Two Types of Identified Ascending Interneurons With Distinct GABA Receptors in the Crayfish Terminal Abdominal Ganglion

Hiroki Miyata, Toshiki Nagayama, and Masakazu Takahata

Animal Behaviour and Intelligence, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Japan

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Miyata, Hiroki, Toshiki Nagayama, and Masakazu Takahata. Two types of identified ascending interneurons with distinct GABA receptors in the crayfish terminal abdominal ganglion. J. Neurophysiol. 77: 1213-1223, 1997. More than half of the identified ascending interneurons originating in the terminal abdominal ganglion of the crayfish received inhibitory sensory inputs from hair afferents innervating the tailfan on the side contralateral to their main branches. Biochemical aspects of this transverse lateral inhibition of ascending interneurons were examined by the use of neurophysiological and pharmacological techniques. Local application of gamma -aminobutyric acid (GABA) and its agonist muscimol into the neuropil induced membrane hyperpolarization of identified ascending interneurons with an increase in membrane conductance. Because the reversal potential of inhibitory postsynaptic potentials (IPSPs) in ascending interneurons elicited by the sensory stimulation and GABA injection was similar, and the sensory-stimulated IPSPs of the interneurons were blocked by GABA and muscimol application, this study strongly suggests a GABAergic nature for transverse lateral inhibition of ascending interneurons. According to the response to the GABAA antagonists bicuculline and picrotoxin, ascending interneurons were classified into two types, picrotoxin-sensitive and picrotoxin-insensitive interneurons. Identified ascending interneurons VE-1 and RO-4 showed a pharmacological profile similar to that of the classical GABAA receptor of the vertebrates. Bath application of both bicuculline and picrotoxin reversibly reduced the amplitudes of IPSPs. The other identified ascending interneurons CA-1, RO-1, and RO-2 were not affected significantly by the bath application of GABAA and GABAB antagonists, although bath application of low-chloride saline reversed the sensory-stimulated IPSPs. IPSPs of the picrotoxin-sensitive interneurons had a rather faster time course and shorter duration in comparison with those of the picrotoxin-insensitive interneurons.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Inhibitory as well as excitatory synaptic interactions are essential to discriminate or enhance sensory cues. One well-known form of sensory filtering is a lateral inhibition that enhances contrast of sensory information. A large monopolar cell of Lumilus eye that is excited by a bright light stimulation inhibits the nearest neighbors to increase visual contrast (Hartline et al. 1956). Lateral inhibition along the animal's body axis also plays an important role when animals utilize directional information for their orientation or escape behavior. For escape, detection of the approach or the strike of a predator is a prerequisite. Water disturbance or mechanical touch to the abdomen and/or tailfan produced by a predator is the cue for performing escape behavior in the crayfish (Wine 1984). The directional information is initially detected by hairs on the tailfan, and the innervating sensory afferents transmit these signals to the CNS. Bilateral coordination within the CNS is therefore necessary to discriminate the transverse direction of the stimulus source.

About 130 ascending interneurons originate in the crayfish terminal abdominal ganglion (Kondoh and Hisada 1986; Reichert et al. 1982) and 30 pairs have so far been identified as unique individuals (Nagayama et al. 1993, 1994; Sigvardt et al. 1982). In most ascending interneurons, the primary neurite originating from the soma crosses the midline and sends an axon anteriorly through the opposite connective into the brain (Aonuma et al. 1994). Many interneurons extend prominent dendritic branches within the hemiganglion contralateral to their somata, that is, ipsilateral to their axon projection, and receive excitatory inputs from the hair afferents. About 60% also receive inhibitory inputs from the tailfan on the opposite side (Nagayama et al. 1993). Thus directional information from the mechanosensory afferents of the tailfan is extracted by ascending interneurons and conveyed to anterior neural centers for abdominal posture and walking systems.

Excitatory mechanosensory inputs to ascending interneurons are directly mediated by the hair afferents through chemical synaptic transmission (Nagayama and Sato 1993). Acethylcholine is an excitatory neurotransmitter released from the sensory afferents (Ushizawa et al. 1996). On the other hand, transverse lateral inhibition of ascending interneurons must be elicited through the central interactions within the terminal abdominal ganglion, because neither the terminal branches of afferents nor the main branches of the most ascending interneurons cross the midline (Kondoh and Hisada 1987; Nagayama and Sato 1993; Nagayama et al. 1993). One of the possible candidates to mediate lateral inhibition of ascending interneurons is the local directionally selective interneuron (LDS), which is a nonspiking interneuron having bilateral arborizations connected by an unbranched, thick-diameter transverse process (Nagayama and Hisada 1988; Reichert et al. 1983). Recent immunologic study has revealed that the somata of LDS showed gamma -aminobutyric acid (GABA)-like immunoreactivity (Nagayama et al. 1996).

