parallel motor pathways from thoracic interneurons of the ventral giant interneurons system of the...

Parallel Motor Pathways from Thoracic lnterneurons of the Ventral Giant lnterneuron System of the Cockroach, Periplaneta americana”

Roy E. Ritzmannt and Alan J. Pollack

Department of Biology, Case Western Reserve University, Cleveland, Ohio 441 06

SUMMARY

The data described here complete the principal components of the cockroach wind-mediated escape circuit from cercal afferents to leg motor neurons. I t was previously known that the cercal afferents excite ventral giant interneurons which then conduct information on wind stimuli to thoracic ganglia. The ventral giant interneurons connect to a large population of interneurons in the thoracic ganglia which, in turn, are capable of excit- ing motor neurons that control leg movements. Thoracic interneurons that receive constant short latency inputs from ventral giant interneurons have been referred to as type A thoracic interneurons (T1,s). In this paper, we demonstrate that the motor response of TIAs occurs in adjacent ganglia as well as in the ganglion of origin for the TIa. We then describe the pathway from T14s to motor neurons in both ganglia. Our observations reveal complex interactions between thoracic interneurons and leg motor neurons. Two parallel pathways exist. TIAs excite leg motor neurons directly and via local interneurons. Latency and amplitude of post-synaptic potentials (PSPs) in motor neurons and local interneurons either in the ganglion of origin or in adjacent ganglia are all similar. However, the sign of the responses recorded

in local interneurons (LI) and motor neurons varies according to the TIA suhpopulation based on the location of their cell bodies. One group, the dorsal posterior group, (DPGs) has dorsal cell bodies, whereas the other group, the ventral median cells, (VMC) has ventral cell bodies. All DPG interneurons either excited postsynaptic cells or failed to show any connection a t all. In contrast, all VMC interneurons either inhibited postsynaptic cells or failed to show any connection. It appears that the TIas utilize directional wind information from the ventral giant interneurons to make a decision on the optimal direction of escape, The output connections, which project not only to cells within the ganglion of origin but also to adjacent ganglia and perhaps beyond, could allow this decision to be made throughout the thoracic ganglia as a single unit. However, nothing in these connections indicates a mechanism for making appropriate coordinated leg movements. Because each pair of legs plays a unique role in the turn, this coordination should be controlled by circuits dedicated to each leg. We suggest that this is accomplished by local interneurons between TIAS and leg motor neurons.

INTRODUCTION

Studies on several motor systems have led investi- gators to assign control of orientation to a popula-

Received July 16, 1990; accepted July 23, 1990 Journal ofNeuroblology, Vol. 21, No 8, pp. 1219-123s (1990) 6 1990 John Wiley & Sons, Inc CCC 0022-3(334/90/08012 19- 17$01.00

* A prehminary account of parts ofthls work has heen pub- lished in abstrdct form (Pollack and Rltzmann, 1989).

t To whom correspondence should be addressed

tion of interneurons that acts as a “democratic congress of cells” in which each neuron has an indcpcndent “vote” on the direction of movement. The total activity within a population represents a vector sum (Georgopoulos, Kettner, and Schwartz, 1988) or weighted average (Lee, Rohrer, and Sparks, 1988) of all the votes. With large populations of cells, such systems can result in fine control of graded directions of movement. The circuit that controls escape turns in the cockroach, Yeriplanela americana, is such a system (Ritz- mann and Pollack, 1988). Cockroach escape ori-

1219

1220 Ritzmann and Pollack

entation is particularly amenable to cellular analysis. Studies focusing upon this system havc identified many of the interneurons that process relevant sensory information and their connections (Ritz- mann, 1984). The resulting organization indicates that this system is controlled by a distributed population of interneurons similar to those proposed for mammalian systems.

The orientation behavior of the cockroach is initiated by gentle wind puffs generated by lunging predators (Camhi and Tom, 1978: Camhi, Tom, and Volman, 1978). These cause a turn away from the attacking predator which is followed by a more random run. The sensory structures that detect the wind are fine hairs located on antenna-like structures called crrci (Gnatzy, 1976). which are located on the rear of the animal’s abdomen. Asymmetri- cal hair sockets confer a directionality upon the sensory neurons that innervate the hairs (Nicklaus, 1965; Westin, 1979). The axons of these neurons synapse onto two populations of giant interneurons, the ventral and dorsal giant interneurons ( G I s and dGIs, respectively) (Callec, Guillet, Pi- chon, and Boistel, 197 I : Daley and Camhi, 1988). Of these. the vGIs have a significant effect on the direction of escape turns (Comer, 1985; Ritz- mann, 198 1). The directionality originally en- coded in the sensory hair movements is main- tained in the interneurons to which they project. Each vGI has a rcproducible wind field, preferring left versus right wind, front versus back wind, or having no directional wind preference at all (Wes- tin, Langberg, and Camhi, 1977).

The VCIS conduct sensory information from the cerci to the motor control centers in the thoracic ganglia. The axons of the vGIs project through the central nerve cord to the brain. As they pass through the thoracic ganglia, each vGI has the opportunity to influence the motor neurons that control leg movements. However, the excitation of motor neurons by vGIs is not direct. Rather this activity passes through at least one more level of interneurons in the thoracic ganglia. In the meso- and metathoracic ganglia (T2 and T3, respectively). a population of interneurons (primarily interganglionic) receives consistently short latency excitation from vGIs (Ritzmann and Pol- lack, 1986; Ritzmann and Pollack, 1988). These cells are referred to as typc A thoracic interneurons (TIAS) (Westin, Ritzmann and Goddard, 1988). So far. 13 individual TIAS have been identified in each half of both T2 and T3, and we believe that their homologues in the prothoracic ganglion (T,) also

receive monosynaptic inputs from vCIS. With their bilateral homologues, this would total 78 interneurons. Given that all individuals have probably not been identified, a conservative estimate would place the entire population at approximately 100 interneurons.

