Transforming growth factor beta1 alters synapsin distribution and modulates synaptic depression in Aplysia.

Transforming growth factor beta1 (TGF-beta1) induces long-term synaptic facilitation and long-term increases in excitability in Aplysia. Here we report that this growth factor has acute effects as well. Treatment of pleural-pedal ganglia with TGF-beta1 for 5 min activated mitogen-activated protein kinase (MAPK) and stimulated the phosphorylation of synapsin in a MAPK-dependent manner. This phosphorylation appeared to modulate synapsin distribution in cultured sensory neurons. Control neurons exhibited a punctate distribution of synapsin along neurites, which appeared to represent high concentration aggregates of synapsin. TGF-beta1-treated sensory neurons showed a significant reduction in the number of these puncta, an effect that was blocked by the MAP/ERK kinase inhibitor U0126. The functional consequence of TGF-beta1 was tested by examining its effects on synaptic transmission at the sensorimotor synapse. Application of TGF-beta1 reduced the magnitude of synaptic depression. This effect was dependent on MAPK, consistent with the hypothesis that TGF-1 mobilizes synaptic vesicles through the phosphorylation of synapsin.

TGF-beta1 in Aplysia: role in long-term changes in the excitability of sensory neurons and distribution of TbetaR-II-like immunoreactivity.

Exogenous recombinant human transforming growth factor beta-1 (TGF-beta1) induced long-term facilitation of Aplysia sensory-motor synapses. In addition, 5-HT-induced facilitation was blocked by application of a soluble fragment of the extracellular portion of the TGF-beta1 type II receptor (TbetaR-II), which presumably acted by scavenging an endogenous TGF-beta1-like molecule. Because TbetaR-II is essential for transmembrane signaling by TGF-beta, we sought to determine whether Aplysia tissues contained TbetaR-II and specifically, whether neurons expressed the receptor. Western blot analysis of Aplysia tissue extracts demonstrated the presence of a TbetaR-II-immunoreactive protein in several tissue types. The expression and distribution of TbetaR-II-immunoreactive proteins in the central nervous system was examined by immunohistochemistry to elucidate sites that may be responsive to TGF-beta1 and thus may play a role in synaptic plasticity. Sensory neurons in the ventral-caudal cluster of the pleural ganglion were immunoreactive for TbetaR-II, as well as many neurons in the pedal, abdominal, buccal, and cerebral ganglia. Sensory neurons cultured in isolation and cocultured sensory and motor neurons were also immunoreactive. TGF-beta1 affected the biophysical properties of cultured sensory neurons, inducing an increase of excitability that persisted for at least 48 hr. Furthermore, exposure to TGF-beta1 resulted in a reduction in the firing threshold of sensory neurons. These results provide further support for the hypothesis that TGF-beta1 plays a role in long-term synaptic plasticity in Aplysia.

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.

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.

A developmental gene (Tolloid/BMP-1) is regulated in Aplysia neurons by treatments that induce long-term sensitization.

Long-term sensitization training, or procedures that mimic the training, produces long-term facilitation of sensory-motor neuron synapses in Aplysia. The long-term effects of these procedures require mRNA and protein synthesis (Montarolo et al., 1986; Castellucci et al., 1989). Using the techniques of differential display reverse transcription PCR (DDRT-PCR) and ribonuclease protection assays (RPA), we identified a cDNA whose mRNA level was increased significantly in sensory neurons by treatments of isolated pleural-pedal ganglia with serotonin for 1.5 hr or by long-term behavioral training of Aplysia. The effects of serotonin and behavioral training on this mRNA were mimicked by treatments that elevate cAMP. The aplysia mRNA increased by serotonin and behavioral training was 41-45% identical to a developmentally regulated gene family which includes Drosophila tolloid and human bone morphogenetic protein-1 (BMP-1). Both tolloid and BMP-1 encode metalloproteases that might activate TGF-beta (transforming growth factor beta)-like molecules or process procollagens. Aplysia tolloid/BMP-1-like protein (apTBL-1) might regulate the morphology and efficacy of synaptic connections between sensory and motor neurons, which are associated with long-term sensitization.

Long-term structural remodeling in Aplysia sensory neurons requires de novo protein synthesis during a critical time period.

