Parallel processing of proprioceptive information in the ... - Springer Link

J Comp Physiol A (1993) 172:389~,00

dkmrlutl of Somme, r and lah, iology .e. Phtn~lology 9 Springer-Verlag1993

Parallel processing of proprioceptive information in the terminal abdominal ganglion of the crayfish P.L. Newland*, T. Nagayama Zoological Institute, Faculty of Science, Hokkaido University, Sapporo 060, Japan Accepted: 30 December 1992

Abstract. The processing of proprioceptive information from the exopodite-endopodite chordotonal organ in the tailfan of the crayfish Procambarus clarkii (Girard) is described. The chordotonal organ monitors relative movements of the exopodite about the endopodite. Displacement of the chordotonal strand elicits a burst of sensory spikes in root 3 of the terminal ganglion which are followed at a short and constant latency by excitatory postsynaptic potentials in interneurones. The afferents make excitatory monosynaptic connections with spiking and nonspiking local interneurones and intersegmental interneurones. No direct connections with motor neurones were found. Individual afferents make divergent patterns of connection onto different classes of interneurone. In turn, interneurones receive convergent inputs from some, but not all, chordotonal afferents. Ascending and spiking local interneurones receive inputs from afferents with velocity thresholds from 2-400~ while nonspiking interneurones receive inputs only from afferents with high velocity thresholds (200-400~ The reflex effects of chordotonal organ stimulation upon a number of uropod motor neurones are weak. Repetitive stimulation of the chordotonal organ at 850~ produces a small reduction in the firing frequency of the reductor motor neurone. Injecting depolarizing current into ascending or non-spiking local interneurones that receive direct chordotonal input produces a similar inhibition. Key words: Crayfish - Reflex - Coordination - Sensory

Introduction Proprioceptors are found at the joints between limb or body segments of both invertebrates and vertebrates and * Present address." Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK Correspondence to.

P.L. Newland

provide information about the relative movements of these joints. Chordotonal organs in the legs of stick insects, locusts and crayfish mediate resistance reflexes of the legs (Mill 1976). In the locust, in particular, the processing of proprioceptive information from a chordotonal organ in the leg, the femoral chordotonal organ, has been extensively investigated and the reflex pathways responsible for resistance reflexes elucidated (Burrows 1987, 1988; Laurent and Burrows 1988). The detailed analysis of these reflex pathways in the locust was made possible because of the accessibility of the chordotonal organ in the leg. In the crayfish we have a detailed understanding of the way in which local circuits control the movements of the terminal abdominal appendages, the uropods (Nagayama et al. 1984). Only recently, however, has such an accessible chordotonal organ in the tailfan been described which provides information regarding the relative movements of the endopodite and exopodite of the uropod (Field et al. 1990). Many of the afferents innervating this chordotonal organ terminate in the 6th abdominal ganglion (Nagayama and Newland 1993) although a few have intersegrnental projections. The uropods are involved in a variety of behaviours. During equilibrium reactions, in which deviation of the body from the upright is detected by the balance organs, the statocysts (Newland and Neil 1987; Yoshino et al. 1980a), and by proprioceptors in the legs (Newland 1989), the uropods adopt asymmetric positions so that they intercept water currents impinging on the body to return the animal to the upright. During escape swimming the uropods provide most of the thrust during the swimming stroke (Webb 1979; Newland and Neil 1990a). The uropods are promoted during the power stroke to maximize this thrust, and remoted during the recovery stroke to reduce hydrodynamic drag. During the power stroke asymmetries in the posture of the uropods again contribute to equilibrium reactions (Newland and Neil 1990b) and steering behaviour (Newland et al. 1992). The uropods are also involved during postural steering (Newland et al. 1992) and avoidance reactions (Nagayama et al. 1986) where the uropods again adopt

390

P.L. Newland, T. Nagayama: Parallel processing in crayfish interneurones

asymmetrical postures that can act against water flow generated by the animal itself during l o c o m o t i o n to redirect movements. In m a n y o f these behaviours the p r o d u c t i o n o f the u r o p o d pattern is only expressed when the a b d o m e n is extended ( T a k a h a t a et al. 1981). The m o v e m e n t s o f the u r o p o d are controlled by the local circuitry within the terminal a b d o m i n a l ganglion, which includes b o t h spiking and nonspiking local interneurones, and ascending intersegrnental interneurones ( N a g a y a m a and H i s a d a 1985; N a g a y a m a et al. 1984, 1992; Reichert et al. 1982). Peripheral and descending inputs mediate and m o d u l a t e particular reflexes depending on internal and external conditions ( B o w e r m a n and Larimer 1974a, b; Larimer and K e n n e d y 1969a; N a g a y a m a and H i s a d a 1987; T a k a h a t a and H i s a d a 1986). The u r o p o d s and their sensory receptors are also involved in eliciting various behaviours. Gentle mechanical stimulation o f the u r o p o d s gives rise to defensive behaviour or l o c o m o t i o n , depending u p o n the age o f the crayfish ( N a g a y a m a et al. 1986). Stronger stimulation can give rise to escape swimming (Wine and K r a s n e 1972). C h o r d o t o n a l organs detect and m o n i t o r the angular changes o f the endopodite and exopodite during m a n y behaviours and could therefore contribute substantially to reflex movements. Given o u r knowledge o f the central projections o f the sensory neurones o f this c h o r d o t o n a l o r g a n and the locations o f several key types o f interneurones, it is n o w appropriate to examine the processing o f proprioceptive i n f o r m a t i o n in the local circuits. We show here that various types o f interneurone in the terminal ganglion receive convergent and divergent patterns o f excitatory inputs directly f r o m the c h o r d o t o n a l afferents and that in turn these interneurones influence the activity o f the u r o p o d m o t o r neurones.