In both the vertebrate and invertebrate CNSs and peripheral nervous systems, GABA is the most widely distributed inhibitory neurotransmitter. Recent immunocytochemical analyses have shown the distribution of GABAergic neurons in the CNS of the crayfish (Elekes and Florey 1987; Mulloney and Hall 1990; Nagayama et al. 1996). Pharmacological studies have been carried out to characterize GABAergic transmission (El Manira and Clarac 1994; Pfeiffer-Linn and Glantz 1991; Takeuchi and Takeuchi 1965). The properties of GABAergic receptors of some identified neurons have also been described physiologically (Cattaert et al. 1992; El Manira and Clarac 1991; Miwa et al. 1990). We have, however, no information about biochemical aspects of transverse lateral inhibition of ascending interneurons in the terminal abdominal ganglion. The aim of this paper is to identify the inhibitory transmitter mediating lateral inhibition and to characterize the receptor properties of the ascending interneurons by the use of electrophysiological and pharmacological studies.

The results show that local application of GABA and its agonist muscimol into the neuropil mimicked inhibitory responses of identified ascending interneurons to the sensory stimulation. The inhibitory response of some ascending interneurons was blocked by the antagonists of vertebrate GABAA receptors but the response of other interneurons was not affected, suggesting that two distinct types of GABA receptors were selectively distributed on the crayfish ascending interneurons.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Crayfish [Procambarus clarkii (Girard)] measuring 8-12 cm in body length from rostrum to telson were used for all experiments. They were obtained from a commercial supplier (Sankyo Labo, Tokyo) and maintained in laboratory tanks supplied with flowing fresh water before use. There were no significant differences in results between males and females.

The nerve chain including the fifth and sixth abdominal ganglia with relevant nerve roots was isolated and pinned, ventral side up, to the floor of a Sylgard-lined perfusion petri dish at the fourth-fifth abdominal connective and the first root of the terminal abdominal ganglion in cooled van Harreveld's solution (van Harreveld 1936). The second root on both sides and the third root on the right side of the terminal ganglion were dissected with the nerve chain for extracellular recording and stimulation. The ventral surface of the terminal ganglion was desheathed by removing the ganglionic sheath surgically.

The spike activity of the motor neurons innervating opener muscles was recorded extracellularly at the bifurcation to the ventral rotator and the abductor exopodite muscles. At least three opener motor neurons innervating the ventral abductor exopodite muscles showed spontaneous discharge (Nagayama et al. 1984). The spike activity of the closer motor neurons was recorded at the bifurcation to the reductor and adductor exopodite muscles (Nagayama et al. 1983). The mechanosensory afferents innervating either the right or left exopodite were stimulated electrically (1-10 V for 0.01-0.05 ms) by suction electrodes placed on the distal portion of the second root sensory bundle on both sides.

Intracellular recording and staining were performed with glass microelectrodes filled with a 3% solution of Lucifer yellow CH (Stewart 1978) dissolved in 0.1 M lithium chloride. The electrodes had resistances of between 100 and 160 MOmega measured in the physiological saline. The terminal ganglion was stabilized on a silver platform to facilitate penetration with glass electrodes. The electrode was inserted into the right lateral neuropil of the ganglion.

After physiological experiments, the penetrated interneurons were stained by iontophoretic injection of Lucifer yellow with the use of 3- to 5-nA hyperpolarizing current pulses 500 ms in duration at 1 Hz for 5-15 min. The terminal abdominal ganglion was removed from the perfusion chamber and fixed in 10% Formalin for 10 min, dehydrated in alcohol series, and cleared in methyl salitylate. The stained interneurons were then photographed in whole mount under a fluorescence microscope for subsequent reconstruction. Ascending interneurons were identified by their gross morphology, pattern of inputs from mechanosensory afferents from both exopodites, and premotor effects on both the closer and opener motor neurons according to criteria based on Nagayama et al. (1993).

The following pharmacological agents were obtained from Sigma: GABA, picrotoxin, bicuculline methiodide (bicuculline), baclofen, and delta -aminovaleric acid hydrochloride (AVA). These drugs were all dissolved in normal crayfish saline to required concentration. A 50% chloride saline was prepared by replacing NaCl in normal saline with equimolar amounts of sodium propionate (Albert et al. 1986).

