Evidence that Synaptic Transmission between Giant lnterneurons and Identified Thoracic lnterneurons in the Cockroach Is Cholinergic Janet L. Casagrand’,*Yt and Roy E. Ritzmann’?* Departments of ’Biology and *Neuroscience, Case Western Reserve University, Cleveland, Ohio 441 06

SUMMARY In the cockroach, a population of thoracic interneurons (TIs) receives direct inputs from a population of ventral giant interneurons ( vGIs). Synaptic potentials in type-A TIs (TI,s) follow vGI action potentials with constant, short latencies at frequencies up to 200 Hz. These connections are important in the integration of directional wind information involved in determining an oriented escape response. The physiological and biochemical properties of these connections that underlie this decision-making process were examined. Injection of hyperpolarizing or depolarizing current into the postsynaptic TI,s resulted in alterations in the amplitude of the postsynaptic potential (PSP) appropriate for a chemical connection. In addition, bathing cells in zero-calcium, high-

magnesium saline resulted in a gradual decrement of the PSP, and ultimately blocked synaptic transmission, reversibly. Single-cell choline acetyltransferase (ChAT) assays of vGI somata were performed. These assays indicated that the vGIs can synthesize acetylcholine. Furthermore, the pharmacological specificity of transmission at the vGI to TI, connections was similar to that previously reported for nicotinic, cholinergic synapses in insects, suggesting that the transmitter released by vGIs a t these synapses is acetylcholine. 01992 John Wiley & Sons, Inc. Keywords: synaptic transmission, giant interneurons, thoracic interneurons, choline acetyltransferase assays, acetylcholine.

INTRODUCTION

rectionally excited by wind (Westin, Langberg, and Camhi, 1977) via cholinergic inputs from cercal sensory afferents (Shankland, Rose, and Donniger, 197 1 ; Harrow, David, and Sattelle, 1982). The vGIs transmit this encoded wind information to a large population of individually identifiable thoracic interneurons through a network of connections (Ritzmann and Pollack, 1988). These interneurons, in turn, are capable of evoking activity in motor neurons, both directly and via local interneurons (Ritzmann and Pollack, 1990). Those thoracic interneurons that receive inputs from the vGIs via constant, short-latency connections are referred to as type-A thoracic interneurons (T1,s). Physiological and morphological evidence indicates that these inputs are direct (Ritzmann and Pollack, 1986; Casagrand and Ritzmann, 1991). The vCI to TI, connections are critical ones in the escape circuit. The TI,s are involved in integrating the directional wind information necessary for or-

The escape circuit of the cockroach, Periplaneta umericanu, is a useful model system for studying, at the single-cell level, the information processing and decision-making events involved in generating a complex behavior (Ritzmann, 1984; Ritzmann and Pollack, 1990). In the escape system of P. americana, there exists a population of wind-sensitive ventral giant interneurons (vGIs) that are diReceived February 4, 1992: accepted May I , 1992 Journal of Neurobiology. Vol. 23, No. 6, pp. 627-643 (1992) (C 1992 John Wiley & Sons, Inc. CCC 0022-3034/92/060627- 17$04.00 Some of these data were previously reported in abstract form (Casagrand and Ritzmann, 1990). * To whom correspondence should be addressed. t Present address: Arizona Research Laboratories, Division of Neurobiology, 6 I I Ciould Simpson Building, University of Arizona. Tucson, A 2 8572 1.

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ienting the animal during escape (Westin, Ritzmann, and Goddard, 1988), and for coordinating wind information with other relevant sensory information (Ritzmann, Pollack, Hudson, and Hyvonen, 1991 ) before a decision to turn is made. Thus, we would like to learn more about the physiological properties that influence the sensory transduction and decision-making processes at these connections. Little is currently known about the nature of transmission between interneurons in insects. This has been due to the unavailability of an accessible, identified, interneuron to interneuron connection, which can be subjected to pharmacological characterization. The vGI to TI, connections offer the opportunity to study transmission between populations of interneurons at the level of single identified cells. Such studies will not only extend our knowledge of neural transmission in insects, but also aid in understanding the integrative processes that result in an oriented escape response. We report here several experiments that provide evidence that the vGI to TI, connections are chemical, and that the likely transmitter utilized by all the vGIs to elicit the synaptic responses in the TIAS is acetylcholine ( ACh).

MATERIALS AND METHODS

B

-YJ \

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Figure 1 ( A ) A diagram of the basic recording setup used in most ofthe experiments. The vCI was stimulated ( S ) via intracellular or extracellular electrodes. A TI, was recorded with an intracellular microelectrode ( R ) in the metathoracic ganglion. ( B ) A camera lucida drawing of one of the Tl,s (Ti, 30 1 ) in whole mount.

Setup and Recording Techniques Prriplanrta umericana were housed in large. plastic garbage cans at 29°C on a 12: 12 h light/dark cycle and had free access to Purina Chick Starter Mash (unmedicated) and water. An animal was prepared by pinning it dorsal side up on a cork platform and opening a window in the cuticle. The gut was ligated at both ends, and removed to expose the nerve cord. Saline was superfused continuously over the nerve cord. The metathoracic ganglion (T,) was desheathed mechanically with a pair of forceps to facilitate exchange of solutions, and was supported by a platform during impalement with microelectrodes. A TI, was impaled either in the soma o r one of its neuropil branches [Fig. 1 ( A ) ] . No difference between neuropil and soma recordings was noted, excepting a slight attenuation in the soma recordings due to electrotonic decrement (Bacon and Murphey, 1984; Shepherd, Kaemper, and Murphey, 1988). The effects of various solutions and bath-applied pharmacological agents upon the amplitudes of excitatory postsynaptic potentials ( EPSPs) evoked by vCI stimulation were tested. We stimulated the vGIs in two ways. In most expenments, the abdominal cord containing the vGIs was stimulated extracellularly with a pair of bipolar silver hook

electrodes at an intensity just sufficient to evoke action potentials in large-diameter axons. The level was adjusted to evoke a single action potential. Hook electrodes placed between mesothoracic (T,) and metathoracic (T,) ganglia monitored this activity. If during the course of the experiment, another axon was recruited, then the data point was discarded. I n several experiments, the vGIs were impaled and stimulated intracellularly. In these experiments, the abdominal cord between the fourth and fifth abdominal ganglia ( A 4 and A,) was placed on a platform and treated with I mg/ml pronase in saline (Sigma type XIV), three times for 5 min each, to facilitate penetration of the microelectrode through the sheath. A vGI was impaled and stimulated with single 400 p s current pulses through a breakaway box on the D C amplifier (Ritzmann and Pollack, 1988). Due to the diameter of the vGls, stimulation required considerable current, and could not be carried out through the bridge circuitry but rather had to be performed through a breakaway box. Although this method of stimulation resulted in large stimulus artifacts recorded in the TI,s, the artifact did not interfere in data analysis. Activity from the stimulated vGI was recorded through hook electrodes

