C-fiber responses in the ventrolateral column of the cat spinal cord.

Saphenous nerve C-fiber volleys generate, in the ventrolateral column of the cat spinal cord, a conducted postsynaptic response revealed by averaging the integrated temporally dispersed axonal discharges. This finding is compatible with the hypothesis that the feline ventrolateral column includes a major pathway related to nociception.

Presynaptic facilitation as a mechanism for behavioral sensitization in Aplysia.

Sensitization is an elementary form of nonassociative learning, related to behavioral arousal, in which a strong stimulus facilitates a reflex response. Studies of the neural circuit of the gill-withdrawal reflex in the isolated abdominal ganglion on Aplysia indicate that short-term sensitization is due to presynaptic facilitation. The facilitation results in a sudden increase in the amount of neurotransmitter released by the sensory neurons at their synapses with motor neurons.

Sensitization in Aplysia: restoration of transmission in synapses inactivated by long-term habituation.

Long-term habituation of a simple withdrawal reflex in Aplysia leads to an inactivation of synaptic transmission between identified sensory and gill motor neurons that persists for more than 3 weeks. A single sensitizing stimulus rapidly reactivates both the depressed behavioral response and the inactivated synaptic transmission. Thus sensitization, a simple competitive form of learning, provides a mechanism whereby changing environmental demands can rapidly override the long-term memory of habituation.

cAMP evokes long-term facilitation in Aplysia sensory neurons that requires new protein synthesis.

Behavioral sensitization leads to both short- and long-term enhancement of synaptic transmission between the sensory and motor neurons of the gill-withdrawal reflex in Aplysia. Serotonin (5-HT), a transmitter important for short-term sensitization, can evoke long-term enhancement of synaptic strength detected 1 day later. Because 5-HT mediates short-term facilitation through adenosine 3',5'-monophosphate (cAMP)-dependent protein phosphorylation, the role of cAMP in the long-term modulation of this identified synapse was examined. Like 5-HT, cAMP can also evoke long-term facilitation lasting 24 hours. Unlike the short-term change, the long-lasting change is blocked by anisomycin, a reversible inhibitor of protein synthesis, and therefore must involve the synthesis of gene products not required for the short-term change.

Habituation and dishabituation of the gill-withdrawal reflex in Aplysia.

A behavioral reflex mediated by identified motor neurons in the abdominal ganglion of Aplysia undergoes two simple forms of shortterm modification. When the gill-with-drawal reflex was repeatedly evoked by a tactile stimulus to the siphon or mantle shelf, the amplitude of the response showed marked decrement (habituation). After a period of rest the response showed spontaneous recovery. The amplitude of a habituated response was facilitated by the presentation of a strong tactile stimulus to another part of the animal (dishabituation). Many characteristics of habituation and dishabituation in Aplysia are similar to those in vertebrates.

Neuronal correlates of habituation and dishabituation of the gill-withdrawal reflex in Aplysia.

We have examinived the nieural correlates of habittuatiotn atid dishabitiuation of tlhe gill-withdrwal reflex in Aplysia. We obtained intracelllular recordings from identified gill motor neurons in the abdominal ganglionz of a semi-intact preparation of Aplysia wlhile we simultaneously recorded behavior responises of the gill. Habituation and dishabituation were not due to peripheral changes in either the sensory receptors or the gill musculature butt were caused by changes in the amplitlude of the excitatory synaptic potentials produced at the gill motor neurons.

Synaptic facilitation and behavioral sensitization in Aplysia: possible role of serotonin and cyclic AMP.

The neural changes accompanying sensitization of the gill-withdrawal reflex in Aplysia are associated with presynaptic facilitation at monosynaptic connections between sensory neurons and motor cells. To analyze the molecular mechanisms underlying the facilitation, the pharmacological actions of serotonin, octopamine, and dopamine were examined. Only serotonin enhanced synaptic transmission between the sensory and the motor neurons. A serotonin antagonist, cinanserin, reversibly blocked the synaptic facilitation. The action of serotonin may be mediated by adenosime 3',5'-monophosphate (cyclic AMP). Exposing the ganglion to dibutyryl cyclic AMP or injecting cyclic AMP into the cell body enhances the synaptic action of a sensory neuron. The mechanism of presynaptic facilitation, therefore, may include activation of one or more serotonergic neurons, which enhance the release of a neurotransmitter by increasing the intracellular concentration of cyclic AMP in the terminals of the sensory neurons.