recorded with an extracellular suction electrode from the proximal portion of the second nerve root, near the antero-proximal edge of the protopodite. Stimulation of the chordotonal organ was carried out by displacing its attachment strand using a fine pin attached to a vibrator (Ling Dynamic Systems), through distances equivalent to a 5~ movement of the exopodite. The vibrator was driven by variable duration, frequency and amplitude ramp and sinusoidal waveforms by a Shoshin OI-8 computer-controlled stimulator. Mechanical stimulation was carried out by touching hairs on the ventral surface of the exo- or endopodite with a fine paintbrush. Intracellular recordings were made from the left half of the terminal abdominal ganglion neuropil with microelectrodes filled with 3% Lucifer Yellow CH (Stewart 1978) dissolved in 0.1 M lithium chloride or with 2 M potassium acetate. Electrode resistance was over 150 MY~ for Lucifer Yellow filled microelectrodes and 20-40 Mf~ for potassium acetate filled electrodes. After physiological examination, interneurones were stained by injection of Lucifer Yellow iontophoretically using 1-5 nA hyperpolarizing current pulses of 500 ms in duration at 1 Hz for 5 to 15 min. The ganglion was then removed and fixed in 10% formalin for 15 min, dehydrated in an alcohol series and cleared in methyl salicylate. It was then photographed in wholemount for subsequent reconstruction using a fluorescence microscope. In the recordings using potassium acetate electrodes, nonspiking local interneurones were identified according to established criteria (Takahata et al. 1981) and ascending intersegmental interneurones were distinguished from spiking local interneurones by the depth of penetration of the electrode. Ascending interneurones were usually recorded 150-250 lain from the ventral surface while spiking local interneurones were usually recorded deeper than 250 ~tm. Physiological recordings were stored on a PCM data recording system for subsequent analysis and display on an IBM compatible microcomputer through a GPIB interface. Signal averaging was performed using a Cambridge Electronics Design 1401 interface and software or on a Tektronix digital oscilloscope. The results are based on recordings from 21 ascending interneurones, 4 nonspiking, and 3 spiking local interneurones that receive direct chordotonal input from 35 crayfish.

Results Materials and methods Adult male and female crayfish, Procambarus clarkii (Girard), of 7-9 cm body length (from rostrum to telson) were used for all experiments. They were obtained from a commercial supplier and maintained in running freshwater aquaria before use. The abdomen was isolated from the thorax and pinned ventral side up in cooled Van Harreveld's (1936) solution. The swimmerets were removed and the terminal (6th) abdominal ganglion was exposed by removing the 6th sternite, the ventral aorta and the connective tissue. The ganglion was stabilized on a silver platform and treated with protease (Sigma type XIV) for 30 s to facilitate penetration of the ganglionic sheath with glass microelectrodes. The exopodite was fixed at an angle of 60~ relative to the endopodite. The soft cuticle of the protopodite overlying the second and third ganglionic roots was removed along with the lateral edge of the protopodite. The underlying hypodermis and connective tissue were removed to expose the chordotonal organ at the proximal ventral edge of the endopodite. The third root was usually cut distal to the chordotonal organ to reduce spontaneous activity from other afferents sensitive to water movement. In some experiments, the third root distal to the chordotonal organ was kept intact to test the response of interneurones to mechanical stimulation of the hairs on the endopodite. The spike activity of the chordotonal afferents was recorded by an oil-hook electrode placed on the third nerve root near the proximal edge of the protopodite. In some experiments, the spike activity of the closer motor neurones was also

Proprioceptive inputs onto ascending interneurones The c h o r d o t o n a l o r g a n that m o n i t o r s m o v e m e n t s o f the exopodite relative to the endopodite ( N a g a y a m a and N e w l a n d 1993) contains a p p r o x i m a t e l y 12 sensory neurones that respond to position, direction, velocity and possibly acceleration (Field et al. 1990) and project in nerve r o o t 3 to the terminal a b d o m i n a l ganglion. W h e n the c h o r d o t o n a l strand was displaced with a sinusoidal stimulus at 0.1 H z it was first stretched (equivalent to an opening o f the exopodite) and then relaxed (equivalent to a closing m o v e m e n t o f the exopodite) to return to the start position. Such a displacement evoked spikes in several sensory neurones, that could be distinguished by their different extracetlularly recorded spike heights in r o o t 3, and led to a burst o f excitatory postsynaptic potentials (EPSPs) in an ascending intersegmental interneurone (Fig. 1A). Superimposing sweeps o f the oscilloscope triggered by spikes in a single sensory neurone f r o m r o o t 3 shows that the EPSPs consistently follow the sensory spikes with a short and constant latency (Fig. 1B). The delay between the afferent spike and the start o f the


