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.     

Temperature-dependent stimulation and inhibition of adenylate cyclase by Aplysia bag cell peptides

The bag cell peptides (alpha-, beta-, and gamma-BCP) are secreted by the neuroendocrine bag cells of Aplysia, and provide feedback modulation of bag cell excitability and cAMP levels. We report here that if 200-500 mM NaCl is included in the assay buffer, the BCPs alter adenylate cyclase activity in a manner consistent with their effects on cAMP levels in intact bag cells. Specifically, beta-BCP and the related peptide A from the atrial gland stimulate the enzyme, while the effects of alpha-BCP(1-7) and gamma-BCP are temperature-dependent, stimulating at 30 degrees C and inhibiting at 15 degrees C. Both stimulation and inhibition require GTP, suggesting mediation by Gs and Gi. The ionic requirements of stimulation and inhibition differ: Cl- is necessary to support stimulation, but not inhibition. Moreover, pertussis toxin blocks inhibition, but does not affect stimulation. These results suggest that the temperature-sensitive mechanism lies upstream from the G-proteins in the signal transduction pathway.     

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.     

Protein kinase C regulates a vesicular class of calcium channels in the bag cell neurons of aplysia

Protein kinase C (PKC) acutely increases calcium currents in Aplysia bag cell neurons by recruiting calcium channels different from those constitutively active in the plasma membrane. To study the mechanism of PKC regulation we previously identified two calcium channel alpha1-subunits expressed in bag cell neurons. One of these, BC-alpha1A, is localized to vesicles concentrated primarily in somata and growth cones. We used antibodies to BC-alpha1A to analyze its expression in the bag cell neurons of juvenile Aplysia at a developmental stage at which PKC-sensitive calcium currents have previously been shown to be low. We find that vesicular BC-alpha1A staining is generally reduced in juvenile bag cell neurons but that its expression level can vary among juvenile animals. In 17 bag cell clusters examined, the percentage of neurons that displayed punctate alphaBC-alpha1A staining ranged from 0 to 85%. Sampling of calcium currents from cells of the same clusters by whole cell patch-clamp techniques revealed that the PKC-sensitive calcium current density is significantly correlated with the degree of vesicular staining. In contrast, no correlation of basal calcium current levels with aBC-alpha1A staining was found. These results strongly suggest that BC-alpha1A, a member of the ABE-subfamily of calcium channels, carries the PKC-sensitive calcium current in bag cell neurons. They are consistent with a model in which PKC recruits channels from the vesicular pool to the plasma membrane.     

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.     

Poststimulation excitability of ventral pallidum self-stimulation neurons.

The degree of neural recovery from refractoriness was inferred in rats self-stimulating with pairs of pulses in the ventral pallidum. The prerecovery intrapair interval varied from 0.5 to 1.0 ms, depending on brain site. At some sites, recovery reached its maximum within less than 1.6 ms whereas, at the majority of sites, a substantial amount of recovery occurred at delays longer than 1.2 ms. The shortest recovery estimates were not fundamentally different from those obtained from sites lying along the medial forebrain bundle. The longest recovery estimates were similar to those obtained from cortical and basal forebrain sites. The differences in recovery noted between sites and the presence of step-like patterns in the recovery curves suggest the presence of neural heterogeneity within the ventral pallidal substrates of reward. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Alteration of protein metabolism in individual, identified neurons from Aplysia

A search for control mechanisms governing protein metabolism in neurons from Aplysia californica has uncovered two examples of altered patterns of newly synthesized proteins: (1) The pattern of newly synthesized proteins in the R2 neuron is altered when protein synthesis occurs at elevated temperatures (22-30 degrees C as compared with 13-15 degrees C). (2) The processing of newly synthesized 12,000 dalton (12k) material to 6-9,000 dalton (6-9k) size in the R15 neuron (Strumwasser, F. and Wilson, D.F. [1976], J. Gen. Physiol., in press) can be blocked by certain ion replacements. If acetate replaces chloride in the incubation medium during the synthesis of 12k material, an early step in the processing, prior to the actual breakdown of 12k material, is blocked. Experiments with RNA-synthesis inhibitors indicate that none of the mRNAs which code for abundantly synthesized protein species in the R2 or R15 neurons have short (less than 4 hr) half-lives. This result has implications for an earlier report of regulation of protein synthesis in the R15 neuron.     

