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Regulation of prodynorphin gene expression in the hippocampus by glucocorticoids
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Regulation of prodynorphin gene expression in the hippocampus by glucocorticoids Linda Thai
a,b Paul H.K.
Lee "'*, Jack Ho a, Harold Suh a and John S. Hong
~
a Laboratory of Molecular and IntegratiL,e Neuroscience, National Institutes ofEnt, ironmental Health Sciences, Research Triangle Park. NC 27709 (USA) and t, Curriculum in Neurobiology, Unit:ersity of North Carolina, Chapel Hill. NC 27514 (USAi (Accepted 26 May 1992)
Key words." Adrenalectomy; Dexamethasone; Hippocampus; Dynorphin; Enkephalin; Gene regulation; Opioid peptide; Steroid hormone
The regulation of prodynorphin gene expression by glucocorticoids in the hippocampus was examined in rats that were adrenalectomized (ADX) either 7, 30, 60 and 90 days prior to sacrifice. Peptide levels in the hippocampus of ADX rats were determined by radioimmunoassay and immunocytochemistry. Prodynorphin (PDYN) mRNA was measured by Northern blot analysis and in situ hybridization. A time-dependent decrease in dynorphin A(I-8) (DYN) levels in the hippocampus (18% at 7 days; 44% at 30 days; 58% at 60 days) of ADX rats was found, which was accompanied by a comparable decrease in the abundance of PDYN mRNA. An in situ hybridization analysis revealed that both the number of positively hybridized cells and the number of silver grains per cell were decreased in the dentate gyrus after ADX. The administration of dexamethasone after surgery reversed the peptide and m R N A attenuation induced by ADX. ADX had no effect on the expression of proenkephalin mRNA or [MetS]-enkephalin immunoreactivity in the hippocampus. Examination of thionin-counterstained tissue showed that the dentate granule cell layer was intact. The decrement of DYN expression in this system is proposed to have resulted from the removal of glucocorticoid input and not dentate granule cell loss. This study provides the strong evidence for a differential susceptibility of these two opioid peptides in the hippocampus to the removal of glucocorticoids. In addition, these data provide support for a potentially selective, glucocorticoidpermissive component in PDYN gene expression.
INTRODUCTION Within the hippocampal formation, dentate granule ceils serve to restrict or amplify signals that originate in extrahippocampal sites and propagate into the hippocampus proper 12. It is also known that dynorphin (DYN), an endogenous opioid peptide, is synthesized by dentate granule cells, and is stored in and released by their axons, the mossy fibers 1'4'14'23. The expression of DYN has been demonstrated to be negatively regulated by glutamatergic transmission. For example, during the normal process of aging, when afferent perforant path fibers are sparse and glutamatergic synaptic activity is lessened, DYN immunoreactivity in the mossy fiber axon terminals has been shown to be increased 9'29. This peptide up-regulation can also be demonstrated by lesioning the perforant path input to the dentate granule cells 22. In contrast, stimulation of
the perforant path causes an increase in glutamate release and suppresses the expression of prodynorphin (PDYN) mRNA 25. Furthermore, perforant path stimulation-induced down-regulation of PDYN mRNA was prevented by pretreatment with a glutamate receptor blocker, y-D-glutamylglycine 26. Thus, DYN regulation in the hippocampus has consistently been shown to be modulated by glutamatergic transmission. The hippocampus appears to be the principal neural target for glucocorticoids because it contains a high concentration of both type I and type II corticosteroid receptors 16. Glucocorticoids, secreted by the adrenal cortex under chronic physiological stress, are capable of damaging or destroying hippocampal pyramidal neurons by sensitizing them to various metabolic insults 1~. Glucocorticoid-induced damage to the hippocampal pyramidal cells is similar to the cell loss during aging, yet the mechanisms underlying the neurotoxic effect of
Correspondence: J.S. Hong, Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Science, Research Triangle Park, NC 27709, USA. Fax: (1) (919) 541-0841. * Present address: Division of Cell Biology, Burroughs Wetlcome Co., Research Triangle Park, NC 27709, USA.
151 this steroid hormone
have not been well character-
i z e d TM. I n c o n t r a s t , d e n t a t e g r a n u l e cells a r e r e s i s t e n t to the toxic effects of glucocorticoids. Recent literature has suggested that glucocorticoids might even be essential f o r t h e s u r v i v a l o f t h e d e n t a t e g r a n u l e cells t3'21. Because of the potent effects of glucocorticoids on the hippocampus, we examined the effect of ADX the expression of PDYN
and proenkephalin
on
g e n e s , in
the rat hippocampus. Here we report that ADX selectively d e c r e a s e s t h e l e v e l s o f D Y N a n d its m R N A
immunoreactivity
in t h e h i p p o c a m p u s . F u r t h e r m o r e ,
this
d e c r e a s e c a n b e b l o c k e d by t h e s y n t h e t i c g l u c o c o r t i coid, d e x a m e t h a s o n e . MATERIALS AND METHODS
Animals Male, 10- to 12-week-old Fischer-344 rats (Charles River, Raleigh, NC) weighing 280-300 g were used. These animals were housed 3 per cage in a room maintained at 21 :t:2°C on a 12:12 h light-dark cycle.
Treatment of animals The animals were randomly divided into 3 groups: sham-operated (CONT), adrenalectomized (ADX) and adrenalectomized plus dexamethasone replacement (ADX+DEXA). The sham-operation or adrenalectomy was performed through a bilateral dorsal incision under methoxyflurane (Metofane, Pitman-Moore, Washington Crossing, NJ) anesthesia as described elsewhere Ix. After surgery, ADX rats received 0.9% saline in addition to 4% sucrose in their drinking water and A D X + D E X A rats received 1 m g / l of dexamethasone (Sigma Chemical Company, St. Louis. MO), a synthetic glucocorticoid, in saline. The completeness of ADX was verified by determining the serum concentration of corticosterone by radioimmunoassay (RIA).
