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Home , Dynorphin, Enkephalin, Neoendorphin

The Journal of Neuroscience March 1966, 6(3): 644-649

Dynorphin- and -like lmmunoreactivity is Altered in Limbic- Regions of Rat Brain After Repeated Electroconvulsive Shock’

T. Kanamatsu, J. F. McGinty,* C. L. Mitchell, and J. S. Hong Laboratory of Behavioral and Neurological Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, and *Department of Anatomy, School of Medicine, East Carolina University, Greenville, North Carolina 27834

In an attempt to determine whether the derived thalamus, whereas the level of hypothalamic /3-endorphin is from participate in the effects of electroconvulsive unaltered (Hong et al., 1979). Our recent study, using in vitro shock (ECS), we used radioimmunoassay and immunocyto- cell free translation or blot hybridization with a rat preproen- chemistry to measure -like immunoreactivity (DN-LI) kephalin A cDNA clone to estimatethe level of mRNA coding in various rat brain regions after repeated ECS treatments. Ten for preproenkephalin A, suggestedthat the increasein hypo- daily ECSs caused a significant increase in (1-8)- thalamic ME-L1 after repeated ECS is due to an increasein LI in most limbic-basal ganglia structures, including hypothal- biosynthesis(Yoshikawa et al., 1985).These observations raise amus (50%), (30%), and septum (30%). No significant the possibility that an increasein enkephalinergicneuronal ac- change was found in the frontal cortex or the neurointermediate tivity is related to ECS-elicited phenomena. lobe of the pituitary. In contrast, 10 ECS treatments depleted It was recently reported that the level of hippocampal dy- DN-LI in hippocampal mossy fibers by 64%. A detailed time- norphin is increasedin amygdaloid-kindled rabbits (Przewlocki course study revealed that a single shock caused a small but et al., 1983)and in rats receiving intra-amygdaloid kainic acid significant increase in hippocampal DN-LI, whereas three con- (Lason et al., 1983). These observations suggestthat, like en- secutive shocks depleted DN-LI by 30%. The maximal decrease kephalin, the metabolic stateof dynorphin storedin hippocam- in DN-LI was reached after six daily ECSs. The level of DN- pal neuronscan changein responseto seizureactivities. In light LI in the partly recovered, but remained lower of these findings, and in an attempt to further examine the than the control value 4, 7, and 14 d after the cessation of six possiblerole of specific dynorphin-containing pathways in re- daily ECSs (SO, 77, and 83% of control value, respectively). In lation to convulsions, we determined the regional brain levels contrast with the ECS-induced depletion of hippocampal dy- of dynorphin A (1-8)-like immunoreactivity (DN-LI) by ra- norphin, 10 daily ECSs caused a significant increase (40%) in dioimmunoassay(RIA), and by changesin immunostainingin- (Mets)-enkephalin-LI in the hippocampus, as well as in other tensity of dynorphin A (l-17)- and -containing limbic-basal ganglia structures. Immunocytochemistry revealed ,using immunocytochemistry (ICC) after repeatedECS. that enkephalin-11 was increased in the perforant pathway, which is presynaptic to the dynorphin-containing mossy fiber Materials and Methods pathway in the hippocampus. These observations suggest that different mechanisms may regulate these two opioid sys- Animals tems in the hippocampus. Moreover, the robust changes of en- Male Fischer 344 rats (Charles River, Wilmington,MA) between10 kephalin and dynorphin in several limbic regions elicited by and 12 weeks of age were used. They were housed, four to a cage,in a repeated ECS suggest that these opioid peptides are associated colony room with a 12 hr light-dark cycle, andmaintained at 21 + 2°C with the behavioral alterations seen in ECS-treated animals. and 50 f 10% humidity. Treatment of animals It has been suggested that endogenous opioid peptides may me- diate electroconvulsive shock (ECS)-elicited behavioral alter- ECS was performed daily using a machine manufactured by Biological ations, such as analgesia, retrograde amnesia, changes in seizure Research Apparatus (Comecio-Varesa, Italy). The current producing maximal tonic and clonic convulsions was delivered to rats via an ear- threshold, or postictal depression (Carrasco et al., 1982; Hola- clip electrode with the following parameters: 85 mA (unless otherwise day and Belenky, 1980; Tortella and Cowan, 1982; Urea et al., specified), 50 Hz, 1 msec pulse interval for a total duration of 1 sec. 198 1, 1983). This notion is supported by our previous report Sham control rats were treated identically, except that they received 0 that repeated ECS increases the (Mets)-enkephalin-like immu- mA. With the exception of the study represented in Figure 3, rats were noreactivity (ME-LI) of certain limbic areas of the rat brain, decapitated for RIA or perfused for ICC (see below) 24 hr after the last such as the septum, amygdala, , and hypo- shock. Radioimmunoassay Received Feb. 7, 1985; revised June 10, 1985; accepted June 11, 1985. Brain regions were dissected according to the method of Glowinski and We thank Drs. Lars Terenius, Richard Miller, and Eckard Weber for their Iversen (1966). The tissue levels of ME-L1 were determined by RIA antisera. Jacqueline Loesche, Kent Slemmons, Johnny Obie, and Billy Scarlett provided excellenttechnical assistance.The secretarialassistance ofLoretta Moore methods as described previously (Hong et al., 1978). In brief, tissue was is greatly appreciated. homogenized in 2 M acetic acid and immersed in boiling water for 5 Correspondenceshould be addressed to J. S. Hong. min. After centrifuging at 25,000 x g for 20 min, the supematant fluid ’ Preliminary results were presented in part at the International Re- was lyophilized. The residue was then reconstituted in RIA buffer so- searchConference, Cambridge, England, 1984. lutions and aliquots were used for RIA. The specificity of the antiserum Cop-fight 0 1986 Society for Neuroscience 0270-6474/86/030644-06$02.00/O against ME has been described in a previous report (Hong et al., 1978).