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EPSP in the interneurone was 2.1 ms. The afferents have a mean conduction velocity of 3.1 + 1.2 m/s (Nagayama and Newland 1993). The activity of the afferent was monitored extracellularly in the 3rd root 3.0 mm from the terminal ganglion. The synaptic sites of the interneurone were a further 400 gm from the edge of the ganglion. Allowing a delay of 1.1 ms for the afferent spikes to conduct over these distances to reach the synaptic sites within the ganglion leaves a value of 1.0 ms for the central latency for this interneurone. Steady depolarizing current (1 nA) injected into the ascending interneurone decreased the amplitude of the EPSP by 25%, while hyperpolarizing current of 1 nA increased the amplitude by 120%, and 2 nA by 135% (Fig. 1C). Taken together, these results suggest that the chordotonal afferents make chemically mediated direct connections with the ascending interneurones. Chordotonal organ afferents make convergent patterns of connections with ascending interneurones (Fig. 2). For example, ascending interneurone NE-1 (Fig. 2A), characterised by its large soma located in the caudal ventral cortex of the terminal ganglion, thick short neurites and a rostrally projecting large diameter axon (Nagayama et al. 1993), receives short latency inputs from chordotonal afferents revealed using signal averaging. In one animal a window circuit was used to trigger the averager selectively from 4 sensory afferents, each with a different extracellular spike height (Fig. 2Bi-iv). Each of these 4 afferents responded during the opening phase of 0.1 Hz sinewave stimuli and each produced short latency EPSPs in NE-1. In another crayfish, only one recorded sensory

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Fig. 1A-C. An ascending interneurone that received direct synaptic input from the exopodite-endopodite CO. A A 5~ sinusoidal displacement of the chordotonal strand (movement) elicited a burst of spikes in the root 3 sensory nerve (r3 sensory) containing the afferents from the chordotonal organ, and a burst of EPSPs in the interneurone (asc. int.). B Six superimposed sweeps triggered from a sensory spike in root 3 show that the EPSPs occur with a constant latency. Dashed arrows indicate EPSPs that followed other spikes from the same afferent with the same latency. C Averaged EPSPs triggered from 1 sensory spike at different membrane potentials of the interneurone. Depolarizing current (+ 1 nA) reduced the size of the potential compared to normal resting potential (rest), while progressive increases in hyperpolarizing current ( - 1 nA and - 2 nA) produced progressive increases in the size of the EPSP. Each trace is an average of 50 occurrences of the EPSP triggered by an afferent spike

afferent made a connection with the interneurone (Fig. 2Ci) while another did not (Fig. 2Cii). These results suggest that many, but not all, chordotonal afferents make connections with this interneurone. The potentials evoked in a different ascending interneurone following imposed chordotonal movements summed as the velocity of the stimulus was increased (Fig. 3). Chordotonal afferents made direct synaptic connections with this ascending interneurone (Fig. 3A) which received short latency EPSPs (Fig. 3B). When the chordotonal strand was moved at 10~ bursts of EPSPs occurred during the opening and closing phases of the ramp (Fig. 3Ci). A group of small amplitude extracellular spikes, that gave rise to the EPSPs in the interneurone, are visible in the root 3 sensory recording during opening (arrows). At 20~ the sensory spikes occurred at a higher frequency, and other units recruited, during the opening movement and the EPSPs evoked in the ascending interneurone summed to produce a larger potential (Fig. 3Cii). At higher stimulus velocities (40~ and 80~ afferents with still larger extracellular amplitudes were recruited in the sensory root recording and the EPSPs in the interneurone were again increased in size (Fig. 3Ciii & iv). The morphology of this interneurone is shown in Fig. 3A. This interneurone and interneurone NE-1 (Figs. 1 and 2) receive inputs from the chordotonal afferents during both the opening and closing movements although their inputs during opening generate larger potentials. The interneurone shown in Fig. 3A has not been previously described in studies of ascending interneurones (Nagayama et al. 1993).

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interneurone. A Drawing of the interneurone (NE-1) in the terminal ganglion as viewed ventrally with anterior at the top. B|-iv Signal averages of EPSPs in the interneurone (asc. int.) triggered from 4 different chordotonal afferents in the same animal. Each trace is an average of 128 occurrences of the EPSPs triggered from their respec-

Pattern of connectivity with local interneurones Spiking local interneurones. Spiking local interneurones also receive direct synaptic inputs from the chordotonal afferents (Fig. 4). A 0.1 Hz sinusoidal movement o f the chordotonal strand with a 5 ~ peak displacement produced a burst o f EPSPs in the interneurone on top of a maintained depolarization throughout the opening phase of the movement (Fig. 4A). Four superimposed sweeps of the oscilloscope triggered from a single chordotonal afferent in root 3 show that the EPSPs follow the sensory spike with a short and constant latency (Fig. 4B). Signal averaging from a second afferent in root 3 shows that it also made a short latency connection with the same spiking local interneurone. Thus, chordotonal afferents make convergent connections with spiking local interneurones. The morphology of this local interneurone is shown in Fig. 4D. Nonspiking local interneurones. Of the 4 nonspiking local interneurones that received proprioceptive inputs none appeared to receive afferent input during low velocity movements (2-40~ of the chordotonal organ strand. Excitatory potentials could only be seen clearly during

tive sensory afferents. Ci, ii Signal averages from the same interneurone in a different crayfish showing that some (Ci), but not all (Cii), chordotonal afferents made connections with the interneurone. Each trace is an average of 650 occurrences of the EPSP triggered from their respective sensory spikes

