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Synaptic regulation of specific protein synthesis in an identified neuron
314
Brain Research, 78 ( t 974) 314-319 c Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Short Communications
Synaptic regulation of specific protein synthesis in an identified neuron
HAROLD GAINER AND JEFFERY L BARKER Behavioral Biology Branch. National Institute o] Chiht Health and Human Development. National Institutes of Health. Bethesda, Md. 20014 (U.S.A,
(Accepted June 15th, 1974)
Studies on the influence of behavioral and synaptic input on the macromolecular metabolism of neural tissue 10 have been limited by the ill-defined relationships existing between the biochemical changes observed and the specific neural inputs presumed to cause these changes. One major problem is that while etectrophysiological techniques allow for single unit analysis, the biochemical analyses have been done on heterogeneous areas of nervous tissue 4 composed of neuronal, glial and connective tissue elements. To improve the biochemical 'signal to noise ratio', we have conducted such a correlative study on a specific identified neuron, RI.~ in Aptysia californica, in which the electrophysiological and metabolic consequences of synaptic stimulation could be determined in the same cell. Furthermore, confiderabte information about the electrophysiology 3.5.~'%~9, synaptic input ~,5,9, and protein metabolism14,16,17,z°,21 of this neuron was available. An earlier study on the effects of synaptic stimulation on R2 of Aplysia z'z detected no changes in specific protein metabolism. In this report, we present evidence for the selective regulation of protein metabolism of R15 by synaptic input. Experiments were performed on isolated abdominal ganglia from Aplysia californica (Pacific Bio-Marine Corp., Venice, Calif.), which had been kept in a well-aerated aquarium of artificial sea water IInstant Ocean) for 3-10 days before use. The animals were maintained in a light-dark cycle (light period: 8.30 a . m . 6.30 p.m.), and incubations were typically begun at 11.00 a.m. The parietio-visceral ganglion was removed from the animal, pinned to Sylgard in a plastic petri-dish and pre-incubated for 1 h in Aplysia saline (composition: 460 m M NaCI, kl0 m M KCt, 11 m M CaC12, 50 m M MgCI2, 50 m M Tris, and 2 mg/ml of glucose, adjusted to a pH of 7.8 with HCI) without isotope. This was followed by a 3-h incubation in fresh saline containing 20 # M of L-[4,5-ZH]leucine (New England Nuclear Corp.; specific activity 33-64 Ci/mmole). Incubations were carried out at room temperature (2t-23 °C) .The l-hour pre-incubation was used as a conditioning period during which the preparation received the same experimental manipulation as was present during the
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3-h isotopic incubation. For example, inhibitory post-synaptic potentials (ipsps) were evoked in R15 by stimulation of the branchial nerve (at a frequency of once every 5 sec) at the beginning of the pre-incubation period and maintained throughout the experiment. The membrane potential of the nerve cells was monitored and recorded using conventional intracellular microelectrode recording techniques. The incubation was terminated by immersion of the ganglion in ice-cold saline, and the appropriate individual neurons were identifieds, dissected out of the ganglion, homogenized in sodium dodecyl sulfate (SDS), and the extracted proteins were separated by SDS polyacrylamide gel electrophoresis 6. Each cell extract was mixed with marker proteins (/3-galactosidase, 130,000 daltons; bovine serum albumin, 68,000; ovalbumin, 43,000; carbonic anhydrase, 29,000; human hemoglobin, 15,500; cytochrome c, 12,000; pancreatic trypsin inhibitor, 6100; and bacitracin, 1411) before electrophoresis, so that each gel separating the proteins of an individual cell was internally standardized with respect to its molecular weight distribution characteristics. The gels were stained with Coomassie MOLECULAR WEIGHT/IO00 150 [
5
68
45
29
I
[
I
15.5 i
12 I
Control (n=7)
6.1
1.4
I
I
Aplysie Cell RI5
44 d (.9
Synaptic Inhibition (n=6)
i~, i!i
~omv 20 s,¢or~s
j~
~2 nr
I
vO
I0
20 50 SLICE NUMBER
40
50
