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.     

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.     

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.     

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.     

Suppression of gill withdrawal reflex behaviours in Aplysia: the role of acetylcholine

Acetylcholine (ACh) dissolved in seawater and perfused through the isolated gill of the Aplysia californica produced suppression of the gill withdrawal reflex (GWR) evoked by tactile stimulation of the gill. This suppression was reversible upon washout and was blocked by co-perfusion of curare and alpha-bungarotoxin. Co-perfusion of atropine did not block the suppression of the GWR produced by ACh. We concluded that the suppressive effects produced by perfusion of ACh through the gill occur as a result of the action of ACh at the nicotinic-like receptors. The role of ACh suppression in the mediation of gill reflex behaviours is discussed.     

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.     

Electrophysiological studies of the gill ganglion inAplysia californica

1. An electrophysiological analysis was made of gill ganglion neurons in Aplysia californica. 2. Gill ganglion neurons behave similarly to neurons in the abdominal ganglion (the central nervous systems; CNS) that are involved with gill withdrawal behaviors. 3. Some gill ganglion neurons are motor neurons much like those in the CNS. 4. Neurons in the gill ganglion are electronically and dye-coupled. In addition, they receive common chemical synaptic inputs from the Int-II network in the CNS. 5. Tactile stimulation of the gill or siphon evokes synaptic activity in gill ganglion neurons whether or not the CNS is present. 6. Pedal nerve stimulation results in synaptic activity in gill ganglion neurons and facilitates synaptic input evoked by tactile stimulation of the gill or siphon. 7. Antibody staining reveals serotonin-like fibers in the branchial nerve close to the gill ganglion but no cell bodies in the ganglion. 8. The gill ganglion may play a role in the mediation of adaptive gill reflex behaviors. It may be one of the loci where the CNS and peripheral nervous system (PNS) interact and form an integrated circuit to mediate gill withdrawal reflex (GWR) behaviors.     

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.     

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     

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.     

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.     

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.     

The abdominal motor system of the crayfish, Cherax destructor. I. Morphology and physiology of the superficial extensor motor neurons

Using extracellular and intracellular stimulation, recording and dye-filling, we identified and studied the superficial extensor motor neurons of the crayfish, Cherax destructor. Functional associations of each neuron were characterised by recording its responses to sensory and abdominal cord inputs, its extensor muscle innervation pattern and its relationships with other neurons. Two clear associations were found among the six neurons of each segment. A medium-sized excitor (no. 3), that innervates a substantial percentage of extensor muscle fibres, and the largest excitor (no. 6), recruited during peak, excitation, were inhibited by input from unknown interneurons that excited the common inhibitor (no. 5). Likewise, these excitors received excitatory input when the inhibitor was silent. Another medium-sized neuron (no. 4) that innervates many muscle fibres was co-active with one of the small excitors (no. 2). The two medium-sized neurons were never active at the same time, and these two groupings may be determined by pre-motor interneurons. The implications of these findings for our understanding of motor control in this system are discussed. Accepted: 21 June 1998     

Salinity regulates claudin mRNA and protein expression in the teleost gill

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.     

Parallel processing in an identified neural circuit: the Aplysia californica gill-withdrawal response model system

The response of the gill of Aplysia calfornica Cooper to weak to moderate tactile stimulation of the siphon, the gill-withdrawal response or GWR, has been an important model system for work aimed at understanding the relationship between neural plasticity and simple forms of non-associative and associative learning. Interest in the GWR has been based largely on the hypothesis that the response could be explained adequately by parallel monosynaptic reflex arcs between six parietovisceral ganglion (PVG) gill motor neurons (GMNs) and a cluster of sensory neurons termed the LE cluster. This hypothesis, the Kupfermann-Kandel model, made clear, falsifiable predictions that have stimulated experimental work for many years. Here, we review tests of three predictions of the Kupfermann-Kandel model: (1) that the GWR is a simple, reflexive behaviour graded with stimulus intensity; (2) that central nervous system (CNS) pathways are necessary and sufficient for the GWR; and (3) that activity in six identified GMNs is sufficient to account for the GWR. The available data suggest that (1) a variety of action patterns occur in the context of the GWR; (2) the PVG is not necessary and the diffuse peripheral nervous system (PNS) is sufficient to mediate these action patterns; and (3) the role of any individual GMN in the behaviour varies. Both the control of gill-withdrawal responses, and plasticity in these responses, are broadly distributed across both PNS and CNS pathways. The Kupfermann-Kandel model is inconsistent with the available data and therefore stands rejected. There is, no known causal connection or correlation between the observed plasticity at the identified synapses in this system and behavioural changes during non-associative and associative learning paradigms. Critical examination of these well-studied central pathways suggests that they represent a 'wetware' neural network, architecturally similar to the neural network models of the widely used 'Perceptron' and/or 'Back-propagation' type. Such models may offer a more biologically realistic representation of nervous system organisation than has been thought. In this model, the six parallel GMNs of the CNS correspond to a hidden layer within one module of the gill-control system. That is, the gill-control system appears to be organised as a distributed system with several parallel modules, some of which are neural networks in their own right. A new model is presented here which predicts that the six GMNs serve as components of a 'push-pull' gain control system, along with known but largely unidentified inhibitory motor neurons from the PVG. This 'push-pull' gain control system sets the responsiveness of the peripheral gill motor system. Neither causal nor correlational links between specific forms of neural plasticity and behavioural plasticity have been demonstrated in the GWR model system. However, the GWR model system does provide an opportunity to observe and describe directly the physiological and biochemical mechanisms of distributed representation and parallel processing in a largely identifiable 'wetware' neural network.     