In terms of directionality, the TIAS can be divided into three subgroups: (1) those which are excited cqually well by left and right wind sources: (2) those which are excited primarily by wind from the left: and ( 3 ) those which are excited primarily by wind from the right (Westin et al., 1988). The left-right bias can be inferred from the presence of large ventral median (VM) branches which are the probable site of vG1 connections. TIAS with VM branches on both sides of the midline receive inputs from vGIs on both sides of the central nervous system (CNS) and consequently are excited equally well from left and right sides (Ritzmann and Pollack, 1988). TIAS with unilateral VM branches are excited only by vGIs on the side ipsilateral to the VM branch and consequently have wind fields biased toward that side. Each cell than can “vote” for a particular direction of turn if it can reach threshold. The ultimate behavior is a sum of activity from all TIAS that reach threshold. For example, if the wind comes from the right side. the nondirectional and right-biased TIAS will have much stronger responses than would those TI,s that are biased to the left side. As a result, the final tally will be distinctly biased toward TIAS with strong inputs from the right side. Assuming that the nght-side-biased TIAs make excitatory connections to motor neurons associated with turns to left. whereas nondirectional cells activate those motor neurons that are needed for any rnovemcnt. the resulting behavior will turn the animal to the left, away from the wind puff.

Although this circuit can explain the left-right decision that must be made to accomplish a suc- cessful escape, it does not account for coordinated leg movements that perform the turn. The turn is a complex movement in which all six legs, each composed of 5 separate segments, must execute Lery spccific movements. In addition, at the time of the wind stimulus, information on the position of the leg joints must be included. This suggests that at least one more level of processing is present in the escape circuit.

In the locust, leg motor neurons are excited by local nonspiking interneurons which play important roles in controlling leg movements (Burrows. 1980). Activity in groups of motor neurons serving

Motor Puthwuj.s from Thoracic Interneurons 1221

$11

V IT

V It

Figure 1 The structures of representative TIA interneurons is shown in whole mount and cross-section. In all three cells, a specific motor branch (MR) projects to the peripheral regions of the ganglion where it provides a site for ovcrlap with both local interneurons and motor neurons that control leg movements. rhe ventral median branch (VM) is the probable site of connection with vGIs. (A) Wholemount of DPG 701 and a drawing of associated cross sections, which are from the region indicatcd by brackets on the whole mounts for this and all other cells. Dye from three adjacent 10-p cross sections were superimposed on one drawing to produce these and subsequent figures. The dorsal intermediate tract (DIT) and ventral intermediate tract (VIT) are indicated for landmarks. (B) Whole mount of DPG 70 1 that originates in TZ and its projection to Tj. Note that a similar motor branch is also present in the adjacent ganglion. (C) Similar motor branch morphology for a VMC interneuron (VMC 7 19) in TZ. Note that for all three cells, the motor branch (MB) is located in the dorsal lateral region ofthe ganglion.


one or more leg segments can be coordinated to make a complex multijointed movement. ln addition, information from proprioceptors can be fac- tored in directly (Burrows. Laurent, and Field, 1988; Laurent and Burrows, 1988) or via another population of spiking local interneurons (Burrows, 1989; Burrows and Pfluger, 1986), which incorpo- rate information that modulates the gain of reflex activity in response to activity in intersegmental interneurons (Laurent and Burrows, 1989). If the TIAS in the cockroach that are responsible for Ieft- right decisions affect leg motor neurons via a similar network of local interneurons, this could account for coordinated leg movements.

The data presented in this paper document the pathway from TIAs to motor neurons. and thus complete the vGI circuit from cercal receptors to motor neurons. Motor activity resulting from activation of TIAS within their ganglion of origin and in adjacent ganglia are described. In addition, we analyze the pathway from TIAS to leg motor neurons, and we ask whether this pathway in- cludes local interneurons similar to those described in locust. If so, separate neural components are responsible for directional decisions and for leg coordination in the orientation movements associated with cockroach escape.

MATERIALS AND METHODS Adult malc Pcriplancta americana were used in all experiments. Animals were housed in large plastic garbage cans at 29°C on a 12:12 1ight:dark cycle and had free access to Purina Chick Starter Mash (unmedicated) and water.

For each experiment, an animal was pinned dorsal side up to a cork platform and the dorsal cuticle was removed from T2 to A3. The gut was either tied anteri- orly and deflected away from the underlying nerve cord or removed entirely. Cockroach saline (Carr and Fourtner, 1980) buffered to pH 7.2 with MOPS was continuously superfused over the nervous system.

Platforms were inserted under T1 and/or T2 to stabi- lize the ganglia for intracellular penetration. The sheath of the thoracic ganglion was softened with 1 mg/ml pro- tease in saline (Sigma type XIV). In experiments in which two interneurons were impaled in the same ganglion, the vibration caused by driving a second electrode through the sheath would dislodge the microelectrode from the first interneuron. We, therefore, found it necessary to remove the sheath from the ganglion in these experiments. To keep the ganglion healthy in the ab- sence of the sheath, we substituted an osmotically balanced saline (Wafford and Sattelle, 1986). Under these conditions the ganglion remained healthy for several hours.

