Aug 20, 1986 - neuropeptides SCPA and SCPB (small cardioactive peptides A and B). Neurons B1 ... cells synthesized the SCPs from radiolabeled precursors and transported the ..... Abrams, T. W., Castellucci, V. F., Camardo, J. S., Kandel,.
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 9794-9798, December 1986 Neurobiology
Release of neuropeptides during intracellular stimulation of single identified Aplysia neurons in culture (small cardioactive peptide/neurotransmitter)
PHILIP E. LLOYD*, SAMUEL SCHACHERt, IRVING KUPFERMANNI, AND KLAUDIUSZ R. WEISSt *Department of Pharmacological and Physiological Sciences, University of Chicago, 947 East 58th Street, Chicago, IL 60637; and tDepartment of Anatomy and Cell Biology and tDepartment of Psychiatry and Physiology, Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons and New York State Psychiatric Institute, 722 West 168th Street, New York, NY 10032
Communicated by Eric R. Kandel, August 20, 1986
Because transmitters, especially peptides, are released in small quantities, the demonstration of neurotransmitter release is often facilitated by using radiolabels. Labeling of traditional transmitters is further facilitated by the fact that their precursors are rapidly taken up at synaptic terminals and used locally to synthesize the transmitters. This is not the case for neuropeptides, since they are synthesized in neuronal somata and shipped to synaptic terminals. This transport can take a considerable length of time, especially when release sites are distant from the somata. The large and identifiable SCP-containing neurons in Aplysia have distant peripheral targets (7). We therefore took advantage of recent progress in the ability to culture identified Aplysia neurons to demonstrate the release of the SCPs. The two largest SCPcontaining neurons in Aplysia (buccal neurons B1 and B2) were used for these experiments. We found that both SCPA and SCPB were released during intracellular stimulation of individual identified B1 and B2 neurons in culture. This release was dependent on calcium concentrations in the medium. Thus, the SCPs satisfy a critical criterion required of a chemical mediator. An additional interesting aspect of this release was that it occurred in the absence of postsynaptic targets.
An important criterion for classifying a subABSTRACT stance as a neurotransmitter is that it is released in an activity-dependent fashion. We have utilized cell culture of individual neurons of Aplysia to demonstrate the release of the neuropeptides SCPA and SCPB (small cardioactive peptides A and B). Neurons B1 and B2 were isolated from the buccal ganglion of Aplysia and maintained in cell culture. The cells grew new processes, which were immunoreactive to antibodies for the neuropeptide SCPB. These processes contained SCPA and SCPB that were detectable by bioassay on snail heart. The cells synthesized the SCPs from radiolabeled precursors and transported the peptides to their neurites. Single cells released SCPs in a calcium-dependent fashion upon intracellular electrical stimulation. Taken together, these results provide critical evidence that SCPs are neurotransmitters. The results also indicate that the cell culture of individual identified neurons can be used to investigate the release of peptides.
The small cardioactive peptides (SCPs) are a class of low molecular weight neuropeptides that have been shown to have potent modulatory actions both in the central nervous system and on peripheral tissues in Aplysia (1-4). To date, two SCPs have been purified and sequences: SCPA (Ala-ArgPro-Gly-Tyr-Leu-Ala-Phe-Pro-Arg-Met-NH2; ref. 5) and SCPB (Met-Asn-Tyr-Leu-Ala-Phe-Pro-Arg-Met-NH2; ref. 6). The carboxyl-terminal amide and seven amino acids are conserved between the two peptides. The two SCPs are present in the same neurons (7), an observation consistent with molecular genetic analyses which indicate that a precursor protein contains the sequences of both peptides (8). The SCPs have been shown to satisfy many of the criteria required of chemical mediators. These peptides are present in and synthesized by individual neurons (7) and are contained in membrane-bound dense core vesicles (9). Immunocytology indicates that the SCPs are not ubiquitously distributed within the nervous system but rather are confined to a select population of neurons. Immunoreactive fibers and varicosities are found at both central and peripheral sites where the SCPs have been shown to potently modulate the efficacy of synaptic transmission (1, 3). The SCPs, however, have not been shown to be released from neurons in a calciumdependent manner. Although there is not a consensus on the precise criteria that must be satisfied to prove that a substance is a neurotransmitter (see ref. 10 for a discussion of these criteria), one criterion that is generally considered to be crucial is that the substance is released during neural activity. An additional corollary of this criterion is that the release should be dependent on extracellular calcium concentrations.
