Development and modulation of endogenous bursting in identified neuron R15 of juvenile Aplysia

Evidence from a variety of both vertebrate and invertebrate preparations has demonstrated that modulation of the intrinsic firing patterns of individual neurons can have a dramatic effect on the functional output of a neural circuit. Although the mechanisms underlying the production and modulation of intrinsic firing patterns have been extensively studied in adult nervous systems, relatively little is known about how these two features of intrinsically active neurons develop. To address these issues, we have examined the development of endogenous bursting and its modulation by neuropeptides in the identified cell R15 of juvenile Aplysia. Confirming Ohmori (1981), we found that the mature parabolic bursting pattern of R15 is absent in early juvenile stages and develops only gradually over the last stage of juvenile development. We have then analyzed the modulatory effects of extracts made from the neurosecretory bag cells of Aplysia on the immature firing pattern of juvenile R15 cells. In the adult, neuroactive peptides released from the bag cells are known to intensify bursting. In juveniles, we have found that bag cell extract (BCE) can induce bursting prematurely as well as intensify immature bursts, whereas control extracts have no effect on the firing pattern of R15. These results show that the ionic currents necessary for the generation of endogenous bursting in R15 are present and can be modulated before the normal developmental expression of the burst pattern.     

Phosphoproteins associated with the regulation of a specific potassium channel in the identified Aplysia neuron R15

The neurotransmitter serotonin (5HT) activates a specific K+ conductance in the identified Aplysia neuron R15. This response to 5HT has been shown previously to be mediated by cAMP and cAMP-dependent protein phosphorylation. We have measured protein phosphorylation within neuron R15 in vivo, following the intracellular injection of [gamma-32P]ATP, and have demonstrated that 5HT modulates the phosphorylation of a number of proteins in R15. The present study was undertaken to determine which of these phosphoproteins are closely associated with, and may be responsible for, the K+ conductance increase. Treatment of neuron R15 with a cAMP analog produces some but not all of the 5HT-induced phosphoprotein changes, indicating that some are not cAMP-dependent and thus can be dissociated from the cAMP-dependent K+ conductance increase. Similar results are obtained by intracellular injection of the adenylate cyclase inhibitor guanosine 5'-O-(2-thiodiphosphate), which completely blocks the 5HT-evoked K+ conductance increase but fails to block some of the 5HT-induced phosphorylation changes. Examination of the phosphoprotein pattern at short times after 5HT application has demonstrated that some of the phosphoprotein changes, but not others, are closely associated in time with the appearance of the physiological response. These and other pharmacological and kinetic experiments have allowed the identification of two phosphoproteins, of Mr = 29,000 and 70,000, which cannot be dissociated from the 5HT-induced K+ conductance increase whatever the experimental manipulation. Thus, one or both of these phosphoproteins may be involved in the regulation of the 5HT-sensitive K+ channel in neuron R15.     

Proteolytic processing of a peptide precursor in Aplysia neuron R14

The large neurons of the mollusc Aplysia are useful for studying the biogenesis of neuropeptides in single cells. Neuron R14 in the abdominal ganglion synthesizes large quantities of a 10-kDa neuropeptide precursor. The amino acid sequence of this precursor has been defined by analysis of the nucleotide sequence of a cDNA clone. We labeled proteins in vivo by microinjection of radioactive amino acids into individual R14 neurons. The labeled peptides were fractionated by high performance liquid chromatography and subjected to Edman degradation, thus enabling us to determine post-translational processing sites. Cleavage of the signal sequence was observed and at two internal sites. Cleavage at these internal sites occurs at basic amino acids and results in three products, a 2.9-, a 4.9-, and a 1.4-kDa peptide. These studies of protein processing serve as a basis for further investigations of the biogenesis and physiological activities of the neuropeptides.     

Intracellular injection of guanyl nucleotides alters the serotonin- induced increase in potassium conductance in Aplysia neuron R15

The effects of the adenylate cyclase inhibitor GDP beta S on the response of Aplysia neuron R15 to serotonin (5HT) were investigated. Previous studies have demonstrated that 5HT causes an increase in K+ conductance in R15 and that the response is mediated by cAMP. At concentrations in the micromolar range, GDP beta S inhibits the stimulation of adenylate cyclase by 5HT in particulate fractions from Aplysia ganglia. When micromolar concentrations of GDP beta S are injected into neuron R15, there is no effect on the resting membrane conductance, but the increase in K+ conductance normally elicited by 5HT is completely inhibited. Furthermore, the decrease in inward current normally elicited by dopamine (DA), which does not appear to involve cAMP, is not affected by micromolar concentrations of GDP beta S. In addition, application of 8-benzylthio cAMP to R15 can evoke an increase in K+ conductance even after the injection of GDP beta S, which indicates that events subsequent to the activation of adenylate cyclase are not inhibited by the GDP analogue. In contrast, when millimolar concentrations of GDP beta S are injected into R15, direct effects on membrane conductance are observed and the response of R15 to 5HT is enhanced. Although these effects of high concentrations of GDP beta S are only poorly understood, the results with micromolar concentrations are consistent with the hypothesis that stimulation of adenylate cyclase is necessary for the 5HT-induced increase in K+ conductance in neuron R15.     