Fig. 1. Comparison of the molecular weight distributions of newly synthesized proteins in control (unstimulated) and synaptically inhibited R15 neurons. The inset shows the typical pattern of spontaneous bursting pacemaker potential activity in the control cells, and the abrupt inhibition of this activity upon stimulation of the branchial nerve (at arrow). Extracts of the R1.5 somas incubated for 3 h under control and experimental conditions in L-[:JH]leticine were separated on I 1% SDS polyacrylamide gels which were then analysed for their radioactive patterns (see text for experimental details). Left ordinate: the radioactivity (counts/rain/gel slice minus background) in each gel slice was plotted relative to the maximum radioactive gel slice in the 60,000-68,000 dalton range. Right ordinate: average absolute counts/min/gel slice found in a given condition (filled circles, control; open circles, inhibited) which corresponded to the relative counts/min/gel slice of 1.0. The lower abscissa represents the gel slice number, and the upper abscissa shows the molecular weights of standard marker proteins corresponding to the various slice positions. Each point on the curves at a given slice number represents the average relative counts/min value of 7 (control) or 6 (inhibited) neurons. The vertical bars at the 12,000 and 6100 dalton points reflect L 1 standard deviation. Synaptic inhibition reduced the relative counts/rain/gel slice of the 12,000 dalton peak from a control value of 3.8 to a value of 2.0 (P 0.001), whereas the apparent decrease in the 6000 dalton region was not statistically significant (P : 0.037).
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blue, destained, sliced, and the radioactivities of the gel slices were counted in it Packard 3320 liquid scintillation counter, as described elsewhere 6, Statistical analyses were performed using a one-tailed. Mann-Whitney U test is. Under control conditions, R15 neurons generated typical bursting pacemaker potential activity 8,5,15.19 throughout the incubation periods (see inse~ in Fig. l). These cells prod uced a characteristic pattern o f [~H] leucin e incor po ration into pro tein dominated by low molecular weight classes (Fig. 1). The graph in Fig | was constructed by plotting the radioactivity in each gel slice relative to the maximum radioactive slice obtained in the 60,000-68,000 dalton range of the gel. Such a normalization procedure permitted the comparison of relative incorporation of [3H]|eucine into specific molecular weight classes of proteins among different cells under various experimental conditions, independent of absolute isotope incorporation rates, precursor permeabilities, and pool sizes'Z.vA6.21.zL Although lipids are known to run on SDS gels in the region corresponding to low molecular weight proteins, various data indicate that the low molecular weight peak of radioactivity in R~.~ ~s m newly synthesized proteins. These data are: (1) preferential incorporation of labeled leucine into low molecular weight proteins is not characteristic of all Aplvsia neurons le. ~., R26.10.z1,2'~ and Lt0 s have very little label on this region of the gelJ. Furthermore. densitometric scans of Coomassie blue stained microgels reveal a large quantity ol protein on the gel corresponding to the highly labeled low molecular weight peak [br R15. but not for R,~H. (2) Using another gel system without SDS (acid gels at pH 2.7) we have been able to demonstrate that a similar 12.000 dalton labeled peak dominates the labeling profile of R15 and not R,~ (Lob and Gainer. unpublished). (3) Extraction of the [3H]leucine labeled TCA-precipitable fraction of R15 with lipid ~olvents does not remove significant radioactivity from the TCA pellet, suggesting that little lipid is synthesized from [3H]teucine on R15 in 3 h. (4) Incubation of Ra5 wi~h a protein synthesis inhibitor, anisomycin (30 #g/ml), inhibits 95-98 %, of [~H]leucine incorporation into protein in this celt. and completely eliminates the low molecular weight peak of radioactivity when the cell extracts are run on SDS gels tunpublished dataL (5) Incubation of the newly synthesized proteins in RI~ with self-digested pronase lor 1 h at 37 C before their gel separation, completely eliminates all evidencc of protein bound radioactivity on the gel. Branchial nerve stimulation (once every 5 sec) evoked ipsps in R ~ , hyperpolarizing the membrane potential of the cell to about 64 mV for the entire experimental period (inset, Fig. 1). The psp evoked is actually biphasic, consisting