Site-specific sensitization of defensive reflexes in Aplysia: a simple model of long-term hyperalgesia

Brief, noxious, electrical or mechanical stimulation of the skin of Aplysia produces enhancement of defensive reflexes triggered at the same site for at least a week after the noxious stimulation. This site-specific behavioral sensitization can be expressed as an increase in duration of the siphon-withdrawal reflex and as an increase in magnitude of the tail-withdrawal reflex. It is unlikely that peripheral factors play a predominant role in the long-term memory. First, long-term enhancement is blocked when the CNS is disconnected from the noxious stimulation site by nerve transection. Second, long-term enhancement is blocked by preventing neural activation at the noxious stimulation site, indicating that persistent physical damage alone is insufficient to cause the enhancement. A role for activity-dependent extrinsic modulation (ADEM) of mechanosensory neurons is suggested by similar site-specific enhancement produced when weak sensory activation is paired with general modulation elicited by strong stimulation of a distant site. Because this pairing represents a form of classical conditioning, site-specific sensitization and cutaneous classical conditioning appear to be closely related in this system. These findings suggest that site-specific sensitization reflects, at least in part, a central, long-term memory of injury. This form of memory may be phylogenetically widespread, and functionally similar to aspects of hyperalgesia. In addition, the close relationship between site-specific sensitization and cutaneous classical conditioning supports the hypothesis that some forms of classical conditioning evolved from mechanisms of sensitization.     

Cellular analog of differential classical conditioning in Aplysia: disruption by the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate.

We previously showed that the associative enhancement of Aplysia siphon sensorimotor synapses in a cellular analog of classical conditioning is disrupted by infusing the Ca(2+) chelator 1, 2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid into the postsynaptic motor neuron before training or by training in the presence of the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (APV). Our earlier experiments with APV used a nondifferential training protocol, in which different preparations were used for associative and nonassociative training. In the present experiments we extended our investigation of the role of NMDA receptor type potentiation in learning in Aplysia to differential conditioning. A cellular analog of differential conditioning was performed with a reduced preparation that consisted of the CNS plus two pedal nerves. A siphon motor neuron and two siphon sensory neurons, both of which were presynaptically connected to the motor neuron, were impaled with sharp microelectrodes. One sensorimotor synapse received paired stimulation with a conditioned stimulus (brief activation of a single sensory neuron) and an unconditioned stimulus (pedal nerve shock), whereas the other sensorimotor synapse received unpaired stimulation. Training in normal artificial seawater (ASW) resulted in significant differential enhancement of synapses that received the paired stimulation. Training in APV blocked this differential synaptic enhancement. A comparison of the present data with the data from earlier experiments that used nondifferential training is consistent with the possibility that differential training comprises competition between the presynaptic sensory neurons. Synaptic competition may contribute significantly to the associative effect of paired stimulation in the differential training paradigm.     

Depletion of serotonin in the nervous system of Aplysia reduces the behavioral enhancement of gill withdrawal as well as the heterosynaptic facilitation produced by tail shock

Noxious stimuli, such as electrical shocks to the animal's tail, enhance Aplysia's gill- and siphon-withdrawal reflex. Previous experimental work has indicated that this behavioral enhancement, known as dishabituation (if the reflex has been habituated) or sensitization (if it has not been habituated), might be mediated, at least in part, by the endogenous monoaminergic transmitter serotonin (5-HT). To assess 5-HT's role in dishabituation and sensitization of Aplysia withdrawal reflex, we treated Aplysia with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). We found that 5,7-DHT treatment significantly reduced the dishabituation of the withdrawal reflex produced by tail shock. Treatment with the neurotoxin also blocked the heterosynaptic facilitation of monosynaptic connections between siphon sensory neurons and their follower cells, which contributes to the behavioral enhancement. Analysis by high-performance liquid chromatography indicated that 5,7-DHT treatment significantly reduced 5-HT levels in the Aplysia CNS. Moreover, the neurotoxic effects of 5,7-DHT appeared to be relatively specific for serotonergic pathways. Thus, 5,7-DHT treatment did not disrupt the ability of nonserotonergic facilitatory interneurons, the L29 cells, to facilitate the connections of siphon sensory neurons. Also, 5,7-DHT reduced 5-HT-dependent, but not dopamine-dependent, histofluorescence in Aplysia central ganglia. Finally, 5,7-DHT does not reduce the levels of the facilitatory peptides SCPA and SCPB within the Aplysia CNS. Our results, together with those of Mackey et al. (1989), indicate that 5-HT plays a major role in mediating dishabituation and sensitization of Aplysia's withdrawal reflex.     