For bath application of GABA antagonists, the perfusion chamber was 2.0 ml in volume and the bathing saline with or without drugs was supplied with the aid of a microtube pump (Eyela MP-3, Tokyorikakikai) at a rate of 2.2 ml/min. The bathing saline and drugs were cooled on ice throughout experiments and the temperature of the solution in which the terminal abdominal ganglion was located was ~18-20°C. After physiological characterization, interneurons were rested for >= 5 min with a continuous perfusion of normal saline. Effect of drugs on the responses of interneurons to the sensory stimulation was measured by changes in the amplitude of inhibitory postsynaptic potentials (IPSPs) that were evoked by electrical stimulation of the second root afferents. The amplitude of IPSPs was measured as the average of 8 or 10 stimulations at 0.3 Hz with the use of a digital storage oscilloscope (Tektronix2440, Tektronix or DS-9121, Iwatsu). The averaged IPSP amplitude before drug application was defined as the control level (100%) and the change in size of IPSPs was compared every 2 or 3 min. Drugs were perfused for 5 min, then washed out with normal saline.

For local application of GABA or its agonists, the tips of micropipettes were broken manually under a microscope to be 2-4 µm OD. GABA, or the agonist muscimol or baclofen, was applied via pressure microinjection from micropipettes into the lateral neuropil of the terminal abdominal ganglion near the recording site. Small quantities of drugs were ejected from the penetrated micropipette by N2 gas pressure controlled by pneumatic picopump (PV830, WPI) at 10-20 psi for 200-1,000 ms.

All the recordings were stored on a digital tape recorder (DTR-1801, Biologic) and displayed on a Gould electrostatic chart recorder (TA240S). The averaged waveform was displayed and stored on an IBM-compatible computer with the use of a GPIB interface. The results are based on 61 stable recordings from ascending interneurons.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Two types of inhibitory responses of ascending interneurons

The inhibitory responses of ascending interneurons to the sensory stimulation on their soma side, that is, the side opposite to their main branches, were classified into two types, fast and slow responses. Under repetitive electrical stimulation of the afferents at 20 Hz, some ascending interneurons showed membrane hyperpolarization only during the stimulation, whereas other interneurons continued to hyperpolarize their membrane after the end of the stimulus (Fig. 1A). In response to single sensory stimulation, the time from the start to peak of IPSPs and the decay to half-amplitude in the former ascending interneurons was shorter than that of the latter interneurons (Fig. 1B). Identified ascending interneuron VE-1, which has its cell body on the ventral rostral portion of the terminal ganglion and a characteristic posteriorly projecting secondary neurite crossing the midline (Nagayama et al. 1993), received IPSPs of relatively short duration (i.e., Fig. 1, A-1 and B-1) when the sensory afferents on the soma side were stimulated. The average of time to peak of IPSPs in VE-1 was ~11.1 ± 4.3 (SE) ms (n = 31). A passage of hyperpolarizing currents injected into VE-1 reduced the amplitude of IPSPs (Fig. 1C-1). Hyperpolarizing current of 3 or 4 nA was usually necessary to reverse IPSPs. Another ascending interneuron, CA-1, which is identified by its two main branches extending within the anterior half of the neuropil (Nagayama et al. 1993), received longer-lasting IPSPs from the same array of sensory afferents (Fig. 1, A-2 and B-2). The time to peak of IPSPs in CA-1 was ~16.5 ± 6.4 ms (n = 11), considerably longer than that of VE-1 (P < 0.002; t-test). Furthermore, IPSPs mediated by sensory stimulation were reversed by a rather weak (usually 2-nA) hyperpolarizing current (Fig. 1C-2). Thus we have focused on both VE-1 and CA-1 to identify the inhibitory transmitter and to characterize the type of receptors of these interneurons.


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FIG. 1. Two types of inhibitory responses of intersegmental ascending interneurons. A: response of interneurons to repetitive electrical stimulation (10 shocks at 20 Hz; up-arrow  down-arrow ) of 2nd root sensory bundle on the side contralateral to the main branches of the interneurons. B: response of interneurons to single sensory stimulation. C: amplitude of inhibitory postsynaptic potentials (IPSPs) depended on the membrane potential of the interneurons. Each record in C-2 is a signal average of 32 sweeps. A-1, B-1, and C-1 were recorded from VE-1; A-2, B-2, and C-2 were from CA-1.

Does GABA mediate the inhibitory response of ascending interneurons?

GABA has been well established as an inhibitory neurotransmitter in arthropod nervous systems with peripheral and central actions. To examine whether GABA acts as an inhibitory transmitter in the transverse lateral inhibition between sensory afferents and ascending interneurons, we applied GABA and its agonists locally into the neuropil area by the use of pressure ejection.

When 1 mM GABA was applied into the neuropil, spontaneous spikes of the ascending interneuron, VE-1, were suppressed completely with a sustained membrane hyperpolarization (Fig. 2A, middle trace). At the same time, tonic spikes of uropod opener motor neurons were also inhibited (Fig. 2A, top trace). Although the amplitude of IPSPs in VE-1 caused by GABA injection was variable (range 8-15 mV, n = 13), partly because of the different position of micropipettes within the neuropil, the membrane hyperpolarization continued after the end of GABA injection. The membrane conductance of VE-1 was increased during GABA-mediated membrane hyperpolarization. The input resistance of VE-1, measured by brief pulses of 1-nA hyperpolarizing current, was reduced to ~30% during the membrane hyperpolarization (Fig. 2B). The amplitude of GABA-mediated hyperpolarization of VE-1 was reduced by the injection of weak (1- to 2-nA) hyperpolarizing current and reversed when >3-nA hyperpolarizing current was injected into VE-1 (Fig. 2C).