of a rapidly decaying depolarizing component followed by a prolonged, hyperpolarizing component. The hyperpotarizing component which predominates is due primarily to an increase in the potassium conductance of the membrane, possibly mediated by dopaminO, ~l. Synaptic inhibition of R~5 did not significantly alter the absolute incorporation of [3H]leucine into cell protein tTable 1), but produced a selective decrease in labeling of low molecular weight (i.e.. 12.000 dalton) proteins ~Fig. 1). Similar effects on R15 were obtained when the ganglia were incubated in media contaimng 10-3 M dopamine, which hyperpolarized the membrane potenual of the cell to around ~--65 mV for the entire incubation period. The data in Table !, dem-
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TABLE 1 S E L E C T I V E M O D U k A T I O N OF P R O T E I N SYNTHESIS IN R 1 5 N E U R O N S
Condition
k
N
Percentage o f total counts/min at specific molecular weights (daltons) * 60,000
12,000
6,000
11.4 =~ 0.4 11.8 ± 0.7 11.8 + 0.7
26.2 ~ 1.1 20.7 ± 1.6'* 16.8 4 0.7**
11.1 :~ 1.0 8.4 5 0.9 8.7 ± 0.6
Total counts/ rain ( x 10 J)
____
Controls Synaptic inhibition Dopamine treated
17 6 6
2.02 =: 0.59 1.96 ± 0.62 2.14 ± 0.34
* The ~/ocounts/rain in the gel for specific molecular weight classes of proteins was obtained by taking the ratio of the sum of counts/rain of 3 gel slices over a given molecular weight region on the gel relative to the total counts/rain in the gel x 100. The data is expressed as means ~ standard errors of the mean for N samples. ** Data significantly different from controls, 0.01 ~-~ P > 0.001. Statistical analyses were done using a one-tailed Mann-Whitney U test.
onstrates that 11.4~o o f the [3H]leucine incorporated into protein in cell R15 was present in the 60,000 dalton region under control conditions, and further that this incorporation was not altered by either synaptic inhibition or dopamine treatment. In contrast, the 12,000 dalton peak which accounted for 2 6 . 2 ~ o f the total protein incorporation in control cells, was significantly decreased by synaptic inhibition (P < 0 . 0 0 1 ) a n d by dopamine treatment (P -- 0.1301). Since the electrophysiological consequences o f both synaptic inhibition and dopamine treatment were to hyperpolarize the membrane potential and eliminate all spontaneous spike activity, it is important to note that no changes in the incorporation patterns o f R15 were observed when the cells' spike activity was abolished by tetrodotoxin, or when the cells were hyperpolarized (to about - - 6 5 mV) throughout the incubation by replacement o f the sodium ions in the media by a variety o f impermeant substitutes (see Table I in ref. 20; confirmed in our laboratory). These data suggest that the selective inhibition o f [3H]leucine incorporation into the 12,000 dalton peak (Fig. 1, Table I) is probably not mediated by either membrane potential hyperpolarization or spike abolition, p e r se. The foregoing results demonstrate that the protein synthesis patterns o f R15 can be selectively modulated by both the physiological synaptic input to the cell and a related pharmacological treatment. The selective decrease in incorporation of [3H]leucine in the 12,0130 dalton peak in R15 produced by the experimental manipulations could reflect a decrease in the synthesis rate or an increase in the breakdown rate o f the 12,000 dalton class o f proteins. We attempted to determine which o f the above possibilities could account for the results by studying the turnover rates o f proteins in R15 cells exposed to the same experimental manipulations as described above. T u r n o v e r rates were determined by first pre-incubating and incubating as above (pulse) and then post-incubating (chasing) in a medium (which contained 1 rnM unlabeled leucine) for various time periods (up to 20 h). Analysis o f control R15 neurons treated in this manner demonstrated a very rapid (ca. 2 h half-time) decay rate o f only one g r o u p o f labeled proteins, i.e., the 12,1300 dalton class. This decay rate
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SHOR'I COMMUNICA]'IONS
was e i t h e r u n c h a n g e d o r d e c r e a s e d (in the case o f d o p a m i n e t r e a t m e n t ) u n d e r the v a r i o u s e x p e r i m e n t a l c o n d i t i o n s tested I G a i n e r and B a r k e r . in press~
Therefore.
