GABA-mediated synaptic inhibition of projection neurons in the antennal lobes of the sphinx moth,Manduca sexta

Responses of neurons in the antennal lobe (AL) of the moth Manduca sexta to stimulation of the ipsilateral antenna by odors consist of excitatory and inhibitory synaptic potentials. Stimulation of primary afferent fibers by electrical shock of the antennal nerve causes a characteristic IPSP-EPSP synaptic response in AL projection neurons. The IPSP in projection neurons reverses below the resting potential, is sensitive to changes in external and internal chloride concentration, and thus is apparently mediated by an increase in chloride conductance. The IPSP is reversibly blocked by 100 microM picrotoxin or bicuculline. Many AL neurons respond to application of GABA with a strong hyperpolarization and an inhibition of spontaneous spiking activity. GABA responses are associated with an increase in neuronal input conductance and a reversal potential below the resting potential. Application of GABA blocks inhibitory synaptic inputs and reduces or blocks excitatory inputs. EPSPs can be protected from depression by application of GABA. Muscimol, a GABA analog that mimics GABA responses at GABAA receptors but not at GABAB receptors in the vertebrate CNS, inhibits many AL neurons in the moth.     

Non-monotonic effects of GABAergic synaptic inputs on neuronal firing

GABA is generally known as the principal inhibitory neurotransmitter in the nervous system, usually acting by hyperpolarizing membrane potential. However, GABAergic currents sometimes exhibit non-inhibitory effects, depending on the brain region, developmental stage or pathological condition. Here, we investigate the diverse effects of GABA on the firing rate of several single neuron models, using both analytical calculations and numerical simulations. We find that GABAergic synaptic conductance and output firing rate exhibit three qualitatively different regimes as a function of GABA reversal potential, E_GABA: monotonically decreasing for sufficiently low E_GABA (inhibitory), monotonically increasing for E_GABA above firing threshold (excitatory); and a non-monotonic region for intermediate values of E_GABA. In the non-monotonic regime, small GABA conductances have an excitatory effect while large GABA conductances show an inhibitory effect. We provide a phase diagram of different GABAergic effects as a function of GABA reversal potential and glutamate conductance. We find that noisy inputs increase the range of E_GABA for which the non-monotonic effect can be observed. We also construct a micro-circuit model of striatum to explain observed effects of GABAergic fast spiking interneurons on spiny projection neurons, including non-monotonicity, as well as the heterogeneity of the effects. Our work provides a mechanistic explanation of paradoxical effects of GABAergic synaptic inputs, with implications for understanding the effects of GABA in neural computation and development.     

Inhibiton and gamma-aminobutyric acid-induced conductance on locust (Schistocerca gregaria) extensor tibiae muscle fibres

• 1.1. Increases in membrane conductance (g_m) were induced by GABA in distal bundles 32, 33 and 34 of extensor tibiae muscles of the locust (Schistocerca gregaria).
• 2.2. Bath application of GABA (10⁻⁵−5 × 10⁻³ M) induced reductions in muscle fibre space constant (λ).
• 3.3. GABA (5 × 10⁻³ M) induced additional membrane conductance of 2.21 ± 0.03 × 10⁻⁶ S/mm, 0.38 ± 0.03 × 10⁻⁶ S/mm and 0.29 ± 0.06 × 10⁻⁶ S/mm on muscle bundles 34, 33 and 32 respectively. The greater sensitivity of muscle fibres in bundle 34 to GABA is due at least in part to a larger number of GABA receptors on bundle 34 muscle fibres.
• 4.4. The decrement of electrotonic potentials in the presence of GABA were measured over distances of both half fibre length and whole fibre length. Good agreement was obtained between changes in space constant produced by GABA using half fibre length and whole fibre length data.
• 5.5. By taking into account changes in space constant induced by GABA it was possible to demonstrate that presynaptic GABA receptors were involved in the inhibition of slow excitatory postsynaptic potentials by GABA.
• 6.6. “Slow” excitatory postsynaptic potentials recorded under current clamp were inhibited in a dose-dependent manner by GABA. This inhibition was not dependent on muscle-fibre GABA sensitivity and could not be completely accounted for by GABA-induced changes in the cable properties of the muscle fibres.

