The development of central nervous system control of the gill withdrawal reflex evoked by siphon stimulation in Aplysia

In older Aplysia, the central nervous system (CNS) (abdominal ganglion) exerts suppressive and facilitatory control over the peripheral nervous system (PNS) which initially mediates the gill withdrawal reflex and its subsequent habituation evoked by tactile stimulation of the siphon. In young animals, both the suppressive and facilitatory CNS control were found to be absent. In older animals, removal of branchial nerve (Br) input to the gill resulted in a significantly reduced reflex latency and, with ctenidial (Ct) and siphon (Sn) nerves intact, a significantly increased reflex amplitude and an inability of the reflex to habituate with repeated siphon stimulation. In young animals, removal of Br had no effect on reflex latency and with Ct and Sn intact, the reflex amplitude latency was not increased and the reflex habituated. Older animals can easily discriminate between different intensity stimuli applied to the siphon as evidenced by differences in reflex amplitude, rates of habituation, and evoked neural activity. On the other hand, young animals cannot discriminate well between different stimulus intensities. The lack of CNS control in young animals was found to be due to incompletely developed neural processes within the abdominal ganglion and not the PNS. The lack of CNS control in young Aplysia results in gill reflex behaviours being less adaptive in light of changing stimulus conditions, but may be of positive survival value in that the young will not habituate as easily. The fact that CNS control is present in older animals strengthens the idea that in any analysis of the underlying neural mechanisms of habituation the entire integrated CNS-PNS must be taken into account.     

Arginine vasotocin,an endogenous neuropeptide of Aplysia,suppresses the gill withdrawal reflex and reduces the evoked synaptic input to central gill motor neurons

The superfusion (15 min) of arginine vasotocin (AVT; 10^?9–10^?12M) over the abdominal ganglion of Aplysia californica suppressed the amplitude of the gill withdrawal reflex evoked by tactile stimulation of the siphon, increased the rate of gill reflex habituation, and decreased the evoked synaptic activity to central gill motor neurons. The suppressive effects of AVT on gill reflex behaviors were not due to toxic effects of the hormone since the effects were completely reversible following washout and 3 h rest. The results obtained with AVT were similar to those previously found using the mammalian neuropeptide arginine vasopressin. AVT may act by increasing the activity of central neurons which exert suppressive control over both gill reflex behaviors and evoked activity to central gill motor neurons.     

The dual effect of enflurane on gill withdrawal reflex ofAplysia

Superfusion of clinical concentrations of enflurane (0.5% or 1.0%), an inhalation anaesthetic, over the abdominal ganglion ofAplysia significantly affected the amplitude of the gill withdrawal reflex evoked by tactile stimulation of the siphon. Enflurane superfusion (0.5%) suppressed the gill withdrawal reflex amplitude (to 46.1% of control; P<0.001 vs control) in eight of ten experiments. In the remaining two experiments, enflurane superfusion of the abdominal ganglion significantly facilitated the gill withdrawal reflex amplitude (174.5% of control;P<0.01). In addition, enflurane superfusion significantly reduced the number of action potentials evoked in central gill motor neurons by the siphon stimulation (to 47.1% of control;P<0.01) in six out of nine experiments. In one of the remaining three experiments, enflurane increased the number of action potentials evoked by the stimulus (to 200.0% of control). In two of the three, enflurane did not alter the numbet of action potentials. Behavioural responses were ‘uncoupled’ from the neuronal responses as a result of enflurane superfusion.     

L9 modulation of gill withdrawal reflex habituation in Aplysia.

Repeated tactile stimulation of the siphon in Aphysia normally results in habituation of the gill withdrawal reflex and a concomitant decrease in the amplitude of the excitatory synaptic input ot gill motor neurons in the abdominal ganglion. It was found, however, that induced low-level tonic activity in motor neuron L9, which does not itself elicit a gill withdrawal movement, prevented habituation of the reflex from occurring. Further, in preparations already habituated, this tonic low-level activity brought about a reversal of habituation. Although tonic L9 activity prevented the occurrence of habituation or brought about its reversal, it did not interfere with the synaptic decremental process which normally accompanies gill reflex habituation. Motor neurons L7 and LDG1 were found not to possess this ability of L9 to modulate gill reflex habituation. Evidence suggests that L9's modulatory effect is mediated in the periphery, in the gill and not centrally in the abdominal ganglion.     