The morphology of identified neurons in the abdominal ganglion of Aplysia californica

The morphology of identified neurons and of one multiaction interneuron (L10) of the abdominal ganglion of Aplysia has been studied using cobalt chloride, injected intracellularly. Cells with little synaptic input, R3-R14, had a relatively poorly developed dendritic tree, whereas the dendrite tree of cells L7 and L10, with extensive synaptic input, was highly complex. Cells L1-L6 and the RB cell cluster were found to have intermediate complexity of synaptic inputs and dendritic morphology. Within cell clusters, individual cells were often morphologically distinct. Identified cells have both invariant and variant axonal branches. Variant axons often project down other than their customary nerve trunks or are supernumerary. Three features of neuropil architecture were encountered. (1) When cells from the same cluster send their axons down the same nerve the axons often run in fascicles. (2) Although an identified cell's dendritic geometry varies from preparation to preparation, its dendrites always occupy approximately the same position in the neuropil. (3) The postsynaptic follower cells of L10 send their main axons through the axonal arborization of L10.     

The role of glutamate reuptake in regulation of glutamate responses in Aplysia neurons

Glutamate elicits several different responses on neurons of isolated ganglia of Aplysia, the most common of which is a hyperpolarization due to conductance increases to either chloride or potassium. We have investigated the actions of aspartate and cysteate on the responses to glutamate. Neither aspartate nor cysteate is potent in activation of glutamate receptors. However both aspartate and cysteate cause a dramatic increase in the response to glutamate when ionophoretically applied before the glutamate application. This potentiating effect of aspartate and cysteate is a result of competition with glutamate for the glutamate transport system, since the potentiation is blocked by cooling and by perfusion with sodium-free sea water. Blockade of glutamate re-uptake by perfusion of sodium-free sea water also causes a significant increase in the response to ionophoretically applied glutamate, which in some neurons may be very large. These results demonstrate that the glutamate reuptake system has an important role in regulation of the responses to glutamate which is similar to that of acetylcholinesterase in regulation of responses to acetylcholine. These observations may be of particular importance in mammalian systems where excess glutamate is associated with neuronal excitotoxicity and cell death.     

Serotonergic neurons differentially modulate the efficacy of two motor neurons innervating the same muscle fibers in Aplysia

Feeding behavior in Aplysia shows substantial plasticity. An important site for the generation of this plasticity is the modulation of synaptic transmission between motor neurons and the buccal muscles that generate feeding movements. We have been studying this modulation in the anterior portion of intrinsic buccal muscle 3 (I3a), which is innervated by two excitatory motor neurons and identified serotonergic modulatory neurons, the metacerebral cells (MCCs). We have shown previously that serotonin (5-HT) applied selectively to the muscle potently modulates excitatory junction potentials (EJPs) and contractions. All the effects of 5-HT were persistent, lasting many hours after wash out. We examined whether the release of endogenous 5-HT from the MCC could produce effects similar to the application of 5-HT. Stimulation of the MCCs did produce similar short-term effects to the application of 5-HT. MCC stimulation facilitates EJPs, potentiates contractions, and decreases the latency between the onset of a motor neuron burst and the onset of the evoked contraction. The effects of MCC stimulation reached a maximum at quite low firing frequencies, which were in the range of those previously recorded during feeding behavior. The maximal effects were similar to those produced by superfusion with approximately 0.1 microM 5-HT. Although the effects of MCC stimulation on EJPs were persistent, they were less persistent than the effects of 0.1 microM 5-HT. Mechanisms that may account for differences in the persistence between released and superfused 5-HT are discussed. Thus activity in the MCCs has dramatic short-term effects on the behavioral output of motor neurons, increasing the amplitude and relaxation rate of contractions evoked by both B3 and B38 and shifting the temporal relationship between B38 bursts and evoked contractions.     