Long-term enhancement produced by activity-dependent modulation of Aplysia sensory neurons

We have investigated long-lasting enhancement of signaling effectiveness in the tail sensory neurons of Aplysia using both intracellular and extracellular stimulation. The pairing of high frequency homosynaptic activation with heterosynaptic modulation produced significantly greater enhancement of monosynaptic connections to identified motor neurons than did homosynaptic activity, heterosynaptic modulation, or test stimuli alone. Enhancement of the monosynaptic excitatory postsynaptic potential produced by pairing persisted for at least 4 hr, and the kinetics of decay of this potentiation indicated a time constant of about 5 hr. Although unpaired stimulation produced much weaker enhancement, both homosynaptic activity and heterosynaptic modulation alone produced enhancement lasting more than 90 min. The results are consistent with the possibility that intrinsic electrical activity can amplify the modulatory effects of a paired extrinsic chemical signal to produce long-term changes in synaptic strength. Paired stimulation also produced a relative enhancement of the excitability of the sensory neuron soma as judged by changes in action potential threshold. The lack of generalized changes in the postsynaptic cell and the observation of pairing-induced long-term changes in action potential threshold in the presynaptic cell soma suggest that long-term enhancement produced by pairing has a presynaptic locus in this system. Since pairing-specific enhancement can encode associations between sensory and motivational events in these cells, this form of plasticity may function as a form of associative memory. Similarities between long-term paired enhancement in this system and associative long-term potentiation in other systems suggest that activity-dependent neuromodulation might be involved in cellular memory in other systems as well.     

Long-term expansion and sensitization of mechanosensory receptive fields in Aplysia support an activity-dependent model of whole-cell sensory plasticity

Long-term changes in peripheral receptive field properties of mechanosensory/nociceptive neurons were investigated 1-3 weeks after noxious stimulation. Noxious stimuli consisted of a deep penetrating cut through the middle of the tail, strong electric shock applied to the tail surface, or a combination of deep and superficial tail stimulation. Action potentials evoked in the tail were monitored with intracellular electrodes in central somata of tail sensory neurons. Three long-term changes in receptive field properties were produced in the region of noxious stimulation: (1) mechanosensory thresholds decreased, (2) receptive field areas increased, and (3) the percentage of cells showing receptive field extension across the tail midline increased. Sizes and shapes of individual receptive fields did not vary during extensive testing of tails perfused with artificial seawater or during testing in cobalt solutions that block synaptic transmission. This stability of receptive field geometry, coupled with the observation that increased peripheral excitability in these cells does not increase receptive field size, suggests that long-term receptive field alterations involve growth of peripheral sensory processes. A model is proposed in which the signaling strength of the entire sensory cell increases in response to trauma of its receptive field. In this model long-term enhancement of central and peripheral sensory responsiveness is selectively triggered by activity dependent extrinsic modulation of the centrally located soma, which accelerates synthesis of growth-associated proteins used in collateral and regenerative sprouting of traumatized peripheral processes.     

Identified serotonergic neurons LCB1 and RCB1 in the cerebral ganglia of Aplysia produce presynaptic facilitation of siphon sensory neurons

Several lines of evidence suggest that 5-HT plays a significant role in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia. Most recently, Glanzman et al. (1989) found that treatment with the 5-HT neurotoxin, 5,7-DHT markedly reduced both synaptic facilitation and behavioral dishabituation. To provide more direct evidence for a role of 5-HT, we have attempted to identify individual serotonergic facilitator neurons. Hawkins (1989) used histological techniques to locate several serotonergic neurons in the ring ganglia that send axons to the abdominal ganglion and are therefore possible serotonergic facilitators. These include one neuron in the B cluster of each cerebral ganglion, which we have identified electrophysiologically and named the CB1 cells. Both glyoxylic acid histofluorescence and 5-HT immunofluorescence indicate that the CB1 neurons are serotonergic. In a semiintact preparation, the CB1 neurons respond to cutaneous stimulation which produces dishabituation and sensitization (such as tail shock) with an increase in firing, which may outlast the stimulation by 15 min. Intracellular stimulation of a CB1 neuron in a manner similar to its response to tail shock produces facilitation of the EPSPs from siphon sensory neurons to motor neurons, as well as broadening of the action potential in the sensory neurons in tetraethylammonium solution. These results strongly suggest that the identified serotonergic CB1 neurons participate in mediating presynaptic facilitation contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia.     

