analysis of proprioceptive inputs to dpg interneurons in the cockroach

Analysis of Proprioceptive Inputs to DPG Interneurons in the Cockroach

MICHELLE MURRAIN and ROY E. RITZMANN Department of Biology, Case Western Reserve University, Cleveland, Ohio 441 06

Received October 7, 1987; accepted April 18, 1988

SUMMARY

In this study we report on morphological and physiological analysis of proprioceptive sensory input to thoracic interneurons. Sensory neurons from leg proprioceptors were filled using cobalt chloride. The morphological location of these sensory neurons was compared with that of the DPG interneurons. The interneurons investigated were found to have morphological overlap with the sensory neurons of the specific proprioceptors, suggesting that they have the potential to receive direct input from these proprioceptors. Individual interneurons were recorded intracellularly and identified by intracellular injection of Lucifer Yellow, and the responses of these cells to mechanical stimulation of specific proprioceptors were analyzed. All of the DPG interneurons tested as well as other interneurons receive input from one or more of these proprioceptors. In addition, DPG interneurons have ipsilateral/contralateral biases in their responses to proprioceptors. Paired stimulation of proprioceptors resulted in enhancement or decrement of the response in the interneurons, depending upon which sensory structures were stimulated together. The results of this study show that proprioceptive information is processed by DPG interneurons.

INTRODUCTION

Sensory information from the legs, specifically proprioceptive information, has been shown to be important in insects for coordination of walking (Pear- son, 1972; MacMillian and Kien, 1983; Bassler, 1977), flight (Kramer and Markl, 1978), and jump (Steeves and Pearson, 1983; Pearson et al., 1980). Proprioceptive input also provides specific information about leg position, as well as behavioral state, i.e., whether the animal is walking or flying.

The escape behavior of the cockroach includes an initial wind-directed turn, followed by a run (Camhi and Tom, 1978). Giant interneurons carry directional wind information from the cercal wind receptors to the thoracic nervous system (Westin et al., 1977). This information initiates the appropriate motor output (Ritzmann and Camhi, 1978; Camhi and Tom, 1978). Both the turn and the run may be influenced by sensory information from the legs. When a cockroach makes a turn, it may start the turn from a variety of leg positions; information about leg position is probably necessary to perform appropriate leg movements to accomplish the turn.

Journal of Neurobiology, Vol. 19, No. 6, pp. 552-570 (1988) 0 1988 John Wiley & Sons, Inc. CCC 0022-3034/88/060552-19$4.00

ANALYSIS OF DPG INTERNEURONS 553

Recently, a group of approximately 30 interganglionic interneurons, named DPG or dorsal posterior group interneurons (Ritzmann and Pollack, 1986), has been identified in each of the thoracic ganglia. These neurons are individu- ally identifiable neurons, with their cell bodies in the dorsal posterior region of the thoracic ganglia. There are eight pairs of neurons in this group known to be intervening interneurons between ventral giant interneurons (vGIs) and leg motor neurons. They were found to receive monosynaptic input from vGIs, and individual members of this group evoke specific motor outputs appropriate to the escape movements (Ritzmann and Pollack, 1986).

We have chosen to investigate these interneurons because they are thought to play an important role in the escape behavior, and proprioceptive inputs to the DPG interneurons may provide critical information on leg position. The sensory structures we investigated included the trochanteral campaniform sensilla (trCS), the trochanteral hair plates (trHP), and the femoral chordotonal organ (feC0). They were chosen because they have been found to be involved in the coordination of leg movements during walking, and also because they are easily accessible for study. The purpose of this study was to determine if sensory information from the legs is received by DPG interneurons and how that information is processed.

MATERIALS AND METHODS

Fills of Sensory Neurons

Neurons that innervate specific sensory structures were filled with cobalt chloride using a technique similar to that used by Hustert (1978) in the locust. To fill sensory neurons from the campaniform sensilla (CS), a small area of the cuticle overlying the CS group was perforated with a fine insect pin, and a 10-20% solution of cobalt chloride was applied. For filling of hair plate (HP) sensory cells, the hairs were shaved level with the cuticle, and a cobalt chloride solution was applied. For filling of sensory neurons from the femoral chordotonal organ (feCO), the nerve just proximal to the connective tissue strand of the feC0, containing just axons from the feC0, was cut and placed into a well containing a 5-10% cobalt chloride solution.