Bipolar silver-wire electrodes were placed under the T2-T3 connectives and insulated with a petroleum jelly/

T2 MOTOR RESPONSE T3 MOTOR RESPONSE

Figure 2 Intracellular stimulation of a TIA in T3 evokes similar motor activity in both Tj and TZ. (A and B) Responses shown were recorded in sequential trials in which DPG 703 was stimulated with a 400-ms depolarization sufficient to evoke activity in the T2-T3 connective. (A) Records from nerve branches 5rl (which contains axons of depressor motor neurons of the leg) and 6Br4 (which contains axons of levator motor neurons) were monitored in Tz. (B) The same nerve branches were monitored but in T3. Note the similarity in the responses. The inset in this and all subsequent records shows the recording situation relative to the interneuron that was stimulated. R,,,, = Recording electrodes on the T2-T3 connective; SDpC = Intracellular stimulating electrode in DPG 703.

Motor Pathways from Thoracic Interneurons 1223

mineral oil mixture. These electrodes were used to monitor stimulus-evoked action potentials in the axons of interganglionic interneurons. In some experiments, extracellular electrodes were also placed on peripheral leg nerves to monitor motor activity extracellularly.

Intracellular microelectrodes were fashioned from single tube filament glass, The tips were filled with 4% Lucifer Yellow CH (LY) and the remainder with 1.0 ,M lithium acetate. Resistances ranged from 40-80 MD.

TIAS were impaled in the neuropile near the mid- point of the thoracic ganglia and were recognized by considerable spontaneous synaptic activity. Local interneurons and motor neurons were impaled in the lateral region of the ganglion. Once the intracellular recordings

were stable, the putative presynaptic TIAs were stimulated with a 400-ms depolarizing current pulse suprathreshold to the level which would produce extracel- Marly recorded T1A action potentials observed in the T2-T3 connective. Electrical traces were recorded on a Hewlett-Packard FM tape recorder. The records were then analyzed with a Nicolet 4094A digital oscilloscope. This allowed us to expand the traces to determine whether a postsynaptic response followed each presynaptic action potential.

At the end of the experiment, dye was iontophoresed into the cells via 5-nA hyperpolarizing current pulses of 1 s on-I s off duration for 5-20 min. The cell was then visualized in vivo by focusing a HeCd (Blue-440 nM)

Table 1 Connections Found Between TIA Interneurons and Local Interneurons or Motor Neurons

TlA Output Connections

Depolarization Inhibition No Connect

TI* LI MN [INK L1 MN LI MN

DPG 30 1 50 1 506 507 508 70 1 Soma

Total VMC

31 1 333 334 510 708 709 717 719 727 737 Soma

Total

2

2 I 7

0

1

1 2 4 0 0

1

I 1 2

I

1 1

1 2 1 1

1 2 1

1 1

0 0 2 1 9

3 1 I

1 8

For each TIA, the number of pairs that showed short latency depolarizing PSPs. short latency IPSPs, or no connection are indicated. They are further separated into subgroups according to whether the postsynaptic neuron of each pair was a local interneuron or motor neuron. In one case, a DPG 701 interneuron was found to excite a neuron in the peripheral region of the ganglion, but the postsynaptic cell failed to fill with dye. As this cell was in the region populated by local interneurons and motor neurons, the pair is included and thc postsynaptic cell is listed as unknown (UNK). The TIAs are separated into the two subpopulations, interneurons with dorsal somata (DPG) and interneurons with ventral somata (VMC). In a few experiments, the dye filled the TIA interneuron incompletely, only revealing the location of the soma. Because this was sufficient to group the TIA in one of the SubpOpUbdtiOnS, they are included under the heading Soma. For the DPGs, this is probably sufficient to indicate that the cell is indeed a TIA. Of the DPGs tested to date, only one has been found which fails to receive inputs from vGIs. This is an auditory cell which has easily recognized recording charactens- tics. The inclusion of the two interneurons in the VMC group based only on soma location is somewhat more tenuous, as some interneurons with ventral somata in this region do not receive inputs from vGIs. Therefore, they may not be TI& Because these two cells failed to show connections. they are not included in the calculations of PSP amplitude and latency. Note, in the totals, that all DPG interneurons either depolarized postsynaptic cells or failed to make connections. whereas all VMC interneurons were either inhibitory or failed to make connections.


laser (Liconix 4210N) on the tissue. The precise loca- tions o f the electrodcs were noted and preliminary drawings were made. The nerve cord was then removed and fixed in 4% paraformaldehyde in saline. dehydrated in alcohol, and cleared in methyl salicylate. Whole mounts wcre observed with a fluorescence microscope, photographed, and drawn with the aid of a drawing tube. The tissue was then embedded in paraplast and sectioned at 10-p intervals for further viewing. This allowed us to identify the precise location of processes of the thoracic interneuron within the ganglion.

A numbering scheme for naming TIs was introduced in an earlier paper (Westin et al., 1988). Under this scheme a three-digit number is assigned to each interneuron. The first digit indicates the relative location of

soma and axons, whereas the last two digits indicate order of discovery. Where the cell was part of a larger population ofsimilar TIs, the number is preceded by the initials that identify that population [e.g., the ventral median cells (VMC) 7191. The gencral classes are as follows: 100-199 are local interneurons; 200-299 have a single axon descending the nerve cord and ipsilatcral to the soma: 300-399 have a single axon descending the nerve cord contralateral to the soma; 400-499 have a single axon ascending the nerve cord ipsilateral to the soma: 500-599 have a single axon ascending the nerve cord contralateral to thc soma; 600-699 have both descending and ascending axons ipsilateral to the soma: 700-799 have both descending and ascending axons contralateral to the soma; 800-899 have ascending

A Area Expanded in

1

U 186 TZ

OPG T3

72-15 CONN. (OPC T3))

k k 0 100 200 300 400 500 600 700 800

B U 186 T2 I] 5 mv

T2-T3 CONN.