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FIG. 2. Effect of gamma -aminobutyric acid (GABA) on the ascending interneuron, VE-1. A: local application of 1 mM GABA into the neuropil elicited the membrane hyperpolarization of VE-1 and the decrease in discharge of the opener motor neurons. B: input resistance of VE-1, measured by a brief injection of 1-nA hyperpolarizing current, was reduced during GABA-mediated membrane hyperpolarization. C: GABA-mediated membrane hyperpolarization of VE-1 was reduced in amplitude by weak hyperpolarizing currents and reversed by strong hyperpolarizing currents.

The identified ascending interneuron CA-1 also showed a sustained membrane hyperpolarization (3-15 mV, n = 8) immediately after pressure injection of 1 mM GABA into the neuropil (Fig. 3A). The inhibitory duration of CA-1 with GABA injection was much longer than that in the case of VE-1. The membrane hyperpolarization of CA-1 continued for >5 s with a 500-ms pulse of GABA injection, whereas hyperpolarization of VE-1 continued for 2.5 s even if a 1-s pulse of GABA was injected (cf. Fig. 2). The membrane hyperpolarization of CA-1 was also accompanied by conductance increases (Fig. 3B).


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FIG. 3. Effect of GABA on the ascending interneuron CA-1. A: local injection of 1 mM GABA elicited the membrane hyperpolarization of CA-1 and suppressed tonic spikes of the opener motor neurons. B: input resistance of CA-1, measured by a brief injection of 1-nA hyperpolarizing current, was reduced during GABA-mediated membrane hyperpolarization. C: interaction between GABA application and the sensory stimulation. Membrane hyperpolarizations induced by the sensory stimulation and GABA application were presented in C-1 as a control. IPSPs elicited by the sensory stimulation were blocked during GABA-mediated membrane hyperpolarization of CA-1 (C-2).

The amplitude of sensory-stimulated IPSPs in CA-1 was reduced during GABA-mediated membrane hyperpolarization (Fig. 3C). Repetitive electrical stimulation of sensory afferents on the side ipsilateral to the soma of CA-1 evoked the membrane hyperpolarization of ~1 mV in CA-1 (Fig. 3C-1, left). The application of a brief pulse (100 ms) of 1 mM GABA produced a long-lasting membrane hyperpolarization of CA-1 that lasted for ~2 s (Fig. 3C-1, right). The IPSP evoked by sensory stimulation was reduced in size to <30% of the initial level just after the peak of GABA-mediated hyperpolarization and then gradually returned to its initial amplitude (Fig. 3C-2).

Effects of GABAergic agonists

Muscimol is a GABAA agonist in both the vertebrates and invertebrates. When 50 µM muscimol was locally applied into the neuropil by the pressure injection, tonic spikes of VE-1 were suppressed with a sustained membrane hyperpolarization depending on the pulse duration of muscimol application (Fig. 4). The duration of membrane hyperpolarization of VE-1 was much longer than that mediated by GABA application (cf. Fig. 2A). Tonic spikes of VE-1 were completely suppressed for ~10 s even if muscimol was applied for 100 ms (Fig. 4A).


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FIG. 4. Effect of the GABA agonist muscimol on the ascending interneuron VE-1. The membrane hyperpolarization of VE-1 was related to the duration of the applied pulses (100 ms in A, 200 ms in B, 500 ms in C) of 50 µM muscimol.

Local application of 50 µM muscimol also mediated membrane hyperpolarization of CA-1 (Fig. 5A, 1st trace). Similar to what occurred in VE-1, the inhibitory effect of muscimol on CA-1 continued for a very long period. Although the amplitude of muscimol-mediated membrane hyperpolarization of CA-1 was decreased by the injection of 1-nA hyperpolarizing current, membrane potential was reversed by the injection of more steady hyperpolarizing currents (Fig. 5A). The size of IPSPs of CA-1 elicited by the sensory stimulation was considerably reduced during muscimol-mediated hyperpolarization (Fig. 5B). Sensory stimulation elicited almost no response to CA-1 just after muscimol application (Fig. 5B, 2nd trace). The inhibitory response of CA-1 to the sensory stimulation gradually recovered (Fig. 5B, 3rd-5th traces), but still smaller than initial response when the membrane potential was returned to the resting level (cf. Fig. 5B, 1st and 5th traces).


