Ganglion Of Aplysia Californica Research Articles

Characterization of neuronal regeneration in the abdominal ganglion of Aplysia californica

Characterization of neuronal regeneration in the abdominal ganglion of Aplysia californica

Characterization of neuronal regeneration in the abdominal ganglion ofAplysia californica

The ability of neurons in the abdominal ganglion of Aplysia to regenerate their axons following branchial nerve crush was studied using retrograde staining and intracellular dye injection. The duration of the gill withdrawal reflex (GWR) was measured prior to and following nerve crush. Three days after crushing the nerve, the duration of the gill withdrawal reflex was reduced to 20% of control levels. There was rapid recovery 19 days after crushing the branchial nerve. The GWR duration returned to control levels by postlesion days 25-27. Some of the behavioral recovery can be attributed to axonal regeneration. Regeneration, as evidenced by retrograde staining, was first observed by postlesion day 15. The number of stained neurons in ganglia with crushes increased until postlesion day 33. The number of stained neurons in experimental animals was always less than that of controls (67+/-9% at postlesion day 56). More axonal regeneration was seen in the hemiganglion ipsilateral to the branchial nerve. Regeneration after 32 days postlesion was 60+/-5% of controls in the ipsilateral hemiganglion, as opposed to 29+/-6% in the contralateral hemiganglion. Regeneration of individual neurons was also demonstrated. Identified neuron R2 was shown by intracellular dye injection and electrical stimulation of antidromic action potentials to have an axon in the branchial nerve in all ganglia allowed to regenerate for longer than 32 days. These results indicate that in Aplysia, despite behavioral recovery, complete axonal regeneration does not occur in a large segment of the neurons in the adult central nervous system.

Classical conditioning, differential conditioning, and second-order conditioning of the Aplysia gill-withdrawal reflex in a simplified mantle organ preparation.

Nonassociative learning was previously examined in a simplified preparation consisting of the isolated mantle organs and abdominal ganglion of Aplysia californica that is advantageous for relating cellular events to behavior (T. E. Cohen, S. W. Kaplan, E. R. Kandel, & R. D. Hawkins, 1997). Results of the current study show that the gill-withdrawal reflex in that preparation also underwent 2 associative forms of learning: classical conditioning and differential conditioning. In addition, the reflex underwent second-order conditioning with either forward or simultaneous pairing of a novel conditioned stimulus (CS2) and a previously conditioned stimulus (CS1). Moreover, extinction of CS1 after simultaneous second-order conditioning was accompanied by a decrease in responding to CS2, suggesting that the conditioning might have involved formation of an association between the CSs. In each of these paradigms, learning in the Aplysia mantle organ preparation resembled learning in vertebrates.

Pre- and postsynaptic actions of nifedipine at an identified cholinergic central synapse of Aplysia.

The effects of the dihydropyridine (DHP) Ca2+ channel antagonist, nifedipine, were studied on the cholinergic synapse between the presynaptic neurones B4/B5 and the postsynaptic neurones B3/B6 located in the buccal ganglion of Aplysia californica. Nifedipine (10 microM) decreased the presynaptic Ca2+ current by 30%-40%. Blockade of DHP-sensitive Ca2+ channels, however, did not affect quantal transmitter release from the presynaptic neurones. Thus, at this synapse, DHP-sensitive Ca2+ channels appear not to be involved in acetylcholine (ACh) release. The postsynaptic response to an ionophoretic application of ACh was decreased by nifedipine, pointing to a blocking action of the drug on the postsynaptic receptor/channel complex. Nifedipine was also found to activate protein kinase C, which in turn induces an increase in the nifedipine-resistant presynaptic Ca2+ influx and in the number of released ACh quanta. These effects of nifedipine could be prevented by a previous application of 1,5-(isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a protein kinase blocker.

Histamine and FLRFamide regulate acetylcholine release at an identified synapse in Aplysia in opposite ways.