The gill withdrawal reflex is suppressed in sexually active Aplysia

In Aplysia, the central nervous system and peripheral nervous system interact and form an integrated system that mediates adaptive gill withdrawal reflex behaviours evoked by tactile stimulation of the siphon. The central nervous system (CNS) exerts suppressive and facilitatory control over the peripheral nervous system (PNS) in the mediation of these behaviours. We found that the CNS's suppressive control over the PNS was increased significantly in animals engaged in sexual activity as either a male or female. In control animals, the evoked gill withdrawal reflex met a minimal response amplitude criterion, while in sexually active animals the reflex did not meet this criterion. At the neuronal level, the increased CNS suppressive control was manifested as a decrease in excitatory input to the central gill motor neurons.     

Transfer of habituation between stimulation sites of the siphon withdrawal reflex in Aplysia californica

Habituation of the siphon withdrawal reflex (SWR) can be evoked by iterative tactile stimuli presented to one of several sites, including the siphon and gill. The SWR evoked at an arbitrary "test" site did not habituate when stimuli were presented at 20-min intervals. However, there was a large decrease in the reflex evoked at the test site when the trial was preceded by 10 repetitive stimuli (interstimuli interval = 30 s) presented to the opposite "habituation" site. Transfer of habituation occurred from gill to siphon stimulation sites, and vice versa. There was a concomitant decrease in the excitatory input evoked in the central siphon motor neurons LDS1 and LDS3. Moreover, transfer of habituation occurred after the abdominal ganglion (central nervous system) was removed. There was little change in the magnitude of the control responses or transfer of habituation after deganglionation. Since transfer of habituation between stimulation sites of the SWR was similar to that reported previously for the gill withdrawal reflex, it was suggested that a common mechanism may underlie the two behaviors.     

Modulation of the Aplysia gill withdrawal reflex by dopamine

The ability of dopamine to modulate gill contractions was tested in Aplysia. When dopamine was perfused through the gill vasculature, gill contractions caused by siphon stimulation (gill withdrawal reflex) and by depolarization of the gill motor neuron L7 were increased in amplitude, as compared with those evoked during seawater perfusion. Habituation of gill movements, brought about by repetitive stimulation of the siphon or of L7, was prevented by dopamine. Despite the absence of reflex habituation, the number of action potentials in central gill motor neurons, evoked by siphon stimulation, showed normal decrement. Dopamine's effects were blocked when the ctenidial nerve was cut or when L7 hyperpolarized. These data suggest that dopamine acts peripherally to increase the efficacy of L7's synaptic transmission onto gill muscle or elements of the gill neural plexus.     

L9 modulation of gill withdrawal reflex habituation in Aplysia

Repeated tactile stimulation of the siphon in Aplysia normally results in habituation of the gill withdrawal reflex and a concomitant decrease in the amplitude of the excitatory synaptic input to gill motor neurons in the abdominal ganglion. It was found, however, that induced low-level tonic activity in motor neuron L₉, which does not itself elicit a gill withdrawal movement, prevented habituation of the reflex from occurring. Further, in preparations already habituated, this tonic low-level activity brought about a reversal of habituation. Although tonic L₉ activity prevented the occurrence of habituation or brought about its reversal, it did not interfere with the synaptic decremental process which normally accompanies gill reflex habituation. Motor neurons L₇ and LDG₁ were found not to possess this ability of L₉ to modulate gill reflex habituation. Evidence suggests that L₉'s modulatory effect is mediated in the periphery, in the gill and not centrally in the abdominal ganglion.     

Dopamine modulation of gill reflex behavior in Aplysia.

We have studied the effects of dopamine on the gill withdrawal reflex evoked by tactile siphon stimulation in the margine mollusc Aplysia. Physiological concentrations of dopamine (diluted in seawater) were perfused through the gill during siphon stimulation series. The amplitude of the reflex was potentiated by dopamine and habituation of the reflex was prevented. This occurred with no change in the activity evoked in central motor neurons. These results lead us to conclude that the dopaminergic motor neuron L9 is modulating habituation in the periphery and that the central nervous system facilitatory control of the peripheral nervous system may act via a dopaminergic pathway.     

