The responses of acetylcholine, γ-aminobutyric acid (GABA), dopamine,octopamine and other putative transmitters on Limulus polyphemus central neurons

1. Intracellular recordings have been made from neurons in the nerve cord of the horse-shoe crab, Limulus polyphemus. The resting potentials range between −30 and −60 mV, while the action potentials range from 2–4 mV up to 60 mV.2. All the cells tested were inhibited by GABA and excited by cholinergic agonists. The action of GABA was reversibly antagonised by picrotoxin while the acetylcholine response was reversibly antagonised by pentolinium and hexamethonium.3. Cells also responded to dopamine, noradrenaline, octopamine, glutamic acid, histamine and 5-hydrosytryptamine (5-HT); certain cells being inhibited by one or more of these compounds while other cells were excited. The actions of dopamine and noradrenaline were always the same, with noradrenaline being about 100 times less potent than dopamine. The action of dopamine was antagonised by metoclopramide and the action of histamine was antagonised by mepyramine.4. The pharmacology of these neurons is compared to insect and crustacean central neurons.     

Neuropharmacological studies on the receptors mediating responses to carbachol, amino acids and octopamine on Limulus and Hirudo central neurons

Intracellular recordings were made from central neurons of Limulus polyphemus and from Retizu cells of the leech, Hirudo medicinalis. The effects of carbachol, amino acids and octopamine were examined on these neurons. Octopamine was found to have a mainly inhibitory effect on a few Limulus neurons. The effects of octopamine were mimicked by clonidine and napthazoline but not by xylazine. Both compounds were slightly more potent than octopamine. Yohimbine, metoclopramide, chlorpromazine and chlordimeform failed to antagonize this octopamine response. The excitatory effect of carbachol was blocked by alpha-bungarotoxin, 10(-7)M. Neither this concentration nor higher concentrations of alpha-bungarotoxin had any effect on L-glutamate excitation. m-Carboxyphenyl derivatives of alanine and glycine acted differentially on Limulus neurons responding to L-glutamate. m- Carboxyphenylglycine only inhibited neurones which showed a biphasic response to L-glutamate while m- carboxyphenylalanine only excited these neurons. Both compounds excited leech Retzius cells, with m- carboxyphenylalanine being about 20 times more potent than m- carboxyphenylglycine . The actions of alpha- ketokainate and allo-alpha- ketokainate were compared to kainate, dihydrokainate and L-glutamate on leech Retzius cells. The equipotent molar ratios for kainate, dihydrokainate , alpha- ketokainate and allo-alpha- ketokainate were 0.0029 +/- 0.0004, 0.021 +/- 0.047, 0.029 +/- 0.005 and 0.14 +/- 0.0093 respectively with L-glutamate as one. All the analogues were more potent than L-glutamate. Quinolinic acid had no glutamate-like activity on either Limulus or Hirudo neurons. Methyltetrahydrofolate was inactive on Limulus neurons but excited leech Retzius cells, being slightly less potent than L-glutamate. Dibutyl cAMP terminated the excitatory actions of kainate on both Limulus and Hirudo neurons. Anisatin , a putative GABA antagonist, was a potent antagonist of GABA inhibition on Limulus neurons.     

The excitatory actions of a series of piperidine dicarboxylates on central neurons of the rat, snail, leech and horseshoe crab and on crab neuromuscular junction

