Skip to main content
Log in

Compartmentalization of cyclic AMP elevation in neurons ofAplysia californica

  • Published:
Cellular and Molecular Neurobiology Aims and scope Submit manuscript

Summary

  1. 1.

    We have measured by radioimmunoassay the amount of total, free, and bound forms of cyclic AMP (cAMP) within the abdominal ganglion and in five identified cell bodies of neurons fromAplysia californica.

  2. 2.

    In the abdominal ganglion the unbound (free) cAMP levels comprised approximately 25–30% of the total cAMP content under the unstimulated condition, i.e., bathed in high-magnesium saline. Under pharmacological conditions that blocked endogenous phosphodiesterase and activated adenylate cyclase, ganglionic free cAMP levels were elevated more than fourfold, while bound cAMP levels more than doubled.

  3. 3.

    Freeze-substitution techniques were employed to facilitate isolation of individual cell bodies either before or after pharmacological manipulation of cAMP levels. The basal, free cAMP content of cells R2, LP1, R15, L11, and L2–L6 was in the range of 10–40 pmol/mg of cell protein, which accounted for approximately one-half of the total cAMP content per cell body. Determinations of individual cell volumes indicated that the basal, free cAMP concentrations ranged from 1 to 6µM.

  4. 4.

    Under the same pharmacological conditions that elevated ganglionic cAMP levels, no changes were measured in either the free or the bound forms of cAMP in isolated cell bodies. Our results indicate that the cAMP elevation was compartmentalized within the neuropilar region of the ganglion, most likely within the processes of the nerve cells.

  5. 5.

    Previous results demonstrated that cAMP injections into the sameAplysia neurons studied here induced a cAMP-activated sodium current,I Na(cAMP). In this report we discuss the possibility that pharmacological elevation of cAMP within neuronal processes may reach concentrations similar to those produced by cAMP injections into somata.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Albano, J., Brown, B., Ekins, R., Tait, S., and Tait, J. (1974). The effects of potassium, 5-hydroxytryptamine, adrenocorticotrophin and angiotensin II on the concentration of adenosine 3′:5′-cyclic monophosphate in suspensions of dispersed rat adrenal zona glomerulosa and zona fasciculata cells.Biochem. J. 142391–400.

    Google Scholar 

  • Aldenhoff, J., Hofmeier, G., Lux, H., and Swandulla, D. (1983). Stimulation of a sodium influx by cAMP inHelix neurons.Brain Res. 276289–296.

    Google Scholar 

  • Alkon, D. (1979). Voltage-dependent calcium and potassium ion conductances: A contingency mechanism for an associative learning model.Science 205810–816.

    Google Scholar 

  • Alkon, D. (1984). Calcium-mediated reduction of ionic currents: A biophysical memory trace.Science 2261037–1045.

    Google Scholar 

  • Bernier, L., Castellucci, V., Kandel, E., and Schwartz, J. (1982). Facilitatory transmitter causes a selective and prolonged increase in adenosine 3′:5′-monophosphate in sensory neurons mediating the gill and siphon withdrawal reflex inAplysia.J. Neurosci. 21682–1691.

    Google Scholar 

  • Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72248–254.

    Google Scholar 

  • Bridger, W., and Henderson, J. (1983).Cell ATP, John Wiley & Sons, New York, pp. 1–6.

    Google Scholar 

  • Bruehl, C., and Berry, R. (1985). Regulation of synthesis of the neurosecretory egg-laying hormone ofAplysia: Antagonistic roles of calcium and cyclic adenosine 3′:5′-monophosphate.J. Neurosci. 51233–1238.

    Google Scholar 

  • Cedar, H., and Schwartz, J. (1972). Cyclic adenosine monophosphate in the nervous system ofAplysia californica. II. Effect of serotonin and dopamine.J. Gen. Physiol. 60570–587.

    Google Scholar 

  • Cedar, H., Kandel, E. R., and Schwartz, J. H. (1972). Cyclic adenosine monophosphate in the nervous system ofAplysia californica. I. Increased synthesis in response to synaptic stimulation.J. Gen. Physiol. 60558–569.

    Google Scholar 

  • Cobd, C., and Corbin, J. (1987). Purification of cAMP-free and cAMP-bound forms of bovine cAMP-dependent protein kinase holoenzyme.Methods in Enzymology (in press).

  • Coggeshall, R. (1967). A light- and electron-microscope study of the abdominal ganglion ofAplysia californica.J. Neurophysiol. 301263–1287.

