Release of Pedal Peptide from Aplysia Neurons in Primary Culture Jon D. Hall and Philip E. Lloyd* Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637
SUMMARY Pedal peptide (Pep) is a modulatory neuropeptide that is predominantly synthesized in a group of neurons on the dorsal surfaces of the pedal ganglia of Aplysiu. Following the determination that Pep is the major peptide selectively present in these neurons in siiu, primary cell culture of single Pep-neurons was used to study the release of this neuropeptide. Individual Pep-neurons were grown in culture where they extended many branched neurites with large varicosities. Immunocytology revealed that
these newly grown varicosities were intensely Pep immunoreactive. Cultured Pep-neurons, grown in a medium containing radiolabeled methionine, synthesized labeled Pep and transported it into their regenerated neurites. Finally, these neurons released radiolabeled Pep in a calcium- and stimulation-dependent fashion. These results, taken together with previous findings, strongly support the proposition that Pep is a transmitter in Aplysiu.
INTRODUCTION
ized by immunocytology and high-pressure liquid chromotography-radioimmunoassay ( HPLCRIA) procedures to a number of neurons in other central ganglia and to varicose axons in many peripheral organs (Lloyd and Connolly, 1989; Pearson and Lloyd, 1989a,b, 1990; Hall and Lloyd, 1990). These studies have shown that Pep meets several of the criteria for classification of a substance as a neurotransmitter. A crucial criterion for the classification of a compound as a neurotransmitter is the release of the compound from a neuron in a stimulation-dependent fashion. Although we have previously demonstrated that Pep-like immunoreactivity could be found in perfusates collected from the foot following pedal nerve stimulation (Hall and Lloyd, 1990). these experiments had several limitations. First, we were unable to find agents that would effectively inhibit proteolysis of the peptide. Second, perhaps as a consequence of the proteolytic activity, insufficient immunoreactive material was recovered to confirm identity by HPLC. Finally, the calcium dependence of release could not be explored because the large size and morphological complexity of the foot precluded modification of extracellular calcium levels. To circumvent
Pedal peptide (Pep)was initially identified in Aplysia nervous tissue because of its highly selective synthesis in pedal ganglia (Lloyd and Connolly, 1989). Pep is synthesized by a discrete group of neurons (termed Pep-neurons) in pedal ganglia and is transported down pedal nerves to varicosities in foot muscle. When the activity of Pep-neurons is recorded in a semiintact animal preparation, they fire tonically at about 1 Hz when the animal is stationary. When the animal locomotes, the firing rate of these neurons increases to about 3 or 4 Hz and occurs in bursts that usually coincide with a phase of the pedal wave. Application of Pep to the foot has significant modulatory effects on foot muscle contractions. Thus. Pep appears to play a role in the regulation of locomotion in Aplysia. Pep also has central actions on specific neurons in the abdominal ganglia. Pep has been localReceived January 22, 199 1: accepted March 15, 1991 Journal of Neurobiology, Vol. 22, No. 6 , pp. 583-589 (1991) 8 1991 John Wiley & Sons, Inc. CCC 0022-303419I/O60583-07$04.00 * To whom correspondence should be addressed.
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these limitations, we took advantage of techniques used in a previous study in which neuropeptide release from single, identified Aplysia neurons was measured in primary cell culture (Lloyd, Schacher, Kupfermann, and Weiss, 1986). In the present study, we report that pedal peptide was the predominant peptide found selectively in Pep-neurons in situ. Individual Pep-neurons from pedal ganglia were grown in culture where they regenerated numerous branched neurites with large varicosities. When these neurons were grown in a medium containing [ 3 5 S ]-methionhe, they synthesized radiolabeled Pep and transported the newly synthesized peptide into their regenerated neurites. Finally, radiolabeled Pep was released from cultured neurons in a calcium- and stimulation-dependent fashion. METHODS
Animals Aplysia culiyornica (50-70 g) were obtained from Marinus, Inc.. maintained in circulating artificial sea water (ASW) tanks at 15"C, and fed dried seaweed at 3-day intcrvals. Animals were immobilized by isotonic MgCI, injection prior to dissection.
Peptide Synthesis by Individual Pep-Neurons in situ Pedal ganglia were pinned with the dorsal surface up in a conical Sylgard labeling dish containing 500 p1 low-Ca*+ ASW (0.5 m M Ca2+,110 m M Mg2+)and desheathed. The bath was then changed over to a labeling medium that consisted of 50% ASW-50% Aplysiu hemolymph containing 1 mM colchicine, 25 U / m L penicillin, 25 pg/ml streptomycin, and 0.1% 2-mercaptoethanol. [ 35S]-Methionine(Amersham) was added to a final concentration of 1 mCi/mL. and the ganglia was incubated at 16°C for 20 h. This was followed by a 4-h chase with ASW containing colchicine, penicillin, streptomycin, 2mercaptoethanol (concentrations as above), and 1 m M cold methionine. Pep-neurons were visually identified (Hall and Lloyd, 1990), and individual somata were manually dissected using the freeze substitution method (Ono and McCaman, 1980). The dissected somata were extracted at I00"C for 10 min in 50 pl0.02 M trifluoroacetic acid (TFA) containing 2 nmol each of synthetic Pep (Applied Biosystems). FMRFamide (Bachem), and SCP, (Peninsula), and filtered for analysis by reversephase high-pressure liquid chromatography (RPHPLC). All RP-HPLC was carried out on a Brownlee C-8, RP 300,4.6 X 220 mm column at a flow rate of 2 mL/min. The extracts from the somata were run through two different modes of RP-HPLC to confirm peptide identity. The first mode used 0.0 1 M TFA as a counterion and a
gradient from 5% CH,CN to 54% CH3CN in 20 min. Fractions werc split, and radioactivity in one aliquot was determined by liquid scintillation counting. Aliquots from fractions containing the radioactive peak were pooled, dried, redissolved in 350 p10.01 M heptafluorobutyric acid (HFBA) containing 2 nmol each of synthetic Pep, FMRFamide, and SCP,, and run on a second mode of RP-HPLC. The second mode utilized 0.01 M HFBA as a counterion and a gradient from 2 1% CH,CN to 24% CH,CN in 2 min. then to 28%CH,CN in 12 min, and lastly to 34% CH3CN in 4 min. Radioactivity in the resulting fractions was determined.