     

Use-dependent block of GABA-activated chloride channels in crayfish muscle fibers by picrate

In crayfish muscle fibers studied with intracellular microelectrodes the protein-binding agent, picrate (2,4,6-trinitrophenolate; 10(-5)-2 X 10(-4) M) was found to have a specific and dose-dependent inhibitory effect on the chloride conductance activated by bath-applied gamma-aminobutyric acid (GABA). A kinetic analysis showed that picrate did not interfere with GABA binding to its receptor. The blocking action of picrate was not increased by lowering the extracellular Cl- concentration which indicates that picrate is not likely to bind to the ionic selectivity site of the postsynaptic Cl- channel. In fibers first exposed to picrate (1-2 X 10(-4) M) and then, in the continuous presence of this drug, to GABA (5 X 10(-4) M), the latter induced a transient increase in the chloride conductance with an apparent rate constant of decay of about 40 sec. It is tentatively suggested that the site of action of picrate is a positively charged amino acid residue that is exposed through the action of GABA and critically involved in the chemical gating of the postsynaptic chloride channel.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage—current relationship of the membrane. By including γ-aminobutyrate (GABA) in the bathing medium, the voltage—current relationship was made linear in the depolarizing direction over a range of 15–20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and asparate functioning synergistically.     

L-glutamate activates excitatory and inhibitory channels in Drosophila larval muscle

Muscle fibers from Drosophila larvae show an L-glutamate-sensitive membrane potential. Bath-applied L-glutamate depolarizes the muscle in the range from 0.5 to 20 microM. Greater concentrations of the agonist repolarize the fibers. The repolarizing effect disappears if chloride is replaced by sulfate in the external medium. Intracellular recordings show the occurrence of depolarizing and hyperpolarizing spontaneous miniature postsynaptic potentials (smpp). Patch-clamp studies indicate the presence of two types of receptor channels: (i) an anion-selective channel activated by both L-glutamate and GABA. In outside out-patches, bathed in symmetrical 140 mM Cl- and 200 microM GABA, the channel displays conductance substates of 40, 80 and 110 pS. In the presence of 200 microM L-glutamate only the 40 and 80 pS substates are observed; (ii) a cation-selective channel activated only by L-glutamate that has a conductance of 104 pS in cell-attached patches (128 mM Na+ outside). The presence of these two types of receptor channels in Drosophila muscle may explain the effect of bath-applied L-glutamate on membrane potential and the presence of inhibitory and excitatory smpp.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster.

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage-current relationship of the membrane. By including gamma-aminobutyrate (GABA) in the bathing medium, the voltage-current relationship was made linear in the depolarizing direction over a range of 15-20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and aspartate functioning synergistically.     

The electrophysiology and pharmacology of lobster neuromuscular synapses

Effects of drugs on resting potential, membrane resistance, and excitatory and inhibitory postsynaptic potentials (e.p.s.p.'s and i.p.s.p.'s) of lobster muscle fibers were studied using intracellular microelectrodes Acetylcholine, d-tubocurarine, strychnine, and other drugs of respectively related actions on vertebrate synapses were without effects even in 1 per cent solutions (10^- w/v). Gamma-aminobutyric acid (GABA) acted powerfully and nearly maximally at 10^-7 to 10^-6 w/v. Membrane resistance fell two- to tenfold, the resting potential usually increasing slightly. This combination of effects, which indicates activation of inhibitory synaptic membrane, was also produced by other short chain ω-amino acids and related compounds that inactivate depolarizing axodendritic synapses of cat. The conductance change, involving increased permeability to Cl^-, by its clamping action on membrane potential shortened as well as decreased individual e.p.s.p.'s. Picrotoxin in low concentration (ca. 10^-7 w/v) and guanidine in higher (ca. 10^-3 w/v) specifically inactivate inhibitory synapses. GABA and picrotoxin are competitive antagonists. The longer chain ω-amino acids which inactivate hyperpolarizing axodendritic synapses of cat are without effect on lobster neuromuscular synapse. However, one member of this group, carnitine (β-OH-GABA betaine), activated the excitatory synapses, a decreased membrane resistance being associated with depolarzation. The pharmacological properties of lobster neuromuscular synapses and probably also of other crustacean inhibitory synapses appear to stand in a doubly inverted relation to axodendritic synapses of cat.     

