Analysis of potentiation in the cerebral ganglion of Aplysia.

Quantal analysis was used to characterize synaptic transmission between A and B neurons in the cerebral ganglion of Aplysia in control and during slow developing potentiation, a form of synaptic plasticity exhibited by these synapses. Control values of mean quantal content (m) and quantal size (q) estimated by the method of coefficient of variation (CV) were m approximately 6, q approximately 56 microV in the solution with Ca2+/Mg2+ = 5/200 and m approximately 18, q approximately 41 microV in the solution with Ca2+/Mg2+ = 55/150. There was a good correlation between an increase in the amplitude of excitatory synaptic potential and an increase in calculated quantal content (m(cv)) during potentiation. A decrease of Ca2+/Mg2+ ratio in the bath solution allowed observation of transmission failures and in some cases regular peaks on excitatory postsynaptic potential amplitude histograms. The latter provided more direct estimate of the quantal size. Induction of the potentiation in this solution, however, became difficult. In cases of successful potentiation induction, probability of failures was less than in control; distances between histogram peaks, reflecting quantal size remained the same. The results obtained in this study support a hypothesis that potentiation of the synaptic transmission between A and B neurons of Aplysia is primarily due to an increase of transmitter release.

Characterization of neuronal regeneration in the abdominal ganglion of Aplysia californica.

The ability of neurons in the abdominal ganglion of Aplysia to regenerate their axons following branchial nerve crush was studied using retrograde staining and intracellular dye injection. The duration of the gill withdrawal reflex (GWR) was measured prior to and following nerve crush. Three days after crushing the nerve, the duration of the gill withdrawal reflex was reduced to 20% of control levels. There was rapid recovery 19 days after crushing the branchial nerve. The GWR duration returned to control levels by postlesion days 25-27. Some of the behavioral recovery can be attributed to axonal regeneration. Regeneration, as evidenced by retrograde staining, was first observed by postlesion day 15. The number of stained neurons in ganglia with crushes increased until postlesion day 33. The number of stained neurons in experimental animals was always less than that of controls (67+/-9% at postlesion day 56). More axonal regeneration was seen in the hemiganglion ipsilateral to the branchial nerve. Regeneration after 32 days postlesion was 60+/-5% of controls in the ipsilateral hemiganglion, as opposed to 29+/-6% in the contralateral hemiganglion. Regeneration of individual neurons was also demonstrated. Identified neuron R2 was shown by intracellular dye injection and electrical stimulation of antidromic action potentials to have an axon in the branchial nerve in all ganglia allowed to regenerate for longer than 32 days. These results indicate that in Aplysia, despite behavioral recovery, complete axonal regeneration does not occur in a large segment of the neurons in the adult central nervous system.

Appearance and maturation of synaptic plasticity during juvenile development in Aplysia.

In the present study we examine the developmental appearance and maturation of synaptic plasticity at the A-B neuron synapse in the cerebral ganglion of Aplysia. In the CNS of juvenile Aplysia 120 days after hatching, the excitatory synaptic connection between A and B cluster neurons is essentially the same as in the adult cerebral ganglion. No differences were observed between the amplitudes of the initial EPSPs in the cerebral ganglia of juveniles and adults. One form of plasticity, low frequency synaptic depression, is also present in juveniles. Another form of activity-dependent plasticity, slow developing potentiation (SDP) appears and matures during the late juvenile stage of development. At 120 days posthatching SDP, evoked by tetanic stimulation, is largely absent. Potentiated EPSPs have a significantly smaller amplitude than in adults. Over the next 80 days SDP undergoes a maturation process. The peak potentiation increases linearly with age from 135 +/- 12% at 125 days to 275 +/- 20% at 188 days. The duration of the potentiation, as measured by the time-constant its decay, also increases linearly from 12.7 +/- 3.4 min to 27.9 +/- 3.9 min. From 120-170 days, < 50% of the A neurons tested exhibited SDP. After 170 days, > 85% exhibited SDP. Changes in the rising phase of the A neuron action potential have been implicated in mediating SDP. At 120 days, the A neuron action potential has a significantly shorter duration (half-width) than in the adult. Between 120 and 200 days, both the duration and rise-time of the A neuron action potential increase linearly. These results confirm findings by other investigators, that different forms of synaptic plasticity develop independently, with depression appearing before potentiation.

Enhanced synaptic transmission at identified synaptic connections in the cerebral ganglion of Aplysia.