(OPG T3)

I , , , , I I I I

460 480 500 520 540

TIME (rnsec)

Figure 3 Intracellular depolarization of a TIA results in action potentials in the TIA and PSPs in a local interneuron (LI 186) in T2. (A) A DPG interneuron was depolarized iiitracellularly for 400 ms. This evokcd action potentials in thc DPG interneuron that were recorded intracellularly through the bridge circuitry (middle trace) and extracellularly through hook electrodes placed under the T2-T3 connective (bottom trace) (see inset for recording set-up). PSPs were recorded in LI 186 (top trace) associated with each of the stimulus-evoked action potentials. (DPG identification was based on location of soma filled with LY). (B) Segment of the record shown in A is expanded to show the consistent relationship between action potcntials in the DPG interneuron and PSPs in LI 186. For this and all subsequent records only a small segment is expanded. However, each evoked action potential was checked on a digital oscilloscope for associated PSPs and, unless otherwise noted, showed similar, albeit often smaller, PSPs associated with them. RL, = Recording microelectrode in a local interneuron. Other labels as per Figure 2.

Motor Pulhwys jinm Thouucic Interneiirons 1225

axons on both sides of the ganglion; 900-999 have descending axons on both sides of the ganglion; cells with numbers higher than 999 have axons in all connectives. Some TIS discussed in this paper have been referred to in the literature by other names. They are the dorsal poste-

cell), 501 (J cell), and 301 (Reverse J).

synaptic connection with motor neurons and local interneurons. In a few favorable preparations. we were able to fill a TIA into an adjacent ganglion [Fig. 1 ( ~ ) ] . those cases, we found that TI,s, which had a distinct MB in their ganglion of ori-

rior group (DPGs) 701 (formerly Lambda), 703 (cross g;n, also had a distinct MB in adjacent ganglia.

RESULTS

Morphology of TIA lnterneurons

Three representative type-A (TI,) interneurons are shown in Figure 1. All three have one or more large branches on the ventral side of the ganglion near the midline. These ventral median branches have been described in previous papers (Ritzmann and Pollack, 1988; Westin ct al., 1988) and arc associated with direct vCI connections. DPG 701 is a particularly large interneuron. Presumed serial homologues are shown in T3 [Fig. l(A)] and T2

[Fig. l(B)]. The cell which originated in T2 was filled into T3, and its projection to that ganglion is also shown. The somata of these interneurons are located in the dorsal posterior region of the ganglion, placing them in a distinct subpopulation of approximately 30 cells per ganglion, which is called the dorsul posterior group. All but onc of the DPG interneurons tested have been shown to rc- ceive direct excitatory inputs from vCIs. The one that did not receive inputs from vGls is an easily recognizable auditory interneuron. The soma of the third interneuron shown in Figure 1 (VMC 719) is located on the ventral surface near the midline of the ganglion. This is part of the second subpopulation of interneurons that receives inputs from vGIs. which is referred to as the ventrd median cell group.

Most of the TIAs (17 of 19) have a distinct branch that projects into the dorsal lateral region of the ganglion, where many branches from motor neurons that control leg movements and from local interneurons that interact with those motor neurons are located (Fig. 1). We refer to this neurite as the motor branch (MB). Because it is located distal to the point of initiation of action potentials, which has been shown to occur near the bend of the main neurite into the anterior-posterior direction (Ritzmann and Pollack, 1988). the MB is part of the T1,’s axon. The two TIAs that do not possess a single distinct MB have multiple branches that also project into the lateral region of the ganglion. This location provides adequate opportunities for

Do TIAS Affect Leg Motor Neurons

To establish that the TIAS are part of the vGI-mediated escape pathway, we needed to demonstrate that not only do they receive inputs from vGls but they also excite motor neurons that control leg movements. This was previously accomplished for one of the subpopulation of T I A S , the DPGs (Ritz- mann and Pollack, 1986). In experiments involving DPG interneurons. depolarizing the TIA through an intracellular microelectrode inserted into the cross-over region of the main neurite evoked activity in motor nerves of the same ganglion contralateral to the cell’s soma (i.e. ipsilateral to its axon). We have now extended these observations to include activation in distant ganglia. An

DEPOURIZE

NORMAL

HY PERPOURIZE -15mv

I , I I 1

0 10 20

TIME (msec)

Figure 4 Evoked PSPs in a T2 motor neuron were af- fected by suprathreshold postsynaptic current injection in a Tz DPG 30 I . Three scparatc trials are shown. (Top trace) Motor neuron had been depolarized with intracellular current injection through the bridge circuit. (Middle trace) No current was injected into the motor neuron. (Bottom trace) Motor neuron was hyperpolarized prior to stimulation of the IIPG interneuron. Note that the evoked PSP was smaller when the motor neuron was depolarized and larger when it was hyperpolanzed. The calibration applies to all three traces.


interneuron that evokes motor activity in its home ganglion (the ganglion in which the cell's soma resides) also excites motor neurons in adjacent ganglia (Fig. 2). The responses were qualitatively similar in different ganglia, that is, TIAS, which excited depressor motor neurons in T3, also excited depressor motor neurons in T2 and vice versa. No evidence of sign reversal has been observed. The strengths of activity in the different ganglia were also similar.

Additional experiments similar in nature were performed on the second subgroup of TI,s (VMC interneurons). However, intracellular depolarization of VMCs consistently failed to evoke motor activity in any recorded leg nerves.

Direct Connections from TIAs

Do TlAs excite motor neurons directly or is there another level of interneurons interposed between TI,s and motor neurons? The experiments described in Figure 2 show only that TIAS can activate motor neurons. They do not establish that direct connections exist. We, therefore, attempted to stimulate TIAs in conjunction with intracellular recordings in neurons located in the lateral regions of the thoracic ganglion, where both motor neurons and local interneurons, which are presynaptic to motor neurons, are located.

The results identified postsynaptic cells for various TI,s. In all, 40 pairs of neurons were studied

A Area Expanded in B

IT IA - MN PAIR1

2 YN FFF. T3

T2-TS CONN.