METHODS Cell Culture Preparation and Bioassay. Buccal ganglia were dissected from small adult Aplysia (30-50 g), incubated briefly with proteolytic enzymes, and surgically desheathed. The cell bodies of neurons B1 and B2, identified by their large size and position within the ganglia, were removed along with a short length of their axons and placed in polylysine-coated dishes containing culture medium (see ref. 11 for details of culture conditions). The cells were maintained in culture for 3-6 days. To determine whether the newly grown neurites contained authentic SCPA and SCPB, cell bodies and neurites were independently dissected from the culture dish (12). Extracts were either directly bioassayed on isolated snail hearts (13) or subjected to reversed-phase high-performance liquid chromatography (RP-HPLC), using procedures previously developed to analyze bioactivity in single neurons (7). RP-HPLC was carried out on an analytical C8 column (Zorbax, DuPont) developed with a gradient from 75% H20/25% CH3CN to 50% H20/50% CH3CN in 25 min. All solutions contained 0.01 M trifluoracetic acid. Immunocytology. Immunocytology of cultured neurons was done using techniques previously described for whole mounts and sections of Aplysia tissue (7, 14). In brief, individual cells or groups of cells growing in culture were fixed for 3 hr with 4% paraformaldehyde in 0.1 M phosphate
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: RP-HPLC, reversed-phase high-performance liquid chromatography; SCP, small cardioactive peptide. 9794
Neurobiology: Lloyd et al. buffer (pH 7.4) containing 30% sucrose. The cells were washed in buffer containing 0.3% Triton X-100 and were dehydrated by a series of ethanol washes. Following rehydration, nonspecific binding sites were blocked by exposing the cells for 0.5 hr to a 1:25 dilution of normal goat serum. The cells were then exposed to a rabbit antiserum, raised to SCPB (8), diluted 1:1000 in buffer containing normal goat serum. After rinsing, the cells were exposed to a 1:25 dilution of anti-rabbit IgG rhodamine-labeled antibody in goat serum. Following a final wash, the cells were cleared with 70% (vol/vol) glycerol. They were viewed with a Leitz microscope equipped for epifluorescence and photographed with Ektachrome or Tri-X film. Staining was blocked by preincubation with either SCPB or SCPA, but not by Phe-MetArg-Phe-NH2 (FMRF-NH2), a molluscan neuropeptide with moderate sequence similarity to the SCPs. As an additional control, immunocytology was carried out on B1 or B2 cells that were cocultured with neurons from the abdominal ganglia that are peptidergic (RUQ neurons, R3-R13; see ref. 15) but do not contain the SCPs in vivo. Radiolabeling and Release of SCPs. Newly synthesized peptides were labeled by incubating the cultured neurons with [35S]methionine (-800 Ci/mmol, Amersham; 1 Ci = 37 GBq). Each culture dish contained a single neuron. The protocol consisted of incubating neurons that had grown in culture for 48 hr with two changes of radioactive methionine in Aplysia hemolymph for 72 hr. This was followed by a 24-hr chase with excess unlabeled methionine in culture medium. The cell body was then penetrated with a microelectrode. Since the cells in vivo typically do not spontaneously fire, only those neurons that did not show spontaneous activity when initially penetrated were used for release experiments. The culture dish was perfused at a rate of 1.5 ml/min (1 bath volume/min). The perfusate contained 10 AM SCPA and SCPB to act as a carrier for released radiolabeled peptides, and in most experiments 3 mM 2-mercaptoethanol was added to reduce oxidation of SCPB (see Results). The SCPs at this concentration were excitatory on B1 and B2 in culture. They increased input resistance and depolarized the neurons a few millivolts but did not induce action potentials in otherwise quiescent neurons. The culture dish was equilibrated for 10 min with a low-calcium solution. In some experiments, this solution was 0.5 mM CaCI2/165 mM MgCl2/10 mM KCl/220 mM NaCl; in other experiments, it was 0.1 mM CaCl2/55 mM MgCl2/10 mM KCl/460 mM NaCl. This equilibration was followed by six periods during which the perfusate was collected. For the initial three 10-min periods, the cells were maintained in low-calcium saline (periods 1, 2, and 3). In the second period, the neuron was stimulated by intracellular current pulses to fire at an overall rate of 2-3 spikes per sec (or roughly 1000-2000 spikes in the 10-min collection period). In the first and third periods, the neuron was hyperpolarized by 5-15 mV to ensure that it fired no action potentials. The culture dish was then equilibrated (10 min) to a medium containing normal divalent cation concentrations (10 mM Ca2+, 55 mM Mg2+) and the same series of three 10-min collection periods was repeated (periods 4, 5, and 6). That is, in periods 4 and 6, the neuron was hyperpolarized, whereas in period 5 the neuron was fired at 2-3 spikes per sec. Each perfusate was collected in an ice-cold tube, acidified with trifluoroacetic acid (final concentration, 0.01 M), and immediately passed through a reversed-phase cartridge (C18; Waters Associates), and the retained material was eluted with 66% CH3CN. This solution was filtered, dried, and run on RP-HPLC as for the bioassay, except that a gradient from 20% CH3CN to 50% CH3CN in 30 min was used. At the end of each experiment, the neurites were separated from the cell body, dissected free of the substrate, and extracted, and the extracts were analyzed by RP-HPLC. This permitted the
Proc. Natl. Acad. Sci. USA 83 (1986)
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amount of labeled peptides released during stimulation to be normalized to the content of the neurites.