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FIG. 5. Effect of the GABA agonist muscimol on the ascending interneuron CA-1. A: brief application (500 ms) of 50 µM muscimol caused a long-lasting membrane hyperpolarization in CA-1. Passage of hyperpolarizing currents reduced and reversed muscimol-mediated IPSPs. B: sensory-stimulated IPSPs of CA-1 were blocked during muscimol-mediated membrane hyperpolarization. Time indicated on each trace: elapsed time after muscimol injection.

Baclofen is known as a potent GABAB agonist in vertebrates. Local injection of baclofen (1 mM) into the neuropil had, however, no observable effect on the membrane potential of any ascending interneurons including both VE-1 and CA-1. Furthermore, tonic spikes of the uropod motor neurons were not changed significantly even if baclofen was injected for 1 s (not shown).

Effects of GABAergic antagonists

Bicuculline acts as a direct competitive antagonist ofGABAA receptors in vertebrates. Bath application of bicuculline usually reduced the amplitude of IPSPs in VE-1 in response to the sensory stimulation (Fig. 6, A and B). Before bath application of 1 mM bicuculline, VE-1 received IPSPs 6-7 mV in amplitude (Fig. 6A, top trace). The IPSP amplitude began to decrease gradually during bath application of 1 mM bicuculline (Fig. 6B). The IPSP amplitude decreased to ~30% of its initial amplitude after ~8 min (Fig. 6A, middle trace), stayed the same for ~15 min, recovered gradually, and was restored to the initial level after ~40 min of washing (Fig. 6A, bottom trace). Inhibitory responses of CA-1 to the sensory stimulation were, however, not significantly affected by bicuculline (Fig. 6C).


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FIG. 6. Effect of the GABAA antagonist bicuculline on the ascending interneurons VE-1 (A and B) and CA-1 (C). A: response of VE-1 to the sensory stimulation. Perfusion of bicuculline (1 mM) for 5 min decreased the IPSP amplitude of VE-1. Each trace consists of 5 superimposed sweeps at 0.3 Hz. Time indicated on each trace: elapsed time after bicuculline application. B: relative change in IPSP amplitude with bicuculline application plotted every 2 or 3 min. The size of IPSPs is expressed as a percentage of the initial IPSP level just before drug application (0 min). All measurements are the averaged IPSPs of either 8 or 10 trials at 0.3 Hz. Thick bar: period of bicuculline application. C: response of CA-1 to the sensory stimulation. Perfusion of bicuculline (1 mM) for 5 min made no observable changes in the IPSP amplitude of CA-1. Each trace is an average taken from 8 IPSPs elicited by sensory stimulation at 0.3 Hz.

Picrotoxin acts as a noncompetitive GABAA antagonist in the vertebrates, presumably because of its ability to block GABA-activated ionophores. Bath application of 50 µM picrotoxin reduced the amplitude of IPSPs in VE-1 in response to the contralateral sensory stimulation (8 of 9 preparations). Before bath application of picrotoxin, the sensory stimulation elicited IPSPs in VE-1 of ~6 mV in amplitude (Fig. 7A, 1st trace). The amplitude of IPSPs decreased within 5 min after the application of 50 µM picrotoxin (Fig. 7, A and B). The IPSP amplitude was decreased to ~30% of its initial level after within 10 min and continued for ~20 min. The IPSP then recovered gradually and was restored to its original amplitude after ~40 min of washing. Remarkable change in resting potential attributable to the application of picrotoxin was not observed. The effect of picrotoxin was dose dependent (Fig. 7C). When 10 µM picrotoxin was applied (n = 2), the amplitude of IPSPs in VE-1 elicited by the sensory stimulation was reduced to 80% of initial amplitude and recovered within 10 min after washing. Application of 20 µM picrotoxin reduced the amplitude of the IPSP to ~60% (n = 3). IPSP amplitude decreased to <20% of the original size at 50 µM picrotoxin (n = 8).


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FIG. 7. Effect of the GABAA antagonist picrotoxin (PTX) on the ascending interneurons VE-1 (A-C) and CA-1 (D). A: response of VE-1 to the sensory stimulation. Picrotoxin (50 µM) perfusion for 5 min decreased the IPSP amplitude of VE-1. Each trace is an average taken from 10 IPSPs elicited by sensory stimulation at 0.3 Hz. Time indicated on each trace: elapsed time after drug application. B: relative change in IPSP amplitude with picrotoxin application plotted every 2 or 3 min. The size of IPSPs is expressed as a percentage of the initial IPSP level just before drug application (0 min). All measurements are the averaged IPSPs of 10 trials at 0.3 Hz. Thick bar: period of bicuculline application. C: semilogarithmic dose-response relationship between the concentration of applied picrotoxin and the decrement of IPSP amplitude of VE-1 (means ± SE). Numbers in parentheses: number of crayfish tested. D: response of CA-1 to the sensory stimulation. Perfusion of picrotoxin (50 µM) for 5 min made no observable changes in the IPSP amplitude of CA-1. Each trace is an average taken from 10 IPSPs elicited by sensory stimulation at 0.3 Hz.