Peptide Content of Neurons in the Pedal Ganglia Neurons from eight pedal ganglia were pooled for these analyses. The dorsal surfaces of the ganglia were desheathed and the Pep-neurons (-40 per ganglion) were removed using the freeze substitution method and extracted in 150 pl0.02 M TFA at 100°C for 10 min. An equal number of neurons similar in size and just anterior to the Pep-neurons were dissected and extracted as controls. Extracts were pooled, filtered, and analyzed using a very broad gradient from 5% CH,CN to 63% CH,CN in 20 min with 0.01 M TFA as a counterion. The broad gradient was used to insure that nearly any peptide would be resolved. Finally, to insure that resolved peptides would be detected, absorption at 206 nm was monitored. At this wavelength, peptide bonds are strongly absorbant. so detection of peptides does not require the presence of aromatic amino acids (Woods and O'Bar, 1970).
Primary Cell Culture Dissociated cell culture of visually identified Pep-neurons from pedal ganglia was carried out as described by Schacher and Proshansky ( 1983), except that 16-mm diameter culture wells (Nunc) were used, and the neurons were maintained at 22°C after initial plating.
lmmunocytology Following 1-3 days in culture, immunocytology of Pepneurons was carried out using a modification of procedures previously described (Pearson and Lloyd, 1989a). The ASW in the culture wells containing neurons was replaced with a fixative solution of 4% paraformaldehyde-30% sucrose in 0.1 M phosphate buffer (PB, pH 7.4) and incubated for 4 h at 22°C. Following fixation, the wells were washed briefly with PB, then in several changes of PB with 1% Triton X-100 and 0.1% sodium azide overnight. They were then incubated for 6 h in a blocking solution that consisted of phosphate-buffered saline (PBS: 0. I4 M NaCl, 0.0 1 M phosphate, pH 7.4), 1% Triton X-100, 0 . l Y ~sodium azide, 1% normal goat serum (Tago) , and 4% skim milk. Pep-neurons were in-
Aplysia Neiiropeptide Release in Culture cubated in primary antiserum (PAS-2) (Pearson and Lloyd, 1989a), diluted 1:500 in blocking solution for 12-16 h. The primary antiserum was washed out with several changes of blocking solution over a 4-h period. This was followed by a 4-h incubation in secondary antiserum, goat anti-rabbit conjugated to fluorescein isothiocyanate (Tago), and diluted 1:2@@in blocking solution. The secondary antiserum was washed out with several changes of PBS, and the preparation was coverslipped and viewed on a Leitz Orthoplan fluorescence microscope.
Incubation of Cultured Pep-Neurons with [ 35S]-Methionine Cultured Pep-neurons to be used in experiments utilizing [ 35S]-methionine were initially plated in a medium consisting of 50% Aplysia hemolymph and 50% methionine-free L 15 containing penicillin-streptomycin-fungizone (PSF) (Gibco) to a final concentration of 1YO. After 1-2 days in culture, [ '5S]-methionine was added to the well to yield a final specific activity of 0.5 mCi/mL. Following 18-20 h in label, the neurons were chased with 30 mL L15 plus I % PSF over 1 h. To determine if the neurons continued to synthesize and transport Pep into their regenerated neurites, the somata and the neurites were separately dissected (Lloyd et al., 1986), extracted, and run through two modes of RP-HPLC to confirm peptide identity asdescribed above. The radioactivity in the resulting fractions was determined by liquid scintillation counting.
Release of [ 35S]-Methionhe-Labeled Pep from Cultured Pep-Neurons For all release experiments only a single neuron was plated per culture well. Release experiments were conducted immediately following the chase period and consisted of three collection periods conducted consecutively. Superfusion of the well was regulated by a peristaltic pump operating at 1 m l l m i n ; this corresponded to two bath volumes per minute. In the first collection period, the culture well containing the neuron was superfused with low-Ca2+ASW for 15-30 min. The neuron was then impaled with an intracellular electrode ( R = 510 MQ) and stimulated to fire five to 10 spikes for I s every 3 s for 10 min. Following the 10-min stimulation period, the neuron was hyperpolarized for 5 min. The superfusate from the entire 15-min period was collected on ice and acidified to 0.0 1 MTFA. The bathing solution was then changed over to normal ASW and allowed to equilibrate for 15-30 min. Following equilibration, the neuron was hyperpolarized to prevent any action potentials from firing for 15 min, and the superfusate from this 15-min period was collected as described above. The neuron was then stimulated to fire as before for 10 min and hyperpolarized for 5 min: the superfusate from this 15min period was collected. All superfused ASW contained 1 mM2-mercaptoethanol and 100 nMsynthetic Pep. A t
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this concentration, synthetic Pep had no discernable effect on the resting membrane potential of cultured Pepneurons. The samples from the three collection periods were individually passed through a C- 18 reverse-phase extraction cartridge (Sep-Pak), and the peptides were eluted with 3 mL 75% CH3CKand 0.0 1 M : TFA. This was then lyophilized and redissolved in 350 ~ 1 0 .10M TFA immediately prior to RP-HPLC analysis using a gradient from 18% CH,CN to 34% CH,CN in 6 rnin with 0.01 M TFA as a counterion. The somata and neurites of the neurons from these experiments were dissected out separately, extracted at 100°C for 10 min in 50 ~1 0.02 M TFA containing 2 nmol synthetic Pep, and run through the samc RP-HPLC gradient as the release samples. Radioactivity in the resulting fractions was then determined.