Identified facilitator neurons L29 and L28 are excited by cutaneous stimuli used in dishabituation, sensitization, and classical conditioning of Aplysia

Tactile or electrical stimulation of the skin can be used to produce dishabituation, sensitization, and classical conditioning of the gill- and siphon-withdrawal reflex in Aplysia. These behavioral effects are thought to involve presynaptic facilitation at the synapses from siphon sensory neurons to gill and siphon motor neurons. Facilitation of PSPs onto the motor neurons can also be produced by intracellular stimulation of single identified neurons in the abdominal ganglion, including L29 and L28. In this paper, we further characterize L29 and L28. First, we show that they are excited by cutaneous stimuli similar to those used to produce dishabituation, sensitization, and classical conditioning and may therefore participate in mediating those behavioral effects. The results are also consistent with a possible role of L29 and L28 in higher-order features of conditioning. Second, we show that 5-HT does not mimic some of the PSPs of L29, in agreement with previous evidence that L29 is not serotonergic. Third, we present 2 types of evidence that L29 acts directly to produce facilitation of the sensory cells: (1) L29 comes into close contact with sensory cells in fluorescent double-labeling experiments, and (2) L29 produces facilitation of sensory cells in dissociated cell culture. Together with the results of the preceding paper (Mackey et al., 1989), these results indicate that facilitation of sensory cell synapses contributing to behavioral enhancement of the reflex can be produced by identified neurons that use 2 different transmitters: 5-HT (the transmitter of CB1) and the unknown transmitter of L29.     

Slow depolarization produced by associative conditioning of Aplysia sensory neurons may enhance Ca2+ entry

Sensory neurons activated by intracellular stimulation immediately before sensitizing tail shock displayed a slow depolarization after the shock. By contrast, sensory neurons exposed to the effects of tail shock alone or unpaired activation and tail shock showed a slow hyperpolarizing response to the shock. A voltage-sensitive Ca2+ conductance that is activated near the resting potential may be modulated by these opposite effects of associative and non-associative training.     

Somatotopic organization and functional properties of mechanosensory neurons expressing sensorin-A mRNA in Aplysia californica

A previous study reported that a peptide, sensorin-A, is expressed exclusively in mechanosensory neurons having somata in central ganglia of Aplysia. The present study utilized in situ hybridization, staining by nerve back-fill and soma injection, and electrophysiological methods to characterize the locations, numbers, and functions of sensorin-A-expressing neurons and to define the relationships between soma locations and the locations of peripheral axons and receptive fields. Approximately 1,000 cells express sensorin-A mRNA in young adult animals (10-30 g) and 1,200 cells in larger adults (100-300 g). All of the labeled somata are in the CNS, primarily in the abdominal LE, rLE, RE and RF, pleural VC, cerebral J and K, and buccal S clusters. Expression also occurs in a few sparsely distributed cells in most ganglia. Together, receptive fields of all these mechanosensory clusters cover the entire body surface. Each VC cluster forms a somatotopic map of the ipsilateral body, a "sensory aplunculus." Cells in the pleural and cerebral clusters have partially overlapping sensory fields and synaptic targets. Buccal S cells have receptive fields on the buccal mass and lips and display notable differences in electrophysiological properties from other sensorin-A-expressing neurons. Neurons in all of the clusters have relatively high mechanosensory thresholds, responding preferentially to threatening or noxious stimuli. Synaptic outputs to target cells having defensive functions support a nociceptive role, as does peripheral sensitization following noxious stimulation, although additional functions are likely in some clusters. Interesting questions arise from observations that mRNA for sensorin-A is present not only in the somata but also in synaptic regions, connectives, and peripheral fibers.     