1. Intracellular recordings were made from Helix, Hirudo and Limulus central neurons and from Eupagurus leg muscle and extracellular recordings from rat lateral geniculate neurons. The actions of 2,3-, 2,4-, 2,5- and 2,6-piperidine dicarboxylates were tested on those preparations for both direct effects and interactions with the response to L-glutamate. 2. All four piperidine analogues were devoid of either reproducible direct or indirect effects on Helix and Limulus neurons save that the 2,5-analogue did mimic the action of L-glutamate on Limulus neurons which were inhibited by glutamate. 3. On Hirudo and rat neurons and on Eupagurus muscle, all four piperidines mimicked the action of L-glutamate, all were weak agonists on rat neurones while on Hirudo neurons the 2,5-analogue was the most potent but on Eupagurus muscle, the 2,3- and 2,6-analogues were the most potent. 4. All four piperidines potentiated the action of L-glutamate on rat and Hirudo neurons and on Eupagurus leg muscle and of muscle ejps. 5. The piperidine analogues indicate that there are differences between the glutamate receptors used in the present study and provide some supportive evidence that the preferred conformation for glutamate on crab leg muscle is partially folded while for Hirudo neurons it is more extended.     

GABA-mediated inhibition of primary olfactory receptor neurons

Applying GABA (1 microM-1 mM) to the soma of cultured lobster olfactory receptor neurons evokes an inward current (V(m) = -60 mV) accompanied by an increase in membrane conductance, with a half-effect of 487 microM GABA. The current-voltage relationship of this current is linear between -100 and 100 mV and reverses polarity at the equilibrium potential for Cl(-). The current is blocked by picrotoxin and bicuculline methiodide, and is evoked by trans-aminocrotonic acid, isoguvacine, muscimol, imidazole-4-acetic acid, and 3-amino-1-propanesulfonic acid, but not by the GABA(C)-receptor agonist cis-4-aminocrotonic acid and the GABA(B)-receptor agonist 3-aminopropylphosphonic. Applying GABA to the soma of the cells in situ reversibly suppresses the spontaneous discharge and substantially decreases the odor-evoked discharge. The effects of GABA on the cell soma in situ are antagonized by both picrotoxin and bicuculline methiodide. Taken together with evidence that GABA directly activates a chloride channel in outside-out patches excised from the soma of these neurons, we conclude that lobster olfactory receptor neurons express an ionotropic GABA receptor that can potentially regulate the output of these cells. Copyright Copyright 1999 S. Karger AG, Basel     

Evidence that glycine and GABA mediate postsynaptic inhibition of bulbar respiratory neurons in the cat.

Experiments were carried out on decerebrate cats to identify transsynaptic mediators of spontaneous postsynaptic inhibition of bulbar inspiratory and postinspiratory neurons. Somatic membrane potentials were recorded through the central micropipette of a coaxial multibarreled electrode. Blockers of type A gamma-aminobutyric acid (GABA-A) and glycine receptors were iontophoresed extracellularly from peripheral micropipettes surrounding the central pipette. Effective antagonism was demonstrated by iontophoresis of agonists with antagonists; application of strychnine antagonized the action of glycine but not GABA, and application of bicuculline antagonized the action of GABA but not glycine. In both types of neurons, iontophoresis of either antagonist depolarized the somatic membrane and increased input resistance throughout the respiratory cycle. Bicuculline preferentially depolarized the somatic membrane in both types of neurons during inactive phases. Strychnine increased the firing rate of inspiratory neurons during inspiration despite maintenance of somatic membrane potential at preiontophoresis levels. Tetrodotoxin reduced the effects of iontophoresed bicuculline and strychnine, suggesting that the action of the antagonists required presynaptic axonal conduction. The present results suggest that presynaptic release of both GABA and glycine contributes to tonic postsynaptic inhibition of bulbar respiratory neurons. GABA-A receptors appear to contribute to inhibition during inactive phases in inspiratory and postinspiratory neurons, whereas glycinergic mechanisms appear to contribute to inspiratory inhibition in inspiratory neurons.     

Convulsant/depressant site of action at the allosteric benzodiazepine-GABA receptor-ionophore complex

Several classes of centrally acting convulsant, depressant, anticonvulsant and anxiolytic drugs modulate GABAergic transmission. The postsynaptic receptor with which these drugs interact is an allosteric complex with distinct binding sites for GABA, benzodiazepines, picrotoxinin and related compounds. Convulsants which inhibit GABA transmission (except bicuculline) inhibit competitively the binding of dihydropicrotoxinin (DHP) or t-butylbicyclophosphorothionate (TBPT) to the picrotoxinin site and prevent the allosteric enhancing effect of depressant drugs on GABA and benzodiazepine binding. Depressant drugs give a mixed inhibition of TBPT binding. The possible topography of the picrotoxinin site and its relationship to convulsant/depressant drug action at the benzodiazepine-GABA receptor-ionophore complex is discussed.     