Innervation pattern of the gill arches and gills of the carp (Cyprinus carpio)

The innervation pattern of the respiratory gill arches of the carp (Cyprinus carpio) is described. The gill region is innervated by the branchial branches of the glossopharyngeal and vagal nerves. Each branchial nerve divides at the level of or just distal to the epibranchial ganglion into: 1) a pretrematic branch, 2) a dorsal pharyngeal branch, and 3) a posttrematic branch. The dorsal pharyngeal branch innervates the palatal organ in the roof of the buccal cavity. The pretrematic and posttrematic branches innervate the posterior and anterior halves, respectively, of the gill arches bordering a gill slit. Each branch splits into an internal and an external part. The internal bundle innervates the buccal side of the gill arch, including the gill rakers. The external bundle terminates in the gill filaments. The epibranchial motor branch, a small nerve bundle containing only motor fibers, circumvents the ganglion and anastomoses distally with the posttrematic branch. The detailed course and branching patterns of these branches are described.     

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.     

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.     

Control of gill reflex habituation and the rate of EPSP decrement of L7 by a common source in the CNS of Aplysia

It was found in older Aplysia that the rate of decrement of the EPSP evoked in L₇ by repeated tactile stimulation of the gill was dependent on the strength of the applied stimulus and that the rate of decrement paralleled the rate of gill reflex habituation. As the stimulus intensity was increased both rates slowed. In contrast, it was found in young Aplysia that the rate of EPSP decrement and the rate of gill reflex habituation were independent of the strength of the stimulus applied to the gill. Neither rate changed as the stimulus intensity was changed. Moreover, L₇'s of young animals are more responsive to tactile stimuli applied to the gill than are L₇'s of older animals and the difference in excitability is not due to any differences in passive membrane properties between the L₇'s in young and older Aplysia. These findings are fully consistent with and supportive of the proposal that a common neuronal source in the parieto-visceral ganglion of Aplysia controls the rate of gill reflex habituation, the synaptic input to L₇, and the rate of decrement of this input, evoked by repeated tactile stimulation of the gill. Additinally, it was proposed that this common source is developed in completely in the young. As a consequence of this incomplete development, young Aplysia exhibit less adaptability to changing stimulus conditions and, in general, less ability to suppress their behavior. It may thus be possible to study directly developmental changes in the nervous system which act to transform juvenile behavior to adult behavior.     

Neuronal control of swimming in the medicinal leech

Summary The cell bodies and function of twelve neurons whose impulse pattern is clearly related to that of the swimming rhythm were identified in the segmental ganglion of the leech. These include excitatory and inhibitory motor neurons of the dorsal and ventral longitudinal muscles and the excitatory flattener motor neuron of the dorsoventral muscles. During swimming the membrane potential of these cells oscillates between a depolarized and a hyperpolarized phase. The activity of this ensemble of cells is sufficient to account for the contractile rhythm of the swimming animal. The following connections were found between these motor neurons. Electrotonic junctions link: (1) bilaterally homologous cells; (2) excitors of the dorsal longitudinal muscles; (3) excitors of the ventral longitudinal muscles; (4) inhibitors of both dorsal and ventral longitudinal muscles. The dorsal inhibitors project via an inhibitory pathway to the dorsal excitors, and the ventral inhibitor projects via an inhibitory pathway to the ventral excitors. The membrane potential oscillation of the excitors is at least partly attributable to the phasic inhibitory synaptic input which they receive from the inhibitors. The excitatory shortener motor neuron of the entire longitudinal musculature is maintained in an inactive state during swimming. This control is achieved by rectifying electrotonic junctions linking this neuron to the dorsal and ventral excitors. These junctions allow passage of only depolarizing current from the shortener to the dorsal and ventral excitors and of only hyperpolarizing current in the reverse direction. Furthermore, both dorsal and ventral inhibitors project via inhibitory pathways to the shortener neuron.We are greatly indebted to Ann Stuart for advice and help in this study, and for communicating to us some unpublished findings. We thank Elizabeth Mullenbach for excellent technical assistance.This research was supported by grant GB 31933 X from the National Science Foundation, and by Public Health Service Research grant GM 17866 and Training Grant GM 01389 from the Institute for General Medical Sciences.