A cellular analysis of inhibition in the siphon withdrawal reflex of Aplysia

Recent behavioral experiments examining the siphon withdrawal reflex of Aplysia have revealed inhibitory effects of strong tail shock, a stimulus commonly used as an unconditioned stimulus in studies of associative and nonassociative learning in Aplysia. We utilized a reduced preparation to perform a cellular analysis of tail shock-induced inhibition in the siphon withdrawal reflex. First, we carried out behavioral studies that showed that the reduced preparation exhibits a siphon withdrawal reflex to water jet stimuli, and that tail shock produces inhibitory behavioral effects comparable to those in the intact animal: (1) strong shock produces transient inhibition of nonhabituated responses, and (2) a habituated response is facilitated by weak shock, but not by strong shock, suggesting that increasing tail shock intensity recruits the inhibitory process that competes with facilitation of habituated reflexes. Next, we carried out cellular studies that showed that the amplitude of the complex EPSP in siphon motor neurons elicited by water jet stimuli to the siphon also exhibits the inhibitory patterns produced by tail shock: (1) the nondecremented complex EPSP (a neural correlate of a nonhabituated siphon withdrawal reflex) is significantly inhibited 90 sec after strong tail shock and recovers to preshock levels 10 min later, and (2) the decremented complex EPSP (a neural correlate of a habituated reflex) is significantly facilitated by weak shock, but is not facilitated by strong shock. In addition to the complex EPSP, we simultaneously examined the monosynaptic connection between siphon sensory neurons and siphon motor neurons. The monosynaptic EPSP does not show the pattern of inhibitory modulation by tail shock exhibited by the siphon withdrawal reflex and the complex EPSP: (1) the nondecremented monosynaptic EPSP is not inhibited 90 sec after strong shock, but tends to be above preshock levels; and (2) the decremented monosynaptic EPSP is facilitated by weak as well as strong tail shock. Our results suggest that an important component of the inhibitory process triggered by strong tail shock is mediated by neural elements presynaptic to the siphon motor neurons. Because modulation of the monosynaptic connection between identified siphon sensory and siphon motor neurons does not parallel the tail shock-induced inhibitory patterns observed in the siphon withdrawal reflex and in the complex EPSP, other synaptic connections are likely to play an important role in mediating tail shock-induced inhibition in the siphon withdrawal reflex.     

Slow depolarization produced by associative conditioning of Aplysia sensory neurons may enhance Ca entry

Sensory neurons activated by intracellular stimulation immediately before sensitizing tail shock displayed a slow depolarization after the shock. By contrast, sensory neurons exposed to the effects of tail shock alone or unpaired activation and tail shock showed a slow hyperpolarizing response to the shock. A voltage-sensitive Ca²⁺ conductance that is activated near the resting potential may be modulated by these opposite effects of associative and non-associative training.     

Localization of potential serotonergic facilitator neurons in Aplysia by glyoxylic acid histofluorescence combined with retrograde fluorescent labeling

A variety of evidence suggests that 5-HT participates in presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the gill- and siphon-withdrawal reflex in Aplysia. Most recently, Glanzman et al. (1989) have shown that the 5-HT neurotoxin 5,7-DHT markedly reduces both the synaptic facilitation and behavioral dishabituation produced by tail shock. To provide more direct evidence for a role of 5-HT, I have used histological techniques to try to locate individual serotonergic facilitator neurons. I first used a modification of the glyoxylic acid histofluorescence technique to map serotonergic and dopaminergic neurons in the CNS of Aplysia. Intracellular fluorescent labeling combined with histofluorescence indicates that the previously identified L29 facilitator neurons are not serotonergic. Nerve transection experiments suggest that most of the perisomatic 5-HT histofluorescence in the abdominal ganglion (the location of the siphon sensory cells) comes from neurons whose cell bodies are located in the pedal or cerebral ganglia. As there are at least 500 serotonergic neurons in those ganglia, I combined retrograde fluorescent labeling with histofluorescence to identify a small subset of those neurons which send processes to the abdominal ganglion and are therefore potential serotonergic facilitators. In the following paper, Mackey et al. (1989) show that stimulation of 2 of those neurons in the cerebral ganglia (the CB1 cells) produces presynaptic facilitation of the siphon sensory cells contributing to dishabituation and sensitization of the withdrawal reflex.     

Dissociation between sensitization and learning-related neuromodulation in an aplysiid species.

