Threshold serotonin concentration required to produce synaptic facilitation differs for depressed and nondepressed synapses in Aplysia sensory neurons

1. The modulatory actions produced by the neurotransmitter serotonin (5HT) in Aplysia sensory neurons (SNs) can be distinguished on the basis of their concentration requirement for 5HT, their activation and recovery kinetics, and their dependence on the relative contribution of different second messenger pathways. In addition, some of the facilitatory mechanisms activated by 5HT appear to be different depending upon the recent activation history of synaptic transmission from the SNs. In this study, we examined the concentration requirements of 5HT-induced facilitation of depressed and nondepressed synapses. 2. In isolated pleural-pedal ganglia, we produced facilitation of monosynaptic connections between tail SNs and motor neurons (MNs), using different concentrations of 5HT. As a measure of each preparation's greatest sensitivity to 5HT, we first determined the lowest 5HT concentration that produced increased excitability in the SNs ("threshold" 5HT). Then, in one series of experiments, we applied 5HT sequentially to the same synapse, first in the nondepressed and then in the depressed state. In a second series, we applied 5HT simultaneously to two SNs connecting to the same MN; one synapse was depressed, the other nondepressed. 3. In both series of experiments, we found that the 5HT concentration required to produce facilitation of depressed excitatory post-synaptic potentials (EPSPs) was invariably lower than the 5HT concentration that produced facilitation of nondepressed EPSPs. In the first series, 'threshold' 5HT (1.6 microM) was sufficient to facilitate the synapse in the depressed state, but not the nondepressed state. However, the nondepressed synapse could still be facilitated by higher concentrations of 5HT (10 microM). In the second series, increased excitability of SNs, facilitation of depressed synapses, and facilitation of nondepressed synapses were progressively recruited as a function of increasing 5HT concentration (4.1, 6.7, and 10-15 microM, respectively). 4. These data are consistent with previous studies suggesting that different cellular mechanisms contribute to the facilitation of depressed and nondepressed synapses. In addition, our results provide a way to experimentally separate the two processes and to analyze them simultaneously and independently. Taking advantage of this dissociation, in future experiments it may be possible to directly compare the relative contributions of different intracellular mechanisms to synaptic facilitation and to relate them to the degree of recent synaptic activation.     

Activation of protein kinase A contributes to the expression but not the induction of long-term hyperexcitability caused by axotomy of Aplysia sensory neurons

Nociceptive sensory neurons (SNs) in Aplysia provide useful models to study both memory and adaptive responses to nerve injury. Induction of long-term memory in many species, including Aplysia, is thought to depend on activation of cAMP-dependent protein kinase (PKA). Because Aplysia SNs display similar alterations in models of memory and after nerve injury, a plausible hypothesis is that axotomy triggers memory-like modifications by activating PKA in damaged axons. The present study disproves this hypothesis. SN axotomy was produced by (1) dissociation of somata from the ganglion [which is shown to induce long-term hyperexcitability (LTH)], (2) transection of neurites of dissociated SNs growing in vitro, or (3) peripheral nerve crush. Application of the competitive PKA inhibitor Rp-8-CPT-cAMPS at the time of axotomy failed to alter the induction of LTH by each form of axotomy, although the inhibitor antagonized hyperexcitability produced by 5-HT application. Strong activation of PKA in the nerve by coapplication of a membrane-permeant analog of cAMP and a phosphodiesterase inhibitor was not sufficient to induce LTH of either the SN somata or axons. Furthermore, nerve crush failed to activate axonal PKA or stimulate its retrograde transport. Therefore, PKA activation plays little if any role in the induction of LTH by axotomy. However, the expression of LTH was reduced by intracellular injection of the highly specific PKA inhibitor PKI several days after nerve crush. This suggests that long-lasting activation of PKA in or near the soma contributes to the maintenance of long-term modifications produced by nerve injury.     