Rhythmic activities of isolated and clustered pacemaker neurons and photoreceptors of Aplysia retina in culture

Each eye of Aplysia contains a circadian clock that produces a robust rhythm of optic nerve impulse activity. To isolate the pacemaker neurons and photoreceptors of the eye and determine their participation in the circadian clock and its generation of rhythmic autoactivity, the retina was dissociated and its cells were placed in primary cell culture. The isolated neurons and photoreceptors survived and vigorously extended neurites tipped with growth cones. Many of the photoreceptors previously described from histological sections of the intact retina were identified in culture, including the large R-type photoreceptor, which gave robust photoresponses, and the smaller tufted, whorled, and flared photoreceptors. The pacemaker neurons responsible for the rhythmic impulse activity generated by the eye were identified by their distinctive monopolar morphology and recordings were made of their activity. Isolated pacemaker neurons produced spontaneous action potentials in darkness, and pacemaker neurons attached to fragments of retina or in an isolated cluster interacted to produce robust spontaneous activity. This study establishes that isolated retinal pacemaker neurons retain their innate autoactivity and ability to produce action potentials in culture and that clusters of coupled pacemaker neurons are capable of generating robust autoactivity comparable to pacemaker neuron rhythmic activity recorded in the intact retina, which was previously shown to correspond to 1:1 with the optic nerve compound action potential activity.     

Developmental regulation of apoptosis in dorsal root ganglion neurons

The survival of dorsal root ganglion (DRG) neurons, both in vivo and in vitro, is dependent on the availability of nerve growth factor (NGF) for a transient period early in development after which these neurons become independent of NGF for survival. The precise molecular mechanism by which developing DRG neurons gain independence from NGF has not been determined. We used an in vitro model of DRG neuronal development to test hypotheses that independence from NGF in mature DRG neurons could be caused by developmental regulation of either elements of the NGF withdrawal signal transduction pathway or of proteins important for activation of the apoptosis output pathway. Interruption of phosphotidylinositol-3 kinase activation, by treatment with the specific inhibitor LY294002, resulted in apoptosis in immature but not mature DRG neurons in a manner similar to that observed with NGF withdrawal. Further downstream along the signal transduction pathway, c-JUN phosphorylation occurred in both immature and mature DRG neurons after NGF withdrawal or treatment with LY294002, despite the fact that the older neurons did not undergo apoptosis. In contrast, the ratio of expression of the proapoptotic gene bax to antiapoptotic gene bcl-xL was many times higher in immature than mature neurons, both in vivo and in vitro. Taken together, these results strongly suggest that developmental regulation of NGF withdrawal-induced apoptosis in DRG occurs via control of the relative level of expression of members of the bcl-2 gene family.     

Insulin receptor in Aplysia neurons: characterization, molecular cloning, and modulation of ion currents

We have isolated the cDNA for a tyrosine kinase receptor that is expressed in the nervous system of Aplysia californica and that is similar to the vertebrate insulin receptor. Binding studies and immunocytochemical staining show that the receptor is abundant in the bag cell neurons. Application of vertebrate insulin to clusters of bag cell neurons stimulates the phosphorylation of the receptor on tyrosine residues, and exposure of isolated bag cell neurons to insulin produces an increase in height and a decrease in duration of the action potentials that can be detected within 15-30 min. These effects were not seen with insulin-like growth factor-1. In voltage-clamped neurons, insulin produces an increase in the amplitude of the voltage-dependent Ca2+ current that can be blocked by preincubation with herbimycin A, an inhibitor of tyrosine kinases. Insulin also enhances a delayed K+ current. We suggest that insulin-like peptides regulate the excitability of the bag cell neurons.     