t h e r e g u l a t o r y effects o b s e r v e d in these e x p e r i m e n t s are p r o b a b l y n o t d u e to a l t e r e d c a t a b o l i c rates b u t m o s t likely r e p r e s e n t c h a n g e s in synthesis rates. T h a t the p r o t e i n class w h i c h c o u l d be m o d u l a t e d in its synthesis also had a high t u r n o v e r rate m a ? be m o r e t h a n c o i n c i d e n c e . It is p o s s i b l e t h a t in relatively s h o r t - t e r m e x p e r i m e n t s such as these, o n l y t h o s e p r o t e i n s w i t h high t u r n o v e r rates will be significantl~ m o d i f i e d by s y n a p t i c i n p u t ~or will be m o d i f i e d sufficiently to be d e t e c t e d by c u r r e n t a n a l y t i c a l m e t h o d s ) . In this regard, the fact t h a t n o n e o f t h e classes o f n e w l y s y n t h e s i z e d proteins in the g i a n t n e u r o n R2 h a d high t u r n o v e r rates ~, w o u l d lead us to suggest t h a t p r o t e i n m e t a b o l i s m in this n e u r o n s h o u l d be relatively r e s i s t a n t to influence by s y n a p t i c input. I n d e e d . the e x p e r i m e n t s o f W i l s o n a n d B e r r y ~'~ s h o w e d t h a t relatively p r o l o n g e d s t i m u l a t i o n o f e x c i t a t o r y s y n a p t i c i n p u t to the A p l y s i a grant n e u r o n R~ p r o d u c e d n o c h a n g e s in the p a t t e r n s o f p r o t e i n m e t a b o l i s m in this cell. It is. t h e r e f o r e , possible that in relatively s h o r t - t e r m e x p e r i m e n t s , s y n a p t i c r e g u l a t i o n o f n e u r o n a l p r o t e i n m e t a b o l i s m m a y be c o n f i n e d e i t h e r to the i n d u c t i o n o f n e w p r o t e i n s 12,In. or to the m o d u l a t i o n o f the o n g o i n g synthesis o f specific p r o t e i n s with relatively r a p i d t u r n o v e r rates.
I ASCHER. P., KEHOE, J S.. ET TAUt:. L.. Effets &injections etectrophoretiques de dopamme sur les neurones d'Aplysie, J. Physiol. Paris), 59 (1967J 331-332 2 ARCH, S.. Polypeptide secretion from the isolated parieto-visceral ganglion of Aplvsia cali/ort#cao J. gen. Physiol.. 59 (1972) 47-59. 3 CARPENTER. D. O.. AND GUNN. R., The dependence of pacemaker discharge ol Ap/ysia neurons upon Na Nand Ca ~~, J. CellPhys'io/., 75 (1970) 121-127. 4 CICERO. T. J.. AND VIOORE. B. W.. Turnover of the brain specific protein, S-10C) Sciem'e. 169 (1970) 1333-1334. 5 FRAZIER. W. T., KANDEL. E. R.. KUPFERMAN. I.. WAZIRI. R.. AND COGGESHALL~ R. ]~,~ Morphological and functional properties of identified cells i n the abdominal ganglion of Aplysia ,'ali/brnica. J. Neurophysiol., 30 (1967) 1288-1351. 6 GAINER. H.. Micro-disc electrophoresis in sodium dodecyl sulfate: An application to the study ol protein synthesis in individual, identified neurons. Analyt. Biochem.. 44 t1971) 589-605. 7 GAINER, H.. Patterns of protein synthesis in individual, identified molluscan neurons~ 8rai, Research. 39 (1972) 369-385. 8 GAINER. H.. AN[) WOLLBERG. Z.. Specific protem metabolism in identifiable netnrons of Aph'sia caliJbrnica, J. Neurobiol., 5 (1974 ~243-261. 9 GtLLER. E.. AND SCHWARTZ,J. H.. Choline acetyltransferase in identified neurons of abdominal ganglion of Aplysia cafiforniea. J. Neurophysiol., 34 (1971) 93-107. 10 GLASSMAN. E.. The biochemistry of learning: an evaluation of the role of RNA and protein. Ann. Rev. Bioehem., 38 11969) 605-646. 11 KERKUT.G. A.. HORN. N.. AND WALKER. R. J., Long-lasting synaptic inhibition and its transmitter in the snail Helix aspersa, Comp. Biochem. Physiol., 30 (1969) 1061-1074 12 KLEIN, D. C.. AND WELLER, J., lndole metabolism i~ the pineal gland: A circadian rhythm in N-acetyltransferase. Science, 169 (1970) 1093-1095. 13 KLErN. D. C.. BERG.G. R.. AND WELLER.J.. Melatonin synthesis: adenosine 3',5"-monophosphale and norepinephrine stimulate N-acetyltransferase, Science, 168 (1970) 979-980. 14 LOll. Y. P.. Protein Synthesis and Neuronal Function in Single ldentih'ed Nem'ons ~)f Aplysut californica, Ph.D. dissertation, Univ. of Pennsylvania, 1973.82 pp. 15 MATHIEU. P. A.. AND ROBERGE. [7. A.. Characteristics of pacemaker oscillatmn~ m /lplvs~a neurons. Canad. J. Physiol. Pharmacol.. 49 t 1971) 787-795. 16 PETERSON. R. P., AND Loft. Y. P.. The role of macromolecules in neuronal function in Apljwia, Prog. Neurobiol.. 2 (1973) 179-203.