Cellular analysis of long-term habituation of the gill-withdrawal reflex of Aplysia californica.

Long-term habituation training in Aplysia californica produces a profound depression in the efficacy of synaptic transmission between mechanoreceptor neurons and gill motor neurons. This depression persists for more than 3 weeks. Thus a critical synaptic site for plasticity underlying long-term habituation is the same as that for short-term habituation. For this simple form of learning, short- and long-term memory share a common locus and aspects of a common mechanism: synaptic depression.

Neuronal mechanisms of habituation and dishabituation of the gill-withdrawal reflex in Aplysia.

The cellular mechanisms of habituation and dishabituation of the gill-withdrawal reflex in Aplysia were studied with an isolated abdominal ganglion connected to a piece of skin from the tactile receptive field of the reflex. By obtaining simultaneous intracellular recordings from both the sensory neurons and one of the main identified motor neurons, we have been able to reduce the reflex to its monosynaptic components. The monosynaptic excitatory postsynaptic potentials showed a profound low-frequency depression when repeatedly elicited and showed heterosynaptic facilitation after application of a strong stimulus to another pathway. Thus, both habituation and dishabituation can be explained in part and perhaps entirely by changes in the efficacy of specific excitatory synapses.

Central and peripheral control of gill movements in Aplysia.

Two types of gill contraction in Aplysia were used to study the relation of peripheral and central pathways in controlling behavioral responses in a mollusk. A weak or moderate tactile stimulus to the mantle elicits gill contraction (gill-withdrawal reflex) as a component of a more extensive withdrawal response; a stimulus applied directly to the gill elicits a localized response of the gill pinnule (pinnule response). Central pathways through the abdominal ganglion are both necessary and sufficient for the gill-withdrawal reflex, and motor neuron L7 makes direct connections with gill muscles, without engaging the peripheral plexus. Peripheral pathways are necessary and sufficient for the pinnule response. As a result of the independence of peripheral and central pathways, habituation by repeated tactile stimulation of one pathway does not affect the responsiveness of the other pathway.

A critical period for macromolecular synthesis in long-term heterosynaptic facilitation in Aplysia.

Both long-term and short-term sensitization of the gill and siphon withdrawal reflex in Aplysia involve facilitation of the monosynaptic connections between the sensory and motor neurons. To analyze the relationship between these two forms of synaptic facilitation at the cellular and molecular level, this monosynaptic sensorimotor component of the gill-withdrawal reflex of Aplysia can be reconstituted in dissociated cell culture. Whereas one brief application of 1 microM serotonin produced short-term facilitation in the sensorimotor connection that lasted minutes, five applications over 1.5 hours resulted in long-term facilitation that lasted more than 24 hours. Inhibitors of protein synthesis or RNA synthesis selectively blocked long-term facilitation, but not short-term facilitation, indicating that long-term facilitation requires the expression of gene products not essential for short-term facilitation. Moreover, the inhibitors only blocked long-term facilitation when given during the serotonin applications; the inhibitors did not block the facilitation when given either before or after serotonin application. These results parallel those for behavioral performance in vertebrates and indicate that the critical time window characteristic of the requirement for macromolecular synthesis in long-term heterosynaptic facilitation is not a property of complex circuitry, but an intrinsic characteristic of specific nerve cells and synaptic connections involved in the long-term storage of information.

A quantitative analysis of 2-D gels identifies proteins in which labeling is increased following long-term sensitization in Aplysia.

Long-term memory for sensitization of the gill- and siphon-withdrawal reflex in Aplysia, produced by 4 days of training, is associated with increased synaptic efficacy of the connection between the sensory and motor neurons. This training is also accompanied by neuronal growth; there is an increase in the number of synaptic varicosities per sensory neuron and in the number of active zones. Such structural changes may be due to changes in the rates of synthesis of certain proteins. We have searched for proteins in which the rates of [35S]methionine labeling are altered during the maintenance phase of long-term memory for sensitization by using computer-assisted quantitative 2-D gel analysis. This method has allowed us to detect 4 proteins in which labeling is altered after 4 days of sensitization training.