    Google Scholar 

  • Connor, J. A., and Hockberger, P. (1982). Direct measurements of cAMP effects on membrane conductance, intracellular Ca2+ and pH in molluscan neurons. InConditioning: Representation of Involved Neural Function (Woody, C. D., Ed.), Plenum, New York, pp. 179–196.

    Google Scholar 

  • Connor, J. A., and Hockberger, P. E. (1984). A novel membrane sodium current induced by injection of cyclic nucleotides into gastropod neurones.J. Physiol. (Lond.)354139–162.

    Google Scholar 

  • Coombs, J., and Thompson, S. (1987). Forskolin's effects on transient K-current in nudibranch neurons are not reproduced by cAMP.J. Neurosci. (in press).

  • Corbin, J., Sugden, P., Lincoln, T., and Keely, S. (1977). Compartmentalization of adenosine 3′:5′-monophosphate and adenosine 3′:5′-monophosphate-dependent protein kinase in heart tissue.J. Biol. Chem. 2523854–3861.

    Google Scholar 

  • Deterre, P., Paupardin-Tritsch, D., Bochaert, J., and Gerschenfeld, H. (1981). Role of cyclic AMP in a serotonin-evoked slow inward current in snail neurones.Nature 290783–785.

    Google Scholar 

  • Deterre, P., Paupardin-Tritsch, D., Bockaert, J., and Gerschenfeld, H. (1982). cAMP-mediated decrease in K+ conductance evoked by serotonin and dopamine in the same neuron: A biochemical and physiological single-cell study.Proc. Natl. Acad. Sci. USA 797934–7938.

    Google Scholar 

  • Earp, S., and Steiner, A. (1978). Compartmentalization of cyclic nucleotide-mediated hormone action.Annu. Rev. Pharmacol. Toxicol. 18431–459.

    Google Scholar 

  • Eppler, C., Palazzolo, M., and Schwartz, J. (1982). Characterization and localization of adenosine 3′:5′-monophosphate-binding proteins in the nervous system ofAplysia.J. Neurosci. 21692–1702.

    Google Scholar 

  • Farmer, R., Harrington, C., and Brown, D. (1975). A simple radioimmunoassay for 3′,5′, cyclic adenosine monophosphate.Anal. Biochem. 64455–460.

    Google Scholar 

  • Fesenko, E., Kolesnikov, S., and Lyubarsky, A. (1985). Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment.Nature 313310–313.

    Google Scholar 

  • Frazier, W. T., Kandel, E. R., Kupfermann, I., Waziri, R., and Coggeshall, R. E. (1967). Morphological and functional properties of identified neurones in the abdominal ganglion ofAplysia.J. Neurophysiol. 301288–1351.

    Google Scholar 

  • Gelperin, A. (1986). Complex associative learning in small neural networks.Trends Neurosci. 9323–328.

    Google Scholar 

  • Giller, E., and Schwartz, J. H. (1971). Acetylcholinesterase in identified neurons of abdominal ganglion ofAplysia californica.J. Neurophysiol. 34108–115.

    Google Scholar 

  • Green, D., and Gillette, R. (1983). Patch- and voltage-clamp analysis of cyclic AMP-stimulated inward current underlying neurone bursting.Nature 306784–785.

    Google Scholar 

  • Harper, J., and Brooker, G. (1975). Femtomole-sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2′,0-acetylation by acetic anhydride in aqueous solution.J. Cyclic Nucl. Res. 1207–218.

    Google Scholar 

  • Hayes, S., Brunton, L., and Mayer, S. (1980). Selective activation of particulate cAMP-dependent protein kinase by isoproterenol and prostaglandin E1.J. Biol. Chem. 2555113–5119.

    Google Scholar 

  • Hayes, J., and Brunton, L. (1982). Functional compartments in cyclic nucleotide action.J. Cyclic Nucl. Res. 81–16.

    Google Scholar 

  • Haynes, L., and Yau, K. (1985). Cyclic GMP-sensitive conductance in outer segment membrane of catfish cones.Nature 31761–64.

    Google Scholar 

  • Hockberger, P., and Connor, J. A. (1983). Intracellular calcium measurements with arsenazo III during cyclic AMP injections into molluscan neurons.Science 219869–871.

