Synaptic connections in the cerebral ganglion ofAplysia

Brain Research, 100 (1975) 209-214 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Synaptic connections in the cerebral ganglion of

STEVEN

M. F R E D M A N

AND

209

Aplysia

BEHRUS JAHAN-PARWAR

Worcester Foundation/br Experimental Biology, Shrewsbury, Mass. 01545 (U.S.A.) (Accepted August 26th, 1975)

The cerebral ganglion of Aplysia appears to be a major center for processing sensory information. The neural pathways from the major sense organs, the anterior and posterior tentacles z,5,8, the eyes 11 and the statocysts 1 converge in this ganglion. Stimulation of the anterior tentacles with natural food ingredients elicits a stereotyped feeding behavior 3,5,7,9,1°. The same stimuli also produce changes in activity in well defined groups of cerebral ganglion neuronsS,L Furthermore, evidence from our laboratory suggests that the feeding response of Aplysia can be classically conditioned 6. We have studied the cellular organization of the cerebral ganglion of Aplysia californica. The ganglion consists of bilaterally symmetrical pairs of neuronal clusters (Fig. 1). The exact location of each cluster, their physiological properties and their peripheral connections have been described elsewhere 8. The biochemical properties of some of these neurons have also been reported 12. This paper describes a neural circuit that involves two pairs of clusters, the A and B clusters, which are symmetrically located in the caudal quadrants of the ganglion. The identification of synaptic connections between neurons in the A and B clusters is of particular interest since these neurons respond to chemical, mechanical, and visual stimulation via pathways from the sense organs of the head regionS,V,s, 11 and the paired activation of these pathways can result in behavioral modification 6. The A and B cluster neurons, although located very close to each other and having similar appearances, differ markedly in their properties. The A cluster neurons are usually silent and fire a brief burst of spikes only upon penetration. The B neurons maintain a steady irregular spontaneous firing pattern. Although the A and B neurons exhibit both inhibitory postsynaptic potentials (IPSPs) and excitatory postsynaptic potentials (EPSPs), in the A ceils it is the former that predominate, while in the B cells it is the latter. There is some common input to both groups of neurons, but o f opposite signs. Similarly, both groups respond to tactile and chemical stimulation of the anterior and posterior tentaclesS,7, 8. Upon touching a tentacle the B neurons give a short burst of spikes, or summated EPSPs. The A neurons receive IPSPs to the same stimulus, but at a slightly longer latency. Occasionally tactile stimulation will produce a biphasic response and a single spike in some A neurons. The isolated, but otherwise intact CNS of Aplysia was used to investigate con-

210

Fig. I. A diagrammatic representation of the cerebral ganglion. The ganglion is bilaterally symmetrical with 7 pairs of neuron clusters, labeled A-G. There is also an eighth, asymmetric cluster in the right rostral region, the H clusters. Abbreviations: C-B, cerebral-buccal connective; C-PL, cerebral-pleural connective; C-P, cerebral-pedal connective; ULAB, upper labial nerve; AT, anterior tentacular nerve; PT, posterior tentacular nerve; O, optic nerve; LLAB, lower labial nerve. nectivity a m o n g n e u r o n s in the c e r e b r a l g a n g l i o n . | n t r a c e l l u l a r c u r r e n t injection into the A n e u r o n s reveals t h a t t h e y m a k e e x c i t a t o r y m o n o s y n a p t i c c o n n e c t i o n s with b o t h ipsilateral a n d c o n t r a l a t e r a l B n e u r o n s (Fig. 2). Since each A and B cluster c o n t a i n s at least 15-25 large n e u r o n s , it is i m p o s s i b l e to test all the p e r m u t a t i o n s o f c o n n e c tions a m o n g all the A a n d B cells. N o n e t h e l e s s , o n e c a n d e m o n s t r a t e t h a t there are e x t e n s i v e i n t e r c o n n e c t i o n s a m o n g these n e u r o n s . T h e p r o c e d u r e for testing was as follows. T h r e e B n e u r o n s o n o n e side were i m p a l e d w i t h m i c r o p i p e t t e s . W i t h a f o u r t h

aI

i

b

!

i L ................................

L ......................

I

Fig. 2. Monosynaptic connections between A and B neurons, a: 3 simultaneously impaled B neurons. Current injected into a contralateral A neuron produces a spike and EPSPs in all 3 B cells. Although the amplitude of the EPSPs varies, the latency is the same for all 3 cells. Time: 20 msec. Gain: A, 20 mV; B, 5 inV. b: repetitive sweeps showing constant latency between an action potential in an A neuron and an EPSP in an ipsilateral B neuron. Note the decrement in the amplitude of the EPSP. Time: 10 msec. Gain: A, 20 mV; B, 2 inV.

211 TABLE I MONOSYNAPTIC

CONNECTIONS

Neurons stimulated

A B

No.

