- Email: [email protected]
Repeated electroconvulsive shocks alter the biosynthesis of enkephalin and concentration of dynorphin in the rat brain
Neuropeptides
5: 557460,
1985
REPEATED ELECTROCONVULSIVE SHOCKS ALTER THE BIOSYNTHESIS CONCENTRATION
OF DYNORPHIN
OF ENKEPHALIN AND
IN THE RAT BRAIN
** *** J. S. Hong*, K.,xoshikawa , T. Kanamatsu*, J. F. McGinty , C. L. Mitchell*, and S. L. Sabol Laboratory of Behavioral,and Neurological Toxicology, NIEHS, NIH, Research Triangle Park, NC 27249, Laboratory of Biochemical Genetics: NHLBI, NIH, Bethesda, MD 20205, Department of Anatomy, School of Medicine, East Carolina University, Greenville, NC 27834 ABSTRACT 5Ten daily electroconvulsive shocks (ECSs) caused a two-fold increase in (Met )-enkephalin-like immunoreactivity (ME-LI) and an 80% increase in the level of mRNA coding for preproenkephalin A in the hypothalamus. These observations suggest that repeated ECSs increase the biosynthesis of hypothalamic ME. Ten daily ECSs also increased dynorphin A (1-8)-like immunoreactivity (DN-LI) in hypothalamus (45%) but not in frontal cortex. Unlike other brain regions, a 64% decrease of DN-LI was found in the hippocampus after 10 daily ECSs whereas a significant increase of ME-L1 (40%) was tbserved. Furthermore, immunocytochemical studies revealed an increase of (Leu )-enkephalin-like immunoreactivity in the perforant pathway and a decrease of DN-LI in the mossy fiber system of the hippocampus after 10 daily ECSs. These studies suggest that alterations in enkephalin and dynorphin in the limbic system may contribute to the behavioral changes observed after repeated ECSs. INTRODUCTION ___We have previously reported that repeated electrocoyvulsive shocks (ECSs), eliciting maximal tonic extensor seizures, increase (Met )-enkephalin-like immunoreactivity (ME-LI) in certain limbic areas of rat brain such as septum, amygdala, nucleus accumbens gnd hypothalamus (1). This result suggests the possible involvement of (Met )-enkephalin (ME) in some ECS-elicited behavioral alterations. Nevertheless, changes in ME-L1 after repeated ECSs did not provide information concerning the dynamic changes of ME-containing neurons since an increase in peptide level could be due to an increase in biosynthesis or due Thus, we addressed this question by estimating to a decrease in utilization. the rate of biosynthesis of ME by measuring the level of mRNA coding for the precursor of ME, preproenkephalin A, using both in vitro cell free translation Secondly, we determinedregional brain levels of dyand blot hybridization. norphin A (1-8)-like immunoreactivity {DN-LI) after repeated ECSs in light of recent reports that brain DN-LI is increased in amygdaloid-kindled rats (2) and in rats receiving intra-amygdaloid kainic acid (3). 557
MATERIALS AND METHODS Male Fischer-344 rats (Charles River, Wilmington, MA) weighing 230-250 g were used. ECS was administered with an Edison electroshock apparatus through ear-clip electrodes as described (1). Rats were shocked once per day for 6 to 10 consecutive days with 150 V at 60 Hz for 1 sec., then sacrificed along with control rats by decapitation 24 hr afterlthe last shock. Levels of hypothalamic mRNA were measured by cell-freg traqslation followed by immunoprecipitation32 using antiserum against ME-Arg -phe and Northern blot hybridization using Plabeled rat preproenkephalin A cDNA as described (4). Brain ME-L1 was determined using a radioimmunoassay described previously (5). AnBantiserum against dynorphin A (l-8), which does not crossreact with ME or (Leu )-enkephalin (LE) but crossreacts with dynorphin A (l-13) (0.02%) and dynorphin A (l-17) (O.Ol%), was used in a radioimmunoassay for determining the brain level of DN-LI. For immunocytochemistry, rats were perfused and brains were prepared as described (6). Frozen sections were incubated with antisera to dynorphin-A (1-17) (provided by L. Terenius, Uppsala, Sweden) or LE (provided by R. J. Miller, U. Chicago) followed by avidin-biotin-peroxidase immunoreagents. Antisera characteristics and the staining procedure have been described (6). RESULTS Effect of repeated ECSs on translation activity and mRNA abundance in hypothalamus. Among various brain regions examined, the hypothalamus showed the largest increase in the level of ME-L1 (Table 1) after 10 daily ECSs. This is cot$stant with our previous observation in Sprague Dawley rats (1). After the Slabeled cell-f&ee tyanslated proteins were immunoprecipitated with an antiserum against ME-Arg -Phe , the carboxy-terminus of preproenkephalin A, and electrophoresed, four protein species (Mr 30,000 (major band), 31,000, 22,000 and 20,000 (minor bands) were