Long-term sensitization training induces persistent changes in both electrophysiological properties and specific structural features of sensory neurons in Aplysia californica. Previously, we found that transient elevation of intracellular cAMP could also modify these features in sensory neurons located in the pleural ganglion. In the present study we examined the role of protein synthesis in structural remodeling induced by cAMP. When applied during the intracellular injection of cAMP, anisomycin blocked increases in both the number of varicosities and the number of branch points in single sensory neurons. Exposure to anisomycin during different time periods, from as early as 12 hr prior to cAMP injection to periods as late as 15 hr after, indicated that the requirement for protein synthesis starts at the time of cAMP injection and extends for at least seven hours afterwards. Because it is metabolized rapidly, cAMP probably triggers a cascade of protein synthesis whose products continue to be synthesized for several hours after cAMP levels have returned to baseline. Thus, the present results suggest that the induction of long-term structural changes in sensory neurons has an extended but finite requirement for protein synthesis.

Modulation of an inhibitory interneuron in the neural circuitry for the tail withdrawal reflex of Aplysia.

1. Serotonin (5-HT), small cardioactive peptide B (SCPB) and FMRFamide have well-established facilitatory and inhibitory effects on sensory neurons and their connections with motor neurons mediating withdrawal reflexes in Aplysia. Little is known, however, about their effects on interneurons contributing to those reflexes. As a first step, we examined the effects of these three transmitters on the identified inhibitory interneuron RP14 in isolated pleural-pedal ganglia. 2. Bath application of 5-HT hyperpolarized RP14, inhibited its spontaneous activity and decreased its excitability. In addition, 5-HT decreased the amplitude of inhibitory postsynaptic potentials produced by RP14 in tail sensory and motor neurons. 3. In contrast, bath application of SCPB increased spontaneous activity in RP14. Subsequent application of 5-HT to the bath, which still contained SCPB, inhibited RP14. Therefore, the effects of SCPB were essentially opposite to those of 5-HT on this inhibitory interneuron. 4. FMRFamide had little effect on RP14. It did not produce an obvious change in its resting membrane potential and produced only a transient increase in its spontaneous activity. 5. These results suggest that various neuromodulators have differential effects on elements of the neuronal circuit underlying the tail-withdrawal reflex of Aplysia. Differential modulation may determine the overall behavioral manifestations associated with sensitization.

Identification and characterization of pleural neurons that inhibit tail sensory neurons and motor neurons in Aplysia: correlation with FMRFamide immunoreactivity.

Neurons on the rostral edge of the ventral surface of the right pleural ganglion were identified as elements of the circuit mediating the defensive tail withdrawal reflex of Aplysia. These neurons produced IPSPs in tail sensory neurons and were classified into two groups, RPI4 and RPI5, according to their affinity for an antibody directed against FMRFamide. RPI4 was not FMRFamide immunoreactive, and RPI5 was. RPI4 and RPI5 were found to have different electrophysiological profiles. The summated IPSPs in sensory neurons produced by RPI4 developed more rapidly and had a shorter duration than those produced by RPI5. In addition, RPI4 produced IPSPs in the tail motor neurons, whereas RPI5 did not. Both RPI4 and RPI5 received excitatory synaptic inputs from stimulation of the pleural-abdominal connective as well as peripheral nerves P8 and P9, which innervate the tail and posterior part of the animal's body. These inputs were sufficient to elicit spikes. In RPI4, the excitatory synaptic inputs were followed by short and transient hyperpolarization, whereas in RPI5, the excitatory synaptic inputs were followed by slow and long-lasting hyperpolarization. Excitatory inputs elicited in RPI4 by stimulation of peripheral nerves appeared to be mediated, at least in part, by activation of tail sensory neurons. Intracellular stimulation of sensory neurons produced EPSPs in RPI4 that appeared to be monosynaptic. These results suggest that inhibitory interneurons underlying the circuit of the tail withdrawal reflex may play roles in mediating or modulating neuronal responses to tail stimulation. By inhibiting tail sensory and motor neurons, these interneurons may reduce the effectiveness of an animal's response to stimulation of the tail.