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17 SCHWARTZ, J. H., CASTELLUCHI, V. F., AND KANDEL, E. R., Functioning of identified neurons and synapses in abdominal ganglion of Aplysia in the absence of protein synthesis, J. Neurophysiol., 34 (1971) 939-953. 18 SIEGEL, S., Non-Parametric Statisties fbr the Behavioral Sciences, McGraw-Hill, New York, 1956, 312 pp. 19 STRUMWASSER, F., Membrane and intracellular mechanisms governing endogenous activity in neurons. In F. D. CARLSON (Ed.), Physiological and Biochemical A,speets t~/"Nervous Integration, Prentice-Hall, New Jersey, 1968, pp. 329 341. 20 STRUMWASSER, F., Neural and humoral factors in the temporal organization of behavior, The Physiologist, 16 (1973) 9-42. 21 WILSON, D. W., Molecular weight distribution of proteins synthesized in single, identified neurons of Aplysia, J. gen. Physiol., 57 (1971) 2 6 4 0 . 22 WILSON, D. W., AND BERRY, R. W., The effect of synaptic stimulation on R N A and protein metabolism in the Re soma of Aplysia, J. Neurobiol., 3 (1972) 369-379.
Brain Research, 78 ( t 974) 314-319 c Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Short Communications
Synaptic regulation of specific protein synthesis in an identified neuron
HAROLD GAINER AND JEFFERY L BARKER Behavioral Biology Branch. National Institute o] Chiht Health and Human Development. National Institutes of Health. Bethesda, Md. 20014 (U.S.A,
(Accepted June 15th, 1974)
Studies on the influence of behavioral and synaptic input on the macromolecular metabolism of neural tissue 10 have been limited by the ill-defined relationships existing between the biochemical changes observed and the specific neural inputs presumed to cause these changes. One major problem is that while etectrophysiological techniques allow for single unit analysis, the biochemical analyses have been done on heterogeneous areas of nervous tissue 4 composed of neuronal, glial and connective tissue elements. To improve the biochemical 'signal to noise ratio', we have conducted such a correlative study on a specific identified neuron, RI.~ in Aptysia californica, in which the electrophysiological and metabolic consequences of synaptic stimulation could be determined in the same cell. Furthermore, confiderabte information about the electrophysiology 3.5.~'%~9, synaptic input ~,5,9, and protein metabolism14,16,17,z°,21 of this neuron was available. An earlier study on the effects of synaptic stimulation on R2 of Aplysia z'z detected no changes in specific protein metabolism. In this report, we present evidence for the selective regulation of protein metabolism of R15 by synaptic input. Experiments were performed on isolated abdominal ganglia from Aplysia californica (Pacific Bio-Marine Corp., Venice, Calif.), which had been kept in a well-aerated aquarium of artificial sea water IInstant Ocean) for 3-10 days before use. The animals were maintained in a light-dark cycle (light period: 8.30 a . m . 6.30 p.m.), and incubations were typically begun at 11.00 a.m. The parietio-visceral ganglion was removed from the animal, pinned to Sylgard in a plastic petri-dish and pre-incubated for 1 h in Aplysia saline (composition: 460 m M NaCI, kl0 m M KCt, 11 m M CaC12, 50 m M MgCI2, 50 m M Tris, and 2 mg/ml of glucose, adjusted to a pH of 7.8 with HCI) without isotope. This was followed by a 3-h incubation in fresh saline containing 20 # M of L-[4,5-ZH]leucine (New England Nuclear Corp.; specific activity 33-64 Ci/mmole). Incubations were carried out at room temperature (2t-23 °C) .The l-hour pre-incubation was used as a conditioning period during which the preparation received the same experimental manipulation as was present during the