found to be related to preproenkephaliq A. The average densities of the Mr 30,000 and 20,000 proteins from poly (A) RNA of ECStreated rats were 71 + 6% and 122 + 21% higher, respectively, than those of control rats. The sum of synthesized Mr 30,000 and 22,000 proteins was increased 79 f 8% after repeated ECSs (4). The effect of ECS on the abundance of preproenkephalin A mRNA was+also determined by blot hybridization. Northern blot analysis of poly (A) RNA from the hypothalamus of control and ECS-treated rats revealed that the rat and human preproenkephalin A cDNA probes hybridized with an apparently single mRNA species with an approximate chain length of 1450 bases (4). ECS significantly increased the abundance of preproenkephalin A mRNA in the hypothalamus (4). A subsequent study using dot-blot hybridization showed that 10 daily ECSs caused a 76 + 7% increase in the level of preproenkephalin A mRNA in the hypothalamus (4). Effects of repeated ECSs on the level of dynorphin A (1-8)-like immunoreactivity Similar to the effect in the hypothamus, but not ever, ECS exerted opposite tides in the hippocampus:
on ME-LI, 10 daily ECSs caused an increase in DN-LI in the frontal cortex (Table 1 and reference 1). Howeffects on the concentration of these two opioid pepa 40% increase of ME-L1 and a 64% decrease of DN-LI.
558
Table 1 LEVELS OF DYNORPHIN A (1-8)-LIKE IMMUNOREACTIVITY AND (MET )-ENKEPHALIN-LIKE IMMUNOREACTIVITY OF VARIOUS BRAIN REGIONS AFTER 10 DAILY ECSs Brain Region
Hippocampus Hypothalamus Frontal Cortex
DN-LI (p mole/g wet wt.7 Sham ECS 16.5 + 0.6 29.4 7- 1.1 4.2 E 0.33
5.9 42.7 4.7
% of Control
+ 0.27 + 1.6 + 0.5 -
1;:: 111
Each value is the Mean + S.E.M. of 7 to 10 rats.
ME-L1 (p mole/g wet wt.) Sham ECS ,722 7 852N; 51
*PcO.O05
239 + 9 1660 + 80 ND-
% of Control 139* ,95*
ND = Not determined.
The increase in ME-L1 in the hippocampus of ECS-treated rats correlated with an increase in the intensity of LE-LI in the perforant pathway (Fig. 1A and 8). The decrease in DN-LI correlated with virtual elimination of DN immunostaining in hippocampal mossy fibers (Fig. 1C and D). All hippocampal cells, including granule cells, in ECS-treated rats appeared to be intact and healthy (Fig. D).
Figure 1. Opioid peptide-ir in coronal sections of rat brains one day after cessation of 6 daily ECS treatments. Left = Normal control. Right = ECStreated. A. Normal distribution of enkephalin-ir in entorhinal cortex (ERC) and in perforant path (PP) and mossy fibers (M) of ventral hippocampus. B. Increased intensity of enkephalin-ir in perforant path and mossy fibers (M) in an ECS-treated rat (level slightly caudal to that of A). C. Normal distribution of dynorphin-ir in hippocampal mossy fibers (M) innervating pyramidal cells (P3). Nissl counterstain. D. Dynorphin-ir in mossy fibers is lost after 6 daily ECS treatments. M = mossy fiber layer. Note integrity of granule (G) and pyramidal (P) cells. Nissl counterstain. 559
DISCUSSION
The present study and a previous report (1) demonstrate that repeated ECSs alter the brain enkephalinergic- and dynorphinergic-, but not B-endorphinergic systems. After 10 daily ECSs, the abundance of hypothalamic preproenkephalin A mRNA was increased by 76-79% as measured by two independent methods, i.e. cellfree translation and dot-blot hybridization (4). This increase in theundance in preproenkephalin A mRNA suggests that the ECS-elicited increase in the hypothalamic level of ME-L1 is due to an increase in the biosynthesis of ME rather than due to a reduction in utilization. Thus, it is possible that repeated ECSs accelerate the turnover of hypothalamic ME-containing neurons. This hypothesis is further supported by our preliminary finding that repeated ECSs reduced both the delta and mu opiate receptor binding sites (Nakata et al., manuscript in preparation) which suggests an increase in the release of opioid peptides following ECS treatment. Repeated ECSs altered the levels of ME-L1 and DN-LI in opposite directions. To our knowledge, this is the first observation where the concentration of these two opioid peptides in the hippocampus was differentially altered. The functional significance of these opposite changes is not clear presently. But, it is interesting to note that hippocampal dynorphin A (1-13) levels are increased in rabbits in which seizures have been produced by intra-amygdaloid injection of kainic acid (3) or amygdaloid kindling (2). The disparity in the seizureinduced changes of dynorphin levels by various treatments may provide an important clue for further understanding the role of this opioid peptide in relation to the seizure phenomenon. REFERENCES 1. 2. 3. 4. 5.