higher velocity stimulation. F o r example, ramp displacements of 200~ resulted in a high frequency burst of sensory spikes in root 3 and compound EPSPs in a nonspiking interneurone (Fig. 5A), Signal averaging revealed an EPSP in the nonspiking local interneurone that followed the afferent spike in root 3 with a short latency (Fig. 5B). The same chordotonal afferent that was recorded extracellularly was later recorded intracellularly in root 3 as it entered the ganglion. The intracellular spike, recorded at a distance of 3.8 mm from the extracellular electrode followed the extraceUular spike with a delay o f 1.9 ms. This then gives a conduction velocity of 2.0 m/s for this particular chordotonal afferent. The nonspiking local interneurone was recorded at a distance of 800 gm from the intracellular afferent recording site. Allowing for the conduction velocity of the afferent, and the distance between the intracellular recording sites of the afferent and interneurone, a central latency between the afferent spike and interneurone EPSP of 0.9 ms can be calculated. Nonspiking local interneurones also receive convergent inputs from chordotonal afferents. Signal averages triggered from 2 different sensory spikes in root 3 show that short latency EPSPs in the interneurone follow each


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2ms Fig. 3A-C. Effects on a different interneurone of moving the chordotonal organ strand with different velocities. A Drawing of the interneurone in the terminal ganglion as viewed ventrally. B The interneurone (asc. int.) received direct short latency inputs from a chordotonal afferent. Each trace is an average of 74 occurrences

triggered from the extracellularly recorded chordotonal afferent spike. Ci-iv Increasing the velocity of the imposed movement recruited progressively more afferents during the movement phase of the stimulus and led to a summation of EPSPs in the interneurone. Stimulus velocities (i), 10~ (ii), 20~ (iii), 40~ and (iv) 80~

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Fig. 4A-D. A spiking local interneurone was excited by afferents from the chordotonal organ. A A 5~ sinusoidal displacement of the chordotonal organ strand evoked a burst of EPSPs and a sustained depolarization in the interneurone (sp. local int.) during the stretch phase of the imposed movement. B Four superimposed sweeps triggered from a sensory spike in root 3 show that the EPSP occur-

red with a short and constant latency. C A signal average triggered from another chordotonal afferent (50 occurrences) shows that this afferent also made a short latency connection with the same interneurone. D Morphology of the spiking local interneurone in the terminal ganglion as viewed ventrally with anterior to the top

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ns. localint. 3ms Fig. 5A-D. Afferents from the chordotonal organ synapse on a nonspiking local intemeurone. A A ramp displacement of the chordotonal strand at 200~ produced a burst of spikes in the sensory nerve and compound potentials in the nonspiking interneurone (ns. local int.). B Signal averages (25 occurrences), triggered from a sensory spike in root 3, of an EPSP in the interneurone and an intracellularly recorded spike in the same afferent in root 3 as it spike (Fig. 5Ci, ii). The morphology of this interneurone (shown in Fig. 5D) is typical of a PL-type nonspiking interneurone (Nagayama and Hisada 1987).

Divergent patterns of connections from the chordotonal afferen ts Sequential recordings of different interneurones in the same crayfish ,made it possible to examine the divergent connections made by the chordotonal afferents. An ascending interneurone received a short latency EPSP from a particular chordotonal afferent (Fig. 6Ai). The same amplitude extracellular sensory spike was also followed by a short latency EPSP in a spiking local interneurone (Fig. 6Aii). Afferents also made divergent connections with ascending interneurones (Fig. 6Bi) and nonspiking interneurones (Fig. 6Bii). However, not all afferents that made connections with a given interneurone made connections with other interneurones that received chordotonal organ input. For example, sequential recordings from another ascending interneurone and a nonspiking interneurone showed that an afferent that produced short latency EPSPs in the local interneurone (Fig. 7A) produced small, long latency potentials in the ascending interneurone (Fig. 7B). Due to their long latency these potentials must be produced through indirect pathways.

3ms entered the ganglion. The neurones were recorded sequentially in the same preparation. The EPSP in the interneurone followed the afferent spike with a short and constant latency. Ci, ii Signal averages triggered from 2 different sensory afferents showing short latency convergent inputs onto this nonspiking local intemeurone. D Morphology of the interneurone in the terminal ganglion as viewed ventrally with anterior to the top

Output effects of proprioceptive interneurones During escape swimming the exopodites promote during abdominal flexion and remote during extension. These movements, o f approximately 50 ~ can occur within periods of 25 ms (Cooke and MacMillan 1985) leading to velocities in excess of 2000~ We know that the CO afferents can code for velocities of over 1000~ (Field et al. 1990). Since the reflex effects of chordotonal stimulation upon the 2nd root motor neurones were weak we used repetitive stimulation using ramp displacements with velocities of 850~ to examine the reflex effects upon uropod motor neurones. Such repetitive stimulation produced only a slight reduction in the spontaneous discharge of the reductor motor neurone (Fig. 8A). Some ascending interneurones, for example, fnterneurone NE-1, that received inputs from sensory afterents in the terminal ganglion have no output effects on uropod motor neurones (Nagayama et al. 1993). Other ascending interneurones which receive proprioceptive inputs do, however, affect the uropod motor neurones. Another type of ascending interneurone, interneurone CI-1 described by Nagayama et al. (1993), was spontaneously active at its normal resting potential and received inputs from low and high velocity threshold afferents. Hyperpolarizing the interneurone by -1 nA, to prevent action potentials revealed potentials related to