Evidence,fi)uACh at \GI to TI Connection placed between A, and A,. With both methods of vGI stimulation, action potentials were evoked at a rate of one per minute. The PSPs recorded in the TI,s were stable when stimulated at this frequency. Although the background synaptic activity fluctuated during the recording, it did not interfere with data analysis. In cases where the spontaneous activity occurred at the same time as the evoked potential, the data point was discarded. Generally, the background activity showed the same trend as the evoked potential. All intracellular and extracellular signals were recorded on VCR tape using a Medical Systems Corporation A / D VCR Adapter. Data were analyzed either on an AT&T 6386 WGS computer using the analogue-todigital hardware and Axotape software (Axon lnstruments, Inc.), or on a Gould Digital Oscilloscope. The tips of the microelectrodes were filled with 4% Lucifer Yellow C H (Aldrich, Molecular Probes) in 0.3 M LiAc and the remainder with I M LiAc. Electrodes had resistances of 50-80 Mohms. Generally, enough dye leaked passively from the electrode throughout the course of the experiment to stain the cells. When possible, cells were further stained at the conclusion of an experiment by iontoplioresing dye (500 m s / s pulses of -5 nA). The preparations were fixed in 4% paraformaldehyde in saline overnight at 4"C, dehydrated through an ethanol series, cleared in methyl salicylate, and viewed in whole mount with a fluorescence microscope to identify the TI, [Fig. 1 (B)]. When vGIs were also filled, the preparation was subsequently embedded in paraplast, and sectioned at 10 pm. The identity of the vG1 was confirmed from the location of its axon in cross section (Westin et al., 1977).

Saline Solutions and Pharmacological Agents The saline used in all experiments to superfuse the nervous system was that of Wafford and Sattelle ( 1986) for desheathed ganglia. This will be referred to as normal saline. In some experiments, a high-magnesium (50 m M ) , zero-calcium saline was used to block chemical transmission (Cam and Fourtner, 1980). The NaCl and sucrose concentrations were reduced accordingly to maintain osmolarity. The following pharmacological agents were obtained from Sigma Chemical Co. (St. Louis, MO) : acetylcholine chloride, atropine sulfate, alpha-bungarotoxin, alpha-cobratoxin, carbamylcholine chloride, eserine hemisulfate, decamethonium bromide, d-tubocurarine chloride, mecamylamine hydrochloride, neostigmine bromide, and nicotine hydrogen tartrate (-). These were dissolved in saline to the required concentrations and bath applied using a peristaltic pump (Rainin) to regulate flow. The flow rate of the pump was set between 2 and 4 mllmin.

629

Choline Acetyltransferase Assay Animals were chilled at 4°C for 10-15 min, and pinned dorsal side up. A window was opened in the cuticle to expose the nerve cord from T, to the terminal abdominal ganglion ( A6). The cord was removed and placed in chilled (4°C) saline (Wafford and Sattelle, 1986), to which I m M Di N a + EDTA had been added (Giller and Schwartz, 1968). The cords were pinned out in sylgard dishes, and T, and A, were mechanically desheathed with forceps. The saline was replaced by filtered 2.5% Neutral Red solution (in saline) for 10-15 min to aid in visualization of cells. The ganglia were washed in chilled saline (with EDTA added), and a microelectrode was used to tease out somata of interest. These were identified on the basis oftheir size and location. Somata were transferred with a pipette to test tubes ( 12 >< 75 mm), in which the assays were run, in a total volume of 10 pl. Bovine serum albumin (BSA) was first drawn up into the pipette tips, which were then rinsed with saline, to prevent cells from sticking to the sides of the pipette tip (Schacher, 1985). After transfer, cells were inspected under the microscope to confirm their transfer, and quickly frozen on dry ice. Cells were not pooled, and thus n refers to assays of individual cells. Choline acetyltransferase ( ChAT) activity ofthe individual somata was assayed by a modification of the method described by Fonnum (1975). which is based upon the reaction between labelled acetyl CoA and unlabelled choline to give labelled ACh as the product. Briefly, 5 pl of reaction mixture (50 m M NaPO, buffer ( p H 6.8), 200 m M NaCI, 0.342 m M eserine sulphate, 34.2 m M choline bromide, 0.58 mg/ml BSA, and 135 pMacetyl CoA) was added to each tube. The radioactive tracer used was 12,500 cpm of 3 C i / m M labelled 'Hacetyl CoA (New England Nuclear). Samples were incubated at 35°C for 30 min. The reaction was stopped by the addition of 2 ml of cold, 10 m M NaPO, buffer (pH 7.3). Samples were transferred to scintillation vials and an extraction solvent ( 2 ml acetonitrile plus 0.5% tetraphenylboron, and 10 ml of toluene scintillation mixture containing 4% butyl-PBD) was added to separate the ACh from the rest of the solution. The tetraphenylboron forms an ionic complex with the ACh that is extracted by the toluene, which also functions as a scintillant. Thus, when the samples were counted (Beckman LS 5000 TD Liquid Scintillation System), the activity of the labelled ACh in the organic phase was counted, while the activity from the labelled acetyl CoA in the aqueous phase was not, as water is not a scintillant. Blanks ( n = 15-25) consisting of saline plus EDTA were also run with each assay. The activity per cell was then calculated from the raw counts based upon the variation in the blanks and the specific radioactivity in the reaction mixture. Whole-ganglion homogenates were also prepared to determine that the assay conditions were appropriate for the enzyme. The metathoracic ganglion was homoge-

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nized in 100 pl of homogenization buffer (50 m M PO., buffer, 160 m M NaCI, 1 m M Di Na' EDTA) (Dagan and Sarne, 1978). and 10 pl aliquots assayed for activity. The results indicated that enzyme activity was choline and concentration dependent, and time dependent up to 30 min. similar to previous reports for invertebrate tissue (Giller and Schwartz. 1968; Dagan and Sarne. 1978).