6.
Hong J, Gillin C, Yang H, Costa E. (1979) Repeated electroconvulsive shocks and the brain content of endorphins. Brain Research 177, 273-278. Przewlock: R, Lason W, Stach R, Kaez D. (1983) Opioid peptides, particularly dynorphin, after amygdaloid-kindled seizures. Regulatory Peptides 6, 385-392. Lason W, Przewlock: B, Stala L, Przewlock: R. (1983) Changes in hippocampal immunoreactive dynorphin and neoendorphin content following intraamygdalar kainic acid-induced seizures. Neuropeptides 3, 399-404. Yoshikawa K, Hong J, Sabol S. (1984) Electroconvulsive shock increases preproenkephalin messenger RNA abundance in rat hypothalamus. Proceedings of the National Academy of Sciences USA (in pressj. Hong J, Yang H, Fratta W, Costa E. (1978) Rat striatal methionine-enkephalin content after chronic treatment with cateleptogenic and non-cateleptogenic antischizophrenic drugs. Journal of Pharmacology and Experimental Therapeutics 205, 141-147. McGinty J, Henriksen S, Goldstein A, Terenius L, Bloom F. (1983) Dynorphin is contained within hippocampal mossy fibers: Immunochemical alterations after kainic acid administration and colchicine-induced neurotoxicity. Proceedings of the National Academy of Sciences USA 80, 589-593.
560
5: 557460,
1985
REPEATED ELECTROCONVULSIVE SHOCKS ALTER THE BIOSYNTHESIS CONCENTRATION
OF DYNORPHIN
OF ENKEPHALIN AND
IN THE RAT BRAIN
** *** J. S. Hong*, K.,xoshikawa , T. Kanamatsu*, J. F. McGinty , C. L. Mitchell*, and S. L. Sabol Laboratory of Behavioral,and Neurological Toxicology, NIEHS, NIH, Research Triangle Park, NC 27249, Laboratory of Biochemical Genetics: NHLBI, NIH, Bethesda, MD 20205, Department of Anatomy, School of Medicine, East Carolina University, Greenville, NC 27834 ABSTRACT 5Ten daily electroconvulsive shocks (ECSs) caused a two-fold increase in (Met )-enkephalin-like immunoreactivity (ME-LI) and an 80% increase in the level of mRNA coding for preproenkephalin A in the hypothalamus. These observations suggest that repeated ECSs increase the biosynthesis of hypothalamic ME. Ten daily ECSs also increased dynorphin A (1-8)-like immunoreactivity (DN-LI) in hypothalamus (45%) but not in frontal cortex. Unlike other brain regions, a 64% decrease of DN-LI was found in the hippocampus after 10 daily ECSs whereas a significant increase of ME-L1 (40%) was tbserved. Furthermore, immunocytochemical studies revealed an increase of (Leu )-enkephalin-like immunoreactivity in the perforant pathway and a decrease of DN-LI in the mossy fiber system of the hippocampus after 10 daily ECSs. These studies suggest that alterations in enkephalin and dynorphin in the limbic system may contribute to the behavioral changes observed after repeated ECSs. INTRODUCTION ___We have previously reported that repeated electrocoyvulsive shocks (ECSs), eliciting maximal tonic extensor seizures, increase (Met )-enkephalin-like immunoreactivity (ME-LI) in certain limbic areas of rat brain such as septum, amygdala, nucleus accumbens gnd hypothalamus (1). This result suggests the possible involvement of (Met )-enkephalin (ME) in some ECS-elicited behavioral alterations. Nevertheless, changes in ME-L1 after repeated ECSs did not provide information concerning the dynamic changes of ME-containing neurons since an increase in peptide level could be due to an increase in biosynthesis or due Thus, we addressed this question by estimating to a decrease in utilization. the rate of biosynthesis of ME by measuring the level of mRNA coding for the precursor of ME, preproenkephalin A, using both in vitro cell free translation Secondly, we determinedregional brain levels of dyand blot hybridization. norphin A (1-8)-like immunoreactivity {DN-LI) after repeated ECSs in light of recent reports that brain DN-LI is increased in amygdaloid-kindled rats (2) and in rats receiving intra-amygdaloid kainic acid (3). 