A Timm’s intensification procedure, as outlined by Bacon and Altman (1977), was carried out on all nerve cords with cobalt fills. The cords were then dehydrated in an alcohol series, cleared with methyl salicylate, viewed using standard light microscopy, and drawn in wholemount. After- wards the cords were embedded in Ladd’s Ultra Low Viscosity Embedding Medium and sectioned to 25 pm.

Stimulation of Sensory Structures

All experiments were performed on adult male American cockroaches, Perzplaneta arnericana, from an ongoing colony, The animal was pinned to a cork platform ventral side up. This allowed for maximal access to the sensory structures on the ventral surface of the legs. Pins were placed around the legs to immobilize them at a 90” angle in relation to the body. Three groups of sensory structures were studied the trochanteral campaniform sensilla (trCS), the trochanteral hair plates (trHP), and the femoral chordotonal organ (feC0). The structures of interest were stimulated using two methods. In the first a small insect pin which was attached to a speaker. The speaker was driven by high-frequency trains from a stimulator, causing the pin to vibrate. The pin contacted either the cuticle of the trochanter to stimulate the trCS or the hairs of the hair plates for direct stimulation of those structures. Also, the pin could be placed under the strand of the feC0 to stimulate the sensory cells of the feC0. The second technique, which allowed much finer control, employed a pin attached to a piezoelectric crystal (PEC). A high-voltage pulse applied across the PEC caused it to bend. This movement either put stress on the trochanteral cuticle,

554 MURRAIN AND RITZMANN

bent the hairs of the hair plates, or lifted the strand of the feC0. The usual stimulus strength was 300400 V (at low amperage) which caused movements in the PE crystal which were just barely detectable under the dissecting microscope. In most cases, activation was limited to phasic fibers. Adaptation of the response was avoided by waiting 20-30 s between stimuli.

The sensory structures were ablated fro specific experiments (see Results) by either scraping deeply into the cuticle overlying one of the groups of campaniform sensilla or shaving off the hairs of the hair plates. These techniques were selective, since the proprioceptors in the trochanter are sufficiently far apart to prevent damage to other structures.

Recording Techniques

The ventral cuticle was cut to reveal the ventral nerve cord. One of two ganglia, either the second or third thoracic ganglion (T, or T3) was raised onto a steel platform. The nerve cord was superfused constantly with cockroach saline (Callec and Sattelle, 1973) buffered with MOPS. The sheath of the ganglion was softened by a 1 mg/ml solution of protease (Sigma).

Electrode tips were filled with a 4% Lucifer Yellow CH (LY) solution (Aldrich), and the shanks were filled with a 2.0 A4 solution of lithium chloride. A WPI M707 microprobe system was used to amplify the intracellular signals. LY was iontophoresed into the cell using 0.5 nA hyperpolarizing current in 1 s pulses at 0.5 Hz. After an experiment, when a cell was filled with LY, it could be viewed in situ by illuminating the ganglion with a Liconix Helium-Cadmium laser (Reichert and Krenz, 1986).

The electrodes were placed on the midline of the ganglion, and the placement of the electrode in relation to the morphology of the cell was verified using the in situ laser technique. It was crucial that the electrode be on the midline for an accurate measurement of any ipsilateral/contralat- era1 bias.

Extracellular recordings were taken from one or both of the nerve 5s that supplied the legs being stimulated. This nerve contains most of the axons of sensory neurons in the leg and all of the axons of the sensory structures stimulated. Grass P511 amplifiers were used for the extracellular recordings.

The intracellular and extracellular traces, along with a monitor of the stimulus, was stored on a Hewlett-Packard 3968a instrument tape recorder. Data was analyzed and plotted using a Nicolet 4094 digital oscilloscope and an HP 7470 plotter. Two measurements were taken: PSP amplitude at its maximum point and area under the PSP. The area was calculated with the aid of a downloadable program on the Nicolet oscilloscope.

When interneurons cells were filled with LY after an experiment, the nerve cord was removed

COXA I

Fig. 1. A ventral view of the leg, showing the sensory structures investigated. The three groups of campaniform sensilla on the ventral surface of the trochanter are stippled. They are groups 11, 111, and IV. The orientation of the sensilla in the groups is indicated by the arrows next to the group. In the upper region of the trochanter near the coxa is the hair plate (HP). The chordotonal organ in the femur (CO) is also shown, and it is an internal sensory structure. A: anterior, P: posterior.

