath application of GABA caused membrane depolarization to –40 mV and elicited action potentials. These results demonstrate that SPn receive distinct functional synaptic inputs arising from the thalamus, cortical plate, and subplate, indicating that SPn are capable of integrating and processing information from cortical and subcortical regions.

Role of GABAB Receptor in the Regulation of Excitatory Synaptic Transmission in Trigeminal Motoneurons

The aim of the present study was to determine if excitatory synaptic transmission onto trigeminal motoneurons is subject to a presynaptic modulation by γ-aminobutyric acid (GABA) via GABA_B receptor in this system. Whole cell recordings were made from trigeminal motoneurons in longitudinal brain stem slices taken from 8-day-old rats. Monosynaptic excitatory postsynaptic potential (EPSP) activity was evoked by placing bipolar stainless steel electrodes dorsal-caudal to the trigeminal motor nucleus. Bath application of the GABA_B receptor agonist, baclofen, produced a marked reduction in the mean amplitude and variance of evoked EPSPs and also increased the portion of transmission failures. It also produced a decrease in the frequency, but not in the mean amplitude, of spontaneous miniature EPSPs. Bath application of GABA_B receptor antagonists 6-hydroxy-saclofen and CGP35348 increased both the amplitude and frequency of miniature EPSP activity. Taken together the above results suggest that the excitatory synaptic inputs onto trigeminal motoneurons are controlled by tonic presynaptic modulation by GABA_B receptor.

Participation of GABA in establishing behavioral hierarchies in the terrestrial snail.

GABA-immunoreactive fibers were observed in the neuropile of each ganglion of Helix lucorum, while GABA-immunoreactive neural somata were found only in the buccal, cerebral, and pedal ganglia. Bath application of 10(-5) M GABA to the preparation "buccal mass-buccal ganglia" elicited a sequence of radula movements characteristic of feeding behavior. Corresponding bursts of activity were recorded in the buccal nerves under GABA application and in the buccal neurons recorded optically. In preparations of isolated central nervous system, the bath applications of GABA (10(-5) to 10(-4) M) elicited no changes in synaptic input of the premotor interneurons involved in the withdrawal behavior. However, a significant decrease in amplitude of the synaptic input and in the number of spikes in responses elicited by the test nerve stimulation was observed in metacerebral serotonergic neurons involved in modulating the feeding behavior. GABA application inhibited the spontaneous spike activity in some pedal serotonergic neurons involved in the network underlying withdrawal responses and evoked bursting activity in the other neurons of this functional group. The effects of GABA application on mechanically isolated serotonergic neurons suggest that the primary effect of GABA is inhibition. Thus, our results give evidence of the putative role of GABA in activating the feeding behavior and in the synergistic suppression of serotonergic modulation of the withdrawal behavior and serotonergic modulation of feeding, which has corresponded to the observed behavioral suppression of withdrawal reactions during feeding.

GABA Enhances Transmission at an Excitatory Glutamatergic Synapse

GABA mediates both presynaptic and postsynaptic inhibition at many synapses. In contrast, we show that GABA enhances transmission at excitatory synapses between the lateral gastric and medial gastric motor neurons and the gastric mill 6a and 9 (gm6a, gm9) muscles and between the lateral pyloric motor neuron and pyloric 1 (p1) muscles in the stomach of the lobster Homarus americanus. Two-electrode current-clamp or voltage-clamp techniques were used to record from muscle fibers. The innervating nerves were stimulated to evoke excitatory junctional potentials (EJPs) or excitatory junctional currents. Bath application of GABA first decreased the amplitude of evoked EJPs in gm6a and gm9 muscles, but not the p1 muscle, by activating a postjunctional conductance increase that was blocked by picrotoxin. After longer GABA applications (5-15 min), the amplitudes of evoked EJPs increased in all three muscles. This increase persisted in the presence of picrotoxin. beta-(Aminomethyl)-4-chlorobenzenepropanoic acid (baclofen) was an effective agonist for the GABA-evoked enhancement but did not increase the postjunctional conductance. Muscimol activated a rapid postsynaptic conductance but did not enhance the amplitude of the nerve-evoked EJPs. GABA had no effect on iontophoretic responses to glutamate and decreased the coefficient of variation of nerve-evoked EJPs. In the presence or absence of tetrodotoxin, GABA increased the frequency but not the amplitude of miniature endplate potentials. These data suggest that GABA acts presynaptically via a GABA(B)-like receptor to increase the release of neurotransmitter.