557
MATERIALS AND METHODS Male Fischer-344 rats (Charles River, Wilmington, MA) weighing 230-250 g were used. ECS was administered with an Edison electroshock apparatus through ear-clip electrodes as described (1). Rats were shocked once per day for 6 to 10 consecutive days with 150 V at 60 Hz for 1 sec., then sacrificed along with control rats by decapitation 24 hr afterlthe last shock. Levels of hypothalamic mRNA were measured by cell-freg traqslation followed by immunoprecipitation32 using antiserum against ME-Arg -phe and Northern blot hybridization using Plabeled rat preproenkephalin A cDNA as described (4). Brain ME-L1 was determined using a radioimmunoassay described previously (5). AnBantiserum against dynorphin A (l-8), which does not crossreact with ME or (Leu )-enkephalin (LE) but crossreacts with dynorphin A (l-13) (0.02%) and dynorphin A (l-17) (O.Ol%), was used in a radioimmunoassay for determining the brain level of DN-LI. For immunocytochemistry, rats were perfused and brains were prepared as described (6). Frozen sections were incubated with antisera to dynorphin-A (1-17) (provided by L. Terenius, Uppsala, Sweden) or LE (provided by R. J. Miller, U. Chicago) followed by avidin-biotin-peroxidase immunoreagents. Antisera characteristics and the staining procedure have been described (6). RESULTS Effect of repeated ECSs on translation activity and mRNA abundance in hypothalamus. Among various brain regions examined, the hypothalamus showed the largest increase in the level of ME-L1 (Table 1) after 10 daily ECSs. This is cot$stant with our previous observation in Sprague Dawley rats (1). After the Slabeled cell-f&ee tyanslated proteins were immunoprecipitated with an antiserum against ME-Arg -Phe , the carboxy-terminus of preproenkephalin A, and electrophoresed, four protein species (Mr 30,000 (major band), 31,000, 22,000 and 20,000 (minor bands) were found to be related to preproenkephaliq A. The average densities of the Mr 30,000 and 20,000 proteins from poly (A) RNA of ECStreated rats were 71 + 6% and 122 + 21% higher, respectively, than those of control rats. The sum of synthesized Mr 30,000 and 22,000 proteins was increased 79 f 8% after repeated ECSs (4). The effect of ECS on the abundance of preproenkephalin A mRNA was+also determined by blot hybridization. Northern blot analysis of poly (A) RNA from the hypothalamus of control and ECS-treated rats revealed that the rat and human preproenkephalin A cDNA probes hybridized with an apparently single mRNA species with an approximate chain length of 1450 bases (4). ECS significantly increased the abundance of preproenkephalin A mRNA in the hypothalamus (4). A subsequent study using dot-blot hybridization showed that 10 daily ECSs caused a 76 + 7% increase in the level of preproenkephalin A mRNA in the hypothalamus (4). Effects of repeated ECSs on the level of dynorphin A (1-8)-like immunoreactivity Similar to the effect in the hypothamus, but not ever, ECS exerted opposite tides in the hippocampus:
on ME-LI, 10 daily ECSs caused an increase in DN-LI in the frontal cortex (Table 1 and reference 1). Howeffects on the concentration of these two opioid pepa 40% increase of ME-L1 and a 64% decrease of DN-LI.
558
Table 1 LEVELS OF DYNORPHIN A (1-8)-LIKE IMMUNOREACTIVITY AND (MET )-ENKEPHALIN-LIKE IMMUNOREACTIVITY OF VARIOUS BRAIN REGIONS AFTER 10 DAILY ECSs Brain Region
Hippocampus Hypothalamus Frontal Cortex
DN-LI (p mole/g wet wt.7 Sham ECS 16.5 + 0.6 29.4 7- 1.1 4.2 E 0.33
5.9 42.7 4.7
% of Control
+ 0.27 + 1.6 + 0.5 -
1;:: 111
Each value is the Mean + S.E.M. of 7 to 10 rats.