IL - /- Post

trCS group 111

. . PD&

Fig. 2. Wholemount views (dorsal side up) and cross-section views of the fills of metathoracic leg proprioceptors. (A-E) 4 t left is the wholemount view, and the lines indicate where the sections at right are taken from. (A) Fill of trCS group 11. Tracts labeled DIT, dorsal intermediate tract; VIT, ventral intermediate tract; LDT, lateral dorsal tract. (B) trCS group 111: DMT, dorsal medial tract. (C) trCS group IV. (D) feC0: Vac, ventral association region. (E) trHP. (F) Areas of arborization of sensory neurons filled. These areas include lateral/dorsolateral neuropil (L/DLN), intermediate neuropil (IN), and the ventral association region.

PO*L / u

F LIDLN

(Continued from previous page.)

from the animal, fixed in 10% formalin solution in cockroach saline, dehydrated in an alcohol series, and cleared in methyl salicylate. The cells were viewed with a Ziess fluorescence microscope and drawn with the aid of a drawing tube. After viewing, the nerve cord was embedded in paraplast and sectioned in 10 pm sections.

RESULTS

Morphological Analysis

Sensory Neurons. To determine the projections of the sensory neurons arising from specific proprioceptive structures on the leg, cobalt fills of the

neurons were carried out. The sensory neurons of the three ventral groups (groups 11, 111, and IV) of trochanteral campaniform sensilla (trCS), the trochanteral hair plate (trHP), and the femoral chordotonal organ (feC0) were studied in both T2 and T,. The location of these sensory structures on the ventral surface of the leg is shown in Fig. 1. There were nine fills of trCS group 11, six of trCS group 11, three of trCS group IV, three of trHP, and three of feC0. Usually multiple sensory neurons were filled in individual experiments. The morphology of these individual sensory neurons were almost identical to each other for CS and HP sensory neurons. However, individual neurons of feC0 tended to have different morphologies. There was little variability between experiments.

All of the sensory neurons that were filled had axons that are located in nerve 5, which is the major peripheral nerve serving the distal leg. Each proprioceptor or group of trCS had characteristic morphologies as seen in wholemount and in crosssection (Fig. 2). No interganglionic projections were observed for any sensory neuron, and all arbors were restricted to the ipsilat- era1 side of the ganglion. The sensory neurons of the trCS and trHP were similar in morphology. The principle areas of arborization for these groups are shown in Fig. 2(F). They both had sets of branches which are located in the lateral and dorsolateral neuropil and ventral branches located laterally and medially. Groups I1 and IV sent a few branches into ventral neuropil but not into the ventral association region. The medial branches were in intermediate neuropil.

The sensory neurons of the feCO branched in different patterns than the trCS and HP [Fig. 2(D)]. They did have arbors in intermediate and dorsolat- era1 neuropil and also had extensive branches in the ventral association region.

Morphology of DPG Interneurons in Comparison to Sensory Neurons. The morphological location of sensory neurons was compared with the DPG interneurons. All of the DPGs investigated showed some degree of morphological overlap with all of these sensory neurons. Overlap was con- firmed by the use of a computer program to analyze and compare morphological location of neurons within the thoracic ganglia. This indicates that most or all of the DPG interneurons have the potential to receive some input from these proprioceptors. Data from physiological analysis indicates that such input does indeed exist.

Control Experiments. A series of control experiments were carried out to determine the basic properties of the response of sensory neurons to the mechanical stimulus (as seen in extracellular recordings) and to determine the specificity of the stimulus. Figure 3 (A-C) shows the responses recorded from nerve 5 (n5) to stimulation of sensory structures. The recording site was about 2 mm distal to the ganglion. Motor reflexes were usually observed in the recordings along with the sensory activity. After cutting n5 proximal to the recording electrodes, which eliminated motor reflexes, HP responses generally consisted of a single sensory spike when stimulated with a PEC or one on and one off spike if stimulated with a speaker [Fig. 3(E)]. CS responses consisted of from one to three spikes [Fig. 3(D)].