RESULTS Pedal Peptide is the Major Peptide Found Selectively in Pep-Neurons in situ
Before carrying out release experiments, we first wanted to examine if Pep was indeed the predominant peptide selectively present in Pep-neurons in pedal ganglia. We investigated this problem in two ways. In the first method, pedal ganglia were labeled with [ 35S]-methionine, and individual Pepneuronal somata were dissected, extracted, and analyzed by two sequential modes of RP-HPLC. Significant incorporation of label was found primarily in the fractions that coeluted with synthetic Pep in both modes (Fig. 1 ) .Two smaller peaks of incorporation were also consistently observed. One of these peaks coeluted with a known break-down fragment of Pep, while the other is unidentified. Thus, Pep is the major methionine-containing peptide synthesized by these neurons. A second method was used to determine if Pepneurons selectively synthesized other peptides that were not detected using [ 35S]-methionine labeling. In this experiment, absorbance detection at low wavelength was used to compare the contents of an extract of pooled Pep-neurons with that of an extract of a similar number of other pedal neurons that are not Pep immunoreactive. Comparison of the absorbance profiles revealed that the major difference was the single large peak of Pep present selectively in the Pep-neuron profile (Fig. 2). A second smaller difference appears to be associated with the Pep fragment (Pearson and Lloyd, 1990). All other absorbance peaks are shared by the two groups of neurons. Therefore. Pep is the predominant peptide selectively present in Pep-neurons in situ. We cannot, however, exclude the possibility
Hall and f.foyd
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TFA
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/
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"
-
c ,
Iep
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extended numerous, highly branched neurites. Neurites regenerated from the initial axon segment and, to a much lesser extent, from the cell soma. These neurites contained many large varicosities and often terminated in growth cones. Immunocytology with an antiserum directed towards Pep revealed that the somata of these neurons maintained Pep-like immunoreactivity after several days in culture [Fig. 3 (A)]. Particularly intense staining was associated with the regenerated varicosities [Fig. 3 (B)] .Other peptidergic/ip/jLYianeurons cocultured with the Pep-neurons did not express Pep-like immunoreactivity (data not shown). To examine if Pep-neurons in culture also continued to synthesize Pep, we labeled individually grown neurons with [ 35S]-methionine.Two consecutive modes of RP-HPLC of extracts from somata and neurites of these neurons showed that authentic Pep was synthesized by the cell bodies (data not shown) and transported to regenerated neurites (Fig. 4 ) . Thus, as is the case for Pep-neurons in situ, cultured Pep-neurons continue to synthesize Pep as their major methionine-containing peptide, and these neurons also retain the ability to transport the peptide. The culture process, there-
36
HPLC Fraction
Figure 1 Pedal peptide is the predominant methioninecontaining peptide synthesized by Pep-neurons in pedal ganglia. Pedal ganglia were desheathed and incubated in [ "S]-methionine for 20 h and chased for 4 h. Individual somata were dissected for analysis by two consecutive modes of RP-HPLC, first using TFA as a counterion (top). Aliquots of fractions corresponding to Pep were pooled and analyzed in a second mode of RP-HPLC using HFBA as a counterion (bottom). Fractions where synthetic Pep was eluted are indicated (retention times: 10 min in TFA, and 9 min in HFBA). The small peaks marked with an asterisk rcpresent a fragment of Pep.
that some of the absorbance peaks common to the two profiles may represent transmitters present in both groups of neurons. Also, it is possible that some extremely hydrophilic peptides may not have been retained on the column and thus would not be detected by this method. Pedal Peptide is Synthesized by PepNeurons in Culture and Transported into Regenerated Neurites
Within 24-48 h of dissociation, Pep-neurons attached to the poly-L-lysine-coated substrate and
0.1
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Figure 2 Pep is the predominant peptide present selectively in Pep-neurons. Pep-neurons (top trace) and control pedal neurons (bottom trace) were dissected from pedal ganglia and run on RP-HPLC. Each trace represents the absorbance profile from approximately 250 pooled neurons. The major differences between the profiles are the Pep peak and a smaller peak (asterisk) with the same retention timc as a Pep fragment. The large peaks that elute approximately 2 min prior to Pep are common to all Aply.~zanervous tissue. An additional 2.5 min of the two profiles after 20 min were nearly identical and were not shown.
Aplysia Neuropeptide Releare in Culture 24
fore, did not initiate the expression of other methionine-containing neuropeptides in these neurons. Pep is Released in a Ca2+- and Stimulation-Dependent Fashion Release experiments were carried out on neurons that were cultured individually in wells. Neurons were incubated in [ 35S]-methionhe to label Pep as described in the previous section. These expenments were divided into three collection periods. During the first period, the neurons were bathed in low-Ca2+ASW and stimulated via an intracellular electrode to examine the Ca2+-dependence of release. For the second collection period, the super-
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Figure 4 Radiolabeled Pep synthesized in the soma of cultured neurons is transported to the regenerated neurites. (Top) Extract from the neurites of a single cultured Pep-neuron grown in [ "S] -methionhe-containing media was run on RP-HPLC with TFA as a counterion. (Bottom) Aliquots of the fractions that coeluted with synthetic Pep were pooled and run on RP-HPLC with IIFBA as a counterion. Gradients and retention times are identical to those in Figure 1.
Figure 3 Cultured Pep-neurons exhibit Pep-like immunoreactivity. ( A ) Fluorescence micrograph of a Pepneuron that was grown for 48 h and subjected to immunocytology with a primary antiserum directed towards Pep and a fluorescein-conjugated secondary antiserum. ( B ) Regenerated neurites contain large varicosities that show intense Pep-like immunoreactivity. Scale bar = 200 pm ( A ) and 20 pm (B) .
fusate was changed to normal ASW, and the neuron was hyperpolarized. In the final collection penod, the superfusate was normal ASW, and the neuron was stimulated to fire as in the low-Ca2+ portion of the experiment. Peptides present in the superfusates from each collection period were extracted, run on RP-HPLC ( Fig. 5 ), and the radioactivity that eluted was determined. There were little or no counts above background in the Pep fractions from the first two collection periods. However in the third period of the experiment there is a marked increase in the counts in the Pep and Pep fragment fractions (Fig. 5 ). A summary of the release experiments is shown in Figure 6. This shows that Pep is released in a Ca2+-and stimulation-de-
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pendent fashion. The amount of labeled Pep released into the superfusate during stimulation in normal ASW represents 4.0 1.9% (S.E.M., n = 5 ) of the total labeled Pep extracted from neurites at the end of each experiment.
