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.     

Age-dependent behavioral changes and physiological changes in identified neurons in Aplysia californica

The gill withdrawal reflex (GWR) to direct gill stimulation was studied in sexually mature Aplysia and in those older by at least two months. The GWR threshold in old Aplysia was five- to sevenfold higher than that in mature animals. In the habituation paradigm, the GWR amplitude decremented rapidly to zero in old animals whereas in mature animals it persisted for at least ten trials. The GWR could not be dishabituated in old animals. The GWR is an age-dependent behavior in that parieto-visceral ganglion suppression of the GWR appears to increase with age. Also the electrophysiological properties of two neurons in the parieto-visceral ganglion were compared in the two age groups: L₇ a neuron which dishabituates the GWR in mature and not in old animals; and R₂ which manifests cytological changes with age. In old animals L₇′s input resistance was lower, the time constant was increased, and the size of the psp evoked by gill stimulation was smaller than those of mature L₇s. Similar membrane changes with age were measured in R₂. Soma size of L₇ was approximately the same in the two age groups as was that of R₂. The physiological parameters of neurons of known function continue to change during postmetamorphic life of Aplysia.     

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.     

Neurophysiological mechanisms underlying habituation of the tentacle retraction reflex in the land slug Ariolimax

Habituation of the tentacle retraction reflex was studied at the following response levels: (1) Muscle tension elicited in the tentacle retractor muscle by repeated stimulation of a cerebral nerve (at 60-sec intervals) declined in parallel with evoked activity of the largest unit in the tentacle retractor nerve. (2) The largest unit in the tentacle retractor nerve (L₄) showed spontaneous recovery and dishabituation. The rate of response decrement was inversely related to the strength of stimulus, and an optimal interstimulus interval ca. 60 s was found. Retention of habituation for 24 h was exhibited. (3) The major retractor motoneurons (L₂, L₃, L₄) all showed habituation, dishabituation, and spontaneous recovery. The decline of L₄ activity was parallelled by a decline in muscle response. (4) Compound EPSPs elicited in the retractor motoneurons by stimulation of sensory pathways showed habituation and dishabituation. (5) Unitary EPSPs elicited by stimulation of cerebral nerves and connectives with minimal stimulus strengths also showed habituation and were unaffected by spontaneously occurring EPSPs. Dishabituation by another pathway was also shown. (6) Depolarization of L₄ by a constant current produced spike trains of constant firing rate and evoked a constant level of muscle tension in repeated trials, suggesting the absence of habituation in a peripheral nerve net or at the neuromuscular junction.     

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.     

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.     

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.     

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.     

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.     

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 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.     

The integration of antagonistic reflexes revealed by laser ablation of identified neurons determines habituation kinetics of the Caenorhabditis elegans tap withdrawal response

Previously, we described the circuitry that underlies the tap withdrawal response of the nematode Caenorhabditis elegans. In response to a light mechanosensory stimulus a worm will withdraw, usually by initiating backward locomotion, but occasionally with increased forward locomotion. The form of an animal's response is a product of the balance between two antagonistic reflexes: backward locomotion (reversals) triggered by anterior mechanosensory input and forward locomotion (accelerations) triggered by posterior mechanosensory input. During habituation of this reflex, the frequency of forward and backward locomotion in response to tap is modulated by both experience and interstimulus interval; reversals are more frequent early in a habituation series and at longer Inter stimulus intervals. Single-cell laser microsurgery was used to study each of the subcomponents of the intact behavior during habituation training. Groups of intact or laserablated worms were habituated at either a 10-s or a 60-s inter stimulus interval and the kinetics of habituation in each group was analyzed. We demonstrate that each component of the behavior habituates and does so with kinetics that are consistent with the decrement observed in the intact animal.Abbreviations ALM anterior lateral microtubule - AVM anterior ventral microtubule - ISI interstimuls interval - NGM nematode growth medium - PLM posterior lateral microtubule - PVC posterior ventral interneuron C     

Habituation and inhibition of the crayfish lateral giant fibre escape response.

1. Decrement of the lateral giant fibre escape response was studied in intact, restrained, crayfish and in those with the ventral nerve cord transected at the thoracic-abdominal level. 2. Taps (delivered at rates of 1 per 5 min to the abdomen) depressed responsiveness to about 50% of its inital value in 10 trials, for both intact and operated animals. 3. With additional stimulation, responsiveness dropped to near zero for both groups. Recovery was negligible 2 h later, but nearly complete after an additional 24 h rest. 4. Protection against response decrement in this situation was obtained by directly activating the cord giant fibres 30 msec prior to the tactile stimulus. The directly-elicited giant fibre spikes which follow the tactile stimulus do not influence the course of response decrement. 5. The results establish the decrement as centrally mediated habituation, and minimize the role of receptor alterations or descending neuronal influences in the behavioural change. 6. A comparison is made between the properties of hibituation and the homosynaptic depression of afferent to interneurone synapses that is presumed to be the physiological mechanism of habituation in this situation.     