In CA-1 (n = 5), by contrast, no significant reduction in the amplitude of IPSPs was observed after the bath application of picrotoxin at the concentration of 50 µM (Fig. 7D). Any significant change in both the amplitude and the duration of IPSPs was observed before and after perfusion of picrotoxin.

The response of the ascending interneurons, picrotoxin-sensitive VE-1 and picrotoxin-insensitive CA-1, was quite different when the sensory afferents were stimulated electrically. Sensory stimulation elicited slower IPSPs to picrotoxin-insensitive interneurons (cf. Fig. 7, A and D). The time to peak of IPSPs in VE-1 was significantly faster (P < 0.003, t-test) than that of picrotoxin-insensitive interneurons (Fig. 8).


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FIG. 8. Histogram of the time from start to peak of IPSPs of the picrotoxin-sensitive interneuron VE-1 (white bars) and the picrotoxin-insensitive interneurons (CA-1, RO-1, and RO-2; black bars).

Bath application of AVA (1 mM), a GABAB receptor antagonist, caused no significant effects on the response of any ascending interneurons to the sensory stimulation (not shown).

Effect of low-chloride saline on the response of ascending interneurons

The possibility that the response of ascending interneurons is related to openings of the chloride channels was examined by the use of low-chloride solution (Fig. 9). After the saline was changed to a low-chloride solution, the resting membrane potential of some interneurons was slightly shifted more negatively (~5 mV) and continued several minutes after washing. This shift of resting potential might affect the response of interneurons to the sensory stimulation, but did not show any direct causality with the change in IPSP amplitude. In VE-1, the sensory-stimulated IPSPs was reversibly reduced in amplitude after the normal saline was changed to a low-chloride saline by replacement of Cl- with sodium propionate (Fig. 9A). Bath circulation of low-chloride solution also affected the response of interneurons insensitive to the picrotoxin. For example, the sensory-stimulated IPSPs of RO-2 (Fig. 9B, top trace) was almost eliminated by changing the saline to the low-chloride solution (Fig. 9B, middle trace) that recovered after washing (Fig. 9B, bottom trace). Similar reduction of IPSPs under low-chloride solution was also observed in CA-1 (not shown).


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FIG. 9. Effect of low-chloride saline on the picrotoxin-sensitive interneuron VE-1 (A) and the picrotoxin-insensitive interneuron RO-2 (B). Each trace is an average taken from 32 IPSPs elicited by sensory stimulation at 1 Hz.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

GABA mediated inhibitory sensory transmission

This study strongly suggests that GABA mediated transverse lateral inhibition of ascending interneurons in response to contralateral sensory stimulation, because 1) microinjection of GABA and its agonist, muscimol, into the neuropil induced the membrane hyperpolarization of identified ascending interneurons including VE-1 and CA-1 (Figs. 2-5), 2) the sensory-stimulated IPSPs in CA-1 were blocked by the local injection of GABA and muscimol (Figs. 3 and 5), and 3) the bath perfusions of bicuculline and picrotoxin, antagonists of GABAA receptors, reversibly reduced the amplitudes of sensory-stimulated IPSPs in VE-1 (Figs. 7 and 8). GABA injection was accompanied by an increase in the conductance (e.g., Fig. 2B), the duration of hyperpolarization in interneurons was related to the duration of the applied pulses of drugs (e.g., Fig. 4). and the falling phase of membrane hyperpolarization after GABA injection was smooth and decayed monotonically. These suggested direct causality between GABA injection and membrane hyperpolarization of the ascending interneurons, although we have not analyzed the effect of GABA application under Ca2+-free saline.

Ascending interneurons seemed to show desensitization in response to the sensory stimulation during GABA or muscimol injection (Figs. 3B and 5B). Furthermore, the reversal potential of IPSPs induced by the sensory stimulation and GABA or muscimol injection was rather similar. IPSPs were usually reversed by hyperpolarizing current of 2 nA in CA-1 (cf. Figs. 1C-2 and 5A) and 3-4 nA in VE-1 (cf. Figs. 1C-1 and 2C). These observations indicate, further, thatGABAergic transmission mediated the pathway for transverse lateral inhibition from hair afferents to contralateral ascending interneurons. Because we could not measure the input resistance of ascending interneurons, the exact level of reversal potentials of IPSPs could not be determined in this study, but it would be some 10-20 mV below the resting potential (55-68 mV).