644 The Journal of Neuroscience Effects of ECS on Brain Systems 645 CONTROL

Figure 1. Dynorphin-like immunoreactivity (DN-LI) in coronal sections of limbic and basal ganglia regions 1 d after six daily ECS treatments (right) or in sham-controls (lefi). A and B, Dynorphin B-L1 in hippocampal mossy fibers (M) of the dentate hilus. Repeated ECS virtually eliminated DN-LI in this pathway. x 27. C and D, Dynorphin A (l-17)-LI in the ventral pallidum (VP) under the anterior commissure (AC) increased after repeated ECS. x 103 (C), x86 (D). E and F, Dynorphin B-L1 in cells and fibers of the medial basal increased after repeated ECS. x 98 (0, x 104 (F). G and H, Dynorphin A (l-l 7)-LI in the zona reticulata (SNzr) increased after repeated ECS. x 4 1. 646 Kanamatsu et al. Vol. 6, No. 3, Mar. 1986

x *

Intact - - l-l cant. Sham .- ECS 20 40 85 I IO I 3 6 IO EC.5 Intensity (mA) Days Fig-we 2. Hippocampal dynorphin (1-8)-like immunoreactivity 1 d Figure 4. Hippocampal (Mets)-enkephalin-like immunoreactivity 1 d after six dailyECSs with different shock intensities. Each value is the after 1, 3, 6, and 10 consecutive daily ECSs. Each value is the mean mean +SEM of eiaht rats. ANOVA (F = 20.5. df= 3.28. D < 0.001). +SEM of eight rats. ANOVA (F = 6.66, df= 6,49, p < 0.005). *p < *p < 0.005, compared to the 0 mA group. ” - 0.05, compared to sham-control groups.