1. The effects of histamine and FLRFamide (Phe-Leu-Arg-Phe-NH2) on acetylcholine (ACh) release were studied in the buccal ganglion of Aplysia californica on an identified synapse (buccal ganglion inhibitory synapse, BGIS) involved in a small neuronal circuit controlling the feeding behaviour. The inhibitory postsynaptic current (IPSC) evoked by a presynaptic spike in the voltage-clamped postsynaptic neurone was decreased by histamine and increased by FLRFamide. 2. Histamine and FLRFamide modified the amplitude of the presynaptic spike. To test if these drugs acted directly on presynaptic calcium influx, we evoked transmitter release by 3 s depolarizations of the presynaptic neurone (to +10 mV) under voltage clamp to avoid modifications of presynaptic membrane polarization induced by changes in presynaptic voltage-dependent K+ and/or Na+ conductances. 3. Statistical analysis of this evoked long-duration (3 s) induced postsynaptic current (LDIPSC) allowed us to calculate the amplitude and the decay time of the miniature postsynaptic current and consequently the number of quanta released by the presynaptic terminal. 4. The amplitude of the LDIPSC decreased during the 3 s presynaptic depolarization. This was not due to a lack of available transmitter, since LDIPSC amplitude could be maintained constant by a 'clamp of the release of ACh' which adequately depolarized the presynaptic neurone, but rather to changes in the calcium influx into the presynaptic neurone. 5. FLRFamide increased more the initial portions of the LDIPSC than the final portions. This effect of FLRFamide was only reduced and delayed by atropine or curare, antagonists of muscarinic-like and nicotinic-like autoreceptors previously demonstrated to be present at the same terminal. Activation of the nicotinic-like receptors, which also increased transmitter release, induced a modification of the shape of the LDIPSC which was completely different from that due to FLRFamide. 6. Histamine decreased the amplitude of the LDIPSC. This effect was more pronounced at the beginning of the response. The effects of histamine were insensitive to curare and atropine, but were completely blocked by cimetidine, a specific histamine receptor antagonist. 7. The modifications of the shape and of the amplitude of the LDIPSC by FLRFamide and histamine suggested that these molecules alter presynaptic influx of calcium. This was confirmed by the analysis of calcium current recorded from the presynaptic neurone: the calcium inward current in the presynaptic neurone was increased by FLRFamide and reduced by histamine, whereas the activation of autoreceptors had no measurable effect on calcium current.(ABSTRACT TRUNCATED AT 400 WORDS)

Activity-related changes in protein phosphorylation in an identifiedAplysia neuron

The relationship between long-term electrical activity and protein phosphorylation was investigated in single, identifiable neurons in the abdominal ganglion of Aplysia californica by the intracellular injection of radiolabeled ATP followed by sodium dodecyl sulfate (SDS) gel electrophoresis. Natural and pharmacological treatments that alter the impulse activity of neurons L6 and R15 for prolonged periods did not appear to affect the phosphorylation of most of the 15 major phosphoproteins examined in these cells. Long-term excitation of L6 induced by the phosphodiesterase inhibitor IBMX correlated with phosphorylation of a 29,000-dalton protein. Long-term inhibition of L6 induced by afterdischarge of peptidergic bag-cell neurons appeared to cause dephosphorylation of a 29,000-dalton protein. Burst augmentation of R15 induced by bag-cell afterdischarge did not cause detectable changes in the phosphorylation of the major proteins we examined. These data are consistent with other studies of neural and nonneural tissues which have found a correlation between activity and the level of phosphorylation of a 29,000-dalton protein.

Calcium of A-currents in perfused Aplysia neurons

Transient outward currents were studied in neurons in the visceral ganglion of Aplysia californica, using intracellular perfusion and voltage-clamp techniques. The early outward currents in response to depolarizations from holding potentials near -90 mV were activated in the range -60 to -20 mV, below the threshold for the delayed outward current. Resting inactivation of the early outward currents was removed by prehyperpolarizations in the range -130 to -70 mV. A-currents produced in this manner were blocked by external application of CoCl2 and augmented by increasing external Ca-concentration. They were also blocked by treatment with 4-aminopyridine. The currents were reduced by treatment with verapamil hydrochloride, further suggesting a role for calcium in the current-generating mechanism. A model with a fourth-power activation process and first-power inactivation process could fit the early outward currents reasonably well. The effect of application of Ca-free, cobalt-containing solution was modeled as a decrease in peak conductance and an increase in the time constants of activation and inactivation.

Cytoplasmic alkalization reduces calcium buffering in molluscan central neurons

The effect of raised cytoplasmic pH (pHi) on intracellular concentration ([Ca2+]i) transients following calcium influx during membrane depolarization was studied in identified neurons in the abdominal ganglion of Aplysia californica. The pHi was monitored with pH-sensitive microelectrodes. Sea water containing 15 mM NH4Cl at pH 7.7 elevated pHi about 0.35 pH units from the normal level of 7.17. These cells have an estimated buffering power of about 60 mM/pH unit. Calcium influx was elicited by depolarizing pulses under voltage clamp and [Ca2+]i transients were monitored with the photoprotein aequorin or the metallochromic dye arsenazo III. Aequorin photo-emissions increased by 21--131% (mean, 48%) and arsenazo III absorbance changes accompanying depolarization increased by 9--33% (mean, 20%) after 30 min in NH4+, corresponding roughly to a 14% increase in [Ca2+]i transients. Calcium-dependent potassium tail currents following a depolarizing pulse were somewhat slower and 4--91% (mean, 38%) large in NH4+. The magnitude and time- and voltage-dependence of the membrane calcium conductance was studied using calcium tail currents following depolarizing pulses. The calcium current was unaffected by NH4+, so the enhanced [Ca2+]i transients must reflect reduced calcium buffering at high pHi. Either reduced cytoplasmic calcium binding or slowed active extrusion of calcium may be responsible for this effect.

Fluorometric determination of octopamine in tissue homogenates by high-performance liquid chromatography.