In the neuromuscular system of the crustaceans, inhibitory transmission mediated by GABA has been well characterized (Albert et al. 1986; Constanti 1978; Shank et al. 1974; Takeuchi and Onodera 1972; Takeuchi and Takeuchi 1965). Inhibitory function of GABA in the CNS of the crustacean has also been examined (Cazelets et al. 1987; El Manira and Clarac 1991; Jackel et al. 1994; Marder and Paupardin-Tritsch 1978; Pfeiffer-Linn and Glantz 1991). Furthermore, distribution of GABAergic neurons in the crayfish CNS has been analyzed immunologically (Elekes and Florey 1987). About 10% of the total number of neurons in the terminal abdominal ganglion present GABA-like immunoreactivity (Mulloney and Hall 1990; Nagayama et al. 1996). Because neither terminal branches of the hair afferents nor main branches of the most ascending interneurons crossed the midline (Kondoh and Hisada 1987; Nagayama et al. 1993), some interneurons with bilateral configuration must be intercalated in the circuit to convey and reverse mechanosensory information from a given side to the opposite side. One of the most potent candidates to mediate transverse lateral inhibition is a bilateral nonspiking local interneuron, LDS, that received direct excitatory inputs from hair afferents on its some side and made inhibitory output connections with several ascending interneurons including RO-4 (Nagayama et al. 1994; Reichert et al. 1983). The combination of intracellular staining of LDS and immunocytochemical labeling reveals that LDS showed GABA-like immunoreactivity (Nagayama et al. 1996).

Recently, however, L-glutamate has also been known to be used as a central inhibitory transmitter in some crab stomatogastric neurons (Marder and Paupardin-Tritsch 1978), crayfish leg motor neurons (Pearlstein et al. 1994), and crayfish swimmeret motor neurons (Sherff and Mulloney 1996). Sherff and Mulloney (1996) have shown that swimmeret motor neurons are sensitive both to GABA and to glutamate and both transmitters elicit chloride currents with similar reversal potentials. Inhibitory responses to GABA of these motor neurons are picrotoxin insensitive, but the glutamate responses are picrotoxin sensitive. Because LDSs that mediate lateral inhibition are known as GABAergic neurons (Nagayama et al. 1996) and the inhibitory response of VE-1 to the sensory stimulation was blocked by theGABAergic antagonist bicuculline (Fig. 6A), GABA is, at least in part, an inhibitory transmitter mediating lateral inhibition. In this study, however, we have not examined the effect of glutamate on the ascending interneurons. At present, therefore, we cannot conclude whether or not GABA is the only inhibitory transmitter in this circuit. Further study to characterize the glutamatergic central interaction in the crayfish terminal abdominal ganglion is essential to clarify this point.

 
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TABLE 1. Summary of effects of GABA and its agonists and antagonists on the response of identified ascending interneurons

Two distinct types of GABA receptors

Table 1 summarizes the results of drugs' effects on ascending interneurons. Similar to VE-1 and CA-1, three other identified ascending interneurons, RO-1, RO-2, and RO-4, were also characterized in this study. The application of picrotoxin had no remarkable effect on RO-1 (n = 2) and RO-2 (n = 3), but had a significant effect on RO-4 (2 of 3 preparations).

Vertebrate GABA receptors have been divided into two major types, GABAA and GABAB receptors, on the basis of the pharmacological evidence and the ionic specificity of the channels. GABAA receptors are directly associated with Cl- channels and antagonized by bicuculline and picrotoxin (Kaila 1994). GABAB receptors are coupled to Ca2+ or K+ channels via second-messenger systems related with G protein (Andre et al. 1986). These channels are activated by baclofen and antagonized by phaclofen and AVA. Furthermore, GABAC receptors have recently been identified in the vertebrate retina (Polenzani et al. 1991; Qian and Dowling 1993).

If we follow this classification, GABAergic receptor on VE-1, and probably RO-4, would coincide with a pharmacological profile of vertebrate GABAA receptors. First, local application of muscimol, the competitive agonist of the GABAA-activated Cl- channel, induced membrane hyperpolarization of VE-1 (Fig. 4), whereas application of baclofen, a GABAB agonist, elicited no noticeable response in VE-1. Second, the amplitude of IPSPs elicited by the sensory stimulation was reversibly reduced by the bath application of bicuculline and picrotoxin, although the inhibitory effect of the bicuculline was considerably weaker (Figs. 6 and 7). By contrast, AVA, an antagonist of GABAB receptors, had no significant effect on the response of VE-1. Finally, the IPSP in VE-1 was reduced in amplitude when the external chloride ions were decreased owing to the consequent positive shift of the Cl- reversal potential (Fig. 9A). GABA receptors that have the same profile as VE-1 and RO-4, that is, sensitive to the GABAA antagonists bicuculline and picrotoxin, have been known, in invertebrates, to be distributed on lobster muscles (Constanti 1978; Shank et al. 1974) and projection interneurons in the antennal lobes of Manduca (Waldrop et al. 1987).