RESULTS Bi and B2 in Culture Extend Neurites That Contain SCPs. Neurons B1 and B2 in tissue culture grew out a new field of neurites with the axon as the primary location of the growth (Figs. 1 and 2A). Within 48 hr the cells had extended many neurites that had varicosities along their length and ended in growth cones (Fig. 1C). Immunocytology of these neurons with an antiserum raised to SCPB but which also binds to SCPA (9) indicated that B1 and B2 cell bodies still contained the peptides after several days in culture. Other cultured peptidergic neurons which do not contain the SCPs in vivo did not show significant staining (Fig. 2B). The newly grown neurites of B1 and B2 were also immunoreactive (Fig. 2C),
FIG. 1. Dark-field photomicrography of neurite outgrowth from B1 neuron during the first 48 hr in culture. (A) Cell body with axon removed from ganglion. (B) Neuritic growth, primarily from the distal axon stump, after 24 hr. (C) Neuritic extension proceeds rapidly, so that by 48 hr, the cell has an extensive neuritic field. Varicosities (arrowheads) separated by narrow necks and growth cones (arrows) are present in large numbers. (Bar = 100 Am.)
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Proc. Natl. Acad. Sci. USA 83 (1986)
Neurobiology: Lloyd et al.
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RETENTION TIME FIG. 3. RP-HPLC of SCP-like bioactivity from extracts of the soma and neurites of a single cultured B1 neuron. SCP-like bioactivity was measured on an isolated snail heart and normalized to synthetic SCPB (the two SCPs are equipotent in this assay). Each sample was collected for 30 sec, and the arrows indicate the retention times of synthetic standards run after the extracts. See Methods for
details of RP-HPLC.
FIG. 2. Immunostaining of cultured B2 and RUQ neurons (soma and peripheral processes). (A) Light microscopic view of B2 and a control peptidergic cell (RUQ cell). (Bar = 50 Am.) (B) Fluorescence microscopic view of the same field as in A, showing immunostaining with an antibody directed to SCPB. Note that the B2 soma but not that of the RUQ neuron was stained. (Bar = 50 Mm.) (C) Higher magnification fluorescence view of SCP-like immunoreactivity in neurites and varicosities of B1. (Bar = 25 Am.)