The function of the proximal synapses of the squid stellate ganglion

In the oxygenated excised squid (Loligo pealii) stellate ganglion preparation one can produce excitation of the stellar giant axons by stimulating the second largest (accessory fiber, Young, 1939) or other smaller preganglionic giant axons. Impulse transmission is believed to occur at the proximal synapses of the stellar giant axons rather than the distal (giant) synapses which are excited by the largest giant preaxon. Proximal synaptic transmission is more readily depressed by hypoxia and can be fatigued independently of, and with fewer impulses than, the giant synapses. Intracellular recording from the last stellar axon at its inflection in the ganglion reveals both proximal and distal excitatory postsynaptic potentials EPSP's). The synaptic delay, temporal form of the EPSP, and depolarization for spike initiation were similar for both synapses. If the proximal EPSP occurs shortly after excitation by the giant synapse it reduces the undershoot and adds to the falling phase of the spike. If it occurs later it can produce a second spike. Parallel results were obtained when the proximal EPSP's arrived earlier than the EPSP of the giant synapse. In fatigued preparations it was possible to sum distal and proximal or two proximal EPSP's and achieve spike excitation.     

GABA and taurine sensitivity of cockroach giant interneurone synapses: Indirect evidence for the existence of a GABA uptake mechanism

The effects of exogenous GABA and taurine were studied on the cercal afferent-giant interneurone synapses (G.I. 2) located in the neuropile of the sixth abdominal ganglion of the cockroach, Periplaneta americana L. The decrease in excitatory synaptic potentials and the increase in postsynaptic membrane conductance due to GABA were enhanced by lowering the temperature of the saline, by using Na⁺ pump inhibitors, Na⁺ free salines or by agents blocking GABA uptake. The action of temperature was studied for taurine. Implications of these results for the identification of a metabolically dependent GABA uptake mechanism into glial cells are discussed.     

GABAB-receptor-mediated suppression of sympathetic outflow from the spinal cord of neonatal rats.

Using a splanchnic nerve-spinal cord preparation in vitro that could spontaneously generate sympathetic nerve discharge (SND), we investigated the roles of intraspinal GABA(B) receptors in the regulation of SND. Despite an age-dependent difference in sensitivity, bath applications of baclofen (Bac; GABA(B)-receptor agonist) consistently reduced SND in a concentration-dependent manner. The drug specificity of Bac in activation of GABA(B) receptors was verified by application of its antagonist saclofen (Sac) or CGP-46381 (CGP). Sac or CGP alone did not change SND. However, in the presence of Sac or CGP, the effects of Bac on SND inhibition were reversibly attenuated. The splanchnic sympathetic preganglionic neuron (SPN) was recorded by blind whole cell, patch-clamp techniques. We examined Bac effects on electrical membrane properties of SPNs. Applications of Bac reduced excitatory synaptic events, induced membrane hyperpolarizations, and inhibited SPN firing. In the presence of 12 mM Mg2+ or 0.5 microM TTX to block Ca2+- or action potential-dependent synaptic transmissions, applications of Bac induced an outward baseline current that reversed at -29 +/- 6 mV. Because the K+ equilibrium potential in our experimental conditions was -100 mV, the Bac-induced currents could not simply be attributed to an alteration of K+ conductance. On the other hand, applications of Bac to Cs+-loaded SPNs reduced Cd2+-sensitive and high-voltage-activated inward currents, indicating an inhibition of voltage-gated Ca2+ currents. Our results suggest that the activation of intraspinal GABA(B) receptors suppresses SND via a mixture of ion events that may link to a change in Ca2+ conductance.     

Golgi Cell-Mediated Activation of Postsynaptic GABA(B) Receptors Induces Disinhibition of the Golgi Cell-Granule Cell Synapse in Rat Cerebellum

In the cerebellar glomerulus, GABAergic synapses formed by Golgi cells regulate excitatory transmission from mossy fibers to granule cells through feed-forward and feedback mechanisms. In acute cerebellar slices, we found that stimulating Golgi cell axons with a train of 10 impulses at 100 Hz transiently inhibited both the phasic and the tonic components of inhibitory responses recorded in granule cells. This effect was blocked by the GABA(B) receptor blocker CGP35348, and could be mimicked by bath-application of baclofen (30 μM). This depression of IPSCs was prevented when granule cells were dialyzed with GDPβS. Furthermore, when synaptic transmission was blocked, GABA(A) currents induced in granule cells by localized muscimol application were inhibited by the GABA(B) receptor agonist baclofen. These findings indicate that postsynaptic GABA(B) receptors are primarily responsible for the depression of IPSCs. This inhibition of inhibitory events results in an unexpected excitatory action by Golgi cells on granule cell targets. The reduction of Golgi cell-mediated inhibition in the cerebellar glomerulus may represent a regulatory mechanism to shift the balance between excitation and inhibition in the glomerulus during cerebellar information processing.     