Patterns of proteins synthesized in the R15 neuron of Aplysia. Temporal studies and evidence for processing

The time-course of changes in the pattern of newly synthesized proteins in the R15 neuron of the parietovisceral ganglion of Aplysia californica has been studied at 14 degrees C. 5% polyacrylamide gels containing sodium dodecyl sulfate (SDS) have been used to separate newly synthesized (leucine-labeled) proteins from the neuron. We have demonstrated that the pattern of newly synthesized proteins from the R15 neuron does not change significantly if 5-h pulses of labeled leucine are given during the first 72 h of in vitro incubation of the excised ganglion. However, the level of leucine incorporation begins to decline somewhere between 17 and 43 h after the ganglion is isolated; at 43 and 69 h the levels of incorporation fell to 29 and 10% of the initial level, respectively. A number of conclusions have been drawn from the use of a sequential, double-label type of experiment in the same cell. There is processing of SDS-soluble, 12,000-dalton (12k) material to 6,000-9,000-dalton (6-9k) material. These materials are the two major peaks on gels after long labeling periods and together account for about 35% of all newly synthesized proteins. After synthesis of 12k material, there is a gradual disappearance of 12k (half-life about 8 h) and simultaneous appearance of 6-9k material on the gels, as the postsynthesis "chase" period of ganglia incubation is increased. The processing of 12k to 6-9k material occurs even in the presence of anisomycin, a protein syntehsis inhibitor, during the chase period. While the rate of 12k to 6-9k conversion can vary from cell to cell, it appears to remain consistent within, and is characteristic of, any individual R15. We detect no circadian rhythm in either the rate of 12k synthesis or the rate of 12k to 6-9k processing with 5-h label periods. These results are discussed in relation to the roles of 12k and 6-9k material in the R15 neuron.     

Water regulation by a presumptive hormone contained in identified neurosecretory cell R15 of Aplysia

Injection of an homogenate of identified neuron R15 into the hemocele of Aplysia produced a weight increase of 3-10% within 90 min. Control injections of several other identified neurons or of seawater, were ineffective. The weight increase occurred even when the animals were maintained in 5% hyperosmotic seawater. The activity of the R15 homogenate was retained after acidification to pH 2 and heating to 100 degrees C; but activity was destroyed by proteolytic digestion with Pronase. Dialysis in cellulose dialysis tubing resulted in a significant loss of aion on Sephadex G-50 (nominal exclusion limits 1,500-30,000 daltons), activity was present in the partially included volumes, but was absent in the totally excluded or totally included volumes. The data support the notion that R15 contains one or more hormones involved in ionic regulation or water balance. The results of bioassays of R15 extracts subjected to different treatments are consistent with the hypothesis that activity is due to one or more stable polypeptides of relatively low molecular weight.     

Decreased Response to Acetylcholine during Aging of Aplysia Neuron R15

How aging affects the communication between neurons is poorly understood. To address this question, we have studied the electrophysiological properties of identified neuron R15 of the marine mollusk Aplysia californica. R15 is a bursting neuron in the abdominal ganglia of the central nervous system and is implicated in reproduction, water balance, and heart function. Exposure to acetylcholine (ACh) causes an increase in R15 burst firing. Whole-cell recordings of R15 in the intact ganglia dissected from mature and old Aplysia showed specific changes in burst firing and properties of action potentials induced by ACh. We found that while there were no significant changes in resting membrane potential and latency in response to ACh, the burst number and burst duration is altered during aging. The action potential waveform analysis showed that unlike mature neurons, the duration of depolarization and the repolarization amplitude and duration did not change in old neurons in response to ACh. Furthermore, single neuron quantitative analysis of acetylcholine receptors (AChRs) suggested alteration of expression of specific AChRs in R15 neurons during aging. These results suggest a defect in cholinergic transmission during aging of the R15 neuron.     

A possible sensory-motor neuron in Aplysia californica

A granule-containing cell is described in the secretory epithelia of the accessory genital mass in Aplysia californica. The ciliated apical process of this cell protrudes into the lumen of the oviduct. The granule-containing basal region of the cell is drawn out into fine processes that resemble axons. Granule-filled dilatations of these axons are found directly under large secretory cells. On this basis, it is suggested that these cells fulfill the morphological criteria for sensory-motor cells, and this data will be used as a basis for microelectrode studies to confirm or deny the above suggestion.     