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Fig. 6A-C. Divergent pattern of connections of chordotonal afferents between ascending and local interneurones. Ai, ii Signal averages (50 occurrences) triggered from the same chordotonal afferent showing divergent outputs onto an ascending interneurone and spiking local interneurone respectively. Bi, ii Another afferent made divergent connections with a different ascending interneurone

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and nonspiking local interneurone. Each trace is an average of 50 occurrences of the EPSPs triggered from their respective sensory spikes. Recordings of the interneurones were made sequentially in the same crayfish. C Summary of the sensory neurone to interneurone connections


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c h o r d o t o n a l stimulation. W h e n the stimulus velocity was increased the EPSPs s u m m e d and gave rise to a spike. R a m p displacements with a velocity o f 100~ gave rise to a c o m p o u n d E P S P in the interneurone (Fig. 8Bi), while velocities o f 850~ gave rise to spikes (Fig. 8Bii). Injecting depolarizing current ( + 1 nA) into this intern e u r o n e increased the rate o f s p o n t a n e o u s discharge and at the same time decreased the s p o n t a n e o u s discharge rate o f r o o t 2 m o t o r neurones (Fig. 8D), including the reductor m o t o r neurone.

Fig. 7A, B. Selectivity of connections. Not all chordotonal afferents made divergent connections with interneurones that received chordotonal input. Superimposed sweeps showing that an afferent with a velocity threshold of 80~ made a short latency connection with a nonspiking local interneurone (A) but not with an ascending interneurone (B). Recordings of the interneurones were made sequentially in the same crayfish

N o n - s p i k i n g local interneurones that received chord o t o n a l input also influenced u r o p o d m o t o r activity. W h e n + 1 n A depolarizing current was injected into a neuropilar process o f a P L - t y p e nonspiking interneurone there was a reduction in the s p o n t a n e o u s rate o f discharge o f the r o o t 2 m o t o r neurones recorded extracellularly (Fig. 9A). Increasing the current injected into the nonspiking interneurone to + 3 n A p r o d u c e d a progressively greater inhibition o f the m o t o r neurones (Fig. 9B).

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Fig. 8A-D. Reflex effects of chordotonal organ stimulation. A Repetitive stimulation (100 cycles at 850~ produced a burst of sensory spikes in root 3 and decreased the firing frequency of the reductor motor neurone spike in root 2 (indicated by arrows). B-D Output effects of an ascending interneurone on root 2 motor neurones. The interneurone was held hyperpolarized by - 1 nA to prevent it from spiking tonically. Bi, ii Increasing the velocity of

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N o identified m o t o r n e u r o n e , i n c l u d i n g 2 fast a n d 2 slow o p e n e r m o t o r n e u r o n e s , 2 fast a n d 2 slow closer m o t o r n e u r o n e s , a t e l s o n - u r o p o d a l i s a n d a dorsal r o t a t o r m o t o r n e u r o n e , received direct i n p u t s f r o m the chord o t o n a l afferents themselves.

Fig. 9A, B. Output effects of a nonspiking local interneurone on root 2 motor neurones. A Injecting + 1 nA depolarizing current into a nonspiking interneurone that receives CO input produced an inhibition of the root 2 motor neurones recorded extracellularly. B Greater depolarizing current (+ 3 nA) produced a greater inhibitory effect on the motor neurones. Note the bridge circuit on the intracellular recording amplifier was not balanced

Convergence of different sensory modalities onto ascendin9 interneurones Some a s c e n d i n g i n t e r n e u r o n e s received i n p u t s f r o m afferents coding different stimulus modalities. F o r example,


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Fig. 10A, B. Convergence of different sensory modalities onto ascending interneurones. Ai Repetitive sinusoidal stimulation of the chordotonal strand at 10 Hz produced a spike in the interneurone followed during subsequent cycles by EPSPs. Stimulation of sensory hairs on the endopodite (All) and exopodite (Aiii) also led to EPSPs and spikes in the interneurone. Bi A different interneurone was stimulated at 5 Hz which led to EPSPs in the interneurone. Bii Stimulation of the sensory hairs on the endopodite had no effect

an ascending interneurone received input during repetitive stimulation of the chordotonal strand at 10 Hz (Fig. 10Ai). Stimulation of exteroceptive hairs on either the endopodite (Fig. 10Aii) or the exopodite (Fig. 10Aiii) also gave rise to bursts of EPSPs and spikes. A different interneurone, however, did not receive inputs from these hairs (Fig. 10B). Repetitive chordotonal stimulation at 5 Hz gave rise only to depolarizing potentials in the interneurone but no spikes (Fig. 10Bi). Stimulation of exteroceptive hairs on the endopodite had no effect (Fig. 10Bii) nor did stimulating hairs on the exopodite (not shown).

time is similar to those measured for connections between exteroceptive afferents innervating tactile hairs on the exopodite and ascending interneurones in the crayfish terminal ganglion (Nagayama and Sato 1993), connections of coxo-basipodite chordotonal afferents with motor neurones in the crayfish 4th thoracic ganglion (El Manira et al. 1991a) and connections between femoral chordotonal afferents and spiking and nonspiking local interneurones (Burrows 1987; Burrows et al. 1988), intersegmental interneurones (Laurent and Burrows 1988) and motor neurones (Burrows 1987) in the locust. All of these connections are thought to be monosynaptic.