GABA and neuroligin signaling: linking synaptic activity and adhesion in inhibitory synapse development

GABA-mediated synaptic inhibition is crucial in neural circuit operations. In mammalian brains, the development of inhibitory synapses and innervation patterns is often a prolonged postnatal process, regulated by neural activity. Emerging evidence indicates that gamma-aminobutyric acid (GABA) acts beyond inhibitory transmission and regulates inhibitory synapse development. Indeed, GABA(A) receptors not only function as chloride channels that regulate membrane voltage and conductance but also play structural roles in synapse maturation and stabilization. The link from GABA(A) receptors to postsynaptic and presynaptic adhesion is probably mediated, partly by neuroligin-reurexin interactions, which are potent in promoting GABAergic synapse formation. Therefore, similar to glutamate signaling at excitatory synapse, GABA signaling may coordinate maturation of presynaptic and postsynaptic sites at inhibitory synapses. Defining the many steps from GABA signaling to receptor trafficking/stability and neuroligin function will provide further mechanistic insights into activity-dependent development and possibly plasticity of inhibitory synapses.     

GABA mediated excitatory responses on Aplysia neurons

γ-Aminobutyric acid (GABA), a known inhibitory neurotransmitter in mammals, can elicit two different types of excitatory response in the nervous system of the marine mollusc, $\text{Aplysia}$. These responses are depolarizing when GABA is applied ionophoretically, and result from either an increase in membrane conductance to Na⁺ or a decrease in conductance to K⁺. In addition, GABA on other neurons causes an inhibitory response similar to that commonly found in other preparations. Although not all neurons have GABA receptors, identified single cells consistently have the same type of response. These observations suggest the possibility that GABA may function in at least some preparations as an excitatory neurotransmitter in addition to its documented inhibitory function.     

Crustacean Neuromuscular Mechanisms: Functional Morphology of Nerve Terminals and the Mechanism of Facilitation

Two aspects of crustacean neuromuscular physiology are discussed:(1) the ultrastructural identification of the excitatory andinhibitory nerve terminals, and (2) the characteristics of,and the possible mechanisms for, facilitation. The first problem was studied in crayfish opener muscle whichhas one excitatory and one inhibitory axon. One of the nerveswas stimulated in the presence of DNP until synaptic transmissionfailed; the preparations were then fixed for electron microscopy.Whenever the excitatory nerve was stimulated, the terminalswith round synaptic vesicles were depleted while nearby terminalswith smaller elongate vesicles were normal. When the inhibitorynerve was stimulated, the converse was true. The possible reasons for the diversity in crustacean neuromuscularproperties are discussed. Large EPSP's with a high quantal content(m), appear to be produced by terminals which are invaded bya propagated spike. Small EPSP's (small m) appear to be producedby terminals which don't spike and which are depolarized bya decrementally conducted potential. There is an inverse relationshipbetween m and the amount of facilitation. The physiologicalbasis for facilitation is discussed; previous hypotheses arefound wanting and a new one is proposed, that of slow depolarization.     

Effects of barbiturates on the inhibitory action of GABA on excitatory response of the stomach to stimulation of vagal afferent fibers in cat

Effects of barbiturates on the inhibitory action of GABA to the hexamethonium-resistant excitatory response of the stomach to stimulation of the vagal afferent fibers were studied in cats. Inhibition of the hexamethonium-resistant excitatory response by GABA were compared under alpha-chloralose, alpha-chloralose-phenobarbital (PhB), and alpha-chloralose-pentobarbital (PB)-anesthesia in cats. The ID50 of GABA on the hexamethonium-resistant excitatory response was not significantly affected by PhB, but reduced by PB. Both picrotoxin and bicuculline antagonized the effects of GABA. The present experiments demonstrated that PB potentiated the inhibitory effect of GABA on the hexamethonium-resistant excitatory response of the stomach, and suggested that the potentiation by PB may be due to activation of GABA-receptor-ionophore complex.     