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3-h isotopic incubation. For example, inhibitory post-synaptic potentials (ipsps) were evoked in R15 by stimulation of the branchial nerve (at a frequency of once every 5 sec) at the beginning of the pre-incubation period and maintained throughout the experiment. The membrane potential of the nerve cells was monitored and recorded using conventional intracellular microelectrode recording techniques. The incubation was terminated by immersion of the ganglion in ice-cold saline, and the appropriate individual neurons were identifieds, dissected out of the ganglion, homogenized in sodium dodecyl sulfate (SDS), and the extracted proteins were separated by SDS polyacrylamide gel electrophoresis 6. Each cell extract was mixed with marker proteins (/3-galactosidase, 130,000 daltons; bovine serum albumin, 68,000; ovalbumin, 43,000; carbonic anhydrase, 29,000; human hemoglobin, 15,500; cytochrome c, 12,000; pancreatic trypsin inhibitor, 6100; and bacitracin, 1411) before electrophoresis, so that each gel separating the proteins of an individual cell was internally standardized with respect to its molecular weight distribution characteristics. The gels were stained with Coomassie MOLECULAR WEIGHT/IO00 150 [
5
68
45
29
I
[
I
15.5 i
12 I
Control (n=7)
6.1
1.4
I
I
Aplysie Cell RI5
44 d (.9
Synaptic Inhibition (n=6)
i~, i!i
~omv 20 s,¢or~s
j~
~2 nr
I
vO
I0
20 50 SLICE NUMBER
40
50
Fig. 1. Comparison of the molecular weight distributions of newly synthesized proteins in control (unstimulated) and synaptically inhibited R15 neurons. The inset shows the typical pattern of spontaneous bursting pacemaker potential activity in the control cells, and the abrupt inhibition of this activity upon stimulation of the branchial nerve (at arrow). Extracts of the R1.5 somas incubated for 3 h under control and experimental conditions in L-[:JH]leticine were separated on I 1% SDS polyacrylamide gels which were then analysed for their radioactive patterns (see text for experimental details). Left ordinate: the radioactivity (counts/rain/gel slice minus background) in each gel slice was plotted relative to the maximum radioactive gel slice in the 60,000-68,000 dalton range. Right ordinate: average absolute counts/min/gel slice found in a given condition (filled circles, control; open circles, inhibited) which corresponded to the relative counts/min/gel slice of 1.0. The lower abscissa represents the gel slice number, and the upper abscissa shows the molecular weights of standard marker proteins corresponding to the various slice positions. Each point on the curves at a given slice number represents the average relative counts/min value of 7 (control) or 6 (inhibited) neurons. The vertical bars at the 12,000 and 6100 dalton points reflect L 1 standard deviation. Synaptic inhibition reduced the relative counts/rain/gel slice of the 12,000 dalton peak from a control value of 3.8 to a value of 2.0 (P 0.001), whereas the apparent decrease in the 6000 dalton region was not statistically significant (P : 0.037).