Ca2+-independent protein kinase C Apl II mediates the serotonin-induced facilitation at depressed aplysia sensorimotor synapses.

At nondepressed Aplysia sensory to motor synapses, serotonin (5-HT) facilitates transmitter release primarily through a protein kinase A pathway. In contrast, at depressed Aplysia sensory to motor synapses, 5-HT facilitates transmitter release primarily through a protein kinase C (PKC)-dependent pathway. It is known that only two phorbol ester-activated PKC isoforms, the Ca(2+)-dependent PKC Apl I and the Ca(2+)-independent PKC Apl II, exist in the Aplysia nervous system. For the first time, we have now been able to functionally determine which isoform of PKC is involved in a particular form of plasticity. We microinjected cultured sensorimotor pairs of neurons with various PKC constructs tagged with the enhanced green fluorescent protein as a reporter for successful plasmid expression. Our results demonstrate that short-term facilitation of depressed synapses is mediated by PKC Apl II. Dominant-negative PKC Apl II, but not dominant-negative PKC Apl I, disrupted the normal kinetics of 5-HT-induced facilitation by completely blocking its rapid onset. This effect was specific to depressed synapses, because dominant-negative PKC Apl II did not inhibit 5-HT-mediated facilitation of nondepressed synapses. Our results suggest that not only different signal transduction pathways but also different isoforms of a specific cascade may mediate physiological responses according to the state of a synapse.

Cloning and characterization of Aplysia neutral endopeptidase, a metallo-endopeptidase involved in the extracellular metabolism of neuropeptides in Aplysia californica.

Cell surface metallo-endopeptidases play important roles in cell communication by controlling the levels of bioactive peptides around peptide receptors. To understand the relative relevance of these enzymes in the CNS, we characterized a metallo-endopeptidase in the CNS of Aplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and physiological levels. The membrane-bound activity cleaved Leu-enkephalin at the Gly3-Phe4 bond with an inhibitor profile similar to that of the mammalian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that the Aplysia and mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific anti-Aplysia NEP (apNEP) antibodies and by the [125I]-labeled NEP-specific inhibitor RB104, demonstrating that the apNEP gene codes for the RB104-binding protein. In situ hybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is present both in the cell bodies and in neurites that travel along the neuropil and peripheral nerves. When incubated in the presence of a specific NEP inhibitor, many neurons of the buccal ganglion showed a greatly prolonged physiological response to stimulation, suggesting that NEP-like metallo-endopeptidases may play a critical role in the regulation of the feeding behavior in Aplysia. One of the putative targets of apNEP in this behavior is the small cardioactive peptide, as suggested by RP-HPLC experiments. More generally, the presence of apNEP in the CNS and periphery may indicate that it could play a major role in the modulation of synaptic transmission in Aplysia and in the metabolism of neuropeptides close to their point of release.

cDNA structure and in situ localization of the Aplysia californica pro-hormone convertase PC2.

The complete cDNA structure of the Aplysia californica pro-protein and pro-hormone convertase PC2 (aPC2) was obtained from a cDNA library of the nervous system. The deduced amino acid sequence revealed that aPC2 exhibits an 85%, 61% and 62% sequence identity to the Lymnaea stagnalis, Xenopus laevis and mouse PC2 homologues, respectively. The deduced stagnalis, Xenopus laevis and mouse PC2 homologues, respectively. The deduced primary sequence suggested a protein of 653 amino acids which includes a 27- and 88-amino acid signal peptide and pro-segment. The signal peptide and the C-terminal segments are the least conserved regions. On Northern blots of nervous system we detected a transcript of 6.8 kb. The in situ hybridization histochemistry on the abdominal ganglion revealed intense labeling of the bag cells. Large peptidergic cells and clusters of sensory and motor neurons also contained high levels of aPC2 mRNA.