(DPC 701 T3)

0 200 400 600 800

I . , . . l , . . . ~ . , , . ~ . . j , I

220 230 240 250 260

TIME (maec)

Figure 5 TIA stimulation evokes PSPs in motor neurons. In this experiment, DPG 70 1 in T3 was stimulated in conjunction with a recording of a motor neuron in T3. Action potentials that resulted from intracellular stimulation of DPG 70 1 were monitored extracellularly in the T2-T3 connective (bottom traces). The depolarization required to raise DPG 70 1 above threshold in this experiment was too great to permit balancing of the bridge. B is an expanded segment of the bracketed region indicated in A. Note that the postsynaptic response for this motor neuron is similar to that recorded in local interneurons and shown in Figure 6. In the inset only the primary branches of the presynaptic cell (DPG 701-dotted lines) are drawn. This was necessary to avoid obscuring the drawing of the postsynaptic cell. Thc complete morphology of DPG 701 is shown in Figure 1. RMw = Recording microelectrode in the motor neuron. Other inset labels as Figure 2.

Motor Pclthu.ays,fiom Thoracic Interneurons 1227

in which the presynaptic cell was positively identified as a TIA (Table 1). Identification of TI,s was based upon morphological properties such as soma location. and presence of VM branches, and previous recordings showing input from vGIs. In 15 of these pairs, electrical data consistent with the

TI L I PAIRS r A-

presence of direct connections were indicated. This was based upon the presence of PSPs that followed presynaptic action potentials at constant short latencies even at very high frequencies. In a typical test, the TIA was depolarized to threshold through the bridge circuit of the DC amplifier. If

Area Expanded in B I INTRAGANGLIONIC PAIR] A I

LI 188 T3

T2-T3 CONN. I . - I

(DPG 701 T3) 1 1 I 1 I I 0 200 400 600 800

f 7 --J- 1 5 rnV B

U 118 T3

T2-T3 CONN.

(DPG 701 T3) 7' I , , , . I . . . . 1 . . . . I . . ,

240 250 260 270

L I

Area Expanded in o INTERGANGLIONIC PAIR

L I

C

U 182 T2 - T2-T3 CONN.

(DPC 701 T3) I I 1 I I

0 200 400 600 800

U 182 T2 J f- -.- -3 5 rnV

D

T2-T3 CONN.

(DPC 701 T3) I I , , , L--- SDPG

210 220 230 240 250

TIME (msec)

Figure 6 PSPs evoked in local interneurons (LIs) are similar to those evoked in motor neurons. Responses evoked in intraganglionic and interganglionic pairs are also similar. (A,B) Recordings from LI I88 (top traces) in T3 in response to intracellular stimulation of DPG 701 in T3 (see insets to right for recording set-up). The area bracketed in A is expanded in B to show the consistent relationship between DPG 701 action potentials in the connective and PSPs in LI 188. (C and D) Results from a similar experiment in which an interganglionic pair was monitored. In this case DPG 701 in T, was stimulated in conjunction with a recording from LI 182 in Tz. Note that the responses in the two pairs are similar. The drawing of DPG 701 in the inset for Ihe intraganglionic pair A and B shows only the primary branches to avoid obscuring the drawing of the motor neuron. The complete morphology of DPG 701 is shown in the lower inset and in Figure 1.

1228 Ritzrnann and Polluck

the bridge could be balanced sufficiently for the depolarizing stimulus, activation of the TIA was indicated by action potentials recorded in the TIA record (Fig. 3 ) . In those instances, latency was measured from the intracellularly recorded action potential to the onset of the PSP. In most cases, the required stimulus was too large to allow balancing of the bridge. In those records, activation was indicated by regularly occurring action potentials in

the extracellular record on one of the T2-T3 connectives or the abdominal cord depending upon the morphology of the particular TIA (e.g., Fig. 5) . Latency was then measured from the extracellular action potential to the onset of the PSP, and an adjustment was made to account for conduction time to the extracellular recording electrodes. Al- though these data cannot completely rule out the presence of interposed cells. such as a small inter-

LI 182 Ant.

A ( J u t - , . LI 183 Ant.

\ d u b 2

Port. DIT

-----+f VIT

Lt I88 Ant.

Post. DIT

VlT

Post.

DIT - V l f

MN Ds Ant.

VIT

Figure 7 The local interneurons and motor neurons that rcccive inputs from TIA interneurons are located in regions that overlap the motor branches of TIA interneurons. Whole mounts and cross sections o f examples o f three local interneurons (A-C) that were shown to receive inputs from TIAs and of D,, a leg motor neuron (D) are presented. In all cases, the brackcts on the whole mount indicates the region from which cross sections were taken, and the location of the DIT and VIT are indicated as landmarks. Note that for all four cells, numerous branches are found in the dorsolateral region of the ganglion, providing sites of overlap with the motor branches of TIAs shown in Figure I .

Molar Pulhwuy.\ from Thorucic Interneurons 1229

neuron with electrical input and output connections, they indicate that. under these conditions, these neurons perform electrically as though they are connected monosynaptically.

In all pairs, postsynaptic activity reliably followed presynaptic activation. Presynaptic cells were typically stimulated for 400 ms at varying stimulus strengths which resulted in presynaptic action potentials at various frequencies. PSPs associated with presynaptic action potentials followed even at very high frequencies. Nine of the pairs were tested at frequencies in excess of 50 Hz, two at 90 Hz and three at 100 Hz. In each case, PSPs followed presynaptic action potentials faith- fully, although they usually declined in amplitude throughout each 400-ms stimulus trial. In spite of this ability to follow at high frequency, the PSPs were influenced by modification of postsynaptic membrane potential in a manner consistent with chemical synapses (Fig. 4). These properties are

similar to those of synapses in locust (Burrows. 1975) and in other synapses in cockroach where there is more extensive evidence for chemical transmission (Casagrand and Ritzmann. 1990).