Disinhibition in Rat Superior Colliculus Mediated by GABACReceptors

The stratum griseum superficiale (SGS) of the superior colliculus contains a high concentration of the recently described GABA(C) receptor. In a previous study, it was postulated that activation of these receptors on inhibitory interneurons functions to disinhibit projection cells that relay visual information to the thalamus and brainstem. To test this model, we used in vitro whole-cell patch-clamp methods to measure effects of GABA and muscimol on EPSCs and IPSCs evoked in rat SGS by electrical optic layer stimulation. The neurons were filled with biocytin for later morphological characterization. As expected, bath applications of GABA and muscimol always strongly depressed evoked PSCs at concentrations of >100 and >1 micrometer, respectively. However, at lower agonist concentrations, which most likely activate GABA(C) but not GABA(A) receptors, effects were not uniform. Evoked responses were suppressed by both agonists in 48% of the neurons, whereas the remaining cells exhibited enhanced responses with increased evoked EPSCs, decreased evoked IPSCs, or both types of change. Most morphologically identified cells with suppressed responses (14 of 17 cells) had morphological characteristics of putative GABAergic interneurons, whereas almost all cells with enhanced responses (8 of 10 cells) had morphological characteristics of projection cells. Finally, all effects of GABA and muscimol at low concentrations were blocked by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid, a specific GABA(C) receptor antagonist, but not by the specific GABA(A) receptor antagonist bicuculline. Taken together, these results indicate that in SGS, GABA(C) receptors are predominantly expressed by GABAergic neurons and that activation of these receptors leads to disinhibition of SGS projection cells.

Development of gill and lung breathing in amphibia

In the 25 morphological stages of larval bullfrog development there exists a gradual transition from gill to lung ventilation associated with a developmental decrease in the contribution of the skin in gas exchange. Bath application of GABA and/or glycine inhibited gill but not lung burst activities of cranial nerve (CN) VII in the premetamorphic (stages 16–19) in vitro tadpole brainstem preparation [1]. It was proposed that the neural basis of gill rhythmogenesis involved network inhibition, whereas lung rhythmicity was pacemaker driven [1]. Bath application of a bicuculline/strychnine solution abolished gill and enhanced lung bursting in stages 16–19 in vitro [1]. Bath application of the GABAB receptor antagonist 2-hydroxy-saclofen disinhibited the lung central pattern generator (CPG) resulting in precocious lung bursting patterns as early as developmental stage 6 [2]. We recorded efferent activity from CNVII and spinal nerve (SN)II in the in vitro tadpole brainstem preparation in three successive developmental groups (3–9; 10–15; 16–19) before and after bath application of a 10 μM bicuculline and 5 μM strychnine solution. We also exposed the brainstem to bath pH7.4, 7.8, and 8.2 before and after bath application of bicuculline/strychnine. Bicuculline/strychnine produced lung ventilatory bursts in all developmental stages tested, indicating the presence of the lung CPG as well as excitatory synapses to respiratory motor neurons as early as stage 3. We also designed an experiment to examine the importance of lung ventilation on the developmental shift from gill to lung bursting. Two groups of tadpoles were hatched from eggs. Control tadpoles had free access to the air-water interface throughout development, whereas tadpoles were denied access to the air-water interface via the placement of Plexiglas 2.5 cm below the surface of the water. CNVII and SNII efferent activities were recorded in vitro at bath pH7.4, 7.8, and 8.2 before and after bath application of 10 μM bicuculline and 5 μM strychnine. Postmetamorphic barrier tadpoles exhibited different motor patterns than stage-matched controls. Tadpoles reared in the barrier condition to stages 20–25 possessed fictive gill and lung ventilatory activities of premetamorphic tadpoles. All barrier tadpole preparations exhibited robust, spontaneous lung burst activity following bath application of bicuculline/strychnine. We propose that developmentally dependent GABA- and glycinergic mechanisms lead to disinhibition of the lung CPG such that early in development gill motor patterns are the dominant respiratory rhythm, whereas in late development the lung CPG is the dominant respiratory rhythm. Denying the tadpole the ability to practice lung breathing during metamorphosis produces a morphologically correct postmetamorphic tadpole with an immature, or premetamorphic respiratory rhythm. We propose that prevention of lung inflation in barrier tadpoles leads to premetamorphic levels of GABA- and glycinergic inhibition, and that this inhibition may be altered by pulmonary stretch receptor feedback in control tadpoles.