An antiserum to dynorphin A (l-8) that does not cross-react with ME or (LetP)-enkenhalin, but does cross-react somewhat with dynornhin A but 10% cross-reactivity to (Mets)-enkephalin), followed by avidin- (l-,13) (6.02%) and dynorphin A (l-l 7) (O.Ol%), was used-in RIA for biotin-peroxidase immunoreagents as described (McGinty et al., 1983). determinine the brain level of DN-LI. The detailed nrocedures for the The third section in a series was stained with Richardson’s methylene RIA of dyn&phin A (l-8) were identical to those used for fi-endorphin blue-azure II Nissl stain (Richardson et al., 1960). In some experiments, described in a previous report (Hong et al., 1983). In brief, lyophilized dynorphin B antiserum (provided by E. Weber, Stanford University) or tissue extracts were reconstituted in RIA buffer solution (0.02 M phos- the dynorphin A (l-8) antiserum used in RIA was used instead of the phate buffer containing 0.15 N NaCl, 0.01% (wt/vol) BSA, 0.1% (wt/ dynorphin A (l-l 7) antiserum, or the (Mets)-enkephalin antiserum used vol) gelatin, and 0.1% Triton X- 100). Y-dynorphin A (l-8) was used in RIA was employed instead of @us)-enkephalin antiserum. However, as a tracer. Free and bound 1Z51-dynorphin A (l-8) were separated by the dynorphin A (l-8) and (Mets)-enkephalin antisera were not as good charcoal suspension. The sensitivity of this RIA is -5 fmol. for ICC as they were for RIA. Immunocytochemical method Statistics Rats were perfused with a 4% solution of paraformaldehyde, and brains A one-way analysis of variance (Winer, 1962) was used to test for overall were prepared for ICC as previously described (see McGinty et al., statistical significance: If a significant overall effect of treatment was 1983). Three serial 50 urn frozen sections were cut at 400 urn intervals observed, post hoc comparisons between group means were made using fiom’the frontal pole through the brain stem. Two out of three adjacent Fisher’s least significant difference test (Miller, 1966). Where only two sections in a series were incubated with an antiserum to dynorphin A groups were compared (Table l), a two-tailed Student’s t test (Winer, (l-l 7) (provided by L. Terenius, Uppsala, Sweden; no detectable cross- 1962) was used to test for statistical significance. A significance of p < reactivity to cr-neoendorphin, (LetP)- or (Mets)-enkephalin, /3-endor- 0.05 was required for rejection of the null hypothesis. phin, ordynorphin A (l-8), and 2%.cross-reactivity with dynorphin A (1-13)) or (LetP)-enkephalin (LE) (which was provided by R. J. Miller, Results University of Chicago; no detectable cross-reactivity to p-endorphin, Dynorphin-like immunoreactivity in various brain * regions after 10 daily ECSs T Ten daily ECSscaused a significant increasein the level of DN- LI (l-8) in hypothalamus, septum, striatum, brain stem, and , but not in frontal cortex or neurointermediate lobe of the pituitary (Table 1). However, the most conspicuouschange in DN-LI (l-8) after ECS occurred in the hippocampus:a 64% m reduction compared with the sham-control group (Table 1). There was a correspondingdecrease of dynorphin A (l-l 7)-like immunoreactivity (55%) in the hippocampus of ECS-treated rats (data not shown). In accordance with the RIA data, ICC revealed increasedintensity of dynorphin A (l-l 7) or dynorphin B immunostaining in hypothalamus, dorsal and ventral stria- turn, ventral pallidurn, and substantia nigra (zona reticulata; Fig. 1), aswell asof the central nucleusof the amygdala,olfactory tubercle, entopeduncular nucleus, and periaqueductalgray. In the hippocampus,DN-LI in the mossy fibers was virtually de- intact pleted (Fig. 1, A and B) in the absenceof any detectablecellular Cont. I 3 6 IO damage(Hong et al., 1985). Days Hippocampal DN-LI (l-8) 24 hr after six daily ECSs with Figure 3. Hippocampal dynorphin A (1-8)-like immunoreactivity 1 d after 1, 3, 6, and 10 daily ECSs. Each value is the mean +SEM of d@$erentshock intensities eight rats. ANOVA (F = 82.8, df = 6,49, p < 0.001). *p < 0.05, **p < To determine whether the ECS-inducedalteration in hippocam- 0.0 1, compared to sham-control groups. pal DN-LI was related directly to the seizure intensity or to a The Journal of Neuroscience Effects of ECS on Brain Opioid Peptide Systems 647 CONTROL

Figure 5. (Leus)-enkephalin-like immunoreactivity (LE-LI) in coronal sections of limbic and basal ganglia regions 1 d after six daily ECS treatments (right) or in sham-controls (Zeft). A and B, LE-LI in the lateral perforant pathway (PP) and mossy fibers (M) of the ventral hippocampus. Repeated ECS increased LE immunostaining, particularly in the perforant path. x 100. C and D, LE-LI in the dorsal striatum (OS) and ventral pallidum (VP) is increased by repeated ECS. AC = anterior commissure. x 35. E and F, LE-LI in the hypothalamus is increased after repeated ECS. F = fomix, ZZZ= third ventricle. x 22. G and H, LE-LI in the dorsal striatum, globus pallidus (GP), and central nucleus (C’) of the amygdala (Ace) is increased after repeated ECS. x 54 (G), x 48 (ZZ). 646 Kanamatsuet al. Vol. 6, No. 3, Mar. 1986