Motor Neurons vs. Local Interneurons as Postsynaptic Neurons

The T14s influence two distinctly different types of postsynaptic neurons. In five of the pairs that showed evidence of direct connection, the postsynaptic cell was a motor neuron (Fig. 5), whereas in nine pairs, the postsynaptic cell was a local interneuron (LI) (Fig. 6). In the one remaining pair, the postsynaptic cell was not filled well enough to establish positively whether it was a motor neuron or a local interneuron.

The Lls that received inputs from TIAs were morphologically similar to several nonspiking local interneurons that have been reported on in

A VMC - LI PAIR

Area Expanded in 6

T2-T3 CONN.

(VMC 713 T2) -R L I

t I 0 200 400 600 BOO

I I I

B

LI 171 T 3

T2-T3 CONN.

(VMC 719 T2) * 1 I I I I I

230 240 250 260 270 280 290

TIME (msec)

Figure 8 Action potentials of VMC interneurons evoke IPSPs in local interneurons. This experiment is similar to those in Figures 5 and 6. However, heru a member of the VMC subpopulation of TIAs was stimulated. In this case, a VMC 719 interneuron in Tl was stimulated and an L I 171 in T, is recorded. Action potentials in VMC 719. monitored in the extracellular record from the T2-T3 connective (bottom trace), arc associated with IPSPs in LI 171 (top trace). The segment indicated in A is expanded i n B to show the consistcnt relationship. The short positive wave preceding each IPSP is probably an artifact resulting from capacitative crosstalk from the presynaptic record. SvhIc = lntracellular stimulating microelectrode in VMC 7 19. Other inset labels are as per Figure 2.


locust (Siegler and Burrows, 1979). Cell bodies were located on the ventral surface in two regions. One region was in the anterior part of the ganglion near nerve 3 [Fig. 7(A)]. The other was in the posterior lateral part of the ganglion between nerve 6 and the posterior connective [Fig. 7(B,C)]. The terminal branches from both groups of local interneurons were found predominantly in the dorsal lateral region of the ganglion [Fig. 7(A-C)]. In this

u 188 A

Nerve 5rl (Depresson)

N . m 6Br4 (Lavatan)

u 183

B N e w 5rl

(Depressors)

N e w 6Br4 (Levaton)

U 183

Nerve 5rl (Depresson)

Nerve 6Br4 ( L m t o n )

area, terminals of the LIs overlap extensively with branches of leg motor neurons [Fig. 7(D)]. In addition, overlap also occurs between the motor branches of the TI,s (Fig. I ) and the branches of both LIs and motor neurons, providing possible sites of synaptic connection. Although we made no at tempt to demonstrate that these local interneurons were nonspiking under all possible conditions, we noted no case of action potentials in any

1 I I I -t

I I I

I .I

I . . . . . . . . . I . . . . . . . . . I . . . . . . . . . I . . . . . . . . . I

0 200 400 600 800

0 ,200 400 600 800 1000 1200 1400 1600

DUE (mrcc)

Figure 9 Local interneurons that receive inputs from TIA interneurons evoke activity in leg motor neurons. In each record (Top trace) is an intracellular record from the local interneuron. It is provided only as a stimulus monitor, (Middle trace) is an extracellular record from nerve branch 5rl which contains depressor motor neurons D, and Df. the (Bottom trace) is an extracellular record from nerve branch 6Br4 which contains levator motor neurons. Upon depolanzing the local interneuron, the bridge went out ofbalance. (A) Stimulation of LI 188 (inset) evoked activity in nerve 5rl probably from D,. No change was seen in the record of 6Br4. (B) Depolarization of LI 183 evoked activity in nerve 6Br4 with no change in nerve 5r l . However, when the same interneuron was depolarized at a time when D, was active (C), the ongoing activity was reduced or completely blocked. Activity in 6Br4 is weaker than that recorded in B. SLI = Intracellular microelectrodes for stimulating local interneurons.

Molor Pathways from Thoracic Interneurons 1231

of the local interneurons that were seen to receive inputs from T1,s. This and the morphological similarity would suggest that these interneurons are, in fact. similar to nonspiking interneurons in locust.

The presence of connections to both motor neurons and local interneurons suggest that at least two parallel pathways exist between TIAs and motor neurons. Where parallel motor pathways have been reported previously, one is usually distinctly stronger than the other (Roberts, Krasne, Hagiwara, Wine, and Kramer, 1982). In our data, no systematic differences were seen between connections involving motor neurons and those involving local interneurons (Fig. 5,6). Mean latency of intraganglionic pairs involving motor neurons was 0.7 t 0.3 ms, whereas mean latency of pairs involving local interneurons was 0.6 k 0.2 ms. In these and all subsequent means IZ 2 3 and the error indicates standard deviation. For interganglionic pairs, mean latency with local interneurons was 1.7 +- 0.3. Only two interganglionic pairs were recorded with motor neurons and both of those had latencies of 1.7 ms. Mean amplitude of TI,-evoked depolarizing PSPs in local interneurons was 2.6 f 0.8 mV. In motor neurons, mean amplitude of evoked depolarizing PSPs was 1.8 & 0.6 mV. Al- though slightly smaller than the amplitude recorded in local interneurons, thcre were not sufficient numbers of motor neuron pairs to determine if this difference was statistically significant.

lnterganglionic vs. lntraganglionic Pairs

Of the pairs studied, eight were interganglionic and seven were intraganglionic (five in T2 and two in T3). It is possible that distant connections would be qualitatively or quantitatively different from connections within the TI,% ganglion of origin. How- ever, this is not the case. The amplitude of depo- lariiing PSPs associated with TIA action potentials were similar for both inter- and intraganglionic pairs (Fig. 6) . Depolarizing PSPs in interganglionic pairs had a mean amplitude of 2.4 t 0.7 mV. The mean amplitude in intraganglionic pairs was 3.2 f 1.5 mV. These differences are well within the range of error due to recording situations. The mean latency for pairs in the same ganglion was 0.7 a 0.3 ms. The mean latency for pairs including cells in adjacent ganglia was 1.7 t- 0.2 ms. Con- duction time between ganglia was measured and found to be 1 .O ms. Thus, the actual synaptic delay is similar in interganglionic and intraganglionic pairs.