The identified A-B neuron synaptic connections in the cerebral ganglion of Aplysia exhibited a novel form of enhanced synaptic transmission. A brief high-frequency train of action potentials (2 s, 10-30 Hz) in the presynaptic A neurons produced a long-lasting increase in the amplitude of excitatory postsynaptic potentials (EPSPs) in B neurons. The increase in synaptic efficacy was termed slow developing potentiation (SDP) since the EPSP amplitude increased slowly with the peak occurring 5 min after the tetanizing train. Peak EPSP amplitudes increased relative to the initial EPSP by an average of greater than 250%. SDP decayed as a single exponential with a time constant of tau = 24 min. The enhanced transmission was neuron specific. Only the connections made by the tetanized A neuron were potentiated. However, potentiation apparently occurred at all the synapses made by the tetanized A neuron. Tetanizing the postsynaptic B neurons neither induced, nor when paired with A neuron tetanization, increased SDP. SDP appears to be primarily due to increased transmitter release by the presynaptic neuron.

Recovery of escape locomotion following a CNS lesion in Aplysia.

The recovery of escape locomotion in Aplysia following a CNS lesion was investigated. The connectives between the cerebral and pleural ganglia were crushed in anesthetized animals, producing a specific behavioral deficit. Animals with lesions failed to initiate escape locomotion in response to tail shock. Tail withdrawal and inking which were also evoked by tail shock were still present. Other behaviors such as normal locomotion and feeding were not impaired. There was gradual recovery from the effects of the lesion. Animals with lesions began to respond to tail shock with weak pedal waves at long latencies after 7-13 days. The responses grew more vigorous and the latencies decreased over subsequent days. Full escape locomotor responses were observed as early as 15 days postlesion. By Postlesion Day 27, all of the animals had completely recovered and gave full escape responses. The mean latency of the escape locomotor response in recovered animals was not significantly different from prelesion control values. In behaviorally recovered animals, retrograde tracing from a point distal to the lesion site stained neurons in the cerebral ganglion. Intracellular dye injections of individual neurons revealed sprouting of new processes. Stimulation of the tail nerve and individual neurons demonstrated synaptic connections between cerebral and pleural ganglia neurons. These results suggest that the observed behavioral recovery was due to pleural ganglia neurons regenerating and forming appropriate synaptic connections in the cerebral ganglion.

Intracellular staining of neurons with nickel-lysine.

A new method of intracellularly staining neurons is described. Nickel-lysine (NL) can be used for both intracellular injection by pressure or iontophoresis and retrograde labelling (axonal backfilling). Once introduced into neurons, NL is reacted with dithiooximide dissolved in dimethyl sulfoxide (DMSO) to produce a blue-black precipitate. Small diameter processes are easily detected. For pressure injections, mixing NL with carboxyfluorescein provides a simple way to gauge how much dye has been injected, in that the latter is readily visible when illuminated with blue light. NL appears to move within neurons by axonal transport. Staining over long distances can be obtained in 12-24 h. NL does not appear to cross electrotonic synapses and remains confined to the neurons into which it has been injected. NL staining is simple, flexible and inexpensive. It has the additional advantage that it is compatible with other staining techniques.

Role of proprioceptive reflexes in control of feeding muscles of Aplysia.

Stretching the muscles of the buccal mass of Aplysia evoked proprioceptive reflexes. These consisted of a direct reflex in which the stretched muscle contracted and a crossed reflex in which the contralateral homolog of the stretched muscle contracted as well. Both reflexes were accompanied by corresponding changes in neural activity in the buccal nerves. The muscle contraction and efferent neural activity were abolished by blocking synaptic transmission in the buccal ganglia. Blocking neuromuscular transmission blocked the contractions but not the stretch-induced afferent neural activity. Proprioceptive responses were obtained from isolated buccal nerve-muscle preparations. Both tonic on- and on-off responses were observed. These responses persisted after blocking synaptic transmission at the muscle, indicating that they were due to afferent fibers rather than peripheral interneurons. Proprioceptive neurons with centrally located cell bodies were found. These included previously identified neurons B4 and B5 as well as small cells. Proprioceptive neurons responded to muscle stretch with peripherally initiated axonal spikes that conducted into the central nervous system (CNS) and preceded their somatic spikes. These responses persisted after blocking synaptic transmission in the CNS. Several motor neurons were found. When intracellularly stimulated, these evoked contractions of their target muscle even after blocking synaptic transmission in the CNS. The motor neurons responded synaptically to stretching the ipsilateral muscle. Some responded to stretching of the contralateral homologous muscle as well. The motor neurons differed in their axonal projections, with some projecting only ipsilaterally, others bilaterally. The majority of motor neurons were inhibited by muscle stretch due to inhibitory monosynaptic input from the proprioceptive cells B4 and B5. The stretch reflex occurred when the motor neurons fired due to postinhibitory rebound. The synaptic organization of the reflex was considered.

Control of extrinsic feeding muscles in Aplysia.