Hippocampal ME-LI 24 hr after I, 3, 6, and 10 daily EC.9 Previously, we reported that repeatedECS increasesME-L1 in several limbic brain regions (Hong et al., 1979). In order to comparethe changesin DN-LI with ME-L1 in the hippocampus, levels of hippocampal ME-L1 from the animals representedin Figure 3 were determined. One or three daily ECSsfailed to alter the level of ME-LI. However, there was a significant in- creasein the levels of this peptide after six and 10 daily ECSs (Fig. 4). ICC revealed that the greatestincrease in hippocampal LE-LI or ME-L1 occurred in the lateral perforant pathway (Fig. 5, A and B). LE-LI or ME-L1 in the mossyfibers was not depleted but showedless consistent increases than had occurred in the perforant pathway. In all other limbic and basalganglia regions examined, ME-L1 or LE-LI was increased.These structures in- cluded the dorsal and ventral striatum, ventral pallidum, hy- pothalamus, and amygdala (Fig. 5, C-H). Days After the Last ECS Hippocampal DN-LI at various time periods after the Fi,qure 6. Hippocampal dynorphin A (l-@-like immunoreactivity at completion of six consecutivedaily ECSs various time periods after the end of six consecutive daily ECSs. Each To determine whether the level of hippocampal DN-LI, after value is the mean KSEM of eight rats. ANOVA (F = 24.1, df= 4,35, repeatedECS, recovered over time, DN-LI was measuredat 1, p < 0.001). *p < 0.05, **p < 0.01, ***p < 0.005, compared to intact 4, 7, and 14 d after the completion of six consecutive daily control groups. shocks.Four days after the shock treatments, the level of DN- LI remained approximately the same as after 1 d of ECS. There was a significant recovery of the level of DN-LI after 7 nonspecific stressfulreaction elicited by the shock treatments, d (77% of the control). However, by day 14, DN-LI was still various shockintensities were administered to different groups lower than the control value (83% of control; Fig. 6). Immu- of rats. Six daily shocksof a subconvulsive intensity (20 mA) nostaining of DN-LI, LE-LI, or ME-L1 in all brain regionsof failed to alter the hippocampal levels of DN-LI. RepeatedECS ECS-treated rats did not differ from that in control brains 7 d with a threshold current of 40 mA, which produced clonic and after the cessationof treatments (data not shown). tonic convulsions, elicited a degreeof reduction in the hippo- campal DN-LI similar to that produced by shockswith a su- Discussion pramaximal intensity (85 mA) (Fig. 2). This study demonstratesthat the metabolic activity of brain dynorphin-containing neurons,like the activity of enkephalin- containing neurons(Hong et al., 1979) changesin responseto HippocampalDN-LI (l-8) 24 hr after 1, 3, 6, and the seizure activity induced by repeated ECS. The opioid neu- 10 daily ECSs rons that respondto ECS-inducedseizure activity appearto be Sincerepeated ECS exerted a robust decreasein DN-LI in the contained largely within limbic-basal gangliastructures that are hippocampus,a detailed study of the time coursewas performed highly interconnected(Nauta, 1979).Changes in opioid peptide to determinethe minimal number of shocksnecessary to induce metabolism in limbic and basal ganglia pathways subsequent the maximal depletion. There was a small but significant in- to ECS may reflect primary involvement in ictal and/or postictal creasein hippocampalDN-LI 24 hr after a singleECS. However, eventsor secondaryinvolvement in ECS-inducedanalgesia, cat- the level of DN-LI started to decrease(by 30%) after three alepsy, , respiratory depression,or mental con- consecutive daily ECSs. The maximal reduction (60-70%) in fusion (Carrascoet al., 1982; Holaday and Belenky, 1980;Tor- the concentration of this opioid peptide was reachedafter six tella and Cowan, 1982; Urea et al., 1981). daily ECSsand did not decreasefurther after 10 shocks(Fig. 3). The most dramatic changein opioid levels after repeatedECS

Table 1. Dynorphin (l-@-like immunoreactivity of various rat brain regions after 10 daily EC,%

Dynorphin (1-8)-like immunore- activity (pmobgm tissue wt f SEM) Percent of Brain region Sham-control ECS control Hippocampus 16.5 + 0.6 5.9 f 0.2*** 36 Hypothalamus 29.4 + 1.1 42.7 k 1.6*** 145 Septum 11.5 f 0.8 15.0 & 0.9** 130 Striatum 55.2 f 1.7 70.1 +- 0.2*** 127 and pons 14.9 lr 0.5 17.1 + 0.5** 115 Spinal cord 5.8 +- 0.2 6.7 + 0.4* 115 Frontal cortex 4.2 k 0.3 4.7 + 0.5 111 Pituitary (neurointermediate lobe) 2.9 k 0.2 2.6 f 0.2 90