Hyperpolarizing

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TI, Nomenclature The T1,s are named according to a numbering system that was introduced in previous papers (Westin et al., 1988; Ritzmann and Pollack, 1990)and is similar to that described by Robertson and Pearson (1983) in the locust. Neurons are assigned a three-digit number, such that thc first number indicates the location of the soma and axons. and the second and third digits represent the order of discovery. When the cells are part of an identified population. the initials of this population precede the number (e.g.. DPG 301 is a member of the dorsal posterior group of cells). The general classes of neurons discussed in this paper belong to the 300, 500, and 700 series. The cells in these classes all have axons contralatera1 to their somata. Neurons in the 300 series have axons that exit the ganglion posteriorly, those from the 500 series have axons that exit anteriorly. and neurons in the 700 series have axons that exit the ganglion both anteriorly and posteriorly.

RESULTS The vGI to TI, Connections Are Chemical

Excitatory postsynaptic potentials (EPSPs) in the TI,s follow action potentials in the vGIs faithfully with constant, short latency (approximately 0.4 ms) at very high frequencies ( u p to 200 Hz), suggesting the possibility that the connection may be electrical (Ritzmann and Pollack, 1986). Many chemical synapses fail when stimulated at high frequencies (Berry and Pentreath, 1976). But this observation is at best suggestive, since chemical synapses in insects have been shown to follow at 125 and 250 Hz (Burrows, 1975; Weeks and Jacobs, 1987). Current Injection. One test of whether a connection is electrical or chemical is to determine whether the amplitude of the PSP is dependent upon the membrane potential of the postsynaptic cell. When hyperpolarizing or depolarizing current was injected into the postsynaptic TI,s, the amplitude of the evoked EPSP was altered in a manner appropriate to a chemical synapse, based upon an

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Time (Mi I I i sec o nd s) Figure 2 Injecting either hyperpolarizing or depolarizing current into TI, 701 through the bridge circuitry alters the amplitude of the EPSP evoked by stimulating vGI 3. The changes in the amplitude of the EPSP were consistent with chemical transmission being mediated by an increased conductance. The evoked EPSP was smaller when TI, 70 I was depolarized, and larger when it was hyperpolarized. The bridge was balanced prior to penetration using a square pulse and checked again after pulling out of the cell. Between 1 and 5 nA were injected into the TIAs with voltage changes of 1-7 mV. The vGI action potcntial was stable throughout the experiment. Traces were superimposed by lining up the resting potential prior to the stimulus.

increased conductance. Hyperpolarizing the T1,s resulted in an increase in the PSP amplitude [consistent with the observation of Ritzmann and Pollack ( 1988)], whereas depolarization caused a decrease in amplitude ( n = 3 preparations) (Fig. 2 ) . Although absolute resting potential values are difficult to access in neuropile recordings, we noted no consistent alterations in resting potential before and after current injection. In chemical synapses, current injected into the postsynaptic cell changes the driving forces on ions that mediate the PSP. Since electrical transmission is due to the direct spread of current from the pre- to the postsynaptic cell, electrical synapses do not show this response to current injection. Block of Synaptic Transmission with Zero-Calcium/High-Magnesium Saline. Another test for chemical synapses is to replace the calcium ions in the solution bathing the cells with magnesium. Chemically mediated synaptic transmission is blocked in the absence of calcium, and magnesium acts as a calcium antagonist. When the saline bathing the T1,s was replaced with an osmotically balanced zero-calcium, high-magnesium ( 50 m M )

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PSP provides further evidence that the vGI to TI, connections are monosynaptic (Burrows, 1975; Berry and Pentreath, 1976; Weeks and Jacobs, 1987). If a spiking interneuron were interposed between the vGIs and TIAS, then the EPSP would rapidly have fallen to zero at the point where the interposed interneuron failed to evoke an action potential, as a consequence of the zero-calcium, high-magnesium saline. These data do not eliminate the possible existence of a nonspiking interneuron interposed between the vGIs and TIA%

Time (Minutea)

Choline Acetyltransferase Assay of Individual vGls

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Time (Milliseconds) Figure 3 Perfusion with zero-calcium, high-magnesium saline resulted in the gradual decrement of the EPSPs evoked in TI, '701 by stimulating vGI 1 intracellularly. Ultimately, this blocked synaptic transmission. Upon washing the preparation with normal saline, the response recovered to its initial level. ( A ) EPSP amplitude is normalized as percent initial response. ( B ) EPSPs are 0.5 min before and 0.5, 1.5, 2.5, and 5.5 min after introduction of zero-calcium, high-magnesium saline. The vGI action potential (top trace) was recorded extracellularly between the meso- and metathoracic ganglia. This example of the vGI action potential was taken prior to introducing zero-calcium saline. Similar stable vGI action potentials were recorded in each trial.

saline, transmission was blocked within 10 min (Fig. 3 ) . The same results were observed whether vGIs were activated by cord stimulation ( n = 6) or by intracellular stimulation of individual vGIs ( n = 2 ) (Fig. 3 ) . After introducing zero-calcium saline, the evoked EPSP decreased gradually in amplitude (Fig. 3 ) , and was eventually completely blocked. The response recovered upon washing the preparation with normal saline. The gradual decrement in the amplitude of the

The next step was to establish the probable identity of the transmitter used at these synapses. Acetylcholine (ACh) is an important excitatory neurotransmitter at sensory connections in insects (Pitman, 1971; Sattelle, 1980). The elements necessary for its synthesis are present in high concentrations in insect central nervous systems (CNS) (Smallman and Pal, 1957; Colhoun, 1958b, 1963), and the ability of insect nervous tissue to synthesize ACh has been clearly demonstrated ( Prescott, Hildebrand, Sanes, and Jewett, 1977). The distribution of choline acetyltransferase (ChAT), the acetylating enzyme catalyzing the synthesis of ACh, correlates well with the distribution of ACh in P. umnevicana (Colhoun, 1958a,b), and is currently considered to be the most reliable marker for cholinergic neurons in insects (Sattelle, Ho, Crawford, Salvaterra, and Mason, 1986). There was evidence to suggest that the GIs could synthesize ACh (Dagan and Sarne, 1978). Cord section between the fifth abdominal ganglion and the terminal abdominal ganglion results in specific degeneration of the GIs anterior to the lesion (Farley and Milburn, 1969; Spira, Parnas, and Bergmann, 39691, since their somata are located in the terminal abdominal ganglion (Daley, Vardi, Appignani, and Camhi, 1981). Dagan and Sarne (1978) utilized this feature to demonstrate a decrease in ChAT activity in lesioned connectives, corresponding to a loss of the GIs. The cord between the fifth abdominal ganglion and the terminal abdominal ganglion was lesioned on one side, and after specific degeneration of the GIs, the connectives on the lesioned side between the fourth abdominal ganglion and the metathoracic ganglion, excluding the ganglia, were assayed for ChAT activity. This value was compared to that obtained from the contralateral, nonlesioned con-