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blue, destained, sliced, and the radioactivities of the gel slices were counted in it Packard 3320 liquid scintillation counter, as described elsewhere 6, Statistical analyses were performed using a one-tailed. Mann-Whitney U test is. Under control conditions, R15 neurons generated typical bursting pacemaker potential activity 8,5,15.19 throughout the incubation periods (see inse~ in Fig. l). These cells prod uced a characteristic pattern o f [~H] leucin e incor po ration into pro tein dominated by low molecular weight classes (Fig. 1). The graph in Fig | was constructed by plotting the radioactivity in each gel slice relative to the maximum radioactive slice obtained in the 60,000-68,000 dalton range of the gel. Such a normalization procedure permitted the comparison of relative incorporation of [3H]|eucine into specific molecular weight classes of proteins among different cells under various experimental conditions, independent of absolute isotope incorporation rates, precursor permeabilities, and pool sizes'Z.vA6.21.zL Although lipids are known to run on SDS gels in the region corresponding to low molecular weight proteins, various data indicate that the low molecular weight peak of radioactivity in R~.~ ~s m newly synthesized proteins. These data are: (1) preferential incorporation of labeled leucine into low molecular weight proteins is not characteristic of all Aplvsia neurons le. ~., R26.10.z1,2'~ and Lt0 s have very little label on this region of the gelJ. Furthermore. densitometric scans of Coomassie blue stained microgels reveal a large quantity ol protein on the gel corresponding to the highly labeled low molecular weight peak [br R15. but not for R,~H. (2) Using another gel system without SDS (acid gels at pH 2.7) we have been able to demonstrate that a similar 12.000 dalton labeled peak dominates the labeling profile of R15 and not R,~ (Lob and Gainer. unpublished). (3) Extraction of the [3H]leucine labeled TCA-precipitable fraction of R15 with lipid ~olvents does not remove significant radioactivity from the TCA pellet, suggesting that little lipid is synthesized from [3H]teucine on R15 in 3 h. (4) Incubation of Ra5 wi~h a protein synthesis inhibitor, anisomycin (30 #g/ml), inhibits 95-98 %, of [~H]leucine incorporation into protein in this celt. and completely eliminates the low molecular weight peak of radioactivity when the cell extracts are run on SDS gels tunpublished dataL (5) Incubation of the newly synthesized proteins in RI~ with self-digested pronase lor 1 h at 37 C before their gel separation, completely eliminates all evidencc of protein bound radioactivity on the gel. Branchial nerve stimulation (once every 5 sec) evoked ipsps in R ~ , hyperpolarizing the membrane potential of the cell to about 64 mV for the entire experimental period (inset, Fig. 1). The psp evoked is actually biphasic, consisting of a rapidly decaying depolarizing component followed by a prolonged, hyperpolarizing component. The hyperpotarizing component which predominates is due primarily to an increase in the potassium conductance of the membrane, possibly mediated by dopaminO, ~l. Synaptic inhibition of R~5 did not significantly alter the absolute incorporation of [3H]leucine into cell protein tTable 1), but produced a selective decrease in labeling of low molecular weight (i.e.. 12.000 dalton) proteins ~Fig. 1). Similar effects on R15 were obtained when the ganglia were incubated in media contaimng 10-3 M dopamine, which hyperpolarized the membrane potenual of the cell to around ~--65 mV for the entire incubation period. The data in Table !, dem-
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317
TABLE 1 S E L E C T I V E M O D U k A T I O N OF P R O T E I N SYNTHESIS IN R 1 5 N E U R O N S
Condition
k
N
Percentage o f total counts/min at specific molecular weights (daltons) * 60,000
12,000
6,000
11.4 =~ 0.4 11.8 ± 0.7 11.8 + 0.7
26.2 ~ 1.1 20.7 ± 1.6'* 16.8 4 0.7**
11.1 :~ 1.0 8.4 5 0.9 8.7 ± 0.6
Total counts/ rain ( x 10 J)
____
Controls Synaptic inhibition Dopamine treated
17 6 6
2.02 =: 0.59 1.96 ± 0.62 2.14 ± 0.34
* The ~/ocounts/rain in the gel for specific molecular weight classes of proteins was obtained by taking the ratio of the sum of counts/rain of 3 gel slices over a given molecular weight region on the gel relative to the total counts/rain in the gel x 100. The data is expressed as means ~ standard errors of the mean for N samples. ** Data significantly different from controls, 0.01 ~-~ P > 0.001. Statistical analyses were done using a one-tailed Mann-Whitney U test.