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HPLC Fraction Figure 5 RP-HPLC profiles of ['%I -methionine-labeled material recovered from the superfusate from each period of a typical experiment. Single Pep-neurons were labeled in [ 35S]-methionine-containing medium for IS20 h and chased for 1 h. Experiments consisted of three collection periods; first, the culture medium was washed out with low-Ca**ASW, and the neuron was stimulated (5-10 Hz for I s every 3 s) for 10 min, then hyperpolarized for 5 min, and the superfusate was collected. Second, the bath was changed to normal ASW and hyperpolarized, and the superfusate collected for 15 min. Finally, the neuron was stimulated in normal ASW with the same paradigm as described above, and the superfusate was collected. All superfusates were run on RPHPLC and the fractions were counted. The gradient used was different from that used in Figures 1 and 4. The fractions where synthetic Pep and the Pep fragment
DISCUSSION Previously, it has been shown that Pep meets a number of criteria for the designation of a substance as a neurotransmitter. It is synthesized selectively by individual neurons, predominantly by a cluster of neurons on the dorsal surface of each pedal ganglion. It is transported down pedal nerves to the foot where it has a significant modulatory effect on foot muscle contraction. It also has central effects on specific neurons in the abdominal ganglia (Lloyd and Connolly, 1989; Pearson and Lloyd, 1989a,b; Hall and Lloyd, 1990). In this study, we add to the evidence supporting the role of Pep as a neurotransmitter in Aplysiu. We have shown that Pep is indeed the major peptide selectively found in these neurons. Also, Pep-neurons in culture continue to synthesize and transport Pep as do their in situ counterparts. Finally, Pep is released when single neurons in culture were stimulated intracellularly in the presence of normal calcium levels. Significant release of Pep was not observed in response to stimulation in low levels of calcium or when the neuron was hyperpolarized in normal levels of calcium. The amount of peptide release seen in these studies is similar to that observed in a similar study examining release of the small cardioactive peptides ( SCPs) from cultured identified buccal neurons B1 and B2 (Lloyd et al., 1986). In that study, release of about 6% of the amount of SCPs in the neurites of the identified buccal neurons was observed over periods similar to those used in this study. Here, we report about 4%of the Pep in neurites was released. These release experiments were facilitated by placing individual neurons in primary cell culture. Our data, taken together with previous findings (Lloyd et al., 1986) suggest that the fidelity of neuropeptide synthesis and transport by Aplysia neurons is maintained in culture. Neurons Bl and B2 continue to synthesize predominantly SCP, and SCP, in culture as they do in situ. Furthermore, the
eluted are indicated. The large, truncated peak with a short retention time primarily represents unincorporated methionine.
Aplysia Neuropeptide Release in Culture 1oc
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tide is released from Pep-neurons in a calcium- and stimulation-dependent fashion. These results provide strong support for the proposition that Pep functions as a neurotransmitter. The present results and those of a previous study suggest that these techniques may prove to be broadly useful in identifying peptide transmitters in identified neurons in Aplysia. We thank W. L. Pearson and M. D. Whim for critical reading of the manuscript. This work was supported by NIH Grant NS-23569 and a Brain Research Foundation grant. J.D.H. is supported by Pharmacological Sciences Training Grant T32GM07 15 1.
50
REFERENCES
7-
0
i
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ASW no stim
ASW
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Figure 6 ’ep is released from single cultured neurons in a stimi tion- and CaZ+-deDendentfashion. The total amount of radioactivity in the Pep fraction and the Pep fragment fraction was determined for each period of five experiments. Values are mean t S.E.M.
two peptides are synthesized in the same ratio as observed in situ. Similarly, Pep is the predominant methionine-labeled peptide synthesized by pedal ganglia Pep-neurons in situ and in culture. These results suggest that this may be a useful procedure for determining the nature of peptides released from other peptidergic neurons in Aplysia. A promising application for this technique might include the very large neurons in the abdominal ganglia of Aplysia for which the sequences of the mRNA that encode peptide precursors have been determined, but the nature of the mature released neuropeptide has not (Taussig, Kaldany, and Scheller, 1984; Shyamala, Fisher, and Scheller, 1986). In this study, we have utilized the technique of individual neuron culture to show that pedal pep-
HALL,J. D. and LLOYD,P. E. (1990). Involvement of pedal peptide in locomotion in Aplysia: modulation of foot muscle contractions. J. Neurobiol. 21:858-868. I., and LLOYD,P. E., SCHACHER,S., KUPFERMANN, WEISS,K. R. ( 1986). Release of neuropeptides during intracellular stimulation of single identified Aplysia neurons in culture. Proc. Nutl. Acad. Sci. USA 83~9794-9798. LLOYD,P. E. and CONNOLLY, C. M. (1989). Sequence of pedal peptide: a novel neuropeptide from the central nervous system of Aplysiu. J. Neurosci. 9:3 12317. ONO,J. K. and MCCAMAN, R. E. ( 1980). Identification of additional histaminergic neurons in Aplysia: improvement of single cell isolation techniques for in randern physiological and chemical studies. ,’c’euro.Ycience 5:8 35-840. PEARSON, W. L. and LLOYD,P. E. ( 1989a). lmmunocytological localization of pedal peptide in the central nervous system of Aplysia. J . Neurosci. 9:3 18-325. PEARSON, W. L. and LLOYD,P. E. ( 1989b). Actions of pedal peptide on neuron L5 in -4plysia. SOC. Neurosci. Abstr. 15736. W. L. and LLOYD,P. E. ( 1990). Distribution PEARSON, and characterization of pedal peptide immunoreactivity in Aplysia. J. Neurobiol. 21:883-892. SCHACHER, S. and PROSHANSKY, E. ( 1983). Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by .4plysia hemolymph and the presence of the initial axon segment. J. Neurosci. 3:24032413. M., FISHER,J. M., and SCHELLER,K. H. SHYAMALA, ( 1986). A neuropeptide precursor expressed in Ap1.vsiu neuron L5. DNA 5:203-208. TAUSSIG, R., KALDANY, K. -R., and SCHELLER,K. H. (1984). A cDNA clone encoding neuropeptides isolated from Aplysia neuron L 1 I . Proc. Natl. Acad. Sci. USA 81:4988-4992. WOODS,A. H. and O’BAR,P. R. (1970). Absorption of proteins and peptides in the far ultraviolet. Science 167: 179-1 81.