Transfer of habituation in Aplysia: contribution of heterosynaptic pathways in habituation of the gill-withdrawal reflex

Habituation of the Aplysia gill-withdrawal reflex (and siphon-withdrawal reflex) has been attributed to low-frequency homosynaptic depression at central sensory-motor synapses. The recent demonstration that transfer of habituation between stimulation sites occurs in this model system has prompted the hypothesis that heterosynaptic inhibitory pathways also play a role in the mediation of habituation behavior. To test this hypothesis, the sites and mechanisms of neural plasticity which underlie transfer of habituation in Aplysia were examined. Transfer of habituation is a reduction in the reflex evoked at one stimulation site (siphon) due to repeated presentation of a stimulus to a second site (gill). Centrally mediated transfer of habituation, measured in a preparation lacking the siphon-gill peripheral nervous system (PNS), was associated with a reduced excitatory response in central motor neurons. Repeated tactile stimulation of the gill did not attenuate the gill response evoked by electrical stimulation of the branchial nerve nor the mechanoreceptor response recorded in LE sensory neurons. In contrast, repeated stimulation of siphon or gill at a site which was "off" the sensory field of a specific mechanoreceptor led to a diminution in synaptic transmission between that sensory neuron and its followers (motor neurons and inter-neurons). These data demonstrate that centrally mediated transfer of habituation results from heterosynaptic modulation of synaptic transmission at the sensory-motor (and sensory-interneuron) synapses. Therefore, habituation behavior in Aplysia is mediated through the conjoint action of homosynaptic and heterosynaptic inhibitory processes.     

In vitro classical conditioning of a gill withdrawal reflex in Aplysia: neural correlates and possible neural mechanisms

An in vitro preparation consisting of the siphon, mantle, gill, and abdominal ganglion undergoes classical conditioning when a weak tactile stimulus (CS) applied to the siphon is paired with a strong tactile stimulus to the gill (UCS). When the stimuli are paired, the CS comes to evoke a gill withdrawal reflex (GWR) which increases in amplitude with training. Only when the stimuli are paired in a classical conditioning paradigm does the CS come to evoke a GWR. With classical conditioning training there is an alteration in the synaptic efficacy between central sensory neurons and central gill motor neurons. Moreover, these changes can be observed in sensory neurons not activated by the CS. The changes observed, as evidence by the number of action potentials evoked in the gill motor neuron do not completely parallel the observed behavioral changes. It is suggested that in addition to changes in the synaptic efficacy at the sensory-motor neuron synapse, other changes in neuronal activity occur at other loci which lead to the observed behavioral changes.     

Habituation and dishabituation mediated by the peripheral and central neural circuits of the siphon of aplysia

The siphon withdrawal response evoked by a weak tactile (water drop) or light stimulus is mediated primarily by neurons in the siphon. Central neurons (abdominal ganglion) contribute very little since the response amplitude and latency are not changed following removal of the abdominal ganglion. Similarly, habituation and dishabituation of this withdrawal response are not different after removal of the abdominal ganglion, indicating that the peripheral neural circuit in the isolated siphon can mediate habituation itself, and thus has many of the properties attributed to central neurons. Responses evoked by electrical stimulation of the siphon nerve habituate, depending upon the stimulus intensity and interval. These habituated responses may be dishabituated by tactile or light stimulation of the siphon. These results show that each neural system, peripheral and central, has an excitatory modulatory influence on the other. Normally adaptive siphon responses must be shaped by the integrated activity of both of these neural systems.     

Habituation and dishabituation mediated by the peripheral and central neural circuits of the siphon of Aplysia.