Identification and characterization of a multifunction neuron contributing to defensive arousal in Aplysia.

1. The tail withdrawal reflex is mediated by a monosynaptic circuit composed of tail sensory and motor neurons, but there appear to be additional neuronal elements that also contribute to the reflex. A newly identified interneuron, called LP117, was located in the pleural ganglion. This neuron formed a parallel excitatory pathway between sensory and motor neurons. The distinguishing feature of LP117 was its ability to elicit a long-lasting (5-100 s) excitatory postsynaptic potential (EPSP) in the motor neuron. 2. Intracellular labeling of LP117 revealed axons projecting to the cerebral and abdominal as well as the pedal ganglia. Simultaneous intracellular recordings confirmed the widely divergent output of LP117 to tentacle motor neurons in the cerebral ganglion, as well as to gill, siphon, and ink motor neurons in the abdominal ganglion. 3. Also receiving input were abdominal neurons L29, which excites LFs motor neurons and facilitates LE sensory neurons, and L25, which is part of the pattern-generating network underlying respiratory pumping. Thus LP117 appears to be a neural element important for the conduction of information about tail stimulation to ganglia that are not innervated by tail sensory neurons themselves. Moreover, the divergent outputs suggest that LP117 is an element of a neural circuit underlying defensive arousal. 4. LP117 produced slow EPSPs in several motor neurons. The long time course of the EPSP could prolong the burst in the motor neuron produced by LP117 itself as well as increase the effectiveness of coincident synaptic input. This suggests that an important function of this interneuron is to extend the duration of the response to tail stimulation in the motor neuron. This could account for the relatively long time course of the motor neuron response to tail stimulation compared with that of the sensory neuron. 5. Sensitization is a form of nonassociative learning that produces changes in the amplitude and duration of reflex responses. It seems unlikely that all of these changes can be attributed to enhanced amplitude of the sensory-motor synapse, however. Therefore LP117 may itself be a site of plasticity for reflexes elicited by tail stimulation.

The role of interneurons in controlling the tail-withdrawal reflex in Aplysia: a network model.

1. The contributions of monosynaptic and polysynaptic circuitry to the tail-withdrawal reflex in the marine mollusk Aplysia californica were assessed by the use of physiologically based neural network models. Effects of monosynaptic circuitry were examined by the use of a two-layer network model with four sensory neurons in the input layer and one motor neuron in the output layer. Results of these simulations indicated that the monosynaptic circuit could not account fully for long-duration responses of tail motor neurons elicited by tail stimulation. 2. A three-layer network model was constructed by interposing a layer of two excitatory interneurons between the input and output layers of the two-layer network model. These interneurons had properties mimicking those of the recently described interneuron LP117, receiving excitatory input from pleural sensory neurons and evoking a biphasic excitatory postsynaptic potential (EPSP) in pedal motor neurons (Cleary and Byrne 1993). The three-layer model could account for long-duration responses in motor neurons. 3. Sensory neurons are a known site of plasticity in Aplysia. Synaptic plasticity was incorporated into the three-layer model by altering the magnitudes of conductance changes evoked in motor neurons and interneurons by presynaptic sensory neurons. In these simulations the excitatory interneurons converted an amplitude-coded input into an amplitude- and duration-coded output, allowing the three-layer network to support a large range of output amplitudes and durations. 4. Synaptic plasticity at more than one locus modified dramatically the input-output relationship of the three-layer network model. This feature gave the model redundancy in its plastic properties and points to the possibility of distributed memory in the circuitry mediating withdrawal reflexes in Aplysia. Multiple sites of control over the response of the network would likely allow a more diverse repertoire of responses.

Inhibitory neuron produces heterosynaptic inhibition of the sensory-to-motor neuron synapse in Aplysia.

We have identified an inhibitory neuron (RPL4) in the right pleural ganglion of Aplysia, which produced hyperpolarization of the sensory and motor neurons involved in the tail withdrawal reflex. Activation of RPL4 significantly reduced the amplitude of excitatory postsynaptic potentials produced in tail motor neurons by action potentials triggered in sensory neurons. This example of heterosynaptic inhibition was due, at least in part, to an increase in membrane input conductance in the motor neuron. Since the synaptic strength of the sensory-to-motor neuron connection has been associated with the strength of the tail withdrawal reflex, RPL4 may contribute to modulation of that reflex.