SUMMARY Pedal peptide (Pep) is a modulatory neuropeptide that is predominantly synthesized in a group of neurons on the dorsal surfaces of the pedal ganglia of Aplysiu. Following the determination that Pep is the major peptide selectively present in these neurons in siiu, primary cell culture of single Pep-neurons was used to study the release of this neuropeptide. Individual Pep-neurons were grown in culture where they extended many branched neurites with large varicosities. Immunocytology revealed that
these newly grown varicosities were intensely Pep immunoreactive. Cultured Pep-neurons, grown in a medium containing radiolabeled methionine, synthesized labeled Pep and transported it into their regenerated neurites. Finally, these neurons released radiolabeled Pep in a calcium- and stimulation-dependent fashion. These results, taken together with previous findings, strongly support the proposition that Pep is a transmitter in Aplysiu.
INTRODUCTION
ized by immunocytology and high-pressure liquid chromotography-radioimmunoassay ( HPLCRIA) procedures to a number of neurons in other central ganglia and to varicose axons in many peripheral organs (Lloyd and Connolly, 1989; Pearson and Lloyd, 1989a,b, 1990; Hall and Lloyd, 1990). These studies have shown that Pep meets several of the criteria for classification of a substance as a neurotransmitter. A crucial criterion for the classification of a compound as a neurotransmitter is the release of the compound from a neuron in a stimulation-dependent fashion. Although we have previously demonstrated that Pep-like immunoreactivity could be found in perfusates collected from the foot following pedal nerve stimulation (Hall and Lloyd, 1990). these experiments had several limitations. First, we were unable to find agents that would effectively inhibit proteolysis of the peptide. Second, perhaps as a consequence of the proteolytic activity, insufficient immunoreactive material was recovered to confirm identity by HPLC. Finally, the calcium dependence of release could not be explored because the large size and morphological complexity of the foot precluded modification of extracellular calcium levels. To circumvent
Pedal peptide (Pep)was initially identified in Aplysia nervous tissue because of its highly selective synthesis in pedal ganglia (Lloyd and Connolly, 1989). Pep is synthesized by a discrete group of neurons (termed Pep-neurons) in pedal ganglia and is transported down pedal nerves to varicosities in foot muscle. When the activity of Pep-neurons is recorded in a semiintact animal preparation, they fire tonically at about 1 Hz when the animal is stationary. When the animal locomotes, the firing rate of these neurons increases to about 3 or 4 Hz and occurs in bursts that usually coincide with a phase of the pedal wave. Application of Pep to the foot has significant modulatory effects on foot muscle contractions. Thus. Pep appears to play a role in the regulation of locomotion in Aplysia. Pep also has central actions on specific neurons in the abdominal ganglia. Pep has been localReceived January 22, 199 1: accepted March 15, 1991 Journal of Neurobiology, Vol. 22, No. 6 , pp. 583-589 (1991) 8 1991 John Wiley & Sons, Inc. CCC 0022-303419I/O60583-07$04.00 * To whom correspondence should be addressed.
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these limitations, we took advantage of techniques used in a previous study in which neuropeptide release from single, identified Aplysia neurons was measured in primary cell culture (Lloyd, Schacher, Kupfermann, and Weiss, 1986). In the present study, we report that pedal peptide was the predominant peptide found selectively in Pep-neurons in situ. Individual Pep-neurons from pedal ganglia were grown in culture where they regenerated numerous branched neurites with large varicosities. When these neurons were grown in a medium containing [ 3 5 S ]-methionhe, they synthesized radiolabeled Pep and transported the newly synthesized peptide into their regenerated neurites. Finally, radiolabeled Pep was released from cultured neurons in a calcium- and stimulation-dependent fashion. METHODS
Animals Aplysia culiyornica (50-70 g) were obtained from Marinus, Inc.. maintained in circulating artificial sea water (ASW) tanks at 15"C, and fed dried seaweed at 3-day intcrvals. Animals were immobilized by isotonic MgCI, injection prior to dissection.
Peptide Synthesis by Individual Pep-Neurons in situ Pedal ganglia were pinned with the dorsal surface up in a conical Sylgard labeling dish containing 500 p1 low-Ca*+ ASW (0.5 m M Ca2+,110 m M Mg2+)and desheathed. The bath was then changed over to a labeling medium that consisted of 50% ASW-50% Aplysiu hemolymph containing 1 mM colchicine, 25 U / m L penicillin, 25 pg/ml streptomycin, and 0.1% 2-mercaptoethanol. [ 35S]-Methionine(Amersham) was added to a final concentration of 1 mCi/mL. and the ganglia was incubated at 16°C for 20 h. This was followed by a 4-h chase with ASW containing colchicine, penicillin, streptomycin, 2mercaptoethanol (concentrations as above), and 1 m M cold methionine. Pep-neurons were visually identified (Hall and Lloyd, 1990), and individual somata were manually dissected using the freeze substitution method (Ono and McCaman, 1980). The dissected somata were extracted at I00"C for 10 min in 50 pl0.02 M trifluoroacetic acid (TFA) containing 2 nmol each of synthetic Pep (Applied Biosystems). FMRFamide (Bachem), and SCP, (Peninsula), and filtered for analysis by reversephase high-pressure liquid chromatography (RPHPLC). All RP-HPLC was carried out on a Brownlee C-8, RP 300,4.6 X 220 mm column at a flow rate of 2 mL/min. The extracts from the somata were run through two different modes of RP-HPLC to confirm peptide identity. The first mode used 0.0 1 M TFA as a counterion and a
gradient from 5% CH,CN to 54% CH3CN in 20 min. Fractions werc split, and radioactivity in one aliquot was determined by liquid scintillation counting. Aliquots from fractions containing the radioactive peak were pooled, dried, redissolved in 350 p10.01 M heptafluorobutyric acid (HFBA) containing 2 nmol each of synthetic Pep, FMRFamide, and SCP,, and run on a second mode of RP-HPLC. The second mode utilized 0.01 M HFBA as a counterion and a gradient from 2 1% CH,CN to 24% CH,CN in 2 min. then to 28%CH,CN in 12 min, and lastly to 34% CH3CN in 4 min. Radioactivity in the resulting fractions was determined.