The extrinsic buccal muscles in Aplysia are responsible for the overall protraction and retraction of the buccal mass during feeding. The six pairs of extrinsic muscles are organized into two groups, consisting of three protractors and three retractors. Insights into how the extrinsic muscles are controlled were obtained by examining the organization of the motor neurons that innervated them. The extrinsic buccal muscles are innervated by cerebral ganglion nerves and neurons. All the muscles examined appear to be multiply innervated. Identified neurons in the cerebral B, E, and G clusters were found to be motor neurons for individual extrinsic muscles. Some extrinsic muscles had both excitatory and inhibitory innervation. Two synergistic muscles, the extrinsic ventrolateral protractor (ExVLP) and the extrinsic dorsal protractor (ExDP), had common excitatory innervation by identified neuron E5. Two antagonistic muscles, the ExVLP and the extrinsic ventral retractor (ExVR), also had common innervation. Identified neuron E1 appeared to be an inhibitory motor neuron for the ExVLP but an excitatory motor neuron for the ExVR. Common innervation provides a simple mechanism for coordinating synergistic and antagonistic extrinsic muscles. On the basis of these data, a model for the control of buccal mass protraction and retraction is proposed. Bursting by extrinsic buccal muscles was coordinated with cyclic activity in the intrinsic muscles of the buccal mass. Antagonistic extrinsic muscles burst antiphasically and synergistic extrinsic muscles burst in phase when the buccal mass was fully protracted and exhibited a series of rhythmic contractions. Additionally, cerebral E cluster neurons burst in phase with stereotyped rhythmic buccal motor neuron discharges recorded from buccal nerves. The cerebral E cluster motor neurons were coordinated by common synaptic input. No monosynaptic connections were observed; homologous neurons in each E cluster received synaptic input with similar but not identical timing, indicating that the interneurons that coordinate the homologous motor neurons are synchronized. The source of the rhythm that drives synaptically mediated cerebral extrinsic muscle motor neuron bursting was in the buccal ganglia. Cutting one cerebral-buccal connective eliminated E neuron bursting on that side but had no effect on homologous neurons on the intact side. This suggests that a single oscillator in the buccal ganglia may coordinate both the extrinsic and intrinsic buccal muscles during feeding.

Cobalt mapping of the nervous system: evidence that cobalt can cross a neuronal membrane.

Exposing intact nerves of Aplysia to exogenous CoCl2 was sufficient to fill neurons by axonal iontophoresis. There were no significant differences between filling intact nerves and nerves whose ends had been cut. Nonneuronal elements were not filled appreciably. These results show that Co2+ can cross intact axonal membranes and suggest that in tracing neuronal pathways Co2+ may not remain confined to neurons whose interiors are directly exposed to it.

Motor program for pedal waves during Aplysia locomotion is generated in the pedal ganglia.

The activity in the nerves innervating the foot of Aplysia was examined during pedal wave generation. Cyclic patterned discharges (i.e. bursting) in the pedal nerves was associated with the pedal wave. Individual units exhibited bursting during pedal wave generation. but fired tonicly when the pedal wave was absent. Bursting in the nerves persisted after the foot was deafferented, confirming previous behavioral results that pedal waves were the result of a centrally generated motor program. Deafferentation caused increased burst durations and decreased spike frequencies within each burst. This suggests that sensory input from the foot excites the oscillator which underlies pedal wave generation and serves to increase the amplitude of the oscillations. Bursting in the nerves persisted after surgically isolating the pedal ganglia, showing that the neural circuitry necessary for pedal wave generation resides in the pedal ganglia. After isolating the pedal ganglia, bursts in the nerves were longer, less vigorous and less frequent. This suggests that input from the other central ganglia affects both the amplitude and the period of the oscillator. The firing of units in homologous pedal nerve branches, showed similar, but not identical patterns with the pedal commisure intact. Individual homologous units differed in both absolute timing and frequency of firing. After cutting the commissure, synchronization of activity in the homologous nerves was lost. This indicates that bilateral coordination of the pedal wave motor programs generated in each pedal ganglion is maintained via the pedal commissure. The possible mechanisms involved are considered.

Role of pedal ganglia motor neurons in pedal wave generation in Aplysia.