Discussion

Selective connections of chordotonal afferents

The chordotonal afferents which monitor the movements of the tailfan exopodite relative to the endopodite make excitatory connections with both spiking and nonspiking local interneurones and ascending interneurones. These connections are thought to be direct and chemically mediated since a) the evoked EPSPs in the interneurones consistently follow the spikes in single chordotonal afferents with a short and constant latency, and b) the amplitude of the EPSPs is dependent upon the membrane potential of the interneurone. Sequential intraceUular recordings from a nonspiking local interneurone and a presynaptic chordotonal afferent in the same crayfish made it possible to calculate the synaptic delay precisely at 0.9 ms. This central delay

The chordotonal afferents make connections with a restricted number of neurones. The probability of encountering any interneurone with inputs from the chordotonal afferents was low (less than 10 % ofinterneurones recorded had inputs from the CO). For example, from stable recordings of over 160 intersegmental interneurones from 35 crayfish only 21 were found to receive inputs from the CO, and most of these could be identified as the 3 types described here. It is possible, however, that other intersegmental interneurones encountered using potassium acetate electrodes were not of these 3 types since their anatomy was not revealed. What is clear is that few interneurones of any type are involved in the processing of information from this proprioceptor,

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which may be due, in part, to the few sensory afferents innervating this receptor. Identification of only the intersegmental interneurones was possible. Few terminal ganglion local interneurones have been identified as unique individuals apart from those that have a particularly large size or unusual morphology. One nonspiking local interneurone that has been identified is the local, directionally sensitive (LDS) interneurone that mediates lateral inhibition of mechanosensory interneurones (Reichert et al. 1983). LDS, however, has a bilateral branching pattern and large diameter processes unlike most of the nonspiking interneurones in the terminal ganglion that are mainly restricted to one half of the ganglion (Nagayama and Hisada 1987), and it is mainly for this reason that it has been possible to identify this interneurone as an individual. Previous detailed studies of both spiking and nonspiking local interneurones in the terminal ganglion were unable to identify interneurones as individuals or as members of a small pool with similar input/output properties and anatomy (Nagayama and Hisada 1985, 1987). Similarly without further more detailed anatomical and physiological studies we are not yet certain as to whether the local interneurones described in this study are unique individuals. The afferents make selective divergent connections with different types of interneurone. All the spiking interneurones that received CO input received direct input during low and high velocity stretches applied to the chordotonal strand. Nonspiking interneurones, on the other hand, only appeared to receive input during high velocity stimulation. Afferents with different velocity thresholds project to different areas in the terminal ganglion, dependent upon their velocity threshold (Nagayama and Newland 1993); those with high thresholds project posteriorly, those with low thresholds project anteriorly and those with intermediate velocity thresholds project in between. From the interneurones that we have so far examined it appears that there is no clear relationship between the branching patterns of the interneurones and their response properties. Interneurones have extensive branching areas that cover much of the afferent map. Thus further studies of the interneurones and afferents are needed to determine if there is any correlation similar to that described in locusts (Burrows and Newland 1993).

Proprioceptive processing in other arthropod systems The coxo-basipodite (CB) chordotonal organ in the walking legs of crustaceans is innervated by about 50 sensory neurones. Stretches applied to the chordotonal organ strand give rise to responses in many leg motor neurones through both local and interjoint reflexes (Clarac et al. 1978). As with the locust femoral chordotonal organ (FCO), the CB afferents make direct, chemically mediated connections with a number of leg motor neurones and are responsible for a resistance reflex movement of the leg (El Manira et al. 1991a, b). The crayfish CB afferents make highly selective monosynaptic connections with particular motor neurones. Only some 4-9 CB afferents out of 50 make connections with each

leg motor neurone (El Manira et al. 1991a). This contrasts with the crayfish uropod CO in which we know that up to 5 out of approximately 12 afferents make connections with a given ascending interneurone, whereas we have found no direct connections between afferents and motor neurones. We do not know how information from CB afferents is processed by interneurones in the thoracic ganglia of crustaceans so that comparisons between this and the processing of CO information in the terminal ganglion are not yet possible. In common with the CB afferents of the crayfish leg, afferents innervating the locust FCO mediate both local (Field and Burrows 1982) and interjoint resistance reflexes (Field and Rind 1981). The FCO afferents make monosynaptic connections with a number of spiking and non-spiking local interneurones (Burrows 1987; Burrows et al. 1988), intersegmental interneurones (Laurent and Burrows 1988) and leg motor neurones (Burrows 1987). Information about the stimulus is preserved within the local circuits so that an appropriate motor response can be made. For example, some spiking local interneurones respond preferentially to one direction of displacement of the FCO while others respond to the opposite direction of movement. The responses of the crayfish local circuit neurones to CO stimulation is not as clear. Interneurones receive input during both directions of movement although depolarization during opening is usually greater than that during closing. The true directional properties of the interneurones remain to be examined in the future. It is not clear why the locust FCO and crayfish CB chordotonal organ patterns of connectivity are so different to the crayfish uropod CO. It may be related to the restricted plane of movement and velocities of leg movements compared to the complex movements and large range of movement velocities of the uropods. Since the uropods are used in a wide range of behavioural acts more subtle and flexible control could be achieved through the many layers of local and intersegmental interneurones.