In contrast with VE-1, inhibitory responses of the interneurons CA-1, RO-1, and RO-2 to the sensory stimulation were not affected significantly under bath application of bicuculline (Fig. 6C) and picrotoxin (Fig. 7D). Because 1) local injection of muscimol caused membrane hyperpolarization of these interneurons (Fig. 5), 2) IPSPs were reversed by the low-chloride solution (Fig. 9B), and 3) the application of GABAB agonist and antagonist had no remarkable effects on the interneurons, GABAergic inhibition to these interneurons was also associated with chloride channels. GABA receptors that showed a profile similar to that of these interneurons have been reported in some invertebrates. GABA-operated chloride channels exhibit an agonist profile resembling that of the mammalian GABAA receptor, but the antagonists bicuculline and picrotoxin fail to block the GABA receptors of Ascaris muscle (Holden-Dye et al. 1988; Wann 1987) and Limulus heart (Benson 1989). Furthermore, many central neurons in the insects are insensitive to bicuculline (Benson 1988; Lees et al. 1987; Satelle 1990). GABA-activated chloride channels in lobster thoracic neurons in culture (Jackel et al. 1994) and crayfish primary afferent neurons (El Manira and Clarac 1991) are also insensitive to bicuculline, although these channels are blocked by picrotoxin. Moreover, picrotoxin-insensitive GABA-gated chloride channels have also been reported in the stomatogastric ganglion of the crab (Cazelets et al. 1987; Marder and Paupardin-Tritsch 1978), the neuromuscular junction of the lobster (Albert et al. 1986), and crayfish swimmeret motor neurons (Sherff and Mulloney 1996). In these systems, GABA-activated chloride channels are not blocked by picrotoxin. In vertebrates, retinal horizontal cells of fish also show a similar profile with insensitivity to picrotoxin (Hankins and Ruddock 1984). Lunt (1991) has therefore suggested that invertebrates have specific binding sites similar to the GABA/muscimol sites of mammalian GABAA receptors, but most of them differ from those of vertebrates by a markedly reduced sensitivity to bicuculline and a weaker-affinity binding to picrotoxin.

Functional difference of two distinct GABA receptors

This study demonstrates that GABA receptors of intersegmental ascending interneurons are classified into two distinct types. One type of receptors had profiles similar to those of vertebrate classical GABAA receptors but another type of receptors did not fit, although both types of receptors were associated with chloride channels. These two types of receptors would be separately distributed on the membrane of particular ascending interneurons.

What is the functional difference between these two groups of ascending interneurons? One obvious difference in response of these two groups of ascending interneurons was the time course of inhibitory responses. The time from start to peak of IPSPs in picrotoxin-insensitive interneurons including CA-1, RO-1, and RO-2 was significantly longer than that of picrotoxin-sensitive VE-1 (Fig. 8). Furthermore, IPSPs recorded in CA-1 were smoother and the half-decay time in CA-1 was much slower (Fig. 1, A and B). When GABA was applied into the neuropil, a similar difference in the time courses of inhibitory responses of two types of the interneurons was also observed. GABA-mediated membrane hyperpolarization of CA-1 was much longer than that of VE-1 (cf. Figs. 2 and 3). Some ascending interneurons have been suggested to respond preferentially to low-frequency waterborne movements, whereas others respond best to more phasic stimuli (Calabrese 1976; Plummer et al. 1986; Wiese et al. 1976). This difference in excitatory responses would be accomplished by the selective connections with sensory afferents that innervated sensory hairs of different sensitivity to water movements (Wine 1984). The rather short duration of IPSPs in picrotoxin-sensitive interneurons suggested that these interneurons could discriminate both the high-frequency and low-frequency waterborne movements to hyperpolarize the membrane after each stimulation. On the other hand, frequency selectivity of the picrotoxin-insensitive interneurons would be somewhat ambiguous, because they responded with a sustained membrane hyperpolarization over the interval of each stimulation (cf. Fig. 1, A-1 and A-2). At the moment, the property of excitatory responses between these two types of ascending interneurons is unknown. Characterization of their frequency selectivity to waterborne movement is necessary to understand the functional difference of these two types of ascending interneurons with distinct GABA receptors.

    ACKNOWLEDGEMENTS

  We are grateful to Dr. H.P.C. Robinson for critical reading of the manuscript.

  This work was supported by Ministry of Education, Science and Culture Grants 08640856 to T. Nagayama and 07640894 to M. Takahata.

    FOOTNOTES

  Address reprint requests to T. Nagayama.

  Received 30 July 1996; accepted in final form 12 November 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society