Each value is the mean + SEM of 7-10 rats. *p < 0.05, **p i 0.01, ***p < 0.001. a pmol/lobe. The Journal of Neuroscience Effects of ECS on Brain Opioid Peptide Systems 649 occurred in hippocampal mossy fibers, in which DN-LI de- References creasedby 70%. In the perforant pathway, which is presynaptic Carrasco,M. A., R. D. Dias,and I. Izquierdo (1982) reverses to dentate granule cells and their axons (the mossyfibers), LE- retrograde amnesia induced by electroconvulsive shock. Behav. Neur- LI/ME-LI increasedsignificantly. The perforant and mossyfiber al Biol. 34: 352-357. pathways are the first two links in a trisynaptic excitatory chain Frenk, H. (1983) Pro- and anticonvulsant actions of and through the hippocampal formation. The opposite effects that the endogenous : Involvement and interactions of multiple are exerted by ECS on the concentration of enkephalin and and non-opiate systems. Brain Res. Rev. 6: 197-2 10. dynorphin peptidesin thesetwo pathways suggestthat different Gall. I C.. I N. Brecha. I H. J. Karten. and K. J. Ghana (198_ 1)I Localization mechanismsare operative in regulating their metabolismin the of enkephalin-like immunoreactivity to ide&fied axonal and neu- ronal populations of the rat hippocampus. J. Comp. Neurol. 198: hippocampussubsequent to seizureactivity. In fact, physiolog- 335-350. ical actions of enkephalinsand in the hippocampus Glowinski, J., and L. L. Iversen (1966) Regional studies of catechol- may be quite different. For example, exogenouslyadministered amines in the rat brain. I. The disposition of ‘H-, ‘H- (and morphine) are uniquely excitatory in the hip- , and )H-DOPA in various regions of the brain. J. Neuro- pocampus(Nicoll et al., 1977) where they display potent con- them. 13: 655-669. vulsant or anticonvulsant actions, depending on the circum- Henriksen, S. J., G. Chouvet, J. F. McGinty, and F. E. Bloom (1982) stances(Frenk, 1983). In contrast, dynorphin does not exert Opioid peptides in the hippocampus: Anatomical and physiological convulsant effectsand is lessconsistently excitatory in the hip- considerations. Ann. NY Acad. Sci. 398: 207-220. pocampus(Henriksen et al., 1982). Holaday, J. W., and G. L. Belenky (1980) Opiate-like effects of elec- troconvulsive shock in rats: A differential effect of naloxone on no- The mechanismunderlying the decreaseof hippocampalDN- ciceutive measures. Life Sci. 27: 1929-l 938. LI is not clear. Since it is possiblethat dentate granule cells are Hong,J. S., H.-Y. T. Yang, W. Fratta, and E. Costa (1978) Rat striatal susceptibleto insults from seizureactivities, we consideredthe (Mets)-enkephalin content after chronic treatment with cataleptogenic possibility that the reduction in DN-LI in the mossy fibers re- and non-cataleptogenic antischizophrenic drugs. J. Pharmacol. Exp. sultedfrom degenerationof the dentate granule cells. However, Ther. 205: 141-147. light-microscopic histologicalevaluation indicated that the den- Hong, J. S., J. C. Gillin, H. Y. T. Yang, and E. Costa (1979) Repeated tate granulecells appeared to be intact after six daily ECSs(Hong electroconvulsive shocks and the brain content of enkephalins. Brain et al., 1985). Furthermore, the time-course study showed a Res. 177: 273-278. gradual, although partial, recovery of DN-LI in the hippocam- Hong, J. S., K. Yoshikawa, and R. W. Hendren (1983) Measurement of fl-endorphin and enkephalins in biological tissues and fluids. In pus within 2 weeks after the cessationof ECS (Fig. 6). These Methods in Enzymology, Vol. 103, M. Conn, ed., pp. 547-564, Ac- observationsdemonstrate that the decreaseof hippocampalDN- ademic, New York. LI after repeatedECS is not due to degenerationof the dentate Hong, J. S., K. Yoshikawa, T. Kanamatsu, J. F. McGinty, C. L. Mitchell, granulecells. Alternatively, the reduction of DN-LI may be due, and S. L. Sabol (1985) Repeated electroconvulsive shocks alter the either to a reduction in biosynthesis,or to an increasein release. biosynthesis of enkephalin and concentration of dynorphin in the rat We are currently developinga blot hybridization method, using brain. 