A rapid, sensitive method for quantitative determination of octopamine (a biogenic amine found in both vertebrate and invertebrate nervous tissue) has been developed using reversed-phase high-performance liquid chromatography. The biogenic amine is extracted with perchloric acid from tissue homogenates and derivatized witho-phthalaldehyde (OPT) prior to chromatography. Separation of the fluorescent octopamine-OPT adduct from other biogenic amines was achieved using a μBondapak C18 reversed-phase column and isocratic elution with a methanol-(0.08 mol/liter) acetic acid (50∶50 by vol, pH 2.9) mobile phase. A variable wavelength fluorometer with an 8-μl flow cell was used for detection (excitation 340 nm, 418 nm secondary filter). Linearity ranged from 500 pg to 30 ng injected onto the column. Recovery of internal standard added to tissue homogenates averaged 65.4% with a standard deviation of 3.1%. The method has been used for the determination of octopamine in ganglia ofAplysia californica.

The amplitude of post-tetanic potentiation of the EPSP RC1-RC15 in aplysia is modulated by environmental parameters

Evidence is presented that the EPSP called RC1-R15, which is recorded from cell R15 of the abdominal ganglion of Aplysia californica upon appropriate stimulation of the right connective, is endogenously active. In previous studies we showed that after repetitive stimulation the amplitude of this EPSP increases and then slowly decays over many minutes, a phenomenon called post-tetanic potentiation (PTP). The rate of endogenous firing of this EPSP varies with time of day and tonicity of the animal's external environment. It is shown that this endogenous firing increases the magnitude of the PTP component of the amplitude of the EPSP. The degree of this potentiation varies with the endogenous firing rate. In general, daytime or a hypertonic external environment increases the firing rate of the EPSP and its degree of potentiation. Since the degree of this PTP reflects time of day and environmental tonicity, it is inferred that it conveys physiologically significant information to the postsynaptic cell R15.

The characterization of histidine decarboxylase and its distribution in nerves, ganglia and in single neuronal cell bodies from the CNS of Aplysia californica.

Abstract—Histamine (HA) is present in substantial quantities in all ganglia of Aplysia californica. Within the cerebral ganglia this amine is known to be concentrated in at least two identified neurons designated C‐2 neurons. In this study a combination of chemical and enzymatic analyses was employed to provide evidence for the existence of a biochemical pathway for HA synthesis in ganglia and individual neurons of this marine mollusk. Examination of extracts of individual neurons dissected from ganglia organ‐cultured in the presence of [3H]histidine showed that every neuron accumulated labelled histidine, but only the HA‐containing C‐2 neurons synthesized and stored labelled HA suggesting that the formation of HA in Aplysia could be catalyzed by the enzyme histidine decarboxylase (HDC). HDC activity was studied with a new microradiometric assay. Many of the properties of the molluscan HDC studied were found to correspond to the vertebrate enzyme. Enzyme activity was inhibited by α‐hydrazino‐histidine but unaffected by concentrations of α‐methyldopa or by 5‐(3,4‐dihydroxycinnamoyl) salicylic acid which produced nearly complete inhibition of aromatic amino acid decarboxylase activity. HDC was measurable in nervous but not other Aplysia tissues assayed. All 5 major ganglia contained HDC activity which spanned a 15‐fold range between the least and most active ganglia. Only 4 of the 13 nerve trunks assayed yielded measurable enzymic activity; these active nerves were associated with the cerebral ganglia which has the highest HDC activity of all measured ganglia. Of the numerous individual neurons assayed for HDC, only the C‐2 cells showed measurable enzyme activity, about 25 pmol/cell/h or 70 μmol/g protein/h. Since the activity of HDC in the HA‐containing neurons was at least three orders of magnitude larger than all other neurons assayed in the cerebral and other ganglia, these data appear to provide a direct metabolic basis for the selective presence of HA in these cells, and they indicate that the cellular presence of HDC provides a useful biochemical marker for the location of HA‐rich neurons in Aplysia.

The effects of delta9-tetrahydrocannabinol on action potentials in the mollusc Aplysia.

The effects of delta9-tetrahydrocannabinol (delta9-THC) on action potentials were examined during intrasomatic recordings from the isolated buccal and parieto-visceral ganglia of Aplysia californica. When added to the saline solution bathing the preparation, the compound (in concentrations 10(-4) - 10(-5) M) caused a reduction in spike overshoot (15-20% of total amplitude) and increased the lability of responses to electrical stimulation. The somatic membrane appeared to be more affected than the axonal membrane. Diffusion barriers in the ganglion probably account for the high degree of variability in drug response, such that both of the characteristic changes were observed in only about 30% of the tests. This is the first report to describe effects of delta9-THC on invertebrate neurones. The results indicate that delta9-THC causes a depression in nerve cell excitability, and these data are consistent with reported effects of THC compounds in mammals.

Ribonucleic acid metabolism of a single neuron: correlation with electrical activity.

The giant neuron of the abdominal ganglion of Aplysia californica incorporates tritiated uridine into RNA at a constant rate at rest. This rate incrceases under synaptic stimulation, the increase being directly proportional to the number of action potentials produced by the neuron. Multineuronal samples from stimulated ganglia faiiled to show an increase in incorporation.