Synthesis of glycoproteins in a single identified neuron of Aplysia californica

Incorporation of L-[³H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [³H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [³H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [³H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [³H]fucose, gel profiles of particulate fractions labeled with [¹⁴C]valine were much more complex.     

Intracellular injection of dopamine enhances acetylcholine responses of neuron R2 in the Aplysia abdominal ganglion

1. At different levels of the holding potential on neuron R2 membrane in the Aplysia depilans abdominal ganglion, dopamine injected intracellularly increases the amplitude of both inward and outward currents recorded in response to the application of acetylcholine (ACh) to the ganglion surface. 2. The addition of dopamine to the external perfused solution produces generation of inward currents and a decrease in the cell response to the ACh. 3. The enhancing effect of injected dopamine on ACh responses is retained after inhibition of acetylcholinesterase (AChE) by a specific organophosphorous inhibitor, compound Gd-42. 4. The modulating effect of injected dopamine on ACh responses is discussed in terms of the existence of intracellular receptors of neurotransmitters in the differentiated cells.     

The cyclic GMP-induced inward current in neuron R14 of Aplysia californica: similarity to a FMRFamide-induced inward current

Injecting cGMP into Aplysia neuron R14 induced an inward current similar to one elicited by application of FMRFamide to the outside of that cell. In contrast, injection of cAMP into R14 caused a long-lasting outward current and conductance increase. Phosphodiesterase inhibitors increased the cGMP and FMRFamide-induced inward currents in R14. The cGMP-induced inward current is voltage dependent and is largely carried by Na+. It is also strongly and inversely dependent on both external [Ca2+] and [Cl-], although these ions are not significant current carriers. Changing external [K+] had no effect. Voltage and ion dependencies of the cGMP-induced inward current are similar to those of an inward current induced by FMRFamide. Thus cGMP may be a second messenger to FMRFamide in producing a slow inward current in R14. cGMP does not appear to be a second messenger to FMRFamide in most Aplysia neurons.     

Differential regulation of a homologous peptidergic neuron in grasshoppers: evolution of a neural circuit

The vasopressin-like immunoreactive (VPLI) neurons of grasshoppers have paired cell bodies in the suboesophageal ganglion and both anterior and posterior running axons. In non-oedipodine grasshopper species (e.g. Schistocerca gregaria), most of their arborisations are distributed in dorsal and lateral neuropil, while in oedipodine species (e.g. Locusta migratoria), the neurons have additional extensive axonal projections in both the optic lobes and proximal portions of the ganglionic peripheral nerves. This study demonstrates that these morphological differences correlate with their physiology. In L. migratoria, VPLI neuron activity is regulated primarily via a spontaneously active interneuron which descends from the brain. This descending interneuron is inhibited by a light-activated brain extraocular photoreceptor. Regulation of VPLI neuron activity by an extraocular photoreceptor is also seen in the other oedipodine grasshopper investigated. In the four non-oedipodines examined (from two subfamilies), we find no extraocular photoreceptor regulation of VPLI neuron activity. Despite this, VPLI neuron in S.␣gregaria does appear to be driven by a descending interneuron homologous to that in L. migratoria. The descending interneuron in both species receives similar mechanosensory input and excites the VPLI neuron via cholinergic synapses. Histamine injection into the medial protocerebrum of both species causes strong inhibition of the descending interneuron. The evolution of the neural circuitry, by which an extraocular photoreceptor comes to regulate the descending interneuron in oedipodine species, is discussed. Accepted: 6 January 1998     

R15 alpha 1 and R 15 alpha 2 peptides from Aplysia: comparison of bioactivity, distribution, and function of two peptides generated by alternative splicing

The mRNA precursor encoded by the R15 gene is alternatively spliced in different neurons to form two related variants, R15-1 and R15-2 mRNA. One of the peptides encoded by the R15-2 mRNA, the R15 alpha 1 peptide, is expressed in the endogenously bursting neuron R15 and mediates some of its central and peripheral synaptic actions. In this study we found that the R15 alpha 2 peptide, which is encoded by the R15-1 mRNA, is synthesized in other neurons in the abdominal ganglion and is also bioactive. The R15 alpha 1 and R15 alpha 2 peptides were found to exert many similar actions on the cardiovascular, digestive, respiratory, and reproductive systems. However, the differences between many of the pharmacological effects of the R15 alpha 1 and R15 alpha 2 peptides indicate that alternative splicing in this system results in two functionally different peptides. Widespread immunoreactivity was found for an antibody directed against the R15 alpha 2 peptide, both in the central nervous system and the periphery. But because of the shared sequence with the R15 alpha 1 peptide, the antibody cross-reacts with the R15 alpha 1 peptide. To distinguish immunocytochemically between the two peptides, we also raised a second antibody that recognizes only the R15 alpha 1 peptide. This antibody labeled the cell body of only one neuron in the central nervous system, R15, although widespread immunoreactivity was found in axons and varicosities in the periphery.     