Peptide Content of Neurons in the Pedal Ganglia Neurons from eight pedal ganglia were pooled for these analyses. The dorsal surfaces of the ganglia were desheathed and the Pep-neurons (-40 per ganglion) were removed using the freeze substitution method and extracted in 150 pl0.02 M TFA at 100°C for 10 min. An equal number of neurons similar in size and just anterior to the Pep-neurons were dissected and extracted as controls. Extracts were pooled, filtered, and analyzed using a very broad gradient from 5% CH,CN to 63% CH,CN in 20 min with 0.01 M TFA as a counterion. The broad gradient was used to insure that nearly any peptide would be resolved. Finally, to insure that resolved peptides would be detected, absorption at 206 nm was monitored. At this wavelength, peptide bonds are strongly absorbant. so detection of peptides does not require the presence of aromatic amino acids (Woods and O'Bar, 1970).
Primary Cell Culture Dissociated cell culture of visually identified Pep-neurons from pedal ganglia was carried out as described by Schacher and Proshansky ( 1983), except that 16-mm diameter culture wells (Nunc) were used, and the neurons were maintained at 22°C after initial plating.
lmmunocytology Following 1-3 days in culture, immunocytology of Pepneurons was carried out using a modification of procedures previously described (Pearson and Lloyd, 1989a). The ASW in the culture wells containing neurons was replaced with a fixative solution of 4% paraformaldehyde-30% sucrose in 0.1 M phosphate buffer (PB, pH 7.4) and incubated for 4 h at 22°C. Following fixation, the wells were washed briefly with PB, then in several changes of PB with 1% Triton X-100 and 0.1% sodium azide overnight. They were then incubated for 6 h in a blocking solution that consisted of phosphate-buffered saline (PBS: 0. I4 M NaCl, 0.0 1 M phosphate, pH 7.4), 1% Triton X-100, 0 . l Y ~sodium azide, 1% normal goat serum (Tago) , and 4% skim milk. Pep-neurons were in-
Aplysia Neiiropeptide Release in Culture cubated in primary antiserum (PAS-2) (Pearson and Lloyd, 1989a), diluted 1:500 in blocking solution for 12-16 h. The primary antiserum was washed out with several changes of blocking solution over a 4-h period. This was followed by a 4-h incubation in secondary antiserum, goat anti-rabbit conjugated to fluorescein isothiocyanate (Tago), and diluted 1:2@@in blocking solution. The secondary antiserum was washed out with several changes of PBS, and the preparation was coverslipped and viewed on a Leitz Orthoplan fluorescence microscope.
Incubation of Cultured Pep-Neurons with [ 35S]-Methionine Cultured Pep-neurons to be used in experiments utilizing [ 35S]-methionine were initially plated in a medium consisting of 50% Aplysia hemolymph and 50% methionine-free L 15 containing penicillin-streptomycin-fungizone (PSF) (Gibco) to a final concentration of 1YO. After 1-2 days in culture, [ '5S]-methionine was added to the well to yield a final specific activity of 0.5 mCi/mL. Following 18-20 h in label, the neurons were chased with 30 mL L15 plus I % PSF over 1 h. To determine if the neurons continued to synthesize and transport Pep into their regenerated neurites, the somata and the neurites were separately dissected (Lloyd et al., 1986), extracted, and run through two modes of RP-HPLC to confirm peptide identity asdescribed above. The radioactivity in the resulting fractions was determined by liquid scintillation counting.
Release of [ 35S]-Methionhe-Labeled Pep from Cultured Pep-Neurons For all release experiments only a single neuron was plated per culture well. Release experiments were conducted immediately following the chase period and consisted of three collection periods conducted consecutively. Superfusion of the well was regulated by a peristaltic pump operating at 1 m l l m i n ; this corresponded to two bath volumes per minute. In the first collection period, the culture well containing the neuron was superfused with low-Ca2+ASW for 15-30 min. The neuron was then impaled with an intracellular electrode ( R = 510 MQ) and stimulated to fire five to 10 spikes for I s every 3 s for 10 min. Following the 10-min stimulation period, the neuron was hyperpolarized for 5 min. The superfusate from the entire 15-min period was collected on ice and acidified to 0.0 1 MTFA. The bathing solution was then changed over to normal ASW and allowed to equilibrate for 15-30 min. Following equilibration, the neuron was hyperpolarized to prevent any action potentials from firing for 15 min, and the superfusate from this 15-min period was collected as described above. The neuron was then stimulated to fire as before for 10 min and hyperpolarized for 5 min: the superfusate from this 15min period was collected. All superfused ASW contained 1 mM2-mercaptoethanol and 100 nMsynthetic Pep. A t
585
this concentration, synthetic Pep had no discernable effect on the resting membrane potential of cultured Pepneurons. The samples from the three collection periods were individually passed through a C- 18 reverse-phase extraction cartridge (Sep-Pak), and the peptides were eluted with 3 mL 75% CH3CKand 0.0 1 M : TFA. This was then lyophilized and redissolved in 350 ~ 1 0 .10M TFA immediately prior to RP-HPLC analysis using a gradient from 18% CH,CN to 34% CH,CN in 6 rnin with 0.01 M TFA as a counterion. The somata and neurites of the neurons from these experiments were dissected out separately, extracted at 100°C for 10 min in 50 ~1 0.02 M TFA containing 2 nmol synthetic Pep, and run through the samc RP-HPLC gradient as the release samples. Radioactivity in the resulting fractions was then determined.