Previous phylogenetic analyses of learning and memory in an opisthobranch lineage uncovered a correlation between two learning-related neuromodulatory traits and their associated behavioral phenotypes. In particular, serotonin-induced increases in sensory neuron spike duration and excitability, which are thought to underlie several facilitatory forms of learning in Aplysia, appear to have been lost over the course of evolution in a distantly related aplysiid, Dolabrifera dolabrifera. This deficit is paralleled by a behavioral deficit: individuals of Dolabrifera do not express generalized sensitization (reflex enhancement of an unhabituated response after a noxious stimulus is applied outside of the reflex receptive field) or dishabituation (reflex enhancement of a habituated reflex). The goal of the present study was to confirm and extend this correlation by testing for the neuromodulatory traits and generalized sensitization in an additional species, Phyllaplysia taylori, which is closely related to Dolabrifera. Instead, our results indicated a lack of correlation between the neuromodulatory and behavioral phenotypes. In particular, sensory neuron homologues in Phyllaplysia showed the ancestral neuromodulatory phenotype typified by Aplysia. Bath-applied 10 microM serotonin significantly increased homologue spike duration and excitability. However, when trained with the identical apparatus and protocols that produced generalized sensitization in Aplysia, individuals of Phyllaplysia showed no evidence of sensitization. Thus, this species expresses the neuromodulatory phenotype of its ancestors while appearing to express the behavioral phenotype of its near relative. These results suggests that generalized sensitization can be lost during the course of evolution in the absence of a deficit in these two neuromodulatory traits, and raises the possibility that the two traits may support some other form of behavioral plasticity in Phyllaplysia. The results also raise the question of the mechanistic basis of the behavioral deficit in Phyllaplysia.     

Serotonin mimics tail shock in producing transient inhibition in the siphon withdrawal reflex of Aplysia

Tail shock-induced modulation of the siphon withdrawal reflex of Aplysia has recently been shown to have a transient inhibitory component, as well as a facilitatory component. This transient behavioral inhibition is also seen in a reduced preparation in which a cellular reflection of the inhibitory process, tail shock-induced inhibition of complex EPSPs in siphon motor neurons, is observed. The biogenic amine serotonin (5-HT) is known to play a role in the facilitatory aspects of sensitization in Aplysia. The aim of this article was to examine whether 5-HT might also contribute to the inhibitory effects of tail shock in the siphon withdrawal reflex. To examine this question, we carried out two kinds of experiments. First, in the isolated abdominal ganglion, we recorded intracellularly from siphon motor neurons and examined the effects of 5-HT on (1) complex (polysynaptic) EPSPs, produced by siphon nerve stimulation, and, simultaneously, (2) monosynaptic EPSPs from siphon sensory neurons. We found that, paralleling the effects of tail shock in the reduced preparation, 5-HT produced transient inhibition of the complex EPSP; the monosynaptic EPSP was facilitated by 5-HT. Second, we examined the behavioral effects of 5-HT on siphon withdrawal in a reduced preparation. We found that 5-HT again paralleled tail shock by producing transient inhibition of the siphon withdrawal reflex. Our results suggest that, in addition to its well-established facilitatory role in reflex modulation in Aplysia, 5-HT might play an important inhibitory role, as well.     

Dishabituation and sensitization emerge as separate processes during development in Aplysia

Until recently, dishabituation and sensitization have commonly been considered to reflect a unitary process: Sensitization refers to a general facilitation produced by strong or noxious stimuli that enhances subsequent responding; dishabituation has been thought to represent a special instance of sensitization in which the facilitation is simply superimposed on a habituated response level. The unitary process hypothesis was based on the observation that both decremented and nondecremented responses are facilitated by a common noxious or strong stimulus. However, this observation does not rule out the possibility that dishabituation and sensitization could reflect separate processes that are activated in parallel by a strong stimulus. Recent cellular experiments by Hochner et al. (1986) suggest that this, in fact, occurs in the sensory neurons of the gill withdrawal reflex in Aplysia. A developmental analysis of learning in the marine mollusc Aplysia permits a direct behavioral test of this hypothesis. If dishabituation and sensitization reflect a unitary process then they should emerge at the same time ontogenetically. On the other hand, if they reflect different processes, then they might emerge according to different ontogenetic timetables. In the present study we examined the temporal emergence of dishabituation and sensitization in the defensive siphon withdrawal reflex in 3 stages of juvenile Aplysia: stage 11, early stage 12, and late stage 12. Animals received one of 2 kinds of training: Dishabituation training, in which the effect of strong tail shock on habituated responses were observed, and Sensitization training, in which the effect of strong tail shock on nondecremented responses was observed. We found that, while dishabituation was present in all stages examined, sensitization did not emerge until several weeks later, in late stage 12. These results were confirmed and extended in a group of animals that were tested twice: first in stage 11, when they showed no sensitization, and again 13 weeks later, in late stage 12, when they then showed significant sensitization. Our analysis of nondecremented responses prior to the emergence of sensitization also revealed an unexpected inhibitory component of tail shock that produces reflex depression. Moreover, there was a clear progression in the net effects of tail shock during development: reflex depression was produced in stages 11 and early stage 12, followed by a transition to reflex facilitation (sensitization) in late stage 12. Finally, when sensitization emerged in late stage 12, the process of dishabituation showed a significant increase compared with previous developmental stages.(ABSTRACT TRUNCATED AT 400 WORDS)     