49 15

AMONG A AND

B CLUSTER

NEURONS

No. o f B neurons recorded Synaptic connections

No. of failures

No. o f preparations

33* 15"*

8 4

8 5

* lpsilateral neurons, 16, contralateral neurons, 17. ** lpsilateral neurons, 8; contralateral neurons, 7.

electrode ipsilateral and contralateral A neurons were impaled sequentially and systematically. This avoided the possibility o f repeated impalement o f the same neuron in the same preparation. Synaptic connections were tested by passing short depolarizing pulses through the electrode in the A neuron. Connections were judged to be monosynaptic on the basis o f PSPs with short and constant latency following at high frequency spikes in the presynaptic cell. This procedure was then repeated with successive groups o f 3, not previously tested B neurons. As shown in Table I extensive m o n o synaptic connections were f o u n d between A cluster neurons and the ipsilateral and contralateral B cluster neurons. These synapses were observed in every preparation where the connections were tested. However, the amplitude o f the EPSP recorded in the B neurons varied considerably, both within a preparation and a m o n g different preparations. I n addition to these excitatory monosynaptic connections, similar connections exist between B neurons (Fig. 3). The m e t h o d o l o g y used to demonstrate this was the same as with the synapses between A and B neurons. Three B neurons were impaled and as m a n y ipsilateral and contralateral B neurons as possible were systematically impaled with a fourth electrode and tested by intraceIlular current injection. F o r each presynaptic cell impaled, synaptic connections made by the first three back on

II2

!

Fig. 3. Monosynaptic connections between B neurons. Repetitive sweeps showing constant latency between an action potential in one B neuron (B1) and an EPSP in another, contralateral, B neuron (B2). Time: 20 msec. Gain: Ba, 20 mV; Bz, 2 mV.

212

B1 B2

Fig. 4. Positive feedback among A and B neurons, lntracellular current injection produced a spike (arrow) in an A neuron which was followed by EPSPs in contralateral B neurons B1 and B2, and a spike in Ba. Subsequent EPSPs in the B cells were probably due to synaptic connections among B cells. The excitation produced was of sufficient magnitude to cause A to fire and re-excite B2 and l~a. The long latency between the spike in the B neurons and the spike in A indicates that the feedback is polysynaptic. A did not fire unless at least one B cell spiked. This was observed repeatedly following activation of the same and other A neurons. Time : 20 msec. Gain : A, 20 mV; B, 10 inV.

to it were also tested. It a p p e a r s that the B cells m a k e reciprocal excitatory m o n o synaptic connections with each other (Table I). As with the A cell to B cell synapses, there were large variations in the a m p l i t u d e o f the E P S P evoked by stimulating B neurons. Failure to d e m o n s t r a t e connections either between A a n d B or B a n d B neurons was usually associated with a p o o r resting potential in the follower cell a n d / o r a noisy recording electrode. Similar testing o f the A cells failed to reveal a n y direct synaptic connections a m o n g them. There do not a p p e a r to be m o n o s y n a p t i c connections either a m o n g ipsilateral A neurons or with those on the c o n t r a l a t e r a l side (20 cells f r o m 5 preparations). N o r does there a p p e a r to be any direct ( m o n o s y n a p t i c ) f e e d b a c k from the B neurons back on to the A cells. However, there m a y be some p o l y s y n a p t i c feedback. This is s u p p o r t e d by the following observations. In one p r e p a r a t i o n where the A to B n e u r o n synapses exhibited b o t h large EPSPs ( a p p r o x i m a t e l y 10 mV) a n d low firing thresholds, a single spike in an A resulting f r o m current injection elicited EPSPs o f sufficient m a g n i t u d e to p r o d u c e a spike in one o f the 3 i m p a l e d B follower cells. This resulted in a series o f EPSPs in all the B neurons, p r e s u m a b l y due to reciprocal excitation a m o n g the B neuron p o p u l a t i o n . F o l l o w i n g this the A n e u r o n fired a b u r s t o f spikes which re-excited the B neurons (Fig. 4). There are several reasons to believe t h a t the burst o f spikes in the A neuron was due to a f e e d b a c k loop. (1) The A neurons are n o r m a l l y silent and do not exhibit action potentials in the absence o f stimulation. (2) Spiking in the A neurons was d e p e n d e n t u p o n at least one B n e u r o n firing. I f the EPSPs in the i m p a l e d B neurons evoked by the initial (driven) spike in the A n e u r o n failed to reach threshold, then the subsequent burst o f spikes in the A n e u r o n were absent. (3) This could be obtained repeatedly with several different A neurons. In addition, the long latency from the B n e u r o n spike to the start o f the burst in the A neuron indicates that several synaptic delays are involved (see Figs. 2 and 3 for c o m parison with a m o n o s y n a p t i c connection.) F u r t h e r evidence for this p r e s u m e d feed-

,L B

(

/d~ j,~"4

213

II

)

!