The siphon withdrawal response evoked by a weak tactile (water drop) or light stimulus is mediated primarily by neurons in the siphon. Central neurons (abdominal ganglion) contribute very little since the response amplitude and latency are not changed following removal of the abdominal ganglion. Similarly, habituation and dishabituation of this withdrawal response are not different after removal of the abdominal ganglion, indicating that the peripheral neural circuit in the isolated siphon can mediate habituation itself, and thus has many of the properties attributed to central neurons. Response evoked by electrical stimulation of the siphon nerve habituate, depending upon the stimulus intensity and interval. These habituated responses may be dishabituated by tactile or light stimulation of the siphon. These results show that each neural system, peripheral and central, has an excitatory modulatory influence on the other. Normally adaptive siphon responses must be shaped by the integrated activity of both of these neural systems.     

Cell and molecular analysis of long-term sensitization in Aplysia

We have found that one cellular locus for the storage of the memory underlying short-term sensitization of the gill and siphon withdrawal reflex in Aplysia is the set of monosynaptic connections between the siphon sensory cells and the gill and siphon motor neurons. These connections also participate in the storage of memory underlying long-term sensitization. In animals that have undergone long-term sensitization, the amplitudes of the monosynaptic connections are significantly larger (2.2x) than the ones in control animals. To study the mechanisms of onset and retention of long-term synaptic facilitation that underly long-term sensitization and the role of protein synthesis in long-term memory, we have developed two types of reduced preparations: the intact reflex isolated from the remainder of the animal, and a dissociated cell culture system in which the monosynaptic component (sensory neurons and motor neurons) of the neuronal circuit mediating the withdrawal reflex is reconstituted. We found that protein synthesis inhibitors, such as anisomycin or emetine, and RNA synthesis inhibitors, such as actinomycin D or alpha-amanitin, blocked long-term facilitation without interfering with short-term facilitation. These results suggest that the acquisition of long-term memory may require the expression of genes and the synthesis of proteins not needed for short-term memory.     

Functions of the LE sensory neurons inAplysia

Mechanosensory neurons which innervate the siphon and have their cell bodies in the LE cluster of the abdominal ganglion ofAplysia have revealed many cellular and molecular processes that may play general roles in learning and memory. It was initially suggested that these cells are largely responsible for triggering the gill-withdrawal reflex evoked by weak siphon stimulation, and that most of this effect is mediated by their monosynaptic connections to gill motor neurons. This implied a simple link between plasticity at these synapses and modifications of the reflex during learning. We review more recent studies from several laboratories showing that the LE cells are not activated by very weak tactile stimuli that elicit the gill-withdrawal reflex, and that an unidentified population of siphon sensory neurons has lower mechanosensory thresholds and produces shorter latency responses. Furthermore, the direct connections between LE cells and gill motor neurons make a minor contribution when the reflex is elicited in pinned siphon preparations by light stimuli that weakly activate the LE cells. Because weak mechanical stimulation of the unrestrained siphon causes little or no LE cell activation, it is unlikely that, under natural conditions, sensitization or conditioning of reflex responses elicited by light siphon touch depends upon plasticity of LE cell synapses onto either motor or interneurons. The LE cells appear to function as nociceptors because they are tuned to noxious stimuli and, like mammalian nociceptors, show peripheral sensitization following nociceptive activation. This sensitization and the profound activity-dependent potentiation of LE synapses indicate that LE cell contributions to defensive reflexes should be largest during and after intense activation of the LE cells by noxious stimulation (with the LE cell plasticity contributing to long-lasting memory of peripheral injury). The LE sensory neurons offer special opportunities for direct tests of this and other hypotheses about specific mnemonic functions of fundamental mechanisms of neural plasticity.     

Neuronal mechanisms of learning in an in vitro Aplysia preparation: sites other than the sensory-motor neuron synapse are involved