The response seen in n5 to stimulation of trCS, trHP, or feCO had different latencies depending upon the structure stimulated. TrHP and feCO tended to

Fig. 3. Responses in sensory axons to mechanical stimulation of individual sensory structures. All responses recorded extracellularly from nerve 5. (A) Responses of trochanteral campaniform sensilla (trCS) sensory neurons. In all records, extracellular recordings are on the top trace, and the monitor of the stimulus is on the bottom trace. (Dark triangles in A, B, and C indicate probable sensory spikes.) (B) Re- sponse of trochanteral hair plates (trHP). (C) Response of femoral chordotonal organ (feC0). (D and E) Control experiments. (Dl) Stim- ulation of trCS, n5 intact. (D2) N5 cut proximal to the recording electrode. Note motor activity is eliminated, just leaving sensory spikes. (El) Stimulation of trHP, n5 intact. (E2) N5 cut (there are two afferent spikes here because the stimulus was a speaker, causing an on and an off stimulation of hair plates.) Bar: 10 ms (A-D) and 5 ms (E).

respond with average latencies from stimulus onset of 4.8 (SE = 0.6, n = 21) and 4.5 (SE = 0.25, n = 18) ms, respectively. TrCS responded with an average latency of 7.1 (SE = 0.58, n = 39) ms. The conduction velocity was calculated by measuring the distance between the sensory structure and the recording site and dividing it by the latency of the response seen in n5. For HP and CS the distance was 5 mm; for feCO the distance was 7 mm. The conduction velocity of the feCO was 1.6 m/s; HP sensory neurons had a conduction velocity of 1 m/s, and the conduction velocity of CS sensory neurons was 0.7 ms. There are two notes about these calculations. One is that this assumes that the length of time for the stimulus to cause the sensory neurons to reach threshold is short. A longer time would decrease the estimate of conduction velocity. A second consideration is that the distance is measured on the cuticle, so the actual length of the nerve is probably longer. With these considerations, the conduction velocity is probably underestimated.

The specificity of the stimulus was determined by either stimulating a proprioceptor and then ablating one or two of the other sensory structures to determine whether the response changed or ablating the sensory structure itself and determining whether the response was totally eliminated. Only in cases where very high stimulus levels were used (about 150-20075 of the high- est level used in normal experiments) was the stimulus not completely elimi-

contrn trHP

Fig. 4. Responses of Lambda cell to stimulation of various proprioceptors. Above is morphology of T3 Lambda cell with electrode placement as indicated. ( A l l Response to stimulation of contralateral trCS in a T2 Lambda cell. In this and all subsequent records the top trace is the intracellular recording from the interneuron, the middle trace (if present) is the extracellular recording from nerve 5, and the bottom trace (if present) is the monitor of the stimulus. The dot over the extracellular trace indicates the probable spike from the afferents. (A2) Re- sponse to ipsilateral trHP. (I31 and B2) Response to ipsilateral and contralateral trHP in another Tz Lambda cell. (Cl) Lack of response to feC0 in a T3 Lambda cell. (C2) Small hyperpolarization observed in a T2 Lambda interneuron. Average of 12 traces. (D) An example of a short

nated. Even then, the resulting response in n5 after ablation of the structure was very small and/or inconsistent. In all of the cases where a normal stimulus level was used comparable to that for normal experiments, the response was totally abolished upon ablation of the sensory structure stimulated.

Interneurons. We recorded from 47 DPG interneurons in T2 and T3. These included eight different identified cells, Lambda (DI-1), DI-3, and Reverse-J (LD-2) in T2 and Lambda, Reverse-J, DI-3, J (LD-l), and Reverse-J posterior (LD-4) in T3. The alternate nomenclature (in parentheses) consists of the tract that the axon is in (DI for dorsal intermediate, LD for lateral dorsal tract), and the cell number (by order of discovery). The names for Lambda, Reverse-J, and J have been used previously in the literature. We also recorded from two other interneurons, which receive monosynaptic input from GIs. These are T cells and an intraganglionic interneuron.

All of the DPG cells from which we recorded responded to one or more of the three proprioceptors studied, and many of the response characteristics are similar. All DPG cells were excited by trCS. Most (six out of seven DPG cell types tested) were excited by trHP. However, most do not respond (four out of seven cell types tested) or respond weakly (two out of seven DPG cell types tested) to feC0. In most cases the response of DPG cells to stimulation of these proprioceptors was subthreshold. We measured maximal PSP amplitude, number of spikes (for suprathreshold responses), and area under the PSP. These measures were always consistent with one another.

There was variability in the latencies measured between the response in n5 and the response in DPGs, with a mean of 4.5 ms (standard deviation = 2.7, n = 40) and a range from 1.1 to 13 ms. This suggests that both short and long latency input from proprioceptors to these interneurons exists.