Central nervous sensitization and dishabituation of reflex action in an insect by the neuromodulator octopamine

Habituation of excitatory synaptic inputs onto identified motor neurons of the locust metathoracic ganglion, driven electrically and by natural stimuli, was examined using intracellular recording. Rapid progressive reduction in amplitude of EPSPs from a variety of inputs onto fast-type motor neurons occurred. The habituated EPSPs were quickly dishabituated by iontophoretic release of octopamine from a microelectrode into the neuropilar region of presumed synaptic action. The zone within which release was effective for a given neuron was narrowly-defined. With larger amounts of octopamine applied at a sensitive site the EPSP became larger than normal, and in many instances action potentials were initiated by the sensitized response. Very small EPSPs onto a motor neuron, which were associated with proprioceptive feedback, and which were originally too small to be detected above the noise, were potentiated to a level of several mV by the iontophoresed octopamine. A DUM neuron (presumed to be octopaminergic) was found, whose direct stimulation was followed by a strong dishabituating and sensitizing action leading to spikes, of inputs to an identified flexor tibiae motor neuron. The action and its time course were closely similar to those evoked by octopamine iontophoresed into the neuropil in the region of synaptic inputs to the motor neuron. It is concluded that DUM (octopaminergic) neurons exert large potentiating actions on central neuronal excitatory synaptic transmission in locusts.     

Influence of GABA-agonists and antagonists on neuroleptic-induced catalepsy in rats.

Direct (muscimol) or indirect (aminooxyacetic acid, diaminobutyric acid, pyrrolidinone) GABA-mimetic compounds significantly potentiate neuroleptic induced catalepsy in rats. In contrast at subconvulsant doses, direct (bicuculline, picrotoxinin and indirect (allylglycine) GABA antagonists antagonized haloperidol-induced catalepsy. The effect of bicuculline and picrotoxinin was biphasic with the lowest doses increasing catalepsy. These results indicate that GABA mechanisms are involved in the induction of catalepsy by neuroleptics.     

Axonal transport of [3H] GABA and [3H] glutamate in excitatory and inhibitory neurons innervating lobster exoskeletal musculature

This paper describes the results of intracellular injections of radiolabelled neurotransmitters and transmitter precursor substances, including glutamate, GABA, aspartate, octopamine, tyramine, tryptophan, and choline, into cell bodies of identified excitatory and inhibitory neurons innervating lobster extensor musculature. The distributions and identities of radioactive substances appearing in axons were examined at various times following injection and in vitro incubation. Injected GABA and glutamate were found in appreciable quantities in both excitatory and inhibitory axons and migrated down axons at an estimated rate of between 16 and 22 mm/day at 12 degrees C, whereas the other substances tested were present in substantially smaller quantities and migrated at an estimated rate of less than 7.5 mm/day at 12 degrees C. Injected GABA, D-glutamate and L-glutamate accumulated proximal to ligatures tied around nerves, whereas neither octopamine nor aspartate accumulated proximal to ligatures. Since GABA is the transmitter substance released by inhibitory neurons and L-glutamate is thought to be released from excitatory nerve terminals, these results are consistent with the suggestion that amino acids serving as neurotransmitters are axonally transported. The specificity of axonal transport does not appear to be restricted to the cognate neurotransmitter, as indicated by the movement of L-glutamate in inhibitory axons and GABA in excitatory axons and of D-glutamate in both excitatory and inhibitory axons, but rather may be relaxed to include substances closely related to the neurotransmitter. Some restrictions, however, are apparently placed on axonal transport of small charged molecules in these neurons in that other substances tested migrated down nerves at a considerably slower rate.     