Sign of Synaptic Connection

The location of the TI, cell body is related to the sign of synaptic transmission in the postsynaptic cell. For the 20 pairs involving DPG interneurons (dorsal somata), 12 evoked depolarizing (probably excitatory) connections (Figs. 5,6; Table l), eight showed no connection, and none showed inhibitory connections. In contrast, in the 20 pairs involving the other subpopulation of TIAs, the VMC interneurons (cells with ventral median somata), none showed depolarizing connections, 17 showed no connection, and three showed distinct inhibitory connections (Fig. 8: Table 1). The latter had similar latencies and ability to follow TI, action potentials to those observed for DPG-mediated depolarizing connections (Fig. 8). This is consistent with the finding that VMC interneurons failed to show posi-

MOTOR NEURON

0 200 400 600 800 - B

U 188 1 L] 10 m"

UOTOR -J 10 mv NEURON

0 200 400 600 800

TIME (mws)

Figure 10 Dual intracellular recording indicates the interaction between LIs and motor neurons. LI 188 (Top trace) was stimulated intracellularly in conjunction with an intracellular recording from a motor neuron (Bottom trace). Both cells were in TZ. (A) LI 188 was depolarized to a relatively small degree resulting in a decrease in the frequency of spontaneously occurring action potentials in the motor neuron. (B) A stronger depolarization in LI 188 resulted in a hyperpolarization in the motor neuron which turned off the ongoing action potentials until the stimulus to LI 188 was turned Off.

1232 Rifzmann and Polluck

tive results in experiments in which motor neurons were monitored extracellularly. In those cases, an inhibitory effect would only be seen as a reduction of ongoing activity. Because motor neurons are only rarely spontaneously active in restrained animals, it is reasonable that we were not able to ob- serve inhibition under those conditions.

Motor Effects of Local lnterneurons

The local interneurons that receive inputs from TI,s influence leg motor neurons. In 13 preparations, we have depolarized local interneurons to determine their influence on leg motor neurons. These preparations included seven morphologically distinct interneurons, three of which were local interneurons that had previously been shown to receive inputs from T I A S . All seven identifiable interneurons had effects on leg motor neurons. Four of these cell types were tested in multiple preparations, and their effects were reproducible. In two preparations each, LI 188 [Fig. 9(A)] and LI 17 1 consistently excited the slow depressor of the coxa (DJ. In three preparations each, LI 183 [Fig. 9(B)] and LI 140 consistently excited levator motor neurons in nerve 6Br4. On occasions when

T3

D, was spontaneously active, depolarization of LI 183 could be seen to inhibit ongoing activity [Fig. 9(C)]. In two preparations, we were able to impale a local interneuron that had previously been shown to receive T I A activation (LIs 183 and 188) simultaneously with a leg motor neuron. Both of these pairs showed negative interactions. Depolar- ization of the local interneuron hyperpolarized the motor neuron and reduced [Fig. lO(A)] or elimi- nated ongoing generation of action potentials [Fig. 1 0( B)]. No distinct inhibitory post-synaptic potentials (IPSPs) were noted. This is not surprising given the presumed nonspiking nature of the local interneurons.

DISCUSSION

Motor Pathway for Escape Circuit With the inclusion of the data reported here, we now have accounted for the major. components of the escape circuit from cercal sensory structures to the motor neurons controlling the ultimate leg movements. A simplified diagram of this circuit is shown in Figure I I . It should be emphasized that in all cases the neurons drawn in the diagram rep- resent populations of neurons rather than single

T2 A6 T I

T-

I.

Figure 11 A summary diagram of the major components of the wind-mediated escape circuit. Each component represents a population of neurons, many of which have been identified as individuals. The expanded image ofT3 shows the details of connections described in this papcr. Similar interactions also occur in adjacent ganglia. Although no connections have been described in TI at this time, there is reason to believe that similar connections also occur there from neurons originating in TI and in other ganglia.

Motor Path ways !‘?om Thomcic Interneurons I233

cells. However, many members of the populations have been identified as individuals. Thc basic components of the circuit begin with sensory neurons from hairs on the cerci, that connect to the vGIs, which originate in the terminal abdominal ganglion and then project the entire length of the ventral nerve cord. In each of the thoracic ganglia, the vGIs connect to TIAS via ventral median branches. TIAS, in turn, connect to motor neurons directly and via local interneurons, which are morphologically similar to the nonspiking interneurons that control motor activity in locusts (Siegler and Burrows, 1979). These connections are made in the TIA’s ganglion of origin as well as in adjacent ganglia. The strengths and synaptic delays of all these connections are approximately the same. Local interneurons that receive inputs from TI,s subsequently influence leg motor neurons by apparently direct excitatory or inhibitory connections.