most notably in structures that appear to be similar to the varicosities (Fig. 1C) observed in central ganglia and peripheral muscles (3, 7) and at growth cones (16). The presence of the SCPs in the newly grown neurites was established by bioassay of crude extracts from dissected neurites. B1 neurites contained 4.9 4.4 pmol (mean SD; 2.9 pmol (n = 3) of SCP-like n = 5) and B2 neurites 4.1 bioactivity. This compares with values of 9.9 2.8 pmol (n = 5) for B1 somata and 5.0 4.5 pmol (n 3) for B2 somata. RP-HPLC of selected extracts confirmed that this bioactivity was due to the presence of authentic SCPA and SCPB (Fig. 3). Labeling experiments demonstrated that at least a portion of the SCPs in the neurites was synthesized and transported into the neurites during the culture period (see below; Fig. 5). Intracellular Stimulation of Bi and B2 in the Presence of ±
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Ca2+ Causes Release of the SCPs. Three complete runs were carried out on B2 neurons and two complete runs on B1 neurons. Individual runs for each neuron are shown in Fig. 4. These results showed that the labeled peptides were released during stimulation in the presence of normal Ca2+ concentrations (period 5). This release is Ca2+-dependent, as no labeled peptides were recovered during stimulation in lowCa2` medium (period 2). The presence of a small amount of labeled peptides in the collection period (period 6) immediately following the stimulation period probably was due to material released near the end of the stimulation period that was not completely washed out of the culture dish. Labeled material with a very brief retention time on this mode of RP-HPLC (see Fig. 5) gradually washed out during the course of the experiment. The rate of this washout was unaffected by stimulation of the neurons in the absence of Ca2+. This material includes unincorporated methionine (5). The amount of labeled SCPA and SCPB recovered after a 10-min stimulation of B1 and B2 neurons in the presence of normal Ca2+ concentrations was 6.2 2.2% (mean SEM; n = 7) of the total labeled peptides remaining in the neurites. The results illustrated in Fig. 4 were similar to the results of the other complete runs and were also consistent with the results from several partial runs. Partial runs were the result of the loss of the intracellular penetrations, perhaps because of the high perfusion rates used in these experiments. To determine whether the two SCPs were synthesized, transported, and released at the same rate, the HPLC profile of radiolabeled material recovered in the perfusate during stimulation in normal Ca2l was compared with the profile of the labeled material in neurites (Fig. 5). The ratio of SCPB to SCPA in the neurites was close to 2 (1.91 0.05; mean + SEM; n = 6). This suggests that in cultured neurons the two peptides are likely to be synthesized and transported at the same rate, since SCPB contains two methionines and SCPA has a single methionine residue. The ratio for released peptides recovered in sea water containing 3 mM mercaptoethanol was less than the expected 2:1 ratio (1.51 0.04; mean SEM; n = 5). Although this raises the possibility that the peptides may be released at different rates, a more likely explanation is that the two peptides were differentially oxidized. We have observed that the amino-terminal methio±
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Neurobiology: Lloyd et al.
Proc. Natl. Acad. Sci. USA 83 (1986)
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FIG. 4. [35S]Methionine-labeled material recovered from perfusates during six 10-min collection periods. SCPs indicates the radioactivity with retention times identical to those of SCPA and SCPB (e.g., see Fig. 5). Remainder indicates the total radioactivity present in all the remaining HPLC samples, which includes unincorporated methionine. Horizontal bars indicate the periods during which perfusate contained normal Ca2+ levels (Ca) and periods during which the neurons were stimulated (STIM). The left pair of graphs represent an experiment on a cultured B1 neuron, and the right pair an experiment on B2. Collection periods: 1, low Ca2", no stimulation; 2, low Ca2 stimulation; 3, low Ca2 no stimulation; 4, normal Ca2 no stimulation; 5, normal Ca2 stimulation; 6, normal Ca2 no stimulation. Note that the SCPs were released during stimulation only in the presence of normal Ca2+ and that stimulation had no effect on the radioactivity in the remainder, which gradually washed out over the duration of the experiment. ,
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nine of SCPB appears to be considerably more prone to oxidation than the carboxyl-terminal methionine residues that the SCPs have in common. In the RP-HPLC system used in this study, the oxidized form of SCPB has the same retention time as SCPA. The occurrence of oxidation in these experiments was inferred from the changes in the apparent ratio of released SCPB to SCPA produced by adding the antioxidant, mercaptoethanol, to the perfusate. In the absence of mercaptoethanol, the ratio was 0.29 0.05 (mean SD; n = 2); with 1 mM mercaptoethanol, the ratio was 0.98 ±
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(n = 1); and with 3 mM mercaptoethanol, the ratio was 1.51 (see above). Perhaps, higher mercaptoethanol concentrations would have further reduced the oxidation, but pilot studies on a neuromuscular system in Aplysia indicated that the efficacy of synaptic transmission declined at mercaptoethanol concentrations .5 mM (K.R.W. and P.E.L., unpublished observations). In addition, the levels of oxidation were quite small, for example, an apparent ratio of SCPB to SCPA of 1.5 is produced by oxidation of only 10% of the SCPB, and a ratio of 1.9 by oxidation of