Identification of two phosphoproteins affected by serotonin in Aplysia sensory neurons

Although control mechanisms of cochlear blood flow (CBF) have been intensively studied since laser Doppler flowmetry was introduced for CBF measurement in animals and humans, the role of adenosine 5'-triphosphate (ATP) in CBF regulation is not known. Since ATP is a potent vasoactive agent in other organs, the aim of this study is to examine ATP-induced changes in CBF and to test whether the nitric oxide pathway is involved in ATP-induced CBF changes. The anterior inferior cerebellar artery (AICA) of anesthetized pigmented guinea pigs was exposed, and ATP was perfused into the AICA. For CBF measurement, the bulla was opened and the 0.7 mm laser probe of a Perimed PF2B flowmeter was positioned on the basal turn of the cochlea. AICA perfusion of an ATP solution caused dose-dependent transient CBF increases. The maximum CBF increase induced was 220% of the baseline. In some animals, CBF showed a dual effect; a transient decrease followed by a longer-lasting increase. The perfusions of sodium nitroprusside (SNP) also resulted in dose-dependent CBF changes. The intravenous application of N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly attenuated ATP-induced CBF increases, and enhanced ATP-induced decreases, but did not affect SNP-induced CBF changes. The ATP-induced CBF responses indicate that ATP plays a role in CBF regulation. The biphasic characteristic of the ATP-induced CBF change suggests the involvement of both P2x- and P2y-subtype purinoceptors. That L-NAME caused attenuation of the ATP-induced CBF increase implies that the ATP-induced CBF increase is mediated by the release of endothelium-derived relaxing factor, nitric oxide, following activation of endothelial P2y-purinoceptors in the cochlear vascular bed and/or cochlear supplying vessels.     

Role of transforming growth factor-beta in long-term synaptic facilitation in Aplysia

The role of transforming growth factor-beta (TGF-beta) in long-term synaptic facilitation was examined in isolated Aplysia ganglia. Treatment with TGF-beta1 induced long-term facilitation (24 and 48 hours), but not short-term (5 to 15 minutes) or intermediate-term (2 to 4 hours) facilitation. The long-term effects of TGF-beta1 were not additive with those of serotonin. Moreover, serotonin-induced facilitation was blocked by an inhibitor of TGF-beta. Thus, activation of TGF-beta may be part of the cascade of events underlying long-term sensitization, consistent with the hypothesis that signaling molecules that participate in development also have roles in adult neuronal plasticity.     

Long-term changes in excitability induced by protein kinase C activation in Aplysia sensory neurons

Protein kinases A (PKA) and C (PKC) play a central role as intracellular transducers during simple forms of learning in Aplysia. These two proteins seem to cooperate in mediating the different forms of plasticity underlying behavioral modifications of defensive reflexes in a state- and time-dependent manner. Although short- and long-term changes in the synaptic efficacy of the connections between mechanosensory neurons and motoneurons of the reflex have been well characterized, there is also a distinct intermediate phase of plasticity that is not as well understood. Biochemical and physiological experiments have suggested a role for PKC in the induction and expression of this form of facilitation. In this report, we demonstrate that PKC activation can induce both intermediate- and long-term changes in the excitability of sensory neurons (SNs). Short application of 4beta-phorbol ester 12,13-dibutyrate (PDBU), a potent activator of PKC, produced a long-lasting increase in the number of spikes fired by SNs in response to depolarizing current pulses. This effect was observed in isolated cell culture and in the intact ganglion; it was blocked by a selective PKC inhibitor (chelerythrine). Interestingly, the increase in excitability measured at an intermediate-term time point (3 h) after treatment was independent of protein synthesis, while it was disrupted at the long-term (24 h) time point by the general protein synthesis inhibitor, anisomycin. In addition to suggesting that PKC as well as PKA are involved in long-lasting excitability changes, these findings support the idea that memory formation involves multiple stages that are mechanistically distinct at the biochemical level.     

Use-dependent decline of paired-pulse facilitation at Aplysia sensory neuron synapses suggests a distinct vesicle pool or release mechanism