Lambda Cell. The DPG interneurons that we characterized most exten- sively were the Lambda cells. We have recorded from 12 T, Lambda cells and 5 T3 Lambda cells. Lambda cells in both ganglia received excitatory input from trCS and trHP on both sides of the body (Fig. 4). There was an ipsilateral/contralateral bias in the response to both trochanteral proprioceptors. In all cases ipsilateral and contralateral refer to the location relative to the interneuron’s soma. The biases for both T2 and T3 homologues were the same. They responded more strongly to contralateral trHP than ipsilateral trHP. However, they responded more strongly to ipsilateral trCS than contralateral trCS. Indeed in some preparations contralateral inputs from trHP resulted in action potentials in Lambda while ipsilateral inputs were subthreshold [Fig. 5(A)]. All comparisons made between ipsilateral/contralateral inputs were made in the same experiment, and all recordings were made on the midline (as verified by subsequent laser illumination in situ).

Figure 4(D) shows an example of a short latency response to trCS (1.8 ms later than the response in n5): Lambda cells generally did not respond to feCO [Fig. 4(C1)], although on one occasion small IPSPs were seen in response to stimulation of the feCO [Fig. 4(C2)].

latency response to stimulation of trCS in a T, Lambda cell. Calibration: Horizontal bar = 10 ms; vertical bar = 5 mV (A and Cl) , 4 mV (B), 0.7 mV (C2). Calibrations for D are marked.

*' 1 A2

CONTRA HP IPS1 HP

L , M e t a t rCS I Fig. 5. (A) Responses of a T2 Lambda cell to stimulation of contralateral and ipsilateral trHP. (Al) Response to stimulation of contralateral trHP. Note that the response is suprathreshold. (A2) Response in the same Lambda cell to stimulation of ipsilateral trHP is subthreshold. (B) Comparison of the responses of a T, Lambda to stimulation of trochanteral CS in the same (meso trCS) and another (meta trCS) ganglion on the ipsilateral side.

Lambda cells, like most DPGs, also responded to proprioceptors in other segments. The responses of a T2 Lambda to stimulation of trCS in T3 are shown in Fig. 5(B). The responses to stimulation of proprioceptors in another segment were always smaller than the responses to proprioceptors in the segment with the interneuron's cell body.

Reverse-J Cell. We have recorded from 5 Reverse-J cells in T3 and 11 in T,. All Reverse-J cells responded to both trHP and trCS. In both ganglia these cells responded more strongly to contralateral CS than to ipsilateral CS. However, in T2 this is based on data from only one experiment [Fig. 6(A)]. In the one preparation in which Reverse-J was tested for trHP ipsilateral/contralateral bias, the response to contralateral trHP was stronger than the response to ipsilateral trHP [Fig. 6(B)]. We do not have sufficient data to make a comparative statement regarding trHP in TB. Reverse-J cells in T3 have not been seen to respond to feC0. In T2, however, Reverse-J cells have been observed to respond with small depolarizations in two out of four experiments [Fig. 6(D)]. An example of a very short latency response to ipsilateral trCS stimulation in a T, Reverse-J cell is shown in Fig. 6(C). The latency is about 1.5 ms. The Reverse-J cells, like other DPG interneurons, also responded to sensory structures in segments other than the one in which their soma resides.

Single trCS Group Experiments. It was of interest to determine whether each individual group of trochanteral CS had similar responses: specifically, whether any group had inhibitory input to the DPG cells which might have been masked by the excitatory input of the others. This was determined by ablating two of three individual groups of campaniform sensilla on the trochanter. Inputs from all three individual groups were investigated, and all of the groups depolarized the DPG cells, although somewhat weaker than the inputs recorded when all structures were intact (Fig. 7). Moreover, the ipsilat-

Post .

f i m contra trCS

brl trHP

contra teCO --

Fig. 6. Responses of the Reverse-J to stimulation of proprioceptors. Above: the morphology of T3 Reverse-J and the electrode placement. (A) Response to ipsilateral and contralateral trCS in a T2 Reverse-J. (B) Response to ipsilateral and contralateral trHP in a different Tz Reverse-J. (C) Example of a short latency response to stimulation of trCS in a T3 Reverse-J. (D) Depolarizing response in a T2 Reverse-J to stimulation of feC0. Horizontal bar = 10 ms; vertical bar = 9 mV (A), 5 mV (B), 8 mV ( C ) , and 10 mV (D).

eral/contralateral bias of the response was consistent with that observed when all three groups were intact.