RESULTS Pedal Peptide is the Major Peptide Found Selectively in Pep-Neurons in situ
Before carrying out release experiments, we first wanted to examine if Pep was indeed the predominant peptide selectively present in Pep-neurons in pedal ganglia. We investigated this problem in two ways. In the first method, pedal ganglia were labeled with [ 35S]-methionine, and individual Pepneuronal somata were dissected, extracted, and analyzed by two sequential modes of RP-HPLC. Significant incorporation of label was found primarily in the fractions that coeluted with synthetic Pep in both modes (Fig. 1 ) .Two smaller peaks of incorporation were also consistently observed. One of these peaks coeluted with a known break-down fragment of Pep, while the other is unidentified. Thus, Pep is the major methionine-containing peptide synthesized by these neurons. A second method was used to determine if Pepneurons selectively synthesized other peptides that were not detected using [ 35S]-methionine labeling. In this experiment, absorbance detection at low wavelength was used to compare the contents of an extract of pooled Pep-neurons with that of an extract of a similar number of other pedal neurons that are not Pep immunoreactive. Comparison of the absorbance profiles revealed that the major difference was the single large peak of Pep present selectively in the Pep-neuron profile (Fig. 2). A second smaller difference appears to be associated with the Pep fragment (Pearson and Lloyd, 1990). All other absorbance peaks are shared by the two groups of neurons. Therefore. Pep is the predominant peptide selectively present in Pep-neurons in situ. We cannot, however, exclude the possibility
Hall and f.foyd
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TFA
0
d
/
72
HFBA
"
-
c ,
Iep
0 X
extended numerous, highly branched neurites. Neurites regenerated from the initial axon segment and, to a much lesser extent, from the cell soma. These neurites contained many large varicosities and often terminated in growth cones. Immunocytology with an antiserum directed towards Pep revealed that the somata of these neurons maintained Pep-like immunoreactivity after several days in culture [Fig. 3 (A)]. Particularly intense staining was associated with the regenerated varicosities [Fig. 3 (B)] .Other peptidergic/ip/jLYianeurons cocultured with the Pep-neurons did not express Pep-like immunoreactivity (data not shown). To examine if Pep-neurons in culture also continued to synthesize Pep, we labeled individually grown neurons with [ 35S]-methionine.Two consecutive modes of RP-HPLC of extracts from somata and neurites of these neurons showed that authentic Pep was synthesized by the cell bodies (data not shown) and transported to regenerated neurites (Fig. 4 ) . Thus, as is the case for Pep-neurons in situ, cultured Pep-neurons continue to synthesize Pep as their major methionine-containing peptide, and these neurons also retain the ability to transport the peptide. The culture process, there-
36
HPLC Fraction
Figure 1 Pedal peptide is the predominant methioninecontaining peptide synthesized by Pep-neurons in pedal ganglia. Pedal ganglia were desheathed and incubated in [ "S]-methionine for 20 h and chased for 4 h. Individual somata were dissected for analysis by two consecutive modes of RP-HPLC, first using TFA as a counterion (top). Aliquots of fractions corresponding to Pep were pooled and analyzed in a second mode of RP-HPLC using HFBA as a counterion (bottom). Fractions where synthetic Pep was eluted are indicated (retention times: 10 min in TFA, and 9 min in HFBA). The small peaks marked with an asterisk rcpresent a fragment of Pep.
that some of the absorbance peaks common to the two profiles may represent transmitters present in both groups of neurons. Also, it is possible that some extremely hydrophilic peptides may not have been retained on the column and thus would not be detected by this method. Pedal Peptide is Synthesized by PepNeurons in Culture and Transported into Regenerated Neurites
Within 24-48 h of dissociation, Pep-neurons attached to the poly-L-lysine-coated substrate and
0.1
A,
10
20 min
Figure 2 Pep is the predominant peptide present selectively in Pep-neurons. Pep-neurons (top trace) and control pedal neurons (bottom trace) were dissected from pedal ganglia and run on RP-HPLC. Each trace represents the absorbance profile from approximately 250 pooled neurons. The major differences between the profiles are the Pep peak and a smaller peak (asterisk) with the same retention timc as a Pep fragment. The large peaks that elute approximately 2 min prior to Pep are common to all Aply.~zanervous tissue. An additional 2.5 min of the two profiles after 20 min were nearly identical and were not shown.
Aplysia Neuropeptide Releare in Culture 24
fore, did not initiate the expression of other methionine-containing neuropeptides in these neurons. Pep is Released in a Ca2+- and Stimulation-Dependent Fashion Release experiments were carried out on neurons that were cultured individually in wells. Neurons were incubated in [ 35S]-methionhe to label Pep as described in the previous section. These expenments were divided into three collection periods. During the first period, the neurons were bathed in low-Ca2+ASW and stimulated via an intracellular electrode to examine the Ca2+-dependence of release. For the second collection period, the super-
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5
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HPLC Fraction
Figure 4 Radiolabeled Pep synthesized in the soma of cultured neurons is transported to the regenerated neurites. (Top) Extract from the neurites of a single cultured Pep-neuron grown in [ "S] -methionhe-containing media was run on RP-HPLC with TFA as a counterion. (Bottom) Aliquots of the fractions that coeluted with synthetic Pep were pooled and run on RP-HPLC with IIFBA as a counterion. Gradients and retention times are identical to those in Figure 1.
Figure 3 Cultured Pep-neurons exhibit Pep-like immunoreactivity. ( A ) Fluorescence micrograph of a Pepneuron that was grown for 48 h and subjected to immunocytology with a primary antiserum directed towards Pep and a fluorescein-conjugated secondary antiserum. ( B ) Regenerated neurites contain large varicosities that show intense Pep-like immunoreactivity. Scale bar = 200 pm ( A ) and 20 pm (B) .
fusate was changed to normal ASW, and the neuron was hyperpolarized. In the final collection penod, the superfusate was normal ASW, and the neuron was stimulated to fire as in the low-Ca2+ portion of the experiment. Peptides present in the superfusates from each collection period were extracted, run on RP-HPLC ( Fig. 5 ), and the radioactivity that eluted was determined. There were little or no counts above background in the Pep fractions from the first two collection periods. However in the third period of the experiment there is a marked increase in the counts in the Pep and Pep fragment fractions (Fig. 5 ). A summary of the release experiments is shown in Figure 6. This shows that Pep is released in a Ca2+-and stimulation-de-
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pendent fashion. The amount of labeled Pep released into the superfusate during stimulation in normal ASW represents 4.0 1.9% (S.E.M., n = 5 ) of the total labeled Pep extracted from neurites at the end of each experiment.