Regional differences in processing of locally translated prohormone in peptidergic neurons of Aplysia californica

Earlier work showed that cell bodies and neurites of the peptidergic bag cell neurons of Aplysia californica contain mRNA for egg-laying hormone. The purpose of the present study was to determine if egg-laying hormone synthesis and prohormone processing is similar in the pleurovisceral connective nerves (containing neurites of bag cell neurons) and the bag cell neuron clusters (containing both cell bodies and neurites of bag cell neurons). Initial experiments confirmed by RT-PCR and sequencing that egg-laying hormone mRNA was present in the pleurovisceral connective nerves. To investigate possible regional differences in translation of mRNA and prohormone processing, clusters were separated from connective nerves and newly synthesized egg-laying hormone-immunoreactive proteins were analyzed. Results showed that synthesis and processing of prohormone occurred in both the clusters and isolated connective nerves; however, the relative abundance of prohormone, processing intermediates, and egg-laying hormone was different. Pulse-chase experiments showed that prohormone was processed more slowly in the connective nerves than in the clusters. These results show that mRNA in isolated neural processes of neuroendocrine cells can be translated, and that the cellular machinery for protein synthesis is present, but processing of the ELH prohormone is significantly compromised.     

Dynamics and metamorphosis of an identifiable peptidergic neuron in an insect

Eclosion hormone (EH) is a 7000 Da peptide that triggers ecdysis behavior in insects. In the moth, Manduca sexta, EH is found in two pairs of ventromedial (VM) cells in the brain which send their axons down the ventral nerve cord to a neurohemal site in the proctodeal nerve in the larva and pupa. During adult development, these cells send axon collaterals to the corpora cardiaca where they form a new release site used for adult eclosion. Studies of bioassayable peptide during the 5th larval instar and the larval-pupal transformation revealed that after depletion at ecdysis, the VM cells showed a transient increase in EH found in their cell bodies and axons. By contrast, their terminals in the proctodeal nerve showed a gradual accumulation of peptide followed by a release of over 90% of the stored material at pupal ecdysis. In situ hybridization analysis on whole mounts of the brains showed that the VM cells always contained EH mRNA with increased accumulation during the larval and pupal molting periods with a slight decline just before ecdysis. High levels of EH mRNA were found in brains of diapausing pupae. During the first two-thirds of adult development, mRNA accumulated to high levels, then slowly declined until ecdysis. EH mRNA levels up to 3 days after adult eclosion. At no time was EH mRNA found in the lateral neurosecretory cell cluster previously reported to produce EH for adult eclosion. 1994 John Wiley & Sons, Inc.     

Ionic permeabilities of an Aplysia giant neuron.

In a giant neuron of Aplysia californica, permeabilities and conductances obtained by measuring net fluxes of Na+, K+ and Cl- with ion-specific microelectrodes were compared with those obtained by measuring transmembrane current and potential changes when the three ions were varied in the external solution. Net fluxes were measured with ion-specific microelectrodes, after blocking metabolic processes, thus allowing movement of ions down their electrochemical gradients. Permeabilities and conductances obtained from the "chemical" measurements (i.e., ion-specific electrodes) were generally comparable to the values obtained from "electrical" measurements. Where discrepancies occurred, they could be explained by showing that some of the assumptions necessary to use the "electrical" method were not quantitatively true in this system. The absolute magnitudes of the permeabilities are significantly less than those found in many axonal preparations. There is also a relatively high PNa/PK ratio. The selectivity of the membrane against ions such as Tris+ and MeSO3 is not good, Tris+ being nearly as permeable as Na+ and MeSO3 about one-half as permeable as Cl-. These properties may be characteristic of somal membranes.     

Sensitivity of a peptide-activated neuron in Aplysia to serotonin and cyclic AMP-relevant agents

Serotonin depolarized Aplysia buccal motoneuron B16. The response could be obtained in high-magnesium/low-calcium medium, indicating a direct effect on B16 and supporting a putative monosynaptic input to B16 from the serotonergic metacerebral neurons. Similar depolarizing effects in high-magnesium/low-calcium medium were obtained in response to 8-bromo cyclic AMP, isobutylmethylxanthine, theophylline and forskolin. Tolbutamide, a putative inhibitor of cyclic AMP-dependent protein kinase, blocked or reversed responses of B16 to egg-laying hormone containing extracts and to serotonin. Serotonin and forskolin significantly increased the cyclic AMP content of buccal ganglia, whereas egg-laying hormone-containing extracts did not.