Paired Stimulation Experiments. The responses seen in the DPG cells show that the trochanteral proprioceptors, and in a few cases the femoral chordotonal organ, had definite effects on these cells. However, in the walking cycle these proprioceptors will often be active at the same time. Any interac- tion in the inputs from proprioceptors could result in a change in propriocep-

A1 m,dLA2r B1 82

b---]-v" ern*. t n s PlV r J - l b8i trCS OW

Fig. 7. (A) Response of T2 Reverse-J cell to stimulation of a single trCS group, group 11. ( A l ) Contralateral trCS group 11. (A2) Ipsilateral trCS group 11. (B) Response of Tz Lambda to contralateral and ipsilateral trCS group 111. (C) Response in another T2 Lambda interneuron to stimulation of contralateral and ipsilateral trCS group IV.

tive information going to the thoracic interneurons. For this reason we exam- ined the effects of pairing activation of different types of proprioceptors. To make certain that the change in response was not due to the order of presenta- tion, in all paired tests the stimuli were presented in a random sequence.

Pairing trHP and trCS resulted in a response that was greater than either response alone in all but one case (four of five). An example of the typical response is shown in Fig. 8.

Unlike pairing trCS and trHP, pairing of either trCS or trHP with feC0 resulted in a decrease in the response in five out of six cases [Fig. 9(A and B)].

contra trCS contra trHP

Fig. 8. Summation of responses when structures are paired in a T, Lambda interneuron. (A) Response to stimulation of contralateral trCS alone. (B) Response to trHP alone. (C) Response to both, which is larger than either alone.

564 MURRAIN AND RITZMANN An contr. l,CS

C A 3 - Fig. 9. Decrease in response when trCS or trHP is paired with feC0. (A) Response to trCS and feCO alone and paired in a T2 Reverse-J cell. (B) Response to trHP alone and trHP plus feCO (very little response, if any) in a T, Reverse-J cell. (C) Input impedance of cell when feCO is stimulated in a T, Lambda cell. Input impedance is measured by constant current/voltage pulses. Note no response in cell, and no change in input impedance. Horizontal bar = 10 ms; vertical bar = 5 mV (A and C) and 2 mV (B).

The decrease is anywhere from 10 to 100% of the response to trCS or trHP; the average decrease was 60%. The one case where a decrease was not observed was in a Lambda cell, where stimulating both trCS and feCO resulted in a 33% enhancement of the response. The decrease in response when feCO is paired with either trochanteral proprioceptor could either be due to direct inhibition of the cell, presynaptic inhibition, or a decrease in the polysynaptic input by inhibition of an intervening cell. It appears that the decrease in response is not through direct inhibition, because there is no change in conductance when the feCO is stimulated [Fig. 9(C)].

Adaptation. A common property of the responses of DPG interneurons to stimulation of proprioceptors was that they decrease after the initial triaI. An example of this adaptation in a T, Reverse-J cell is shown in Fig. 10. Although this was a consistent finding, the average adaptation of the response was only 60% of the initial amplitude, with a large degree of variability (SE = 4.6, n = 20). In only one of these 20 cases did the response adapt to zero. On average, 80% of the total adaptation took place between the first and second trial. Thus, even after the cell reaches its adapted state, the proprioceptive structures still provided a significant input.

Other Cells. All other DPGs from which we recorded were excited by stimulation of trCS, and some had been observed to respond to trHP. Exam- ples of these DPG interneurons and others excited by these proprioceptors are shown in Fig. 11. Another DPG, a DI-3 (Dorsal-Omega) interneuron (in T2), is one of the few DPG interneurons that has been tested with vGIs and failed to

Q - L c

t \ \ \

l D 0 1 7 3 a 3 d 5

T r l - 1 N u m b - r

Fig. 10. An example of adaptation in one T, Reverse-J cell. Horizontal axis is trial number, with 5 trials delivered. The interstimulus interval is 1 s. Vertical axis is percent of initial trial. Solid line is EPSP amplitude, dashed line is area.

show evidence of a connection. It appears to be sound responsive, much like a similarly shaped interneuron in the locust (the G interneuron) (Romer and Marquart, 1984; Pollack and Ritzmann, unpublished observations). The LD-4 cell [Fig. l l (C)] , unlike most DPG interneurons, responded strongly to stimulation of feC0. Also unlike most DPG cells, this cell appeared not to respond to trHP.