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.     

Neuromuscular transmission in an insect visceral muscle

The electrical properties of the muscles of locust oviduct have been examined using intracellular recordings. The muscle cells are both dye and electrically coupled. They possess a wide array of spontaneous electrical activity ranging from slow oscillations of membrane potential to action potentials. In addition to possessing spontaneous electrical activity, certain regions of the oviduct are under motor control. The amplitude of evoked excitatory junction potentials (EJPs) increased step wise revealing innervation from a maximum of three motor units. These EJPs underwent summation and facilitation, and reached a critical threshold at which point the membrane revealed an active response. Bath applied glutamate, aspartate, proctolin, and octopamine were tested for their ability to alter resting potential and EJPs. L-glutamate (1.6 X 10(-5) M and above) produced a dose-dependent depolarization of membrane potential accompanied by a reduction in amplitude of EJPs. Although L-aspartate resulted in similar effects, the concentrations required were higher than those for glutamate. Proctolin (6.3 X 10(-11) M-6.0 X 10(-9) M) resulted in a dose-dependent depolarization but had little or no effect on amplitude of EJPs. Application of D, L-octopamine (3.2 X 10(-5) M-1.7 X 10(-4) M) induced a small hyperpolarization and a reduction in amplitude of EJP. It is suggested that contractions of locust oviduct appear to be regulated by a combination of a classical neurotransmitter such as glutamate, along with the neuromodulators octopamine and proctolin.     

Effects of GABA on acutely isolated neurons from the gustatory zone of the rat nucleus of the solitary tract

Responses of acutely isolated neurons from the rostral nucleus of the solitary tract (rNST) to GABA receptor agonists and antagonists were investigated using whole-cell recording in current clamp mode. The isolated neurons retain their morphology and can be divided into multipolar, elongate and ovoid cell types. Most rNST neurons (97%), including all three cell types, respond to GABA with membrane hyperpolarization and a reduction in input resistance. The GABA(A) receptor agonist muscimol reduces neuronal input resistance in a concentration-dependent manner, whereas the GABA(B) receptor agonist baclofen had no effect on any of the neurons tested. The GABA and muscimol reversal potentials were both found to be -75 mV Both the GABA competitive antagonist picrotoxin and the GABA(A) receptor antagonist bicuculline block the effect of GABA in a concentration-dependent manner. These results suggest that GABA activates all neurons in the rNST and that inhibitory synaptic activity is important in brainstem processing of gustatory and somatosensory information.      

Electrophysiological effects of GABA on cat pancreatic neurons

In mammalian peripheral sympathetic ganglia GABA acts presynaptically to facilitate cholinergic transmission and postsynaptically to depolarize membrane potential. The GABA effect on parasympathetic pancreatic ganglia is unknown. We aimed to determine the effect of locally applied GABA on cat pancreatic ganglion neurons. Ganglia with attached nerve trunks were isolated from cat pancreata. Conventional intracellular recording techniques were used to record electrical responses from ganglion neurons. GABA pressure microejection depolarized membrane potential with an amplitude of 17.4 +/- 0.7 mV. Electrically evoked fast excitatory postsynaptic potentials were significantly inhibited (5.4 +/- 0.3 to 2.9 +/- 0.2 mV) after GABA application. GABA-evoked depolarizations were mimicked by the GABA(A) receptor agonist muscimol and abolished by the GABA(A) receptor antagonist bicuculline and the Cl(-) channel blocker picrotoxin. GABA was taken up and stored in ganglia during preincubation with 1 mM GABA; beta-aminobutyric acid application after GABA loading significantly (P < 0.05) increased depolarizing response to GABA (15.6 +/- 1.0 vs. 7.8 +/- 0.8 mV without GABA preincubation). Immunolabeling with antibodies to GABA, glial cell fibrillary acidic protein, protein gene product 9.5, and glutamic acid decarboxylase (GAD) immunoreactivity showed that GABA was present in glial cells, but not in neurons, and that glial cells did not contain GAD, whereas islet cells did. The data suggest that endogenous GABA released from ganglionic glial cells acts on pancreatic ganglion neurons through GABA(A) receptors.     