We have characterized paired-pulse facilitation at Aplysia sensory neuron-to-motoneuron synapses. This simple form of very short-term synaptic plasticity displayed an unusual feature: it decreased dramatically with repeated testing. Synaptic depression at these synapses and this use-dependent decrease in paired-pulse facilitation occurred independently of each other. Paired-pulse facilitation was inversely correlated with the size of the initial synaptic connection and was absent at stronger synapses. The use-dependent decrease in paired-pulse facilitation occurred at the same rate at large synapses as at small synapses, although the initial paired-pulse facilitation at large synapses was substantially smaller. Rates of synaptic depression were also independent of initial synaptic strength. Paired-pulse facilitation was blocked by presynaptic EGTA injection, but not by postsynaptic EGTA or BAPTA injection. These results indicate that presynaptic Ca2+ influx plays a critical role in paired-pulse facilitation. However, the persistence of the decrease in paired-pulse facilitation for longer than 15 min suggests that Ca2+ from the first paired action potential produces facilitation via a modulatory mechanism rather than by summating with Ca2+ influx during the second paired action potential in activating the Ca2+ binding sites that initiate exocytosis. This modulatory mechanism may not involve protein phosphorylation because paired-pulse facilitation was unaffected by the protein kinase inhibitors H7 and KN-62. These findings further suggest that release by the second paired action potential occurs at sites distinct from those that mediate release by the first action potential.     

Isolation and long-term survival of adult human sensory neurons in vitro

OBJECTIVE: To determine whether adult human dorsal root ganglion neurons can be isolated and maintained in long-term tissue culture, where they would extend processes. METHODS: Dorsal root ganglia were removed from adult human organ donors within 2 hours of clamping the aorta. They were then treated with enzymes for one hour, triturated to dissociate the neurons and their satellite cells, and the individual neurons were then plated in tissue culture dishes in medium containing serum. RESULTS: Isolated adult human dorsal root ganglion neurons survive in vitro for more than 2 1/2 months, in the absence of exogenously supplied neurotrophins. where they remain electrically excitable and extend processes, CONCLUSIONS: Isolated adult human dorsal root ganglion neurons survive in culture for more than 2 1/2 months, extend processes, and remain electrically excitable, without exogenous neurotrophins. These results suggest that, adult human sensory neurons do not require exogenous neurotrophins for survival and process outgrowth, or that sufficient factors were provided by the small number of satellite cells in the cultures. In addition, the neurons survive well in spite of an initial period of up to 14 hours of hypoxia, between the time the aorta was clamped and when the plated neurons were placed in an incubator with the appropriate O2/CO2 environment.     

Mutation in the phosphorylation sites of MAP kinase blocks learning-related internalization of apCAM in Aplysia sensory neurons

The synaptic growth that accompanies 5-HT-induced long-term facilitation of the sensory to motor neuron connection in Aplysia is associated with the internalization of apCAM at the surface membrane of the sensory neuron. We have now used epitope tags to examine the fate of each of the two apCAM isoforms (membrane bound and GPI-linked) and find that only the transmembrane form is internalized. This internalization can be blocked by overexpression of transmembrane constructs with a single point mutation in the two MAPK consensus sites, as well as by injection of a specific MAPK antagonist into sensory neurons. These data suggest MAPK phosphorylation at the membrane is important for the internalization of apCAMs and, thus, may represent an early regulatory step in the growth of new synaptic connections that accompanies long-term facilitation.     

Ontogeny of serotonergic neurons in Aplysia californica

Although the functions of serotonin in adult Aplysia have been the focus of numerous investigations, our understanding of the roles played by this neurotransmitter during development is very incomplete. In the previous study (Marois and Carew [1997a] J. Comp. Neurol. 386:477-490), we showed that identified serotonergic cells are present very early during the ontogeny of Aplysia. In order to gain insight into the possible functions that these serotonergic cells may exert, we have used immuno-electron microscopy in this study to examine the projection patterns and target tissues of the serotonergic cells during the larval development of Aplysia. The results indicate that the larval serotonergic cells have numerous and precise connections to non-neuronal and neuronal target tissues: Serotonergic cells innervate the ciliated cells of the velum, numerous muscle systems, possibly visceral organs, and several cells in the central nervous system. Repeated observations of one serotonergic contact onto an undifferentiated neuron in the abdominal ganglion over a short developmental time span suggest that the serotonergic input may trigger axonogenesis in the postsynaptic cell. Apart from this possibility, we suggest that the innervation patterns of the larval serotonergic cells essentially fulfill the same primary function attributed to the adult serotonergic cells, that of modulating ongoing physiological and behavioral activity.     