A local interneuron with a dorsal soma is presented in Fig. 11(B). This interneuron was found to receive monosynaptic input from ventral giant interneurons (Ritzmann and Pollack, 1986).

The T cell [Fig. 11(D)] is another intervening interneuron between GIs and motor neurons, although it received input from dorsal GIs and not ventral GIs. These cells are thought to be a small population of cells with very similar morphology (Ritzmann and Pollack, unpublished observations). At least some representatives of these cells responded to both trCS and trHP.

DISCUSSION

Sensory Neurons

In our studies, we have investigated the morphology of sensory neurons which innervate a variety of proprioceptors located on the legs of cockroaches. These sensory neurons have been described by Collin (1985), who filled trHP, trCS, and feC0 sensory neurons. However, in this study we have identified the sensory neurons for individual groups of trochanteral CS, and we investigated the location of sensory neurons terminal arborization in relation to morphological landmarks in the ganglion.

I i~ Post.

Ant. D

Fig. 11. Examples of other interneurons. (A) The morphology of a DPG, DI-3 (Dorsal-Omega). (B) The morphology of an intraganglionic interneuron with a dorsal soma. (C) Morphology of the LD-4 (Re- verse-J posterior) cell in TS. (D) Morphology of the T cell(s) in TB, which have ventral somata.

As is the case in locusts (Hustert et al., 1981), the trochanteral proprioceptors arborize in dorsolateral and intermediate neuropil, and the femoral chordotonal organ arborizes in those areas as well as the ventral association region. This suggests some compartmentalization of proprioceptive inputs. Other workers (Altman and Tyrer, 1977; Hustert et al., 1981; Johnson and Murphey, 1985) have seen that the central arborizations of mechanoreceptors are different depending not only on the type of structure but also upon the location of that structure. It is possible that sensory neurons involved in different sets of processes arborize in different areas, and interneurons receive this information at morphologically specified regions. Altman (1980) has suggested that insect ganglia are organized into areas of pure sensory processing (ventral association regions), and areas of sensorimotor integration (intermediate and dorsal neuropils). It is important to note that the feCO arborizes in the same areas as the trCS and trHP, which suggests that the feCO is involved in some of the same behavioral processes as the trochanteral proprioceptors. However, the feCO also arborizes in some differnt regions of neuropil, suggesting a role in behavioral processes in which the trochanteral proprioceptors may not be involved.

Proprioceptive Input to Thoracic Interneurons

A summary of the responses of the Lambda and Reverse-J cells is given in Fig. 12. We have observed sensory inputs, specifically from proprioceptors, to a group of interneurons in the thoracic ganglia of the cockroach. These include inputs from the trochanteral campaniform sensilla, trochanteral hair plates, and the femoral chordotonal organ. All of the DPG interneurons receive some excitatory input from the trochanteral proprioceptors. There are three types of biases seen in the responses: the first is an ipsilateral/contralateral bias and stimulation of specific sensory structures on one side or the other which give consistently stronger responses for a particular interneuron. The second is a proprioceptive bias; the response to trCS and trHP is always stronger than the response to feC0, which is most often small or nonexistent, In addition, there is a segmental bias in the inputs; stimulating sensory structures in segments other than the segment that the cell body is in always resulted in a weaker response than stimulating sensory structures in the same segment.

The ipsilateral/contralateral bias, as well as the proprioceptive bias, probably serve to relate specific information to the interneurons. In the turning movements which initiate escape, information about other segments may be important but not as important as information from the same segment.

Individual trCS groups on the ventral surface of the trochanter appear to contribute fairly equally to the response when all are stimulated. Moreover,

LAMBDA A B

REVERSE-J

Fig. 12. Summary of the responses of the Lambda and Re- verse-J cells. (A) Tz and T3 Lambda cells; (B) T, and T3 Re- verse-J cells.

the ipsilateral/contralateral biases are consistent, suggesting no real differences in input.

In making comparisons between inputs from different segments, it is diffi- cult to compare cells from different preparations because of possible differences in the quality of the intracellular recordings as well as the quality of the sensory stimulation. In all of the experiments, any comparisons made between sensory structures on different legs, or segments, were made in the same preparation, and no comparisons were made between different preparations.