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Cusugrund und Ritzmunn

nectives. A marked decrease in ChAT activity was noted in the nerve cord anterior to the lesion, corresponding to the loss ofthe GIs, suggesting that the GIs could synthesize ACh. However, a direct analysis of the GIs had not been performed. Individual vGI ( 1-3) somata were, therefore, assayed for ChAT activity, along with the large, dorsal unpaired median (DUM) neurons and unidentified neurons from the ventral midline surface of the metathoracic ganglion with similar soma diameters for comparison. The soma of vGI 4 is smaller than for vGIs 1-3, medially located, and could not be reliably identified. The activity of the vGIs ( x = 89.5 k 54.3 (S.E.) pmol/cell/h, n = 25) was significantly higher than that observed in the DUM neurons or ventral somata ( p < 0.0 1, Tukey’s Test for Honestly Significant Difference), indicating that they can synthesize ACh. Some activity was observed in both the DUM somata ( x = 28.3 ? 6.7 pmol/cell/h, n = 1 1 ) and the ventral midline cell somata ( x = 29.9 k 7.8 pmol/cell/h, n = 13). This activity may represent some ability by these cells to synthesize acetylcholine, or may have been due to the presence of contaminating glial cells or neuronal processes adhering to the somata (Giller and Schwartz, 197 1 ). Some of the small DUM neurons, for example, receive inputs from the vCls (Pollack, Ritzmann, and Westin, 1988), and the large D U M neurons are known to respond to iontophoretic application of ACh (Kerkut, Pitman. and Walker, 1969a,b).Nonetheless, the activity in the vGIs was three times that in the DUM neurons and the ventral midline somata. The mean activity calculated for the vGIs in these experiments may be an underestimate. The vGI values ranged from 21 to 231 pmol/cell/h, with several of the VGISexhibiting activity of 150 pmol/cell/h (as did one additional, unidentified neuron from the ventral lateral surface). It is possible that some of the vGIs were damaged, since they were more difficult to tease free than the DUM and ventral midline cell somata, and may subsequently have displayed lower activities (Giller and Schwartz, 197 1; Marder, 1974). It has been noted that, as ChAT is a soluble enzyme, damaged cells contain smaller amounts ofthe enzyme (Giller and Schwartz, 197 I ) . However, no attempt was made to discount cells that had possibly been damaged. This would account for the larger variation in values observed for the vGIs than for the DUM cells (16-41 pmol/cell/h) or for the ventral midline cell somata ( 18-48 pmol/cell/h). Even so, vGIs

1-3 clearly display ChAT activity, thus confirming previous suggestive data (Dagan and Sarne, 1978). Pharmacological Specificity of vGI to TI, Connections

The pharmacological responses of the vGI to TI, connections to nine different cholinergic agents were tested and are summarized in Table 1. Acetylcholine Agonists. Perfusion with nicotine ( 1o - M ~ , n = 3; 5 x 10-4 M , n = 1; 10-5 M , n = I ; lop6M , n = 1; lo-’ M , n = 1 ) produced a rapid depolarization of the TI,s (30 1, 50 I , 5 15, 701 ), and a concomitant reduction in the EPSP evoked by both extracellular cord stimulation ( n = 5 ) and intracellular stimulation of vGIs ( n = 1) [Fig. 4(A,B)], preceded by a burst ofactivity in the TI,. At a concentration of M nicotine, a depolarization of 35 mV was noted. At higher concentrations, depolarizations of 50-65 mV were observed, and the membrane potential approached the ACh reversal potentials extrapolated by David and Pitman ( 1982) for the fast coxal depressor motor neuron (Df), and by Callec ( 1974)for a GI. Repolarization, and subsequent reappearance of the EPSP, upon wash was observed in two preparations [example in Fig. 4(A,B)]. Recordings in the cells of the other preparations were lost before recovery. Upon wash, repolarization began after approximately 5 min, and recovered slowly. No consistent recovery in the evoked EPSP was noted for approximately 20 min, with spontaneous activity appearing several minutes before the evoked activity. Recovery continued slowly thereafter, with the EPSP returning to 75% of the initial amplitude after approximately 55 rnin of washing. Bath application of M carbamylcholine ( n = 1, TI, 30 1 ), a nicotinic and muscarinic receptor agonist (after a previous exposure of 29 rnin to M nicotine followed by an 85 min wash), also resulted in an immediate depolarization of TI, 30 1, but to a lesser degree (approximately 20 m V ) . Repolarization began upon wash with normal saline after 50 rnin of perfusion with carbamylcholine. An initial increase in the amplitude of the EPSP was noted 15 rnin after application of carbamylcholine, which then gradually decremented. Acetylcholine Antagonists. Perfusion with the nicA4 ( M otinic antagonist d-tubocurarine at 9 X = l,TI,701), 1 0 - 4 M [ n = 5,TIAs301(2),501,

Evidencefiir ACIi at vCI to TI Connection

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Table 1 Summary 0 1 the Actions of Cholinergic Agents on the Response of the TI,s to vCI Stimulation

Agent Nicotine

Carbamylcholine Eserine

d-Tubocurarine

Atropine Mecamylamine Decamethonium

alpha-Bungarotoxin alpha-Cobratoxin

Concentration

N

10-4 M 5 x 10-4 M 10-5 M 10-6 M 10-4 M 10-4 M 10-6 A4 10-7 M

3 (1) 1 1 1 1 2 1 2

9 x 10-4 M 10-4 M 5x 10-5~ 10-5 M 5 x 10-4 M 10-4 M 10-4 M 10-5 M 5x 10-3~ 10-3 M 10-4 M 10-6M 10-6 M

TI, Membrane Potential

Effect of EPSP Amplitude’

Depol. Depol. Depol. Depol. Depol. Depol. No change Hyperpol.

Blocked Blocked Blocked Blocked Transient Inc., Blocked 30% Dec. 25 f 5% Inc., then dec. to 60% 90% Dec. 80 f 5 % Dec. 87 f 5% Dec. 25% Dec. 75% Dec. 60 f 26% Dec. 80% Dec. 60% Dec. Blocked 62.5 I 8% Dec.