Neural correlates of habituation of the proleg withdrawal reflex in larvae of the hawk moth, Manduca sexta

The larval proleg withdrawal reflex of the hawk moth, Manduca sexta, exhibits robust habituation. This reflex is evoked by deflecting one or more mechanosensory planta hairs on a proleg tip. We examined neural correlates of habituation in an isolated proleg preparation consisting of one proleg and its segmental ganglion. Repeated deflection of a single planta hair caused a significant decrease in the number of action potentials evoked in the proleg motor nerve (which carries the axons of proleg retractor motor neurons). Significant response decrement was seen for interstimulus intervals of 10 s, 60 s and 5 min. Response decrement failed to occur in the absence of repetitive stimulation, the decremented response recovered spontaneously following a rest, and electrical stimulation of a body wall nerve facilitated the decremented response (a neural correlate of dishabituation). Adaptation of sensory neuron responses occurred during repeated hair deflections. However, when adaptation was eliminated by direct electrical stimulation of sensory neurons, the response in the proleg motor nerve still decreased significantly. Muscle recordings indicated that the response of an identified proleg retractor motor neuron decreased significantly during habituation training. Thus, habituation of the proleg withdrawal reflex includes a central component that is apparent at the level of a single motor neuron. Accepted: 20 December 1996     

Inhibitor of protein synthesis blocks longterm behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia

To study the effect of protein synthesis inhibition on long-term sensitization of the gill- and siphon-withdrawal reflex of Aplysia, we have developed an isolated reflex preparation in which we could expose the inhibitor to only that part of the central nervous system involved in mediating the reflex and not to the other parts of the animal's central nervous system, thus minimizing the possible systemic side effects. We have found that long-term sensitization can be obtained in the isolated gill reflex, and that this long-term process, but not the short-term process, is blocked selectively by anisomycin, a reversible inhibitor of protein synthesis. Moreover, to obtain this blockade of long-term sensitization, this drug need only be applied during the training procedure.     

Two types of plasticity in the tentacle withdrawal reflex of Helix aspersa are dissociated by tissue location and response measure

The tentacle withdrawal reflex of the terrestrial snail Helix aspersa was studied in vitro. The reflex is evoked by mechanical stimulation of the nose. Lesion experiments showed that 45% to 75% of the response amplitude is attributable to peripheral pathways alone. The central contribution increases with increasing stimulus intensity.Repeated stimulation produced pure habituation at low stimulus strengths, and habituation mixed with intrinsic sensitization (warm-up effect) at high stimulus strengths. The simultaneous occurrence of habituation and sensitization is consistent with the dual process theory of plasticity. Additional results differentiate the two processes. Habituation can occur without the CNS, whereas intrinsic sensitization requires the CNS. Also, the two processes are differentially effective in their influences on response amplitude and duration: habituation is more effective in determining response amplitude, while sensitization is more effective in determining response duration.Although the establishment of sensitization requires the CNS, 81% of the memory for intrinsic sensitization was localized to the periphery, by lesion experiments. Extrinsic sensitization, caused by stimulation of the medial lip nerve, had similar behavioural effects and a similar memory locus. Both types of sensitization appear to be caused by neuromuscular facilitation mediated by a central pathway.Abbreviations CNS central nervous system - PNS peripheral nervous system - S-R stimulus-response - TRM tentacle retractor muscle     

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.     

Age-related deficits of long-term memory in the crab Chasmagnathus

Short and long-term memory in adult crabs Chasmagnathus granulatus of different age are evaluated in two learning paradigms: habituation to a visual danger stimulus and appetitive conditioning. No difference between young, middle-aged and aged animals is found in short-term habituation with 15 training trials. A good level of retention of the habituated response at 24 h is exhibited by young and middle-aged crabs but a poor one by aged crabs. When the training-to-testing interval is lengthened to 48 h or the training session reduced to 7 trials, young and middle-aged crabs continue to show long-term habituation but aged individuals exhibit no retention at all. As regards appetitive conditioning, young, middle-aged and aged crabs present similar short-term memory with 5 training trials and similar long-term memory when tested at 24 h, but an age-related deficit in long-term retention is exhibited when the intersession interval is lengthened to 48 h or the training reduced to 3 trials. Thus, a reduction of long-term memory related to age is demonstrated in the crab Chasmagnathus. Since it is shown in two different learning paradigms, the possibility of explaining the deficit in terms of a failure in memory mechanisms due to aging rather than as a consequence of ontogenetic shift in the crab's behavior is discussed.     

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