5: 557-560. cDNA coding for prodynorphin, for quantitating the level of Lason, W., B. Przewlocka, L. Stala, and R. Przewlocki (1983) Changes prodynorphin mRNA as an index for the biosynthesisof this in hippocampal immunoreactive dynorphin and con- peptide. tent following intra-amygdalar kainic acid-induced seizures. Neuro- peptides 3: 399-404. -derived peptidesare present both in the per- McGinty, J., S. Henriksen, A. Goldstein, L. Terenius, and F. Bloom forant path and, to a lesserextent, in mossyfibers (Gall et al., (1983) Dynorphin is contained within hippocampal mossy fibers: 1981; McGinty et al., 1983). The enkephalin-like immuno- Immunochemical alterations after kainic acid administration and col- reactivity in the perforant pathway after repeatedECS wascon- chicine-induced neurotoxicity. Proc. Natl. Acad. Sci. USA 80: 589- sistently enhanced,whereas the enkephalin-like immunoreac- 593. tivity in the mossy fiber pathway was lessobviously and less Miller, R. G. (1966) Simultaneous Statistical Inference, McGraw-Hill, consistently increased.This observation leaves open the pos- New York. sibility that repeated ECS may also alter the metabolism of Nauta, H. J. W. (1979) A proposed conceptual reorganization of the enkephalin differently in thesetwo pathways. The mechanism basal ganglia and telencephalon. Neuroscience 4: 1875-l 88 1. Nicoll, R., G. S&ins, N. Ling, F. Bloom, and R. Guillemin (1977) underlying the increasein hippocampal ME-L1 is not known at Neuronal actions of and enkephalins among brain regions: present.However, in view of the reported increasein the level A comparative microiontophoretic study. Proc. Natl. Acad. Sci. USA of mRNA coding for proenkephalin in the hypothalamus after 74: 2584-2588. repeatedECS (Yoshikawa et al., 1985), it is likely that the Przewlocki, R., W. Lason, R. Stach, and D. Katz (1983) Opioid pep- ECS-elicited increasein hippocampal ME-L1 is due to an in- tides, particularly dynorphin, after amygdaloid-kindled seizures. Reg. creasein the biosynthesisof this peptide. Experiments are cur- Peptides 6: 385-392. rently being conducted to examine this possibility. Richardson, K. D., L. Jarret, and E. H. Finke (1960) Embedding in This study, and our previous reports (Hong et al., 1979, 1985; epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 35: 3 13-323. Yoshikawa et al., 1985), provide neurochemicalevidence that Tortella, F. C., and A. Cowan (1982) EEG, EMG and behavioral the metabolismof opioid peptidesderived from prodynorphin evidence for the involvement of endorphin systems in postictal events and proenkephalinchanges in responseto repeatedECS. To our after electroconvulsive shock in rats. Life Sci. 31: 881-888. knowledge,this is the first observation that the samemetabolic Urea, G., J. Yitzhaki, and H. Frenk (1981) Different opioid systems perturbation can differentially alter the concentration of pep- may participate in post electroconvulsive shock (ECS) analgesia and tides derived from thesetwo opioid families. This differential catalepsy. Brain Res. 219: 385-396. effect was unique to the hippocampus;in all other affected re- Urea, G., A. Nof, B. A. Weissman, and Y. Same (1983) Analgesia gionsof the brain, seizureactivity led to increasedlevels of both induced by electroconvulsive shock: Brain enkephalins may mediate opioid peptide families. The disparity in the seizure-induced tolerance but not the induction of analgesia. Brain Res. 260: 271- 277, changesin ME-L1 and DN-LI in the hippocampusmay provide Winer, B. J. (1962) Statistical Principles in Experimental Design. an important clue for further understandingthe roles of these McGraw-Hill, New York. opioid peptidesin relation to seizure-elicited behavioral alter- Yoshikawa, K., J. S. Hong, and S. L. Sabol (1985) Electroconvulsive ations,such as changein the seizurethreshold or lossof memory shock increases preproenkephalin messenger RNA abundance in rat (Carrascoet al., 1982; Tortella and Cowan, 1982). hypothalamus. Proc. Natl. Acad. Sci. USA 82: 389-593.