Aminergic neuron systems of lobsters: morphology and electrophysiology of octopamine-containing neurosecretory cells

In the American lobster (Homarus americanus) the biogenic amines serotonin and octopamine appear to play important and opposite roles in the regulation of aggressive behavior, in the establishment and/or maintenance of dominant and subordinate behavioral states and in the modulation of the associated postural stances and escape responses. The octopamine-containing neurosecretory neurons in the thoracic regions of the lobster ventral nerve cord fall into two morphological subgroups, the root octopamine cells, a classical neurohemal group with release regions along second thoracic roots, and the claw octopamine cells, a group that selectively innervates the claws. Cells of both subgroups have additional sets of endings within neuropil regions of ganglia of the ventral nerve cord. Octopamine neurosecretory neurons generally are silent, but when spontaneously active or when activated, they show large overshooting action potentials with prominent after-hyperpolarizations. Autoinhibition after high-frequency firing, which is also seen in other crustacean neurosecretory cells, is readily apparent in these cells. The cells show no spontaneous synaptic activity, but appear to be excited by a unitary source. Stimulation of lateral or medial giant axons, which excite serotonergic cells yielded no response in octopaminergic neurosecretory cells and no evidence for direct interactions between pairs of octopamine neurons, or between the octopaminergic and the serotonergic sets of neurosecretory neurons was found.     

Volatile anesthetics gate a chloride current in postnatal rat hippocampal neurons.

A volatile anesthetic-gated current was characterized in patch-clamped cultured postnatal rat hippocampal neurons. In this preparation, the major volatile anesthetics, isoflurane, halothane, and enflurane, open an anion-selective conductance. This volatile anesthetic-gated current exhibits anion selectivity with a chloride-to-acetate permeability ratio of 15, shows outward rectification well described by the constant field equation, and is activated in a dose-dependent fashion with half-maximal response to isoflurane at 0.8 mM (0.032 atm). The current persists in the absence of external Ca2+ and is not blocked by strychnine, a glycine antagonist. However, the gamma-aminobutyric acidA (GABAA) antagonists, bicuculline and picrotoxinin, and the nonspecific anion channel blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), completely block the response. These observations suggest that volatile anesthetics, like several other general anesthetics such as barbiturates, steroids, and etomidate, have a GABA-mimetic effect on vertebrate central neurons in culture. It is not clear whether this GABAA-gating property is a prerequisite for all general anesthetics. However, under normal physiological conditions of low intracellular Cl-, it is likely that drugs with both direct GABA agonist and GABA modulatory properties will produce overall depression of the central nervous system by increasing the normal inhibitory synaptic influence and by directly hyperpolarizing neurons.     

The GABA postsynaptic membrane receptor-ionophore complex

Summary The function of the inhibitory neurotransmitter, -aminobutyric acid (GABA), has been implicated in the mode of action of many drugs which excite or depress the central nervous system. Many convulsant agents appear to block GABA action whereas anticonvulsants enhance GABA action. Some of these drug effects involve altered GABA-mediated synaptic transmission at the level of GABA biosynthesis, release from nerve endings, uptake into cells, and metabolic degradation. A greater number of agents of diverse classes appear to affect GABA action at the postsynaptic membrane, as determined from both electrophysiological and biochemical studies. The recently developedin vitro radioactive receptor binding assays have led to a wealth of new information about GABA action and its alteration by drugs. GABA inhibitory transmission involves the regulation, by GABA binding to its receptor site, of chloride ion channels. In this GABA receptor-ionophore system, other drug receptor sites, one for benzodiazepines and one for barbiturates/picrotoxinin (and related agents) appear to form a multicomponent complex. In this complex, the drugs binding to any of the three receptor categories are visualized to have an effect on GABA-associated chloride channel regulation. Available evidence suggests that the complex mediates many of the actions of numerous excitatory and depressant drugs showing a variety of pharmacological effects.     