Depol.. No Change No Change No Change Depol. No Change No Change No Change No Change

* *

No Change No Change

**

No Effect No Effect

Note: Depol. = Depol.arization; Hyperpol. = hyperpolarization; Inc. = increase; Dec. = decrease. Numbers in parentheses indicate how many of the total number involved intracellular stimulation of the vGIs. * Changes in membrane potential varied. ** In one experiment, a 60% initial increase was followed by a decrease to 70% of the initial value. I Dec. and Inc. indicate a decrease and increase in amplitude, respectively. Blocked indicates that the agent completely eliminated the evoked EPSP.

701 ( 2 ) ] , 5 X lO-’M[n = 3, TIAs 701 ( 2 ) , 7031 and 10 M ( n = 1, T I A 70 1 ) reduced the PSP (Fig. 5 ) , in a concentration-dependent manner to between 10% and 50% of its initial amplitude in 3040 min. Upon wash, the EPSP was generally observed to return to between 70% and 100% of its initial response level. Figure 6(A,B) shows the time course of the response of two TI,s to M and 5 X lo-’ M d-tubocurarine, respectively. No consistent changes in resting potential were observed with application of this or any other ACh antagonist. The rise time of the evoked EPSP increased significantly with the application of d-tubocurarine ( t test), indicating an increase in postsynaptic input impedance. This provides evidence that the decline in EPSP amplitude associated with this agent was not a result of a decrease in postsynaptic input impedence resulting from physical degradation of the preparation. Atropine, which has some actions on the nicotinic receptor ionophore complex in the insect ner-

vous system (Dudai, 1978; David and Sattelle, 1984), has been demonstrated to inhibit fast cholinergic neurotransmission in insects ( Sattelle, 1980), although at higher concentrations than reported for the muscarinic ACh receptor isolated from housefly nervous tissue extracts (Haim, Nahum, and Dudai, 1979). Similar to findings in Manduca (Trimmer and Weeks, 1989), the effects of atropine were more variable than those of d-tubocurarine. At a concentration of lop4M, reductions of the EPSP to lo%, 40%, and 80% of the initial amplitude were noted in different preparations ( n = 4, TIAS 30 1, 70 1, 703). At a higher concentration, 5 X l o p 4M , a reduction of 75% ( n = 1, TI, 30 1 ) was observed. The decrement became apparent after 3 min of beginning perfusion of atropine, and reached its lowest level after 20 min. In the cases where cells were held through the washout, full recovery was noted after 30-35 min. The rise time of EPSPs increased in the presence of atropine, although the change was not significant.

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After 4 h of washing with normal saline, the EPSP had only recovered to 60% of its initial value. Decamethonium, a nicotinic antagonist (Sattelle, 1980), resulted in an initial increase in EPSP amplitude at all concentrations tested. At 5 X M , the response subsequently blocked in 10-15 min [ n = 5, TI,s 301 ( 2 ) , 701 ( 2 ) , unidentified TI, ( I ) ] . Perfusion with lop3A4 resulted in a decrement to 45% of the initial response ( n = 2, TI,s 30 1,70 1 ) . Upon wash with normal saline, the dec-

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Figure 4 ( A ) Perfusion with lo-’ -34nicotine produced a rapid depolarization of the TI,. Repolarization began approximately 5 min after wash began. The DC offset was zeroed before penetrating a cell, and values were read off the DC amplifier at I-min intervals corresponding to the EPSP stimulus points. ( B ) The time course of the effects of nicotine on EPSP amplitude is shown. EPSPs were evoked in the TI, by extracellular stimulation of vGIs. EPSP amplitude recovered upon wash with normal saline. Data are normalized as percent initial response. The missing data points on the graph represent the inability to evoke vCI action potentials, due to an increased stimulus threshold. This occurred at higher concentrations of nicotine and was reversible upon wash with normal saline. It is possible that if extremely high stimulus currents had been used, activity could have been evoked, but this was not attempted to avoid damaging the cells. Note that the EPSP was already blocked by this time.

Mecamylamine, a nicotinic antagonist, at 1 0-5 M ( n = 1, TI, 507) resulted in a reduction of the EPSP to 40% of its initial response in 80 min. Only partial recovery (60%of initial ) was observed upon washing for 70 min, at which time the cell recordM ( n = 1 ), ing was lost. At a concentration of a decrement of 80% was observed after approximately 85 min of perfusion with niecamylamine.

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Time (Milliseconds) Figure 5 d-Tubocurarine ( M ) decreases EPSP amplitude in TI,s. EPSPs were evoked in TI, 501 by intracellular stimulation of vGI 2. The dotted lines represent the amplitude of the EPSP in normal saline. The upper trace in A is the vGI spike recorded extracellularly from the A,-A, connectives. A similar action potential was recorded on all subsequent trials. ( B ) The response o f t h e TI, was decreased to 20% after 10 min exposure to d-tubocurarine, with no significant change in the membrane potential. ( C ) A 70% recovery in EPSP amplitude occurred after 21 min of wash in normal saline, at which time the recording was lost.

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Snake alpha-Toxins Do Not Block Trunsmission. Snake alpha-toxins are often potent blockers of nicotinic ACh receptors in the cockroach (Carr and Fourtner, 1980; Sattelle et al., 1980, 1983). However, there have been reports of alpha-bungarotoxin-insensitive, nicotinic ACh receptors in insects (see Discussion). At the vGI to TI, connections, no change in the PSP evoked in the TI,s was noted after perfusion of lop6 M alpha-bungarotoxin for 10, 65, or 85 min ( M = 3; TI, 701 with vG1 1, TI, 50 1 with vGI 2, TI, 70 1 with cord stimulation). Figure 7 illustrates that no decrement in the amplitude of the EPSP recorded in TI, 501 evoked by stimulating vGI 2 resulted when the ganglion was perfused for 85 rnin with alpha-bungarotoxin. Similarly, exposure of TI, 701 to lop6M alpha-cobratoxin ( n = 1 ) for 20 min did not result in a reduction in the EPSP evoked by cord stimulation. The rise time of evoked EPSPs was not affected by either or these agents.