!

6"

)

T' Fig. 5. Schematic representation of the synaptic connections among A and B neurons. All the synapses are excitatory. For simplicity only one A neuron and two B neurons on each side have been shown. The dashed line represents the feedback pathway which has an unknown number of neurons. Although only one neuron per side has been indicated, the pathway is probably polysynaptic with at least two neurons involved.

back came from other preparations. In these, A neurons held hyperpolarized exhibited EPSPs at long but constant latencies following spikes from current injection into B neurons. These EPSPs appeared to be greatly attenuated, having amplitudes of 1 mV or less, slow rise times, and long durations. This indicates that the source of the input was at a considerable distance from the recording electrode. Again, the long latency implies that there were several interposed synapses. The normally low amplitude of these EPSPs has made studying the connections difficult. It has not yet been possible to determine if the excitatory feedback is c o m m o n to all the A and B neurons or just to a few. To summarize, there are excitatory monosynaptic connections among two pairs of neuron clusters in the cerebral ganglion. Neurons in both A clusters synapse on all or almost all of the neurons in the B clusters. All or almost all of the B cluster neurons synapse on each other. None of the A cluster neurons are directly interconnected, but there appears to be a polysynaptic feedback pathway from some of the B cells back on to the A cells (Fig. 5). At the present time, we do not know the function of this circuit. One possibility is that it is involved in m o t o r control of the anterior body region, such as the defensive withdrawal reflex initiated when the oral veil region or the tentacles are touched, or head waving in response to chemical stimuli. There is evidence to support this hypothesis. Jahan-Parwar and Fredman s found that electrical stimulation of the pedal nerves produced antidromic action potentials in some of the A and B neurons. Filling the main branches of the anterior, medial and posterior pedal nerves with cobalt by axonal iontophoresis 4 resulted in some filled somas in the A and B clusters (Fredman and Jahan-Parwar, unpublished). This indicates that some of these neurons could potentially be motoneurons. Similar connections between groups of motoneurons have been related to the behavior of other molluscs is. Experiments in progress utilizing recording and stimulating the same clusters of neurons in intact animals

214 s h o u l d p r o v i d e the i n f o r m a t i o n n e e d e d to d e t e r m i n e the b e h a v i o r a l role p l a y e d by this n e u r o n a l circuit. T h i s w o r k was s u p p o r t e d by P H S G r a n t s N S l 1 4 5 2 a n d NS12483 to B.J.P.

1 DIJKGRAAF,S., UND HESSELS,H. G. A., Llber Bau und Funktion der Statocyste bei der Schnecke

Aplysia limacina, Z. vergl. Physiol., 62 (1969) 38-60. 2 FREDMAN,S. M., AND JAHAN-PARWAR,B., Central origin of cerebral ganglion nerves in Aplysio, Fed. Proc., 34 (1975) 405. 3 FRINGS, H., AND FRINGS, C., Chemosensory bases from food-finding in Aplysia/uliana (Mollusca, Opisthobranchia), Biol. Bull., 128 (1965) 211-217. 4 ILLS, J. F., AND MULLONEY,B., Procion yellow staining of cockroach motor neurons without the use of microelectrodes, Brain Research, 30 (1971) 397-400. 5 JAHAN-PARWAR,B., Behavioral and electrophysiological studies on chemoreception in Aplysia, Amer. Zook, 12 (1972) 525-537. 6 JAHAN-PARWAR, B., Classical conditioning, sensitization and pseudoconditioning in Aplysia, Proc. 3rd Ann. Meeting Soc. Neurosci., (1973) 239. 7 JAHAN-PARWAR,B., Chemoreception in gastropods. In D. DENTON (Ed.), Olfaction and Taste, 1Iol. V, Academic Press, New York, 1975, pp. 333-343. 8 JAHAN-PARWAR,B., AND FREDMAN, S. M., Cerebral ganglion of Aplysia: cellular organization and origin of nerves, Submitted. 9 KUPFERMANN, 1., Feeding behavior in Aplysia: a simple system for the study of motivation, Behav. Biol., 10 0974) 1-26. l0 PRESTON, R. J., AND LEE, R. M., Feeding behavior in Aplysia californica: role of chemical and tactile stimuli, J. comp. physiol. Psychol., 82 (1973) 368-381. 11 ROSSNER,K. L., Central projections of the ,4plysia visual system, Comp. Biochem. PhysioL, 48A (1974) 609-615. 12 WEINREICH,D., WEINER, C., AND MCCAMAN,R., Endogenous levels of histamine in single neurons isolated from CNS of Aplysia californica ,Brain Research, 84 0975) 341-345. 13 WILLOWS,A. O. O., DORSETT,O. A., AND HOYLE, G., The neuronal basis of behavior in Tritonia. III. Neuronal mechanism of a fixed action pattern, J. Neurobiol., 4 (1973) 255-285.