    Google Scholar 

  • Hockberger, P., and Connor, J. A. (1984a). Alternation of calcium conductances and outward current by cyclic adenosine monophosphate (cAMP) in neurons ofLimax maximus.Cell. Mol. Neurobiol. 4319–338.

    Google Scholar 

  • Hockberger, P., and Connor, J. A. (1984b). Dose-dependent effects of intracellular cyclic AMP on nerve membrane conductances and internal pH. InPrimary Neural Substrates of Learning and Behavioral Change (Farley, J., and Alkon, D., Eds.), Cambridge University Press, New York, pp. 337–358.

    Google Scholar 

  • Kaczmarek, L. K., and Strumwasser, F. (1981). The expression of long lasting afterdischarge by isolatedAplysia bag cell neurons.J. Neurosci. 1626–634.

    Google Scholar 

  • Kandel., E., and Schwartz, J. (1982). Molecular biology of learning: Modulation of transmitter release.Science 218433–443.

    Google Scholar 

  • Kauer, J., and Kaczmarek, L. (1985). Peptidergic neurons ofAplysia lose their response to cyclic adenosine 3′:5′-monophosphate during a prolonged refractory period.J. Neurosci. 51339–1345.

    Google Scholar 

  • Kirk, M., and Scheller, R. (1986). Egg-laying hormone ofAplysia induces a voltage-dependent slow inward current carried by Na+ in an identified motoneuron.Proc. Natl. Acad. Sci. USA 833017–3021.

    Google Scholar 

  • Kirk, M., Thompson, S., and Scheller, R. (1984). A voltage clamp analysis of egg-laying hormone and serotonin effects on the identified motoneuron B16 ofAplysia.Soc. Neurosci. Abstr. 11641.

    Google Scholar 

  • Kononenko, N. (1981). Ionic mechanism of the transmembrane current evoked by injection of cyclic AMP into identifiedHelix pomatia neurons.Neurophysiology (USSR)12339–343.

    Google Scholar 

  • Kononenko, N., Kostyuk, P., and Shcherbatko, A. (1983). The effect of intracellular cAMP injections on stationary membrane conductance and voltage- and time-dependent ionic currents in identified snail neurons.Brain Res. 268321–338.

    Google Scholar 

  • Levitan, I. (1978). Adenylate cyclase in isolatedHelix andAplysia neuronal cell bodies: Stimulation by serotonin and peptide-containing extract.Brain Res. 154404–408.

    Google Scholar 

  • Levitan, I. B., and Norman, J. (1980). Differential effects of cAMP and cGMP derivatives on the activity of an identified neuron: Biochemical and electrophysiological analysis.Brain Res. 187415–429.

    Google Scholar 

  • Levitan, I., Begstroem, E., and Simonet, M. (1978). Adenylate cyclase inHelix andAplysia ganglia: Characteristics of its stimulation by a peptide-containing nervous system extract.J. Neurochem. 311353–1359.

    Google Scholar 

  • Levitan, I., Lemos, J., and Novak-Hofer, I. (1983). Protein phosphorylation and the regulation of ion channels.Trends Neurosci. 6496–499.

    Google Scholar 

  • Lowry, O., Rosebrough, N., Farr, A., and Randall, R. (1951). Protein measurement with the folin phenol reagent.J. Biol. Chem. 193265–275.

    Google Scholar 

  • Macknight, A., and Leaf, A. (1977). Regulation of cellular volume.Physiol. Rev. 57510–573.

    Google Scholar 

  • Miller, W. H., and Nicol, G. (1979). Evidence that cyclic GMP regulates membrane potential in rod photoreceptors.Nature 28064–66.

    Google Scholar 

  • Nicol, G., and Miller, W. (1978). Cyclic GMP injected into retinal rod outer segments increases latency and amplitude of response to illumination.Proc. Natl. Acad. Sci. USA 755217–5220.

    Google Scholar 

  • Ocorr, K., and Byrne, J. (1984). Characterization of phosphodiesterase fromAplysia pleural ganglia.Soc. Neurosci. Abstr. 10897.

    Google Scholar 

  • Ocorr, K., Walters, E., and Byrne, J. (1985). Associative conditioning analog selectively increases cAMP levels of tail sensory neurons inAplysia.Proc. Natl. Acad. Sci. USA 822548–2552.

    Google Scholar 

  • Ono, J., and McCaman, R. (1980). Identification of additional histaminergic neurons inAplysia: Improvements of single cell isolation techniques forin tandem physiological and chemical studies.Neuroscience 5835–840.