120 100

80 160 40

20

0

Figure 6 The time course of 5 X lo-, A4 and M d-tubocurarine effects on vGI-evoked EPSPs recorded in T1,s is shown. In both cases. the EPSPs declined in amplitude when d-tubocurarine was introduced into the saline and recovered with wash in normal saline. The EPSPs in A were evoked by intracellular stimulation of vGI 2 (same preparation as in Fig. 5 ) , those in B were evoked by extracellular stimulation of an unknown vGI. N o change in resting membrane potential was observed in either experiment. Note the different time scales in A and B.

remented responses recovered to their initial amA4, increases in the amplitude of plitudes. At the PSPs ( 130%, I60%, M = 2, TI, 70 1 ) were noted. However, the 130%increase at this concentration was followed after 25 min by a reduction to 60% initial response. (The microelectrode came out of this cell before washout.) In the case where the cell was held throughout the wash, the potentiated response at lop4 M also returned to its initial response level. As with d-tubocurarine, there was a significant increase in the rise time of evoked EPSPs in the presence of decamethonium.

>I

/

Saline Before

B

0

2

4

8

8 10 12 14 18 18 20

Time (Milliseconds) Figure 7 alpha-Bungarotoxin ( M ) failed to affect EPSP amplitude. No effect was recorded in the amplitude of TI, 501 EPSPs evoked by intracellular stimulation of vGI 2. Responses were monitored for 83.5 min. The upper trace in ( A ) is the vG1 action potential recorded extracellularly from the A,-A, connectives. A similar action potential was recorded in all trials. The dotted lines indicate the amplitude of the initial EPSP. Variation in EPSP duration is attributable to normal variation during the long recording period.

Cholinesterase Inhibitors. The cholinesterase inhibitor, eserine, at M initially resulted in a significant increase in the PSPs in two preparations ( T I A S = 301, SO 1 ). A 30% increase in the amplitude of the PSPs was observed during the first 10 min of perfusion with eserine. In one preparation, the recording was lost at this time. In the other, the PSP subsequently decreased to 60% of its initial amplitude. At a concentration of lop6M eserine, a 30% decrease in the amplitude of the PSP was observed in a TI,. Perfusion of eserine at A4 resulted in the rapid depolarization of two TI,s, and a loss of the evoked EPSP. The cells became silent and depolarized, similar to nicotine effects. It was, therefore, difficult to determine whether the microelectrode had become dislodged from the cell. However, in one preparation, the cell continued to depolarize for several minutes after the cell became silent, until finally reaching a plateau level. This would indicate that the electrode was still in the cell at the time it became silent. No repolarization or return of either evoked or spontaneous activity was noted in the TI, after 92 min of wash. Thus, either the cell did not recover in this time or the electrode became dislodged at some point. The effects of eserine are not always reversible (Yamasaki and Narahashi, 1960). In both preparations, the stimulus threshold increased. In one preparation, after the TI, became silent, there was a burst of activity in the extracellular recording, followed by silence. Upon wash with normal saline, this activity returned, and the stimulus threshold also decreased. The reason for this temporary loss of activity is unknown, although the ability of acetylcholinesterase inhibitors, including eserine, to block axonal conduction has been reported (Dettbarn, 1960; Rosenberry, 1975). All vGls Release the Same Transmitter

The ChAT assays indicated that vGIs 1-3 can synthesize ACh, and experiments in which the nerve cord containing the G I s was stimulated to evoke a PSP in the TI,s implied that all vGI input is blocked by cholinergic drugs. We wanted to confirm these observations by stimulating each of the vGIs ( 1-4) directly, and demonstrating for all of the vGIs that cholinergic antagonists block the EPSPs evoked in TI,s. All four vGIs (1-4) were, therefore, tested by intracellular stimulation in the presence of d-tubocurarine ( n = 6). GIs 2 and 4 were also tested with decamethonium ( n = 3 ) , and

vGI 2 with atropine ( n = 1 ). The time course and degree of block of the PSPs were comparable for each of the vGIs, and were similar to those from the cord stimulation experiments.

DISCUSSION

The vGI to TI, synapses are capable of following high-frequency trains of action potentials (>200 Hz) faithfully (Ritzmann and Pollack, 1988). vGls have been shown to respond to wind puffs with maintained spike trains of 290-350 Hz and instantaneous frequencies as high as 900 Hz (Westin et al., 1977). This is an interesting feature ofthe vGI to TI, connections, since, generally, chemical synapses begin to fail when stimulated at high frequency (>200 Hz) (Berry and Pentreath, 1976: Camhi, 1984). Nevertheless, chemical connections able to follow at frequencies of 12.5 and 250 Hz have been described between the wing stretch receptors and flight motor neurons in the locust (Burrows, 197S), and hair afferents and proleg motor neurons in Mandiica x x t u (Weeks and Jacobs, 1987), respectively. Further work on the response characteristics of the TI,s and their consequences for the transduction of sensory information will prove enlightening. Acetylcholine is known to play an important role in synaptic transmission at sensory connections in insects (Pitman, 1971; Sattelle, 1980; Harrow et al., 1982). However, its role in transmission between interneurons has not been established. Our data are consistent with the notion that the likely transmitter at all vGI to TI, connections is ACh. ChAT assays of individual vCI somata indicate that vGIs ( 1-3) can synthesize ACh. This is in agreement with previous nerve cord assays (Dagan and Sarne, 1978). The pharmacological results observed both for extracellular and intracellular stimulation of vCls ( 1-4) also support the contention that the vGIs release the same transmitter. Populations of morphologically similar cells often display cytochemical homogeneity in invertebrates (Anderson, Halpern, and Keshishian, 1988; Watson and Burrows, 1987; Goodman, O’Shea, McCaman, and Spitzer, 1979; Stuart, Blair, and Weisblat, 1987 ) . Nevertheless, it was possible that different vGI to TI, pairs might have utilized different transmitter types. No case is presently described in the literature where this occurs. However, this being one of the first examples where two large populations of identified interneurons (pre-