low Ca2+
*
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HPLC Fraction Figure 5 RP-HPLC profiles of ['%I -methionine-labeled material recovered from the superfusate from each period of a typical experiment. Single Pep-neurons were labeled in [ 35S]-methionine-containing medium for IS20 h and chased for 1 h. Experiments consisted of three collection periods; first, the culture medium was washed out with low-Ca**ASW, and the neuron was stimulated (5-10 Hz for I s every 3 s) for 10 min, then hyperpolarized for 5 min, and the superfusate was collected. Second, the bath was changed to normal ASW and hyperpolarized, and the superfusate collected for 15 min. Finally, the neuron was stimulated in normal ASW with the same paradigm as described above, and the superfusate was collected. All superfusates were run on RPHPLC and the fractions were counted. The gradient used was different from that used in Figures 1 and 4. The fractions where synthetic Pep and the Pep fragment
DISCUSSION Previously, it has been shown that Pep meets a number of criteria for the designation of a substance as a neurotransmitter. It is synthesized selectively by individual neurons, predominantly by a cluster of neurons on the dorsal surface of each pedal ganglion. It is transported down pedal nerves to the foot where it has a significant modulatory effect on foot muscle contraction. It also has central effects on specific neurons in the abdominal ganglia (Lloyd and Connolly, 1989; Pearson and Lloyd, 1989a,b; Hall and Lloyd, 1990). In this study, we add to the evidence supporting the role of Pep as a neurotransmitter in Aplysiu. We have shown that Pep is indeed the major peptide selectively found in these neurons. Also, Pep-neurons in culture continue to synthesize and transport Pep as do their in situ counterparts. Finally, Pep is released when single neurons in culture were stimulated intracellularly in the presence of normal calcium levels. Significant release of Pep was not observed in response to stimulation in low levels of calcium or when the neuron was hyperpolarized in normal levels of calcium. The amount of peptide release seen in these studies is similar to that observed in a similar study examining release of the small cardioactive peptides ( SCPs) from cultured identified buccal neurons B1 and B2 (Lloyd et al., 1986). In that study, release of about 6% of the amount of SCPs in the neurites of the identified buccal neurons was observed over periods similar to those used in this study. Here, we report about 4%of the Pep in neurites was released. These release experiments were facilitated by placing individual neurons in primary cell culture. Our data, taken together with previous findings (Lloyd et al., 1986) suggest that the fidelity of neuropeptide synthesis and transport by Aplysia neurons is maintained in culture. Neurons Bl and B2 continue to synthesize predominantly SCP, and SCP, in culture as they do in situ. Furthermore, the
eluted are indicated. The large, truncated peak with a short retention time primarily represents unincorporated methionine.
Aplysia Neuropeptide Release in Culture 1oc
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tide is released from Pep-neurons in a calcium- and stimulation-dependent fashion. These results provide strong support for the proposition that Pep functions as a neurotransmitter. The present results and those of a previous study suggest that these techniques may prove to be broadly useful in identifying peptide transmitters in identified neurons in Aplysia. We thank W. L. Pearson and M. D. Whim for critical reading of the manuscript. This work was supported by NIH Grant NS-23569 and a Brain Research Foundation grant. J.D.H. is supported by Pharmacological Sciences Training Grant T32GM07 15 1.
50
REFERENCES
7-
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i
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ASW no stim
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Figure 6 ’ep is released from single cultured neurons in a stimi tion- and CaZ+-deDendentfashion. The total amount of radioactivity in the Pep fraction and the Pep fragment fraction was determined for each period of five experiments. Values are mean t S.E.M.
two peptides are synthesized in the same ratio as observed in situ. Similarly, Pep is the predominant methionine-labeled peptide synthesized by pedal ganglia Pep-neurons in situ and in culture. These results suggest that this may be a useful procedure for determining the nature of peptides released from other peptidergic neurons in Aplysia. A promising application for this technique might include the very large neurons in the abdominal ganglia of Aplysia for which the sequences of the mRNA that encode peptide precursors have been determined, but the nature of the mature released neuropeptide has not (Taussig, Kaldany, and Scheller, 1984; Shyamala, Fisher, and Scheller, 1986). In this study, we have utilized the technique of individual neuron culture to show that pedal pep-
HALL,J. D. and LLOYD,P. E. (1990). Involvement of pedal peptide in locomotion in Aplysia: modulation of foot muscle contractions. J. Neurobiol. 21:858-868. I., and LLOYD,P. E., SCHACHER,S., KUPFERMANN, WEISS,K. R. ( 1986). Release of neuropeptides during intracellular stimulation of single identified Aplysia neurons in culture. Proc. Nutl. Acad. Sci. USA 83~9794-9798. LLOYD,P. E. and CONNOLLY, C. M. (1989). Sequence of pedal peptide: a novel neuropeptide from the central nervous system of Aplysiu. J. Neurosci. 9:3 12317. ONO,J. K. and MCCAMAN, R. E. ( 1980). Identification of additional histaminergic neurons in Aplysia: improvement of single cell isolation techniques for in randern physiological and chemical studies. ,’c’euro.Ycience 5:8 35-840. PEARSON, W. L. and LLOYD,P. E. ( 1989a). lmmunocytological localization of pedal peptide in the central nervous system of Aplysia. J . Neurosci. 9:3 18-325. PEARSON, W. L. and LLOYD,P. E. ( 1989b). Actions of pedal peptide on neuron L5 in -4plysia. SOC. Neurosci. Abstr. 15736. W. L. and LLOYD,P. E. ( 1990). Distribution PEARSON, and characterization of pedal peptide immunoreactivity in Aplysia. J. Neurobiol. 21:883-892. SCHACHER, S. and PROSHANSKY, E. ( 1983). Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by .4plysia hemolymph and the presence of the initial axon segment. J. Neurosci. 3:24032413. M., FISHER,J. M., and SCHELLER,K. H. SHYAMALA, ( 1986). A neuropeptide precursor expressed in Ap1.vsiu neuron L5. DNA 5:203-208. TAUSSIG, R., KALDANY, K. -R., and SCHELLER,K. H. (1984). A cDNA clone encoding neuropeptides isolated from Aplysia neuron L 1 I . Proc. Natl. Acad. Sci. USA 81:4988-4992. WOODS,A. H. and O’BAR,P. R. (1970). Absorption of proteins and peptides in the far ultraviolet. Science 167: 179-1 81.