Reflex effects of chordotonal stimulation Stimulation of the chordotonal organ produces very weak reflex effects on exopodite motor neurones. Repetitive stimulation at velocities likely to be experienced during the tail-flip (Webb 1979) produced a reduction in the spontaneous firing frequency of the reductor motor neurone. Current injection into some of the interneurones encountered in this study produced similar effects on the same motor neurone. We did not find any clear output effects of spiking local interneurones on the opener and closer motor neurones. The spiking local interneurones are known, however, to be presynaptic to nonspiking local interneurones that control the movements of the uropods (Nagayama and Hisada 1991) so that their effects on the motor pattern, given that it is rather weak, may be masked by the effects of other interneurones. Chordotonal afferents, therefore, have the potential to have a wide range of effects on interneurones and motor neurones of the local circuits of the terminal ganglion and during slow postural movements may con-

P.L. Newland, T. Nagayama: Parallel processing in crayfish interneurones tribute to setting the general level of excitability o f the local circuit neurones. The uropods are controlled by m a n y muscles which are innervated by m o t o r neurones with somata in the terminal ganglion. Our present study was concerned mainly with the pools of m o t o r neurones that move the expodite and as such would be involved in local reflexes. These m o t o r neurone pools contain m a n y m o t o r neurones producing similar movements of the exopodite. F o r example, Larimer and Kennedy (1969b) and Wine (1984) showed that there are 6 m o t o r neurones that open the exopodite, just over half o f which are tonic. The closer m o t o r neurone pool contains 2 fast and 2 slow m o t o r neurones. We recorded from most of these m o t o r neurones as well as 2 others that are involved in moving the protopodite, the dorsal rotator and the telson uropodalis, but none were found to receive direct CO input. This contrasts greatly with the locust F C O and crayfish CB chordotonal organ in which powerful connections are made directly between the afferents and m o t o r neurones, suggesting that the role of the uropod CO is not primarily to mediate local reflexes of the exopodite. High velocity stimulation of the chordotonal strand often leads to action potentials in m a n y interneurones. Some o f these interneurones (for example, N E 1) do not appear to have output connections with uropod m o t o r neurones in the terminal ganglion ( N a g a y a m a et al. 1992). An ultrastructural analysis (Sato et al. 1993) failed to find any output synapses from N E 1 in the terminal ganglion, consistent with physiological observations. The m o r p h o l o g y of interneurone N E - 1 is distinct from other interneurones in the terminal ganglion in that it has a large diameter soma, neurites and axon and can be clearly recognisable in different animals ( N a g a y a m a et al. 1992). Zucker (1972) showed that an interneurone of this type with a large diameter axon in the abdominal connectives, and in a similar position, makes a direct electrical connection with the lateral giant fiber. The lateral giant fibres are responsible for mediating the short latency rapid tail-flip of decapod crustaceans. In an earlier paper we showed that the intrasegmental CO afferents had conduction velocities in the range 1.6-7.3 m/s (Nagayam a and Newland 1993). These conduction velocities are 2 times greater than those of afferents innervating tactile hairs on the uropods, 0.7-3.4 m/s ( N a g a y a m a and Sato 1993), and CO afferents of the CB leg joint, 0.4-3.5 m/s (El Manira et al. 1991a). These fast conduction times of the uropod CO afferents again suggest that this CO organ m a y be implicated in the tail-flip escape system where short latencies and rapid transmission are essential. That interneurone N E - 1 makes connections with L G and that m a n y interneurones only spike during high velocity movements certainly support this hypothesis and suggest that an examination of other elements in the escape circuitry such as the m o t o r giant and flexor inhibitor m o t o r neurones m a y reveal the precise role of this CO in the control of behaviour. Acknowledgements. We thank Malcolm Burrows, Fred Kuenzi, Tom Matheson and David Parker for their valuable comments on the manuscript. PLN was supported by a visiting fellowship from

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Hokkaido University, a Royal Society Research Grant and by a NIH grant (NS16058) to Malcolm Burrows. TN was supported by a grant from the Japanese Ministry of Education, Science and Culture (03740365). This work was also supported by a Human Frontiers Science Program grant to Malcolm Burrows and Mituhiko Hisada.