Evidtwce for ACh at G I lo TI Connection

and postsynaptic) have been studied, we felt that the synaptic properties should be tested across the vGI population. In addition, it was possible that the TI,s expressed different receptor subtypes with different physiological and pharmacological properties, and that this feature coiild have preserved the encoded wind information. Such receptor diversity can affect the transduction process, as well as the threshold sensitivities to a transmitter, such that one neuron will respond sooner than another, and also the sign of responses to the same inputs (cf Schofield, Shivers, and Seeburg, 1990). Thus, the response and transduction properties of neurons to the same inputs can be made flexible by utilizing different receptor subtypes on different neurons. Again, the pharmacological responses and the time courses of their actions were similar for all the TIAS, suggesting that they express the same receptor types, and indicating that some other mechanism, such as selective connectivity, or relative timing or weights of inputs is employed to preserve the encoded sensory information. The pharmacological specificity exhibited by the vGI to TI, connections was similar in most respects to that previously described for nicotinic ACh receptors in insects (Shankland et al., 1971; Carr and Fourtner, 1980; David and Sattelle, 1984; Trimmer and Weeks, 1989). However, several differences in the actions of the cholinergic ligands were noted, which may be explained by secondary effects of drugs, multiple sites of action, differential accessibility, receptor density, or receptor subtype diversity. The results of perfusion of cholinergic ligands upon the EPSPs evoked in TIAsby vGI stimulation are summarized in Table 1. The pharmacological evidence, thus, suggests that the transmitter utilized by the vGIs to elicit the synaptic responses in the TIAS is ACh. These studies do not rule out the possibility that the vGIs also co-localize another transmitter( s ) . However, to date it appears that co-localized transmitters generally have different time courses, one fast and one slow (Adams and O’Shea, 1983; Bishop, Wine, Nagy, and O’Shea, 1987; Whim and Lloyd, 1990). This was not observed to be the case for the vGIs. Cholinergic Agonists

Nicotinic ACh receptors appear to predominate in insects (Dudai and Ben-Barak, 1977; Breer, 1981; Lummis and Sattelle, 1985), and are the only receptors for which a functional role has been demon-

637

strated. Consistent with a nicotinic ACh receptor (Sattelle, McClay, Dowson, and Callec, 1976; Carr and Fourtner, 1980), nicotine was a more effective agonist than carbamylcholine at the vGI to TIA synapses. The perfusion of nicotine resulted in the rapid depolarization of the TIAS,with a subsequent decrease in the synaptic response, which was reversible upon wash. This decrease in the synaptic response is most likely a function of both the depolarization of the postsynaptic cell, and the nicotine competing with any endogenously released ACh. The observed effects of nicotine were similar to those previously described at insect nicotinic ACh receptors (Flattum and Shankland, 197 I ; Trimmer and Weeks, 1989), although the depolarizing effect was more rapid than that reported at the hair plate to slow coxal depressor motor neuron (Ds) connection in the metathoracic ganglion (Carr and Fourtner, 1980),and the TIASwere much slower to repolarize than other cockroach neurons (Sattelle et al., 1976; Carr and Fourtner, 1980; David and Sattelle, 1984). However, in Manduca sexta, the decreased synaptic transmission between antennal afferents and central interneurons in the antennal lobe was not observed to be reversed by prolonged washing (Hildebrand, 1979). The vGI to TI, connection did recover, although to only 70% of its initial level after 30-50 min of washing. Carbamylcholine, a nicotinic and muscarinic agonist that is less effective than nicotine (David and Sattelle, 1984), depolarized the TI,, but to a lesser extent than nicotine. The amplitude of the PSP also increased, then decreased. A similar finding was reported at the cercal afferent to GI connection for lower concentrations of carbamylcholine, whereas at higher concentrations, the PSP rapidly decremented (Sattelle et a]., 1976). It has been proposed that carbamylcholine, in addition to mimicking ACh at the receptor site, can act presynaptically to stimulate the release of ACh ( Volle and Koelle, 1961). The increase observed at the vGI to TI, connection may thus be due to presynaptic actions of this drug. Cholinergic Antagonists

The effective concentrations and relative potencies of the cholinergic antagonists d-tubocurarine, mecamylamine, atropine, and decamethonium were consistent with those reported previously at nicotinic ACh receptors in insects (Shankland et al., 197 1;Carr and Fourtner, 1980; David and Sattelle, 1984; Blagburn and Sattelle, 1987; Trimmer and

638

Chsugrund and Ritzmunn

Weeks, 1989). d-Tubocurarine was approximately as potent as mecamylamine, and more effective than atropine; least effective was decamethonium. In addition to its blocking effects, decamethonium also produced an initial increase in the PSP amplitude at all concentrations tested before the response decreased, and a sustained increase at lower concentrations. Thesleff ( 1955) and Changeux ( 1966) have demonstrated that decamethonium can inhibit acetylcholinesterase ( AChE). The potentiating effect of decamethonium at the vGI to TI, connections upon initial perfusion and at low concentrations may be a result of this secondary action. It is also possible that this effect was the result of the inhibition of presynaptic “mixed” autoreceptors, of the type that have been biochemically characterized in insects (Eldefrawi and O’Brien, 1970; Jewess, Clarke, and Donnellan, 1975; Clarke and Donnellan, 1975; Mansour, Eldefrawi, and Eldefrawi, 1977; Cattell, Harris, and Donnellan, 1980; Harris, Cattell, and Donnellan, 198 I ). There is some evidence for presynaptic nicotinic (Blagburn and Sattelle, 1987) and muscarinic (Breer and Knipper, 1984; Hue, Lapied, and Malecot, 1989) autoreceptors in insects. However, the existence of a “mixed” receptor type has yet to be demonstrated zn vivo, and remains controversial (Sattelle, 1985). The results with snake toxins were not consistent with some previous results in insects. Snake alpha-toxins are highly effective at blocking ( 1 ) the cercal afferent to GI connection in the terminal abdominal ganglion of the cockroach (Harrow, David, and Sattelle, 1979; Sattelle et al., 1980, 1983), and ( 2 ) the extrasynaptic responses of the GIs ( Harrow and Sattelle, 1983) and ( 3 ) the metathoracic motor neuron, Df, (Sattelle et al., 1980; David and Sattelle, 1984) to iontophoretically apM. In plied ACh at concentrations of lo-’ to addition, the connection between the hair plate afferents and motor neuron Ds in the metathoracic ganglion of P. americana is blocked within 8 min by perfusion of 10p6 A4 alpha-bungarotoxin (Carr and Fourtner, 1980),and cercal afferent to GI connection within 50 min (Harrow et al., 1979). It is possible that the lack of any observed effect of either alpha-cobratoxin or alpha-bungarotoxin for periods up to 85 min on the vGI to TI, connections was due to the fact that the toxins did not penetrate to the synaptic regions. Insect ganglia possess a diffusion barrier( s), not associated with the nerve sheath, which discriminates against large, charged, and polar molecules ( Eldefrawi, Toppo-