It is important to point out that the bias of the interneurons seen at the site of spike initiation is what determines the functional importance of the input. The morphology of the cell can affect the bias in the cell. Reverse-J, for example, receives a stronger input on the contralateral side as compared with the ipsilateral side, and since the site of spike initiation is probably on the contralateral side (near the axon; Ritzmann and Pollack, unpublished observations), this bias is enhanced.

Monosynaptic Versus Polysynaptic Input

When proprioceptors were stimulated, the time between the response in n5 and the responses in DPG cells varied from 1.1 to 13 msec. For most DPGs investigated (including Lambda and Reverse-J cells in both T, and T3), there are numerous examples of very short latency responses (<2 ms), although most responses had a latency in the range of 2.5-6.5 ms. This suggests that the inputs could be both mono- and polysynaptic. Even though estimates of conduction velocity and synaptic delay may not be very precise, the 2 ms bench- mark takes this imprecision into account. Morphological evidence of overlap suggests that it is at least possible for these inputs to be monosynaptic. For various possible roles that these proprioceptors may play in the escape system, monosynaptic inputs are not essential. This information would be useful even at the longer observed latencies. Moreover, proprioceptive input via local interneurons would allow for more processing of leg sensory information, which might be useful in the escape system.

Role of Proprioceptive Information in the Escape Behavior

Prior to this work, proprioceptive information was believed to play a role in the escape system. Some of that expectation was based on physiological evidence: the latency of the escape response was decreased if the animal was walking as compared with an animal standing still (Camhi and Nolen, 1981). However, it was largely intuitive: if the animal is going to perform a specific turn, information on the condition and position of the legs is probably necessary to determine the motor output.

Proprioceptive input can play two roles in the escape system. The first is general potentiation of the system. Excitation of proprioceptors increases when the animal is walking (Zill and Moran, 1981), which, because of their input to intervening interneurons, will probably result in increased levels of depolarization in those neurons. This might be compared with the role played by proprioceptors in the locust jump (Pearson et al., 1980). Although in this case there does not appear to be a specific gating effect, the input from pro-

prioceptors will increase the general level of activity, increasing the probability of activating the DPG interneurons, and that activity will be coupled to the wind-evoked activity, leading to activation of specific DPGs.

Even in a standing animal, load on several proprioceptors will provide some excitation leading to an increased baseline level of depolarization in the thoracic interneurons. One of the observations made in this study was that the responses to proprioceptors almost always adapt to about 60% of the original amplitude. However, they rarely adapt completely. This means that some proprioceptive input will effect the thoracic interneurons even when the response is fully adapted.

In addition to tonic state conditions, proprioceptors might also monitor specific information about leg position. Because of the biases found in the responses, especially the ipsilateral/contralateral bias, this information could provide some specificity about leg position. Since the biases of the Lambda cell to stimulation of trCS and trHP are opposite to each other, the position of the legs is reflected in the level of depolarization in this interneuron. The trCS are most active when the legs are extended, when there is the most weight on them (Zill, personal communication), and the trHP are most active when the legs are flexed (Wong and Pearson, 1976). When the leg is flexed the opposite leg will be in antiphase, and therefore extended. This means that the trHP and trCS will either depolarize a Lambda cell strongly in one extreme position or weakly in the other.

Proprioceptive input to the thoracic interneurons must be viewed in the context of interactions among various structures at any given time. In most cases, the response to both trCS and trHP are decreased when feCO is active, so that the level of input a cell would get in an extreme position will be less than without feCO input. In the Reverse-J cell, and in most cells except the Lambda cell, the input rom feCO will tend to make the level of depolarization greater in the midrange of leg position. In the Lambda cell, the input will have no effect on the positional information that the Lambda cell gets; in fact, if pairing trCS and feCO results in an enhancement of the trCS response, as has been observed, then a sharper difference in levels of depolarization between flexed and extended positions would result.

SUMMARY

We have determined that the DPG interneurons respond to leg proprioceptors and have specific biases in those responses. Based on the previous evidence of DPG interneurons probably playing a role in the escape behavior (Ritzmann and Pollack, 1986), these data would indicate a role for proprioception influencing the escape behavior.

We thank Dr. Charles Fourtner for critically reviewing this manuscript and Alan Pollack for technical assistance. This work was funded hy NIH grant NS-17411 to R.E.R.

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EXP. BhZ. 66:173-183

analysis of proprioceptive inputs to dpg interneurons in the cockroach

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