The contribution of facilitation of monosynaptic PSPs to dishabituation and sensitization of the Aplysia siphon withdrawal reflex.

To examine the relationship between synaptic plasticity and learning and memory as directly as possible, we have developed a new simplified preparation for studying the siphon-withdrawal reflex of Aplysia in which it is relatively easy to record synaptic connections between individual identified neurons during simple forms of learning. We estimated that monosynaptic EPSPs from LE siphon sensory neurons to LFS siphon motor neurons mediate approximately one-third of the reflex response measured in this preparation, which corresponds to siphon flaring in the intact animal. To investigate cellular mechanisms contributing to dishabituation and sensitization, we recorded evoked firing of LFS neurons, the siphon withdrawal produced by stimulation of an LFS neuron, the complex PSP in an LFS neuron, and the monosynaptic PSP from an "on-field" or "off-field" LE neuron to an LFS neuron during behavioral training. Unlike the simplified gill-withdrawal preparation (Cohen et al., 1997; Frost et al., 1997), in the siphon-withdrawal preparation we found no qualitative differences between the major cellular mechanisms contributing to dishabituation and sensitization, suggesting that dissociations that have been observed previously may be attributable to transient inhibition that does not occur for this component of the reflex. Furthermore, in the siphon-withdrawal preparation, all of the various cellular measures, including monosynaptic PSPs from either on-field or off-field LE neurons, changed approximately in parallel with changes in the behavior. These results provide the most direct evidence so far available that both dishabituation and sensitization involve multiple mechanisms, including heterosynaptic facilitation of sensory neuron-motor neuron PSPs.     

Serotonin and cyclic adenosine 3':5'-monophosphate modulate the potassium current in tail sensory neurons in the pleural ganglion of Aplysia

Tail sensory neurons in the pleural ganglion that mediate the afferent portion of the tail withdrawal reflex in Aplysia californica undergo heterosynaptic facilitation of transmitter release during sensitization. As in the siphon sensory neurons, the transmitter serotonin produces facilitation and also elicits a slow, decreased conductance excitatory postsynaptic potential (EPSP) in these neurons. Using voltage clamp and biochemical analyses, we have found that the slow EPSP in the pleural sensory neurons is due to a decrease in a potassium conductance identical to the S potassium current characterized in siphon sensory neurons. Like the S current, the current modulated by serotonin in the pleural sensory neurons is a non-inactivating potassium current, and it contributes to both the resting and action potentials. The current reverses in 120 mM external K+ at -20 mV, close to the predicted Nernst equilibrium potential. Intracellular cesium blocks the serotonin response, but the current is not blocked by equimolar substitution of barium for calcium, nor by 50 mM tetraethylammonium chloride. The effect of serotonin is cAMP dependent, since serotonin elevates cAMP and both cAMP injection and forskolin mimic the serotonin response. These results indicate that the mechanism associated with sensitization of the siphon-gill withdrawal reflex, a slow decreased potassium conductance, is also a component of the neuronal circuitry underlying modulation of another reflex, the tail withdrawal reflex. Therefore, two distinct populations of neurons subserving similar behavioral functions have related biophysical and biochemical properties.     