A dissociation between the ability to print and write cursively in lower-case letters

This paper reports the case of a patient with a peripheral spelling impairment who is much more severely impaired at writing in lower-case letters than in upper-case letters. This pattern can be observed when writing both words and single letters of the alphabet. Despite this, his problems in writing lower-case letters are no longer present when he is writing cursively. This case therefore indicates that the ability to print letters in lower-case can be selectively impaired in the absence of similar problems in printing upper-case letters or in writing lower-case letters cursively. In terms of the model of writing put forward by Ellis (1982, 1988), this suggests that allographic level representations for print handwriting styles can be functionally dissociated from allographic representations for cursive styles.     

Heterologous expression of the Kv3.1 potassium channel eliminates spike broadening and the induction of a depolarizing afterpotential in the peptidergic bag cell neurons

The bag cell neurons of Aplysia are a cluster of cells that control egg laying behavior. After brief synaptic stimulation, they depolarize and fire spontaneously for up to 30 min. During the first few seconds of this afterdischarge, the action potentials of the bag cell neurons undergo pronounced broadening. Single bag cell neurons in culture also show spike broadening in response to repeated depolarizations. This broadening is frequency-dependent and associated with the induction of a depolarizing afterpotential lasting minutes. In some neurons the depolarizing afterpotential is sufficient to trigger spontaneous firing. To test the possibility that spike broadening during stimulation is required to trigger the depolarizing afterpotential, we eliminated frequency-dependent broadening by heterologous expression of the Kv3.1 potassium channel. This channel has rapid activation and deactivation kinetics and no use-dependent inactivation. Expression of Kv3.1 prevented spike broadening and also eliminated the depolarizing afterpotential. Measurements of the integral of calcium current during voltage commands, which simulated the action potentials of the control neurons and those expressing Kv3.1, indicate that spike broadening produces up to a fivefold increase in calcium entry. Manipulations that limit calcium entry during action potentials or chelation of intracellular calcium using BAPTA AM prevented the induction of the depolarizing afterpotential. We conclude that spike broadening is essential for the induction of the depolarizing afterpotential probably by regulating calcium influx and suggest that one of the physiological roles of spike broadening may be to regulate long-term changes in neuronal excitability.     

Nitric oxide inhibits the dopamine-induced K+ current via guanylate cyclase in Aplysia neurons

Nitric oxide (NO) is produced by the enzyme nitric oxide synthase (NOS) and has been implicated in inter- and intracellular communication in the nervous system. The present study was undertaken to assess the effects of sodium nitroprusside (SNP) and hydroxylamine (HOA), NO donors, on a dopamine (DA)-induced K+ current in identified Aplysia neurons using voltage-clamp and pressure ejection techniques. Bath-applied SNP (10-25 microM) reduced the DA-induced K+ current without affecting the resting membrane conductance and holding current. The DA-induced K+ current also was inhibited by the focal application of 200 microM HOA to the neuron somata. The DA-induced K+ current suppressing effects of SNP and HOA are completely reversible. Pretreatment with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), a specific inhibitor of NO-stimulated guanylate cyclase, and hemoglobin (50 microM), a nitric oxide scavenger, decreased the SNP-induced inhibition of the DA-induced current. In contrast, intracellular injection of 1 mM guanosine 3',5'-cyclic monophosphate (cGMP) or bath-applied 3-isobutyl-1-methylxanthine (IBMX; 50 microM), a non-specific phosphodiesterase inhibitor, inhibited the DA-induced current, mimicking the effect of the NO donors. These results demonstrate that SNP and HOA inhibit the DA-induced K+ current and that the mechanism of NO inhibition of the DA-induced current involves cGMP-dependent protein kinase.