Modulation of cholinergic transmission in the neuronal network of the gill and siphon withdrawal reflex in Aplysia.

Inhibitory interneurons are important elements of the network underlying the gill and siphon withdrawal reflex in Aplysia, and a large component of this inhibition is cholinergic. In this study, we investigated one key identified cholinergic inhibitory interneuron of the network, neuron L16, and studied some properties of its synaptic transmission and its modulation. We found that a slow inhibitory postsynaptic potential evoked in sensory neurons by L16 has two components. An earlier inhibitory postsynaptic potential component is sensitive to curare (100 microM) and has a reversal potential near the Cl- equilibrium potential (-54.5 mV). A later inhibitory postsynaptic potential component is sensitive to tetraethylammonium (0.5-1 mM); it is decreased by membrane hyperpolarization and becomes undetectable near the K+ equilibrium potential (between -80 and -90 mV). Input to sensory neurons from L16 can be altered by two neuromodulators of the reflex, the small cardioactive peptide and serotonin. Small cardioactive peptide (10 microM) facilitates the connections between L16 and the sensory neurons, while serotonin (5-10 microM) inhibits them. Part of the effect of serotonin on the transmission between L16 and the sensory neurons is due to a postsynaptic mechanism, since responses to acetylcholine application in these cells are decreased by serotonin. These results indicate an additional site of synaptic plasticity in the withdrawal reflex network, the inhibitory cholinergic transmission, by two major neuromodulatory transmitters, small cardioactive peptide and serotonin.

Serotonin persistently activates the extracellular signal-related kinase in sensory neurons of Aplysia independently of cAMP or protein kinase C.

Activation of the extracellular signal-related kinase is important for long-term increases in synaptic strength in the Aplysia nervous system. However, there is little known about the mechanism for the activation of the kinase in this system. We examined the activation of Aplysia extracellular signal-related kinase using a phosphopeptide antibody specific to the sites required for activation of the kinase. We found that phorbol esters led to a prolonged activation of extracellular signal-related kinase in sensory cells of the Aplysia nervous system. Surprisingly, inhibitors of protein kinase C did not block this activation. Serotonin, the physiological transmitter involved in long-term synaptic facilitation, also led to prolonged activation of extracellular signal-related kinase, but inhibitors of protein kinase A or protein kinase C did not block this activation. We examined whether the protein synthesis-dependent increase in excitability stimulated by phorbol esters was dependent on phorbol ester activation of extracellular signal-related kinase, but increases in excitability were still seen in the presence of inhibitors of extracellular signal-related kinase activation. Our results suggest that prolonged phosphorylation of extracellular signal-related kinase in the Aplysia system is not mediated by either of the classic second messenger activated kinases in this system, protein kinase A or protein kinase C and that extracellular signal-related kinase is not important for phorbol ester induced long-term effects on excitability.

Cloning and tissue distribution of the Aplysia Na+ channel alpha-subunit cDNA.

Voltage-gated Na+ channels generate the depolarizing inward current that is critical for the initiation and conduction of action potentials. To study the roles of Na+ channels in neuronal signaling, we have begun the molecular analysis of Na+ channels in Aplysia californica. We have isolated cDNAs that encode a neuronal Na+ channel alpha-subunit, which we have named SCAP1. DNA sequence analysis of the SCAP1 cDNA revealed an open reading frame that predicts a protein of 1,993 amino acids, which is highly similar to other members of the Na+ channel alpha-subunit gene family. RNase protection assays carried out on various Aplysia tissues indicated that SCAP1 is expressed predominantly in the nervous system. All of the nonneuronal tissues tested were negative with the exceptions that low levels of expression were observed in ovotestis and parapodium, probably due to the presence of small numbers of neurons within these tissue preparations. Southern blot hybridization at reduced stringency indicated that the genome of Aplysia contains more than one Na+ channel alpha-subunit gene.

Some principles emerging from the study of short- and long-term memory.

Recent studies indicate that in invertebrates short-term memory for various forms of learning involves covalent modifications of pre-existing proteins. By contrast, long-term memory utilizes genes and proteins not required for short-term memory.