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.

Neural and molecular bases of nonassociative and associative learning in Aplysia.

A model that summarizes some of the neural and molecular mechanisms contributing to short- and long-term sensitization is shown in Figure 14. Sensitizing stimuli lead to the release of a modulatory transmitter such as 5-HT. Both serotonin and sensitizing stimuli lead to an increase in the synthesis of cAMP and the modulation of a number of K+ currents through protein phosphorylation. Closure of these K+ channels leads to membrane depolarization and the enhancement of excitability. An additional consequence of the modulation of the K+ currents is a reduction of current during the repolarization of the action potential, which leads to an increase in its duration. As a result, Ca2+ flows into the cell for a correspondingly longer period of time, and additional transmitter is released from the cell. Modulation of the pool of transmitter available for release (mobilization) also appears to occur as a result of sensitizing stimuli. Recent evidence indicates that the mobilization process can be activated by both cAMP-dependent protein kinase and protein kinase C. Thus, release of transmitter is enhanced not only because of the greater influx of Ca2+ but also because more transmitter is made available for release by mobilization. The enhanced release of transmitter leads to enhanced activation of motor neurons and an enhanced behavioral response. Just as the regulation of membrane currents is used as a read out of the memory for short-term sensitization, it also is used as a read out of the memory for long-term sensitization. But long-term sensitization differs from short-term sensitization in that morphological changes are associated with it, and long-term sensitization requires new protein synthesis. The mechanisms that induce and maintain the long-term changes are not yet fully understood (see the dashed lines in Fig. 14) although they are likely to be due to direct interactions with the translation apparatus and perhaps also to events occurring in the cell nucleus. Nevertheless, it appears that the same intracellular messenger, cAMP, that contributes to the expression of the short-term changes, also triggers cellular processes that lead to the long-term changes. One possible mechanism for the action of cAMP is through its regulation of the synthesis of membrane modulatory proteins or key effector proteins (for example, membrane channels). It is also possible that long-term changes in membrane currents could be due in part to enhanced activity of the cAMP-dependent protein kinase so that there is a persistent phosphorylation of target proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

cAMP induces long-term morphological changes in sensory neurons of Aplysia.

Long-term sensitization of defensive reflexes in the marine mollusc Aplysia has been correlated with biophysical changes in the somata of sensory neurons that mediate the reflexes and with morphological changes in their axonal processes. The biophysical changes can also be mimicked by intracellular injection of cAMP. In this report we demonstrate that cAMP induces long-term structural changes in pleural sensory neurons, providing a mechanism for this form of memory storage.

Serotonin acts in the synaptic region of sensory neurons in Aplysia to enhance transmitter release.

An important mechanism that contributes to sensitization in Aplysia is heterosynaptic facilitation of the synaptic connections between sensory neurons (SNs) and motor neurons (MNs). Heterosynaptic facilitation, in turn, is associated with broadening of the spike in the SN. Spike broadening is readily observed in recordings from somata of SNs, and from growth cones of SNs in culture, but broadening in synaptic terminals has only been inferred. Intracellular recordings were made from somata of SNs and from somata of follower MNs. Additional recordings were made from the axons of SNs as they enter the neuropil in the pedal ganglion. Serotonin (5-HT) broadened action potentials in axons of SNs and enhanced excitatory postsynaptic potentials (EPSPs) in the MNs, even after the axons of SNs were surgically separated from their somata. These results indicate that both heterosynaptic facilitation and spike broadening in the axon are due to the local action of 5-HT and can occur independently of modulation of membrane properties in the soma.

Inositol trisphosphate and activators of protein kinase C modulate membrane currents in tail motor neurons of Aplysia.