Cellular correlates of long-term sensitization in Aplysia

Although in vitro analyses of long-term changes in the sensorimotor connection of Aplysia have been used extensively to understand long-term sensitization, relatively little is known about the ways in which the connection is modified by learning in vivo. Moreover, sites other than the sensory neurons might be modified as well. In this paper, several different biophysical properties of sensory neurons, motor neurons, and LPl17, an identified interneuron, were examined. Membrane properties of sensory neurons, which were expressed as increased excitability and increased spike afterdepolarization, were affected by the training. The biophysical properties of motor neurons also were affected by training, resulting in hyperpolarization of the resting membrane potential and a decrease in spike threshold. These results suggest that motor neurons are potential loci for storage of the memory in sensitization. The strength of the connection between sensory and motor neurons was affected by the training, although the connection between LPl17 and the motor neuron was unaffected. Biophysical properties of LPl17 were unaffected by training. The results emphasize the importance of plasticity at sensory-motor synapses and are consistent with the idea that there are multiple sites of plasticity distributed throughout the nervous system.     

Binding of serotonin to receptors at multiple sites is required for structural plasticity accompanying long-term facilitation of Aplysia sensorimotor synapses

Long-term changes in the efficacy of Aplysia sensorimotor synapses accompany nonassociative and associative forms of behavioral plasticity. This synapse expresses long-term facilitation either with repeated applications of 5-hydroxytryptamine (5-HT) or with a single pairing of tetanus in the sensory neuron (SN) and bath application of 5-HT. We examined whether structural changes in the SN accompany all forms of long-term synaptic enhancement and the locations at which 5-HT must bind receptors to evoke long-term functional and/or structural changes. Pairing tetanus with one application of 5-HT evoked both functional and structural changes after 24 hr only when 5-HT application was temporally paired with the tetanus and activated receptors on both the SN cell body and terminal region. Repeated application of 5-HT to the terminal region alone failed to evoke any long-term change. Repeated applications of 5-HT to the SN cell body alone evoked a change in synaptic efficacy at 24 hr but failed to increase SN varicosities. Repeated applications of 5-HT to both the SN cell body and the terminal region evoked increases in both synaptic efficacy and the number of SN varicosities at 24 hr. The results indicate that different external stimuli can evoke equivalent forms of long-term synaptic facilitation with or without structural changes in the SNs. Changes in the number of SN varicosities can accompany different forms of long-term facilitation and require the activation of 5-HT receptors at multiple sites.     

A pair of reciprocally inhibitory histaminergic sensory neurons are activated within the same phase of ingestive motor programs in Aplysia

Previous studies have shown that each buccal ganglion in Aplysia contains two B52 neurons, one in each hemiganglion. We now show that there are two B52 neurons in a single buccal hemiganglion and four cells in an animal. We also show that the B52 neurons are histamine-immunoreactive and use reverse phase HPLC to show that the histamine-immunoreactive substance is authentic histamine. Previous studies have shown that the B52 neurons make numerous inhibitory synaptic connections with neurons active during the radula closing/retraction phase of ingestive motor programs. A computational model of the Aplysia feeding central pattern generator has, therefore, suggested that the B52 neurons play a role in terminating closing/retraction. Consistent with this idea we show that both B52 neurons fire at the beginning of radula opening/protraction. We also show that both B52 neurons are sensory neurons. They are depolarized when a flap of connective tissue adjacent to the buccal commissural arch is stretched. During ingestive feeding this is likely to occur at the peak of closing/retraction as opening/protraction begins. In the course of this study we compare the two ipsilateral B52 neurons and show that these cells are virtually indistinguishable; e.g., they use a common neurotransmitter, make the same synaptic connections, and are both sensory as well as premotor neurons. Nevertheless we show that the B52 neurons are reciprocally inhibitory. Our results, therefore, strikingly confirm theoretical predictions made by others that neurons that inhibit each other will not necessarily participate in antagonistic phases of behavior.     

Nitric oxide acts directly in the presynaptic neuron to produce long-term potentiation in cultured hippocampal neurons

Nitric oxide (NO) has been proposed to act as a retrograde messenger during long-term potentiation (LTP) in the CA1 region of hippocampus, but the inaccessibility of the presynaptic terminal has prevented a definitive test of this hypothesis. Because both sides of the synapse are accessible in cultured hippocampal neurons, we have used this preparation to investigate the role of NO. We examined LTP following intra- or extracellular application of an NO scavenger, an inhibitor of NO synthase, and a membrane-impermeant NO donor that releases NO only upon photolysis with UV light. Our results indicate that NO is produced in the postsynaptic neuron, travels through the extracellular space, and acts directly in the presynaptic neuron to produce long-term potentiation, supporting the hypothesis that NO acts as a retrograde messenger during LTP.     