Classical conditioning of the gill withdrawal reflex can be demonstrated in two different in vitro Aplysia preparations. The data obtained show that as conditioning of the gill withdrawal reflex proceeds there are changes in synaptic efficacy at the central sensory-motor neurone synapse. These changes in synaptic efficacy, however, are not necessary nor are they sufficient for the observed changes in gill reflex behaviour. Changes must be occurring at other loci within the nervous system to mediate the associative learning. We hypothesized, based on data obtained from one type of in vitro preparation, that changes occur in the ability of the motor neurone to elicit a gill withdrawal response as a result of classical conditioning training. In order to test this hypothesis we depolarized an identified gill motor neurone before and after classical conditioning and found that the motor neurone's ability to elicit a gill movement was facilitated following classical conditioning training. In control preparations that received an explicitly unpaired stimulus paradigm (which does not lead to classical conditioning of the reflex) there was a decrease in the efficacy of a gill motor neurone to elicit a gill withdrawal response. There are a number of possible sites within the integrated central (CNS) and peripheral (PNS) nervous systems where changes could occur to bring about the alterations in motor neurone efficacy. Our results suggest that changes in neuronal activity which underlie learning occur at multiple sites within the nervous system and that a complete understanding of the mechanisms of associative learning can only be obtained when all of these sites are taken into account.     

Widespread anatomical projections of the serotonergic modulatory neuron,CB1, inAplysia

Although sensitization-related changes in the neural circuitry of withdrawal reflexes inAplysia are well studied, relatively few studies address the organization of the modulatory components of sensitization. In particular, it is not known whether individual modulatory loci can simultaneously influence multiple reflex circuits. There is, however, evidence that a single modulatory transmitter, serotonin, plays a pivotal role in facilitating different reflex circuits during sensitization. Furthermore, it is known that activation of a pair of serotonergic neurons, the CB1s, produces heterosynaptic facilitation of the sensorimotor connections of one of these reflex circuits. These data together raise the possibility that the CB1s may produce sensitizing changes in the neural elements of multiple reflex systems simultaneously. In the present study, we utilized immunocytochemistry and intracellular labeling to obtain anatomical evidence of CB1's possible role in modulating multiple reflex circuits. We found that two distinct neurons satisfy previously published physiological criteria for CB1. One of these, CB1, is immunoreactive to serotonin. The second cell, here named CB2, has a different neuroanatomy and is not serotonin immunoreactive. Focusing on CB1, we found (1) profuse fine processes given off by its axons in the posterior neuropil of the cerebral ganglion, (2) extensive branching and fine processes in the pleural ganglion, and (3) a branch of CB1 that projects into the pedal ganglion. These three observations are consistent with the hypothesis that, in addition to its already established role in modulating the siphon withdrawal circuit, CB1 may also modulate synaptic connections between (1) the sensory and motor neurons of the tentacle withdrawal reflex (2) the sensory neurons and interneurons of the tail and tail-elicited siphon withdrawal reflex, and (3) the sensory and motor neurons of the tail withdrawal reflex. These observations support further physiological investigations of a possible global role of CB1 in modulating the tail and tentacle withdrawal reflexes.     

Endogenous peptides work at multiple sites in the nervous system in the control of gill behaviors in Aplysia

The suprafusion of two endogenous neuropeptides, arginine vasotocin (AVT) and small cardioactive peptide B (SCPB), over the abdominal ganglion of Aplysia californica significantly affects the ability of a central gill motor neuron to elicit a gill withdrawal response. Gill motor neurons L7 or LDG1 were depolarized to produce the same number of action potentials (APs) on each trial. When AVT (10(-6)M) was suprafused, the motor neurons' ability to elicit a gill movement was suppressed; while SCPB (10(-6)M) superfusion facilitated the response. Neither peptide altered the passive membrane properties of the motor neurons nor did they affect the duration of their APs. These results are consistent with the hypothesis that the peptides act via central control neurons which exert both suppressive and facilitatory control over gill reflex behaviors and associated neural activity.     

FMRFamide prevents habituation and potentiates the gill withdrawal reflex in the isolated gill preparation of Aplysia

Perfusion of the endogenous neuropeptide, FMRFamide, through the isolated gill of Aplysia facilitated the amplitude of the gill withdrawal reflex (GWR) evoked by tactile stimulation of the gill. The GWR was facilitated in a dose-dependent manner. The facilitation of the GWR produced by FMRFamide perfusion was reversible. In addition to facilitating GWR amplitude, FMRFamide perfusion could also prevent habituation of the reflex. It is hypothesized that FMRFamide may play a role in the peripheral nervous system (PNS) in the gill in the mediation of behavioral state and modulation of adaptive gill behaviors.     

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. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 160–172, 1998