onstrates that 11.4~o o f the [3H]leucine incorporated into protein in cell R15 was present in the 60,000 dalton region under control conditions, and further that this incorporation was not altered by either synaptic inhibition or dopamine treatment. In contrast, the 12,000 dalton peak which accounted for 2 6 . 2 ~ o f the total protein incorporation in control cells, was significantly decreased by synaptic inhibition (P < 0 . 0 0 1 ) a n d by dopamine treatment (P -- 0.1301). Since the electrophysiological consequences o f both synaptic inhibition and dopamine treatment were to hyperpolarize the membrane potential and eliminate all spontaneous spike activity, it is important to note that no changes in the incorporation patterns o f R15 were observed when the cells' spike activity was abolished by tetrodotoxin, or when the cells were hyperpolarized (to about - - 6 5 mV) throughout the incubation by replacement o f the sodium ions in the media by a variety o f impermeant substitutes (see Table I in ref. 20; confirmed in our laboratory). These data suggest that the selective inhibition o f [3H]leucine incorporation into the 12,000 dalton peak (Fig. 1, Table I) is probably not mediated by either membrane potential hyperpolarization or spike abolition, p e r se. The foregoing results demonstrate that the protein synthesis patterns o f R15 can be selectively modulated by both the physiological synaptic input to the cell and a related pharmacological treatment. The selective decrease in incorporation of [3H]leucine in the 12,0130 dalton peak in R15 produced by the experimental manipulations could reflect a decrease in the synthesis rate or an increase in the breakdown rate o f the 12,000 dalton class o f proteins. We attempted to determine which o f the above possibilities could account for the results by studying the turnover rates o f proteins in R15 cells exposed to the same experimental manipulations as described above. T u r n o v e r rates were determined by first pre-incubating and incubating as above (pulse) and then post-incubating (chasing) in a medium (which contained 1 rnM unlabeled leucine) for various time periods (up to 20 h). Analysis o f control R15 neurons treated in this manner demonstrated a very rapid (ca. 2 h half-time) decay rate o f only one g r o u p o f labeled proteins, i.e., the 12,1300 dalton class. This decay rate
318
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was e i t h e r u n c h a n g e d o r d e c r e a s e d (in the case o f d o p a m i n e t r e a t m e n t ) u n d e r the v a r i o u s e x p e r i m e n t a l c o n d i t i o n s tested I G a i n e r and B a r k e r . in press~
Therefore.
t h e r e g u l a t o r y effects o b s e r v e d in these e x p e r i m e n t s are p r o b a b l y n o t d u e to a l t e r e d c a t a b o l i c rates b u t m o s t likely r e p r e s e n t c h a n g e s in synthesis rates. T h a t the p r o t e i n class w h i c h c o u l d be m o d u l a t e d in its synthesis also had a high t u r n o v e r rate m a ? be m o r e t h a n c o i n c i d e n c e . It is p o s s i b l e t h a t in relatively s h o r t - t e r m e x p e r i m e n t s such as these, o n l y t h o s e p r o t e i n s w i t h high t u r n o v e r rates will be significantl~ m o d i f i e d by s y n a p t i c i n p u t ~or will be m o d i f i e d sufficiently to be d e t e c t e d by c u r r e n t a n a l y t i c a l m e t h o d s ) . In this regard, the fact t h a t n o n e o f t h e classes o f n e w l y s y n t h e s i z e d proteins in the g i a n t n e u r o n R2 h a d high t u r n o v e r rates ~, w o u l d lead us to suggest t h a t p r o t e i n m e t a b o l i s m in this n e u r o n s h o u l d be relatively r e s i s t a n t to influence by s y n a p t i c input. I n d e e d . the e x p e r i m e n t s o f W i l s o n a n d B e r r y ~'~ s h o w e d t h a t relatively p r o l o n g e d s t i m u l a t i o n o f e x c i t a t o r y s y n a p t i c i n p u t to the A p l y s i a grant n e u r o n R~ p r o d u c e d n o c h a n g e s in the p a t t e r n s o f p r o t e i n m e t a b o l i s m in this cell. It is. t h e r e f o r e , possible that in relatively s h o r t - t e r m e x p e r i m e n t s , s y n a p t i c r e g u l a t i o n o f n e u r o n a l p r o t e i n m e t a b o l i s m m a y be c o n f i n e d e i t h e r to the i n d u c t i o n o f n e w p r o t e i n s 12,In. or to the m o d u l a t i o n o f the o n g o i n g synthesis o f specific p r o t e i n s with relatively r a p i d t u r n o v e r rates.
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