Activation and recovery of the PGE2-mediated sensitization of the capsaicin response in rat sensory neurons

Pro-inflammatory prostaglandins are known to enhance the sensitivity of sensory neurons to various modalities of stimulation, including the excitatory chemical agent, capsaicin. In this report, we examined the capacity of prostaglandin E2 (PGE2) to enhance the capsaicin response recorded from sensory neurons isolated from embryonic rats and grown in culture. Previous work demonstrated that the cyclic adenosine 3',5'-monophosphate pathway mediates initiation of the PGE2-induced sensitization, however, little is known about the pathways regulating the recovery from sensitization. Therefore, we examined the neuronal transduction cascades that control the duration of sensitization. Treatment with PGE2 enhanced the capsaicin-evoked current by two- to threefold, however, this sensitization was transient even in the continued presence of prostaglandin. The duration of sensitization produced by PGE2 was related inversely to the extracellular Ca2+ concentration with the shortest recovery times observed in cells exposed to 2 mM Ca2+-Ringer. Inclusion of the Ca2+ chelator, bis-(o-aminophenoxy)-N, N,N',N'-tetraacetic acid, in the recording pipette greatly lengthened the period of sensitization. Pretreatment with either the nitric oxide synthase inhibitor, nitro-L-arginine methyl ester (L-NAME), or the inhibitor of the cyclic guanosine 3', 5'-monophosphate (GMP)-dependent protein kinase, KT-5823, before the application of PGE2 increased the duration of sensitization even in the presence of 2 mM Ca2+. In contrast, after attaining maximal sensitization in 2 mM Ca2+-Ringer containing L-NAME, the addition of either nitric oxide donors (3-morpholinosydnonimine or s-nitroso-n-acetylpenicillamine) or 8-Br-cyclic GMP led to a rapid decrease in the level of sensitization. In the absence of sensitization, nitric oxide-cyclic GMP modulating agents had no effect on the capsaicin-evoked current. Therefore, these results suggest that capsaicin-induced elevations in intracellular Ca2+ levels lead to an enhanced production of cyclic GMP, via the nitric oxide pathway, that ultimately activates cyclic GMP-dependent protein kinase. This protein kinase inactivates or terminates the sensitization produced by PGE2 by an as yet unidentified mechanism.     

Feeding behavior in Aplysia californica: Role of chemical and tactile stimuli.

Performed 5 experiments concerned with feeding. Exp. I was exploratory in nature, and using time-lapse cinematography, it was found that the approach to food (seaweed) involved an initial arousal and then a slow movement toward the food with considerable pausing and head waving. In Exp. II a -maze apparatus was used to show that Aplysia will consistently turn toward the highest concentration of food chemical. Exp. III showed that food-aroused Ss proceeded upstream against a slow current, while Ss unaroused proceeded up or down stream. Exp. IV and V showed that chemical stimulation of the rhinophores, anterior tentacles, and dorsal head area resulted in feeding responses; and tactile stimulus-response relationships varied markedly with the state of arousal. (24 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Attention and habituation: Odor preferences, long-term memory, and multiple sensory cues of novel stimuli.

Investigation of and habituation to novel stimuli are part of exploratory behavior of rodents. They are necessary for assessing the environment in seeking food and sexual partners and in avoiding predators. Six experiments tested male and female Mongolian gerbils (Meriones unguiculatus) in a stimulus-elicited investigation paradigm. Findings indicate that (1) Ss initially showed preference for odors of strange male bedding and for odors of home cage bedding and then habituated. There was no preference between these 2 odors, although they could be discriminated. The complex odor stimulus elicited more response than any of the components tested. (2) Memory of an object or odor was demonstrated up to 4 wks later. (3) Sensory deprivation by blinding, anosmizing, or removing somatosensation of the upper snout made only small differences in investigation. The removal of any 2 of these sensory inputs produced more interference with the response, but all Ss investigated the stimulus. It is concluded that attention to novelty and habituation after repeated exposure are very robust behaviors and are mediated through multiple sensory channels. (41 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)