References Bowerman RF, Larimer JL (1974a) Command fibres in the circumoesophageal connectives of crayfish. I. Tonic fibres. J Exp Biol 60:95-117 Bowerman RF, Larimer JL (1974b) Command fibres in the circumoesophageal connectives of crayfish. II. Phasic fibres. J Exp Biol 60:119-134 Burrows M (1987) Parallel processing of proprioceptive signals by spiking local interneurones and motor neurones in the locust. J Neurosci 7:1064-1080 Burrows M (1988) Responses of spiking local interneurones in the locust to proprioceptive signals from the femoral chordotonal organ. J Comp Physiol A 164:207-217 Burrows M, Newland PL (1993) Correlation between the receptive fields of locust interneurones, their dendritic morphology, and the central projections of mechanosensory neurones. J Comp Neurol (In press) Burrows M, Laurent GJ, Field LH (1988) Proprioceptive inputs to non-spiking local interneurons contribute to local reflexes of a locust hindleg. J Neurosci 8:3085-3093 Clarac F, Vedel JP, Bush BMH (1978) Intersegmental reflex coordination by a single joint receptor organ (CB) in rock lobster walking legs. J Exp Biol 73:2946 Cooke IRC; MacMillan DL (1985) Further studies of crayfish escape behaviour. I. The role of the appendages and the stereotyped nature of non-giant escape swimming. J Exp Biol 118:351-365 El Manira A, Cattaert D, Clarac F (1991a) Monosynaptic connections mediate resistance reflex in crayfish (Procambarus elarkii) walking legs. J Comp Physiol A 168:337-349 E1 Manira A, Dicaprio RA, Cattaert D, Clarac F (1991b) Monosynaptic interjoint reflexes and their central modulation during fictive locomotion in crayfish. Europ J Neurosci 3:1219-1231 Field LH, Burrows M (1982) Reflex effects of the femoral chordotonal organ upon leg motor neurones of the locust. J Exp Biol 101:265-285 Field LH, Rind RC (1981) A single insect chordotonal organ mediates inter- and intra-segmental leg reflexes. Comp Biochem Physiol 68:99-102 Field LH, Newland PL, Hisada M (1990) Physiology and structure of three new uropod proprioceptors in the crayfish Procambarus clarkii. J Exp Biol 154:179-200 Larimer JL, Kennedy D (1969a) The central nervous control of complex movements in the uropods of crayfish. J Exp Biol 51 : 135-150 Larimer JL, Kennedy D (1969b) Innervation patterns of fast and slow muscle in the uropods of crayfish. J Exp Biol 51:119-133 Laurent G, Burrows M (1988) A population of ascending intersegmental interneurones in the locust with mechanosensory inputs from a hind leg. J Comp Neurol 275:1-12 Mill PJ (1976) Structure and function of proprioceptors in the invertebrates. Chapman and Hall, London Nagayama T, Hisada M (1985) Crayfish local bilateral spiking interneurones: role in contralateral uropod pattern formation. Zool Sci 2:641-651 Nagayama T, Hisada M (1987) Opposing parallel connections through crayfish local non-spiking interneurons. J Comp Neurol 257:347-358 Nagayama T, Hisada M (1991) Sensory processing of crayfish nonspiking local interneurones in response to mechanical stimulation of the tailfan. Zool Sci 8:1044

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Nagayama T, Newland PL (1993) A sensory map based on velocity threshold of sensory neurones from a chordotonal organ in the tailfan of the crayfish. J Comp Physiol A 172:7-15 Nagayama T, Sato M (1993) The organization of exteroceptive information from the uropods to ascending interneurones of the crayfish. J Comp Physiol A 172:281-294 Nagayama T, Takahata M, Hisada M (1984) Functional characteristics of local non-spiking interneurons as the premotor elements in crayfish. J Comp Physiol A 154:499-510 Nagayama T, Takahata M, Hisada M (1986) Behavioural transition of crayfish avoidance reaction in response to uropod stimulation. Exp Biol 46:75-82 Nagayama T, Isogai Y, Sato M, Hisada M (1993) Intersegmental ascending interneurones controlling uropod movements of the crayfish Procambarus clarkii. J Comp Neurol (In press) Newland PL, Neil DM (1987) Statocyst control of uropod righting reactions in different planes of body tilt in the Norway lobster, Nephrops norveyicus. J Exp Biol 131:301-321 Newland PL (1989) The uropod righting reaction of the crayfish Procambarus clarkii (Girard): an equilibrium response driven by two largely independent reflex pathways. J Comp Physiol A 164: 685-696 Newland PL, Neil DM (1990a) The tail flip of the Norway lobster, Nephrops norvegicus. I. Giant fibre activation in relation to swimming trajectories. J Comp Physiol A 166:517-527 Newland PL, Neil DM (1990b) The tail flip of the Norway lobster, Nephrops norve#icus. II. Dynamic righting reactions induced by body tilt. J Comp Physiol A 166:529-536 Newland PL, Cattaert D, Neil DM, Clarac F (1992) Steering reactions as adaptive components of the tail-flip in the spiny lobster Jasus lalandii. J Exp Biol 164:261-282 Reichert H, Plummer M, Hagiwara G, Roth RL, Wine JJ (1982) Local interneurons in the terminal abdominal ganglion of the crayfish. J Comp Physiol 149:145 162

Sato M, Nagayama T, Hisada M (1993) Distribution of synapses on two types of ascending interneurons in the crayfish, Procambarus clarkii (Girard). Cell Tissue Res (In press) Stewart WW (1978) Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell 14:741-759 Takahata M, Hisada M (1986) Sustained membrane potential change of uropod motor neurons during the fictive abdominal movement in crayfish. J Neurophysiol 56:702-717 Takahata M, Nagayama T, Hisada M (1981) Physiological and morphological characterization of anaxonic non-spiking interneurones in the crayfish motor control system. Brain Res 226:309-314 Takahata M, Yoshino M, Hisada M (1981) The association of uropod steering with postural movement of the abdomen in the crayfish. J Exp Biol 91 : 341-345 Van Harreveld A (1936) A physiological solution for freshwater crustaceans. Proc Soc Exp Biol Med 34:428-442 Webb PW (1979) Mechanics of escape responses in crayfish (Orconectes virilis). J Exp Biol 79:245-263 Wine JJ (1984) The structural basis of an innate behavioural pattern. J Exp Biol 112:283-319 Wine JJ, Krasne FB (1972) The organisation of escape behaviour in the crayfish. J Exp Biol 56:1-18 Yoshino M, Takahata M, Hisada M (1980) Statocyst control of the uropod movement in response to body rolling in crayfish. J Comp Physiol 139:243-250 Zucker RS (1972) Crayfish escape behavior and central synapses. I. Neural circuit exciting lateral giant fiber. J Neurophysiol 35 : 599-620