Parallel Pathways from Tl,s to Motor Neurons

The presence of direct TIA-to-motor neuron connections in parallel with polysynaptic connections via local interneurons would be surprising but for the fact that a similar type of organization occurs in other arthropod systems. Many instances have been reported for feedfonvard pathways that may potentiate elements, but usually do not by them- selves provide enough strength to account for the entire behavior. For example, in crayfish escape, giant interneurons make weak direct connections to flexor motor neurons. whereas the majority of flexor activation occurs via a segmental giant interneuron (Roberts et al., 1982). In the locust flight system, descending movement detection interneurons make weak connections to motor neurons, whereas stronger connections occur in a polysynaptic pathway that allows for gating by the flight oscillator (Reichert and Rowell, 1985). How- ever, in the cockroach escape system, the strength of the connections between TIAs and LIs and between TIAS and motor neurons appears to be similar.

Sign of Motor Output of Tl,s

Another potentially surprising finding was the systematic differences in sign between the subpopulations of TI,s. TIAS with dorsal somata (DPGs) consistently depolarized postsynaptic cells,

whereas TIAS with ventral somata near the midline (VMCs) either inhibited postsynaptic cells or failed to evoke any discernable response. Again. there is a precedent for this. In the locust flight system, Pearson and Robertson ( 1987) reported that all interganglionic interneurons with dorsal somata excited postsynaptic cells, and all interganglionic interneurons with ventral somata near the midline were inhibitory in nature.

This finding has important implications to the ultimate understanding of the cockroach escape system. The organization is complex in that it in- volves several large populations of interneurons. One of the populations, the TTAs, is made up of approximately 100 interneurons. This large number of interneurons makes a complete understanding of the circuit difficult. However, the data pre- sentcd here and in previous papers suggest that morphological properties can be used to segregate the TIAS into functional subgroups. The pattern of input connections to TIAS is indicated by VM branches on each TIA. The presence of VM branches on left, right, or both sides of the CNS indicates the presence of vGI connections on those sides (Ritzmann and Pollack, 1988). The VM branch is also correlated to the bias found in wind fields of T1,s probably as a consequence of the pattern of vGI connections (Westin et al., 1988). We can now further divide these subpopulations into cells with dorsal somata and cells with ventral somata. Again, a functional correlate exists. this time on the output side, relative to the sign of synaptic connection. With this information, the approximately 100 TIAs can be divided into at least six functional subgroups on the basis of easily recognized morphological cues.

Need for Populations of Interneurons

Why is such complexity necessary for this system? The cockroach escape circuit was once thought to be a model for simple neural pathways involving direct connections from giant interneurons to motor neurons. However, it now appears to have not one but two populations of interneurons interposed between them. Certainly, the inclusion of these interneurons will increase the time needed to effect an escape movement. Because speed is cru- cial in escape systems, as evidenced by thc extensive use in many animals of giant interneurons to span long distances, the presence of these populations is puzzling. Perhaps the answer is that the circuit, as constructed, has the minimum number of elements for this particular behavior.

1234 Rifzmann and Pollack

Our data are consistent with a hypothetical form of neural organization in which the decision on the direction of the turn is made by a population of interneurons that is distinct from the interneurons responsible for producing coordinated leg movements. The decision made by the TIAs re- garding direction of the turn appears to result from the total activity of the approximately 100 TIAs in the thoracic ganglia. Each TIA has a set of preferred wind directions that is established by connections from specific vGIs (Westin et al., 1988; Ritzmann and Pollack, 1988). In this way, the system has a similar organization to models that have been proposed for control of mammalian orientation movements (Georgopoulos et al., 1988).

In the cockroach, a problem occurs in the tim- ing of activity in the thoracic ganglia. The information on wind direction is conducted from posterior to anterior in the vGIs. Thus, a delay occurs between the time the information arrives at T3 and the time it amves at the more anterior ganglia. Although it is a brief interval, this delay could result in a metachronal movement rather than a unified turn. The interganglionic projections of the T I A S could eliminate this problem. With these connections, motor neurons in T2 arc influenced by TIAs in both T2 and in T3. The increase in activation could serve to decrease the time needed to ultimately reach threshold in T2 motor neurons. Neurons located in more posterior ganglia would be excited earlier, but those in more anterior ganglia would ultimately receive more excitation. As a result, cells in all three thoracic ganglia might reach threshold at the same time. Although the motor neurons in T3 would also be influenced by descending projections from TZ, this would occur later than for the motor neurons of TZ. The T2-to- T3 influence would be delayed by the time taken for the vGIs to reach and activate TlAs in T2, in addition to the conduction time within TIA axons returning to T3. As a result, it would have little effect on the initial turn. However, it could serve to maintain a high level of motor activation for subsequent running movements.

Once a decision is made on direction, the movements made by each leg must be executed. The demands on this part of the control system are different from those of the decision-making network. Whereas a unified decision on the direction of turn must be made in the three thoracic ganglia, the control of each leg movement must occur sepa- rately, because the movement made by legs in each thoracic segment is heterogeneous (Camhi and

Levy, 1988; Nye and Ritzmann, 1990). Metatho- rack legs primarily provide power to overcome inertia. Meso- and prothoracic legs provide differ- ential activity that directs the turning movement. Information on the angle of joints at the onset of movement may vary in importance from leg to leg and from joint to joint within legs. Thus, proprioceptive inputs may play a more serious role in some of the legs than in others. As a result, the task of generating coordinated and appropriate leg movements would be more effectively carried out by local circuitry dedicated to each leg. Hence, a local interneuron population is present to fulfill this task. If the neural organization described here is, in fact, the minimal requirement that an animal needs to execute an orientation turn utilizing jointed appendages, we would expect to find the components represented in more complex systems.

The authors thank Drs. Joanne Westin and Hillel Chiel and Ms. Janet Casagrand for critically reviewing the manuscript. This work was supported by NIH grant NS- 174 1 1 to R.E.R.

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parallel motor pathways from thoracic interneurons of the ventral giant interneurons system of the...

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