zada, Salpeter, and O’Brien, 1968; Pitman, 197 I ). Such a barrier(s) would be more effective in preventing diffusion of the large, charged snake alphatoxin molecules than the other cholinergic antagonists tested. Although the cercal afferent to GI connection is located deep within the neuropil, as are the vGI to TI, connections, the terminal abdominal ganglion is smaller than the metathoracic ganglion. The cercal afferent to GI connections, thus, may be more accessible to the toxins than the synaptic regions of the TI,s. Similarly, the extrasynaptic receptors of the Df motor neuron are on the surface of the metathoracic ganglion, and the hair plate to Ds motor neuron connection is peripherally located in the metathoracic ganglion, where the ganglion is not as thick. However, lanthanum. of a similar effective size as alpha-bungarotoxin, was shown to have equal access to the somata and axonal processes of the Df motor neuron and the DUM neurons in the desheathed metathoracic ganglion (Lane, Swales, David, and Sattelle. 1982). Nonetheless, although the response of the Df motor neuron to iontophoretically applied ACh was blocked by alpha-bungarotoxin, the toxin was ineffective in blocking the responses of the metathoracic DUM neurons, suggesting that differential access of alpha-bungarotoxin to the two cells types in the metathoracic ganglion of P. arncricana did not account for the differences in their sensitivity to the toxin. There have been other reports of alpha-bungarotoxin-insensitive nicotinic ACh receptors in invertebrates (Goodman and Spitzer, 1979, 1980; Magazanik, 1976; Marder and Paupardin-Tritsch, 1980: Trimmer and Weeks, 1989), as well as in vertebrates (Triggle and Triggle, 1976; ContiTronconi et al., 1985). In vertebrates, nicotinic ACh receptors are known to constitute a receptor family, whose members differ in their subunit composition and pharmacological specificity. Different combinations of alpha-subunits with the beta,-subunit result in pharmacologically distinct subtypes of the nicotinic ACh receptor (Wada et al., 1988; Boulter et al., 1987). These receptors differ in their sensitivity to the blocker alpha-bungarotoxin. This pharmacological difference is a result of a toxin binding amino acid sequence that overlaps the ACh binding site. In insects, it is also becoming apparent that nicotinic ACh receptors are not a homogeneous class. alpha-Like subunits, which do not bind alpha-bungarotoxin (Bossy, Ballivet, and Spierer, 1988 ). and apparently nonalpha subunits of the ACh receptor

Gvidence for Ac‘h at vGI to TI Conneclion ( Hermans-Borgmeyer et al., 1986) have been iso-

lated from Drosophila. The sequences of the subunits from Drosophilu are highly conserved with the neural nicotinic ACh receptor subunits of vertebrates (Bossy et al., 1988).Zn vivo pharmacological studies within the metathoracic ganglion of the cockroach, Periplaneta americanu, and embryonic locust, Schistocerca gregaria, have demonstrated that different identified neurons express cholinergic receptors that seem to differ in their sensitivities to alpha-bungarotoxin (Goodman and Spitzer, 1979, 1980; Lane et al., 1982; Lees, Beadle, and Botham, 1983). Although alpha-bungarotoxin had no effect at low concentrations, higher concentrations (greater than lop5M ) reduced the sensitivity of the metathoracic DUM neurons to iontophoretically applied ACh (Goodman and Spitzer, 1979, 1980; Sattelle et al., 1980). A similar finding was reported in Manduca (Trimmer and Weeks, 1989), where it was suggested that at this high concentration, contamination by other toxin components (Ravdin and Berg, 1979) could account for the effect. More work needs to be done to determine whether the apparent insensitivity of the TI,s to snake alpha-toxins results from the expression of receptor subtypes different from those present on the GIs, and the Df and Ds motor neurons. AChE Inhibitors Although an initial increase in the PSP amplitude was observed with the AChE inhibitor, eserine, at M , the PSP subsequently decreased, as it did at 10-6 M . At 10-4 ,%Z, eserine resulted in the rapid depolarization of 7’1,s and a loss of the evoked EPSP. Similarly, the cholinesterase inhibitors eserine and prostigmine at lop4 M to lop5 A4 both caused a decrease in the PSP at the cockroach cercal afferent to GI synapse with no apparent facilitatory action (Roeder, Kennedy, and Samson, 1947; Roeder, 1948; Yaniasaki and Narahashi, 1958), A.4 to Mblocked transmisand eserine at sion between the hair plate and the Ds motor neuron (Carr and Fourtner, 1980). These results may be due to a second action of eserine in addition to its acetylcholinesterase inhibiting actions. Postsynaptic, “curarizing” effects of eserine have been reported in several invertebrate and vertebrate preparations (Roeder et al., 1947; Eccles and MacFarlane, 1949; Yamasaki and Narahashi, 1958; Ehrenpreis, 1967; Levitan and Tauc, 1972; Carr and Fourtner, 1980). It has been suggested that this inactivation of the postsynaptic membrane by

639

eserine is faster and more potent than the actions on the acetylcholinesterase enzyme (Tauc and Gerschenfeld, 1962). In addition, high concentrations of eserine have been shown to inhibit alphabungarotoxin binding sites in Drosophilu ( Schmidt-Nielsen, Gepner, Teng, and Hall, 1977). Another explanation has also been offered for a similar decrease in the PSP at the cercal afferent to GI connection in the cockroach due to perfusion of eserine (Hue, Lapied, and Malecot, 1989). It was observed that this decrease could be reversed by application of atropine at low concentrations ( l o p 7 M ) , and it was, therefore, suggested that these results were due to a direct effect of an elevation in ACh at the synaptic cleft on presynaptic muscarinic autoreceptors, which have been proposed to regulate ACh release. The mechanism responsible for the apparent block of synaptic transmission between the vGIs and TI,s is not known. However, the fact that effects were observed would appear to be consistent with cholinergic transmission. The authors would like to thank Drs. Story Landis and Mahendra S. Rao for help with the ChAT assays and Dr. Eve Marder for useful discussions o n the ChAT assays. Drs. Edmund Arbas, Richard Levine, and David Morton and Mr. Alan Pollack critically reviewed the manuscript. This work was supported by NIH grant NS I74 1 1 to R.E.R.

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Evidence that synaptic transmission between giant interneurons and identified thoracic interneurons in the cockroach is cholinergic.

In the cockroach, a population of thoracic interneurons (TIs) receives direct inputs from a population of ventral giant interneurons (vGIs). Synaptic ...
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