Neuromodulation of flight initiation by octopamine in the cockroach Periplaneta americana

We have tested the effect of a known insect neuromodulator, octopamine, on flight initiation in the cockroach. Using minimally dissected animals, we found that octopamine lowered the threshold for windevoked initiation of flight when applied to either of two major synaptic sites in the flight circuitry: 1) the last abdominal ganglion, where wind-sensitive neurons from the cerci excite dorsal giant interneurons, or 2) the metathoracic ganglion, where the dorsal giant interneurons activate interneurons and motoneurons which are involved in producing the rhythmic flight motor pattern in the flight muscles (Fig. 2).Correlated with this change in flight initiation threshold, we found that octopamine applied to the last abdominal ganglion increased the number of action potentials produced by individual dorsal giant interneurons when recruiting the cereal wind-sensitive neurons with wind puffs (Figs. 3, 4, 5) or with extracellular stimulation of their axons (Fig. 6). Octopamine increases the excitability of the giant interneurons (Figs. 7, 8). Also, when we stimulated individual dorsal giant interneurons intracellularly, the number of action potentials needed to initiate flight was reduced when octopamine was applied to the metathoracic ganglion (Fig. 9).Abbreviations EMG electromyogram - dGIs dorsal giant interneurons - GI giant interneuron - A6 sixth abdominal ganglion - T3 third thoracic ganglion - EPSP excitatory postsynaptic potential     

Neuropharmacological spectrum of muscimol

Muscimol was tested in comparison with a series of reference compounds in a variety of situations in which GABA-related drugs are known to have an effect. Muscimol blocked the convulsions and/or lethality due to picrotoxinin, strychnine and a low dose of bicuculline. It was inactive against higher dosis of bicuculline, metrazole or electroshock convulsions. Muscimol reduced both the basal and the picrotoxin-induced multi-unit activity of the neurons of the dorsal Deiters' nucleus ; although active at low doses, the maximum effect of muscimol was relatively weak. Muscimol potentiated neuroleptic-induced catalepsy, and this effect was bicuculline sensitive ; it did not induce catalypsy in the presence of sulpiride. At high doses muscimol blocked apomorphine-induced stereotyped behaviour. It is proposed that muscimol is a GABA agonist of high affinity but of relatively low efficacy as based on its spectrum of neuropharmacological activities “in vivo”.     

Modulatory effects of proctolin on a crab ventilatory muscle

Proctolin enhances nerve-evoked, phasic contractions of a selected respiratory muscle of the shore crab, Carcinus maenas, but has no effect on muscle tonus. Proctolin also increases the work and power output of this muscle. These effects are functionally appropriate in view of previous reports that proctolin stimulates the ventilatory rhythm. They also suggest that proctolin exerts coordinated modulatory control at the central and peripheral levels of the gill ventilatory system. In contrast, serotonin, dopamine and octopamine have no effect on this muscle.     

Ionic mechanisms of depolarization responses in Helix pomatia neurons to glutamate application

Reversal potentials of transmembrane ionic currents induced by glutamate were determined in various D neurons ofHelix pomatia. Two types of neurons were found with mean reversal potentials of –10.6±1.2 and –40.0±0.6 mV. Neurons of the first group responded under ordinary conditions to glutamate application by a volley of action potentials. Neurons of the second group did not generate action potentials under the same conditions during glutamate application. With an increase in the dose of mediator the amplitude of D responses in these neurons increased only up to a certain limit, without reaching the critical depolarization level of the cell; a fall in the external chloride ion concentration led to a decrease in their reversal potential. The possible ionic mechanisms of glutamate-dependent depolarization responses of these groups of neurons are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 6, pp. 572–577, November–December, 1982.