Presumptive pedal ganglia motor neurons involved in pedal wave generation in the foot of Aplysia were examined. Pedal motor neurons fired tonicly in the absence of pedal waves, but exhibited bursting with constant phase angles during pedal wave generation. Motor neurons which burst both in and 180 degrees out of phase with the pedal wave were recorded. The relative latency of neurons firing at large phase angles was more variable than that of neurons firing at small phase angles. Pedal neurons did not make monosynaptic connections among themselves. Their firing both during and in the absence of pedal waves was due to synaptic input, presumably from interneurons which generate the oscillations underlying the pedal wave. Motor neurons firing in phase had common synaptic input of the same sign, while some motor neurons firing out of phase had common synaptic input of opposite sign. There also appeared to be polysynaptic connections among pedal motor neurons. Evidence suggesting polysynaptic feedback from the motor neurons to the oscillator interneurons was found. The results are consistent with a multineuronal oscillator in which different elements drive different groups of motor neurons. The pedal wave is the result of cyclic activation of the motor neurons by the oscillator network. Pedal motor neurons evoked several different types of tension changes in the foot when intracellularly stimulated. Fast and slow tension increases were observed. Some caused decreased tension in the foot when stimulated suggesting that they might be inhibitory motor neurons. Other neurons appeared to be involved in the central control of foot tonus. Pedal motor neurons had conduction velocities ranging from 36 to 136 cm.sec-1 and were found to have axons in more than one foot nerve branch. This property may contribute to the longitudinal spread of the pedal wave.

Intra- and interganglionic synaptic connections in the CNS of Aplysia.

Synaptic connections were found between two groups of neurons in the CNS of Aplysia, the cerebral ganglion A and B cluster neurons which are involved in the control of pedal and parapodial movements and neurons in the pleural ganglion which has been shown to modulate locomotion. The A and B neurons made synaptic connections in both the cerebral and pleural ganglia. Pleural neurons had synaptic connections among themselves and with A and B neurons. The A neurons made excitatory monosynaptic connections with the B neurons and a minimum of 6 pleural neurons including the left giant cell (LGC). All of the A neuron synapses found were excitatory. The B neurons received excitatory synaptic input from two other groups of neurons in the cerebral ganglion and both excitatory and inhibitory input from pleural neurons. The latter were identified on the basis of their synaptic connections with the LGC and A neurons. The B neurons and LGC had several common synaptic inputs. The A neurons received monosynaptic input from only 2 pleural neurons. Complex synaptic circuits between A and B neurons and pleural neurons were found. These included recurrent inhibition of B neurons by A neurons via a pleural interneuron, feedforward summation of A neuron synaptic input to the LGC, and reciprocal excitatory synaptic connections between B and the pleural neurons. The activity of the B neurons was modulated by direct inhibitory and excitatory synaptic connections from pleural neurons. The A neurons were modulated primarily by a polysynaptic pathway through the B neurons. The modulation of cerebral A and B neurons by pleural neurons is consistent with behavioral results obtained studying locomotion.

Control of pedal and parapodial movements in Aplysia. II. Cerebral ganglion neurons.

1. Intracellular stimulation of individual neurons in the two symmetrical A neuron clusters of the cerebral ganglion evoked contractions of both the foot and parapodia. Electrical stimulation of pedal and parapodial nerves caused antidromic action potentials in A neurons. Units recorded in the nerves followed the driven somatic spike 1:1. This suggests that the A neurons are presumptive pedal and parapodial motor neurons.2. Individual A neurons evoked both bilteral and unilateral contractions of the parapodia or split foot. Contractions in the parapodia were independent of those in the foot. An individual A neuron caused contractions in either the foot or the parapodia, but not both. Sequential transection of parapodial nerves had only a slight effect until a key nerve was cut. The contractions produced by a single A neuron on one side were then abolished. These data suggest that the motor fields of the A neurons are well defined within the foot or the parapodia. 3. Parapodial contractions produced by individual A neurons are not dependent on the excitation of follower motor neurons. Blocking synaptic transmission by the addition of CoCl2 did not eliminate the contractions produced by driving individual A neurons. This is consistent with the A neurons being motor neurons. 4. Intracellular stimulation of individual neurons in the symmetrical B neuron clusters of the cerebral ganglion also evoked pedal and parapodial contractions. Electrical stimulation of the pedal and parapodial nerves elicited antidromic spikes in these neurons. Individual B neurons caused contractions in both the foot and parapodia. This suggests that the B neurons are motor neurons with very large motor fields. 5. Filling the pedal and parapodial nerves with cobalt primarily filled the cell bodies of neurons located in the pedal and pleural ganglia. The somata of A and B neurons were also occasionally filled. This is consistent with the electrophisiological results. 6. Other neurons also evoked parapodial contractions. Intracellular stimulation of neurons in the pedal and pleural ganglia caused parapodial contractions in intact animals. Some of these neurons were excited by stretching the parapodia or touching the tentacles. 7. The B neurons are strongly excited by tactile stimulation of the tentacles. Since they can cause pedal and parapodial contractions they may mediate reflex contractions elicited by tentacular stimulation. Stretching the parapodia only occasionally caused the A neurons to fire. This makes it unlikely that they make a major contribution to pedal and parapodial proprioceptive reflexes. These reflexes are probably controlled by neurons in the pedal and pleural ganglia.