Localization of GABA- and glutamate-like immunoreactivity in the cardiac ganglion of the lobster Panulirus argus

Wholemount immunohistochemical methods were used to examine the localization of γ-aminobutyric acid (GABA) and glutamate within the cardiac system of the Caribbean spiny lobster Panulirus argus. All of the GABA-like immunoreactivity (GABAi) in the cardiac ganglion originated from a single bilateral pair of fibers that entered the heart via the two dorsal nerves. Each GABAi axon bifurcated upon entering the ganglion and gave rise to varicose fibers that surrounded the somata and initial segments of the five large motor neurons. The four small posterior cells did not appear to receive somatic contacts. Double-labeling experiments in which individual motor neurons were injected with Neurobiotin showed that their dendritic processes, which project to muscle bundles adjacent to the ganglion and are thought to respond to stretch, were also accompanied by branches of the GABAi fibers. Glutamate-like immunoreactivity (GLUi) was present in each of the motor neuron cell bodies. In some preparations, GLUi was also detected in large caliber fibers in the major ganglionic nerves. These fibers gave rise to more slender branches that innervated the cardiac muscle bundles. GLUi was also found in the small cell bodies and in fibers surrounding motor neuron somata. Taken together, these findings support previous electrophysiological, pharmacological and anatomical studies indicating that GABA mediates extrinsic inhibition and that glutamate acts as a neuromuscular and intraganglionic transmitter in this system. While axosomatic contacts may play a major role in both transmitter systems, the GABAergic inhibition also appears to involve substantial axodendritic synaptic signaling.     

Perisomatic GABA release and thalamocortical integration onto neocortical excitatory cells are regulated by neuromodulators

Neuromodulators such as acetylcholine, serotonin, and noradrenaline are powerful regulators of neocortical activity. Although it is well established that cortical inhibition is the target of these modulations, little is known about their effects on GABA release from specific interneuron types. This knowledge is necessary to gain a mechanistic understanding of the actions of neuromodulators because different interneuron classes control specific aspects of excitatory cell function. Here, we report that GABA release from fast-spiking (FS) cells, the most prevalent interneuron subtype in neocortex, is robustly inhibited following activation of muscarinic, serotonin, adenosine, and GABA(B) receptors--an effect that regulates FS cell control of excitatory neuron firing. The potent muscarinic inhibition of GABA release from FS cells suppresses thalamocortical feedforward inhibition. This is supplemented by the muscarinic-mediated depolarization of thalamo-recipient excitatory neurons and the nicotinic enhancement of thalamic input onto these neurons to promote thalamocortical excitation.     

The ionic mechanism associated with the action of putative transmitters on identified neurons of the snail, Helix aspersa

Intracellular recordings were made from identified neurons in the suboesophageal ganglionic mass of the snail, Helix aspersa. The ionic mechanisms associated with acetylcholine excitation and inhibition, dopamine excitation and inhibition, gamma-aminobutyric acid (GABA) excitation and inhibition and serotonin excitation were investigated. Acetylcholine excitation was found to involve an initial increase in sodium conductance while acetylcholine inhibition was a pure chloride event which reversed at membrane potentials more negative than the chloride equilibrium potential. Dopamine excitation appeared to involve only an increase in sodium conductance while serotonin excitation involved an increase in conductance to both sodium and calcium ions. Dopamine inhibition was associated with an increase in potassium conductance but failed to reverse at membrane potentials more negative than the potassium equilibrium potential. GABA excitation involved conductance increases to both sodium and chloride ions while GABA inhibition was a pure chloride event. An attempt was made to estimate the degree of co-operativity of the putative transmitters with their receptors using log-log and Hill plots. The slopes of the line for the log-log plots for acetylcholine excitation and inhibition were 0.88 and 1.1, respectively, suggesting the interaction of one molecule of acetylcholine with the receptor. The slope of the log-log plot for dopamine inhibition was 0.46 while that for serotonin excitation was 0.75. The Hill plots for GABA excitation and inhibition were 1.64 and 1.42, respectively, suggesting that two molecules of GABA are required for receptor activation.