    Google Scholar 

  • Ono, J., and McCaman, R. (1984). Immunocytochemical localization and direct assays of serotonin-containing neurons inAplysia.Neuroscience 11549–560.

    Google Scholar 

  • Owen, G., and Torre, V. (1981). Ionic studies of vertebrate rods.Curr. Topics Membrane Transp. 1533–57.

    Google Scholar 

  • Paine, P., Moore, L., and Horowitz, S. (1975). Nuclear envelope permeability.Nature 254109–114.

    Google Scholar 

  • Pease, D. (1966). Preservation of unfixed cytological detail by dehydration with “inert” agents.J. Ultrastruct. Res. 14356–378.

    Google Scholar 

  • Pellmar, T. (1981). Ionic mechanism of a voltage-dependent current elicited by cyclic AMP.Cell. Mol. Neurobiol. 187–97.

    Google Scholar 

  • Pinto, L., and Brown, J. (1984). Pressure injection of 3′,5′-cyclic GMP into solitary rod photoreceptors of the tiger salamander.Brain Res. 304197–200.

    Google Scholar 

  • Schaffer, W., and Weissmann, C. (1973). A rapid, sensitive, and specific method for the determination of protein in dilute solution.Anal. Biochem. 56502–514.

    Google Scholar 

  • Shannon, J., and Macy, M. (1973). Freezing, storage, and recovery of cell stocks. InTissue Culture: Methods and Application (Kruse, P., and Patterson, M., Eds.), Academic Press, New York, pp. 712–718.

    Google Scholar 

  • Siegelbaum, S., Camardo, J., and Kandel, E. (1982). Serotonin and cyclic AMP close single K+ channels inAplysia sensory neurones.Nature 299413–417.

    Google Scholar 

  • Stein, C., and Weinreich, D. (1984). Determination of glutathione and ATP in ganglia and individual neurons ofAplysia californica.J. Neurochem. 421170–1174.

    Google Scholar 

  • Steiner, A., Parker, C., and Kipnis, D. (1972). Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides.J. Biol. Chem. 2471106–1113.

    Google Scholar 

  • Swandulla, D., and Lux, H. D. (1984). Changes in ionic conductances induced by cAMP inHelix neurons.Brain Res. 305115–122.

    Google Scholar 

  • Szent-Gyorgyi, A. (1951).Chemistry of Muscle Contraction, Academic Press, New York, pp. 144–146.

    Google Scholar 

  • Terasaki, W., and Brooker, G. (1977). Cardiac adenosine 3′:5′-monophosphate free and bound forms in the isolated rat atrium.J. Biol. Chem. 2521041–1050.

    Google Scholar 

  • Treistman, S. (1981). Effect of adenosine 3′,5′-monophosphate on neuronal pacemaker activity; a voltage clamp analysis.Science 21159–61.

    Google Scholar 

  • Treistman, S., and Levitan, I. (1976). Alteration of electrical activity in molluscan neurones by cyclic nucleotides and peptide factors.Nature 26162–64.

    Google Scholar 

  • Turchini, J., and Malet, P. (1965). Long conservation of histoenzymatic activities of fresh tissues in glycerol.J. Histochem. Cytochem. 13405–406.

    Google Scholar 

  • Udenfriend, S., Böhlen, Dairman, W., Leimgruber, W., and Weigele, M. (1972). Fluorescamine: A reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range.Science 178871–872.

    Google Scholar 

  • Woodruff, M., and Bownds, M. (1979). Amplitude, kinetics, and reversibility of a light-induced decrease in guanosine 3′,5′-cyclic monophosphate in frog photoreceptor membranes.J. Gen. Physiol. 73629–653.

    Google Scholar 

  • Woodruff, M., Bownds, D., Green, S., Morrisey, J., and Shedlovsky, A. (1977). Guanosine 3′,5′-cyclic monophosphate and the in vitro physiology of frog photoreceptor membranes.J. Gen. Physiol. 69667–679.

    Google Scholar 

  • Yau, K., McNaughton, P., and Hodgkin, A. (1981). Effect of ions on the light-sensitive current in retinal rods.Nature 292502–505.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hockberger, P., Yamane, T. Compartmentalization of cyclic AMP elevation in neurons ofAplysia californica . Cell Mol Neurobiol 7, 19–33 (1987). https://doi.org/10.1007/BF00734987

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00734987

Key words

Navigation