ME-L1 (p mole/g wet wt.) Sham ECS ,722 7 852N; 51
*PcO.O05
239 + 9 1660 + 80 ND-
% of Control 139* ,95*
ND = Not determined.
The increase in ME-L1 in the hippocampus of ECS-treated rats correlated with an increase in the intensity of LE-LI in the perforant pathway (Fig. 1A and 8). The decrease in DN-LI correlated with virtual elimination of DN immunostaining in hippocampal mossy fibers (Fig. 1C and D). All hippocampal cells, including granule cells, in ECS-treated rats appeared to be intact and healthy (Fig. D).
Figure 1. Opioid peptide-ir in coronal sections of rat brains one day after cessation of 6 daily ECS treatments. Left = Normal control. Right = ECStreated. A. Normal distribution of enkephalin-ir in entorhinal cortex (ERC) and in perforant path (PP) and mossy fibers (M) of ventral hippocampus. B. Increased intensity of enkephalin-ir in perforant path and mossy fibers (M) in an ECS-treated rat (level slightly caudal to that of A). C. Normal distribution of dynorphin-ir in hippocampal mossy fibers (M) innervating pyramidal cells (P3). Nissl counterstain. D. Dynorphin-ir in mossy fibers is lost after 6 daily ECS treatments. M = mossy fiber layer. Note integrity of granule (G) and pyramidal (P) cells. Nissl counterstain. 559
DISCUSSION
The present study and a previous report (1) demonstrate that repeated ECSs alter the brain enkephalinergic- and dynorphinergic-, but not B-endorphinergic systems. After 10 daily ECSs, the abundance of hypothalamic preproenkephalin A mRNA was increased by 76-79% as measured by two independent methods, i.e. cellfree translation and dot-blot hybridization (4). This increase in theundance in preproenkephalin A mRNA suggests that the ECS-elicited increase in the hypothalamic level of ME-L1 is due to an increase in the biosynthesis of ME rather than due to a reduction in utilization. Thus, it is possible that repeated ECSs accelerate the turnover of hypothalamic ME-containing neurons. This hypothesis is further supported by our preliminary finding that repeated ECSs reduced both the delta and mu opiate receptor binding sites (Nakata et al., manuscript in preparation) which suggests an increase in the release of opioid peptides following ECS treatment. Repeated ECSs altered the levels of ME-L1 and DN-LI in opposite directions. To our knowledge, this is the first observation where the concentration of these two opioid peptides in the hippocampus was differentially altered. The functional significance of these opposite changes is not clear presently. But, it is interesting to note that hippocampal dynorphin A (1-13) levels are increased in rabbits in which seizures have been produced by intra-amygdaloid injection of kainic acid (3) or amygdaloid kindling (2). The disparity in the seizureinduced changes of dynorphin levels by various treatments may provide an important clue for further understanding the role of this opioid peptide in relation to the seizure phenomenon. REFERENCES 1. 2. 3. 4. 5.
6.
Hong J, Gillin C, Yang H, Costa E. (1979) Repeated electroconvulsive shocks and the brain content of endorphins. Brain Research 177, 273-278. Przewlock: R, Lason W, Stach R, Kaez D. (1983) Opioid peptides, particularly dynorphin, after amygdaloid-kindled seizures. Regulatory Peptides 6, 385-392. Lason W, Przewlock: B, Stala L, Przewlock: R. (1983) Changes in hippocampal immunoreactive dynorphin and neoendorphin content following intraamygdalar kainic acid-induced seizures. Neuropeptides 3, 399-404. Yoshikawa K, Hong J, Sabol S. (1984) Electroconvulsive shock increases preproenkephalin messenger RNA abundance in rat hypothalamus. Proceedings of the National Academy of Sciences USA (in pressj. Hong J, Yang H, Fratta W, Costa E. (1978) Rat striatal methionine-enkephalin content after chronic treatment with cateleptogenic and non-cateleptogenic antischizophrenic drugs. Journal of Pharmacology and Experimental Therapeutics 205, 141-147. McGinty J, Henriksen S, Goldstein A, Terenius L, Bloom F. (1983) Dynorphin is contained within hippocampal mossy fibers: Immunochemical alterations after kainic acid administration and colchicine-induced neurotoxicity. Proceedings of the National Academy of Sciences USA 80, 589-593.
560