1. We have investigated how activation of the inositol lipid second messenger pathway may contribute to modulation of membrane currents in tail motor neurons of Aplysia. Specifically, we examined the effects of injected inositol 1,4,5-trisphosphate (IP3) and analogues of diacylglycerol (DAG), both of which are products of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). 2. Injection of IP3 produced an outward current associated with an apparent increase in membrane conductance. Ion substitution experiments, the sensitivity of the response to low concentrations of TEA and its attenuation by intracellular injections of EGTA suggest that the current produced by injection of IP3 is a calcium-activated K+ current (IK,Ca). 3. The response to IP3 was mimicked by intracellular injection of Ca2+. Injection of Ca2+ produced an outward current that was associated with an apparent increase in input conductance of the membrane. The same manipulations that affected the response to IP3 (see above) also affected the response to injections of Ca2+. 4. Injections of activators of protein kinase C (PKC) produced a relatively slow inward current. The inward current has not been fully analyzed, but it does not appear to be due to the actions of any single conventional ion channel. 5. Activators of PKC attenuated responses to subsequent injections of IP3 indicating that one component of PIP2 hydrolysis can attenuate the other. 6. The results suggest that hydrolysis of inositol phospholipids is a mechanism for regulation of membrane properties in tail motor neurons of Aplysia.

Inositol 1,4,5-trisphosphate alters bursting pacemaker activity in Aplysia neurons: voltage-clamp analysis of effects on calcium currents.

1. The left upper-quadrant bursting neurons (cells L2, L3, L4, and L6) of the abdominal ganglion of Aplysia display a regular burst-firing pattern that is controlled by cyclic changes of intracellular Ca2+ that occur during the bursting rhythm. The characteristic bursting pattern of these neurons occurs within a range of membrane potentials (-35 to -50 mV) called the pacemaker range. 2. Intracellular pressure injection of inositol-1,4,5-trisphosphate (IP3) altered the bursting rhythm of the left upper-quadrant bursting (LUQB) cells for up to 15 min. Injection of IP3 induced a brief depolarization that was followed by a long-lasting (2-15 min) hyperpolarization. The hyperpolarizing phase of the response was accompanied by prolonged interburst intervals. 3. When cells were voltage-clamped at potentials within the pacemaker range, injection of IP3 generally induced a biphasic response that had a total duration of 2-15 min. An initial inward shift in holding current (Iin), which lasted 5-120 s, was followed by a slow outward shift in holding current (Iout). 4. At membrane potentials more negative than -40 mV, Iin was associated with a small and relatively voltage-independent increase in membrane conductance. Iin was not blocked by bath application of tetrodotoxin (TTX) or Co2+. Although Iin was activated by injection of IP3, we were unable to block it by iontophoretic injection of ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetra-acetic acid (EGTA) sufficient to block the Ca2+-activated inward tail current (IB). The ionic mechanism that produces Iin has not been analyzed. 5. In normal bathing solution, Iout was present at membrane potentials more positive than approximately -50 mV. Iout was not blocked by 50 mM tetraethylammonium (TEA), which is known to block Ca2+-activated K+ currents (IK,Ca) in these cells. However, it was blocked by 30 mM Co2+, which blocks ICa. These results indicate that a steady-state ICa is necessary for the generation of Iout following injection of IP3, suggesting that Iout is due to inactivation of ICa and not to activation of a K+ conductance. 6. Intracellular iontophoresis of EGTA abolished Iout indicating that elevation of intracellular Ca2+ is necessary.(ABSTRACT TRUNCATED AT 400 WORDS)

Movement of newly synthesized membrane by fast transport along the axon of an identified Aplysia neuron.

In order to ascertain the form in which newly synthesized membrane is moved by fast axonal transport, we examined the distribution of label along an axon of the identified giant serotoninergic neuron (GCN) in the cerebral ganglion of Aplysia californica. Membrane glycoproteins were labeled by intrasomatic injection of 3H-fucose, and segments of GCN's axon in the posterior lip nerve containing the transported organelles were examined at 1, 5, 15, and 24 hours by quantitative electron-microscopic autoradiography. To show that membrane which is rapidly transported is contained only in discrete organelles rather than in continuous sheets of axoplasmic reticulum, we systematically varied conditions of fixation. We found that we could distinguish vesicles from axoplasmic reticulum most reliably in axons fixed with 2% formaldehyde and 2% glutaraldehyde in cacodylate buffer. At short times after intrasomatic injection of 3H-fucose, dense-cored vesicles and multivesicular tubules were the only axonal organelles labeled.