Associative pairing involving monocular stimulation selectively mobilizes a subclass of GABAergic interneurons in the mouse visual cortex

Levels of γ‐aminobutyric acid (GABA) and its synthesizing enzyme in cerebral cortex are regulated by sensory experience. Previously we found that associative pairing of vibrissae stimulation and tail shock results in upregulation of GABAergic markers in the mouse barrel cortex. In order to ascertain whether GABAergic upregulation also accompanies associative pairing in other sensory modalities, we examined the mouse visual cortex after analogous training with visual stimulus. During pairing, visual stimulus (CS) was coupled with a tail shock (UCS). We examined the density of cells expressing glutamic acid decarboxylase (GAD) and parvalbumin (PV) in monocular and binocular segments of the primary visual cortex (V1). The auditory cortex was used as a control. After monocular training, the density of cells expressing GAD rose significantly in the monocular segment of V1 contralateral to the stimulated eye, compared with the opposite hemisphere. This effect was due to the association of CS and UCS, as no changes were found after visual stimulation alone or in the auditory cortex. No changes were noted in the density of PV⁺ neurons, so the effect was attributed to GAD⁺/PV^? neurons. Mobilization of a specific subclass of GABAergic cells, observed after associative pairing in the somatosensory and visual cortices, may reflect the necessity to restrict the activity of circuits involved in sensory association. J. Comp. Neurol. 516:482–492, 2009. © 2009 Wiley‐Liss, Inc.     

Serotoninergic varicosities make synaptic contacts with pleural sensory neurons of Aplysia.

Serotonin is a modulatory neurotransmitter that produces many of the cellular changes associated with sensitization of reflexes in Aplysia. These changes have been carefully documented in sensory neurons located in the abdominal ganglion that mediate the gill-siphon withdrawal reflex and in sensory neurons located in the pleural ganglion that mediate the tail-siphon withdrawal reflex. Although serotonin appears to be necessary for sensitization, there is no direct evidence that serotoninergic neurons make synaptic contacts with sensory neurons. In this study, the immunoperoxidase technique was used to label serotonin-immunoreactive neurites surrounding the cell bodies of sensory neurons in the pleural ganglion. Serotonin-immunoreactive neurites had varicosities whose mean short axis diameter was 1.1 +/- 0.6 microns (mean +/- S.D.). The shape of the size distribution was skewed toward larger sizes, however, suggesting that there were multiple subpopulations of varicosities. One subpopulation was that of varicosities located at branch points whose average short axis diameter was larger than normal (1.7 +/- 0.5 microns). Serotonin-immunoreactive varicosities were directly apposed to the sensory neurons without intervening glial cells. In most contacts, serotonin-immunoreactive neurites invaginated into the plasma membranes of the sensory neurons. There were also a few contacts onto spinelike processes, but these were flat rather than invaginated. Serotoninergic neurons whose activity produces changes in the electrophysiological properties of sensory neurons have been identified, but this study provides the first direct evidence for synaptic connections between serotoninergic neurons and sensory neurons in Aplysia.     

Enhanced nociceptive behaviour following conditioning injection of formalin in the perioral area of the rat

The possible existence of long-term modifications in response to a transient nociceptive conditioning stimulation was investigated in the rat in three experiments. (1) A nociceptive conditioning stimulus was delivered in the form of a s.c. formalin injection (conditioning injection) in the left upper lip. Evaluation of the nociceptive behaviour triggered by another formalin injection (testing injection) made in the controlateral right upper lip was carried out in distinct groups of rats 7, 14 or 28 days after the conditioning. An enhanced nociceptive response at days 7 and 14 and a return to the baseline at day 28 were observed. (2) A similar protocol was developed with formalin used for both conditioning and testing but an anaesthetic blockade of the infraorbital nerve was performed just before the conditioning injection to suppress the initial barrage. The change observed at day 7 was suppressed by the nerve block. (3) A conditioning nociceptive stimulus was applied either ipsilaterally to the right lower lip or to the tail. An increased nociceptive response was observed when the conditioning stimulus was applied to the same side as the test stimulus but no increase in the formalin test response was detected when the conditioning stimulus was applied to the tail. These results indicated that, after a single formalin injection in the left upper lip, a hyperexcitability developed that depended on the initial barrage, lasted for at least 2 weeks, was no longer present at 4 weeks and might rely on a segmental mechanism. The hypothesis of a central sensitization triggered by an initial barrage and maintained by an ongoing input induced from the periphery is discussed.