Radioimmunoassays The tissue levels of [MetS]-enkephalin (ME) and DYN A(1-8) were determined by RIA as described in detail elsewhere 1°. Briefly, animals were decapitated at 4 different time points: 7, 30, 60, or 90 days post surgery and assayed at different time. Brain regions were immediately dissected and frozen on dry-ice. Tissue was homogenized in 2 M acetic acid at 4°C. Homogenates were heated in boiling water for 5 min and centrifuged at 15,000× g for 20 rain. Supernatants were lyophilized to dryness. The residues were reconstituted in RIA buffer and incubated either with, 125I-labeled DYN or 125I-labeled ME, together with, the corresponding antiserum for 18 to 24 h at 4°C. The antiserum against DYN A(1-8) had the following cross-reactivities: DYN A(1-13), 0.02%; DYN A(1-17), 0.01%; ME, 0.01%. The cross-reactivities of the ME antiserum with other opioid peptides were: DYN A(1-8), < 0.06%; [LeuS]-enkephalin 0.47%; and ~-endorphin, 0.2%. Blood samples from the trunk were taken immediately after decapitation. The standard method of corticosterone assays is described elsewhere 6. Briefly, serum were separated by centrifugation (9,681× g). Samples from the CONT group were diluted 50-fold prior to assay. The cortisol antibody was purchased from Radioassay Systems Laboratories, Inc. (Carson, CA).
Immunocytochemistry DYN and ME immunostaining were measured by immunocytochemistry (ICC) in CONT, ADX and ADX + DEXA rats (30, 60 and 90 days after surgery). Eight animals/group were used for this experiment. Rats were anesthetized with 4% chloral hydrate and perfused transcardially with 30 ml of 1% paraformaldehyde per 100 g
body weight before fizing with 70 ml of 4% paraformaldebyde per 100 g body weight at 4°C. Brains were then removed and post-fixed in 4% paraformaldehyde for 24 h. Forty-~m-thick vibratome sections were then incubated, free-fioating, in 2% normal goat serum, followed by incubation with either the DYN A(1-8) or ME antiserum. The avidin-biotin immunoperoxidase method with 3,Y-diaminobenzidine tetrahydrochloride, as the chromagen was used 19. Sections were then mounted onto gelatin-coated slides and coverslipped with Permount.
Northern blot analysis Total RNA was extracted from the hippocampus, striatum and frontal cortex using the guanidinium thiocyanate procedure described in detail elsewhere 5. This extract was further purified by repeated water extractions followed by ethanol precipitation, air dried, and dissolved in water. The content of total RNA was quantified by measuring absorbance at 260 nm. For blot analysis, total RNA was denatured with dimethyl sulfoxide, electrophoresed on a 1.5% agarose gel containing 6% formaldehyde, and transfered to a nylon membrane. A 32p-labeled PDYN cRNA probe was used for PDYN mRNA measurement as described 25. The blot was incubated with a prebybridization buffer at 60~C for 12-15 h. The [32pkRNA probe was then added to the incubation mixture and hybridized with the blots at 60°C for 24 h. After hybridization, the blots were washed, air-dried and exposed to Kodak XAR-5 film at -70°C. Autoradiograms were quantified by scanning densitometry.
In situ hybridization histochemistry In situ hybridization histochemistry was performed as described 2°. After decapitation, brains were immediately frozen on powdered dry-ice and the dorsal hippocampus was sectioned coronally at 10 ~ m on a cryostat. Frozen sections were thawed and mounted onto gelatin-coated slides and post-fixed with 4% paraformaldebyde for 10 rain. Brain sections were pretreated with acetic anhydride, delipidated in a graded series of ethanol and chloroform, rebydratod, and dried. A cocktail of two 33 mer synthetic oligodeoxynucleotide probes complementary to bases 618-652 and 691-723, of rat PDYN cDNA were used for hybridization. These two fragments each contains a 64% randomly distributed GC rich region of the PDYN gene. The proenkephalin probe is 39 mer, 56% GC-rich on the transcribing region of the proenkephalin gene (5' AAG, CGC, TAC, CAG, CCA, GAT, GCA, AAG, TCT, CAG, GAA, CTG, CGC, 3'). The probes were labeled with [35S]dATP using terminal deoxynucleotidyl-transferase (Bethesda Research Laboratory, Grand Island, NY), with a specific acitivity of 2x106 cpm/~l. The sections were then hybridized with the ass-labeled probe (5 x 105 cpm/slide) at 370C for 20 h. For post-hybridization washing, the slides were dipped 4 times into 1 × SSC (0.15 M NaC1/0.015 M sodium citrate, pH 7.0) at 60°C, once for 15 rain, and once in 1 × SSC at room temperature for 1 h. The slides were then dried under a vacuum overnight and dipped in Kodak NTB-2 emulsion (2:1 with water) and exposed for 3 weeks before developing in D-19 solution.
Statistics The RIA data for DYN and ME were subjected to a one-way analysis of variance (ANOVA) to test for overall statistical significance. Comparisons between individual groups were made using Fisher's least significant difference test 24. A level of P < 0.05 was considered significant. RESULTS
Effects o f adrenalectomy on body weight and blood corticosterone level R e s u l t s f r o m all h i s t o l o g i c a l s t u d i e s w e r e
pooled
from three separate experiments. The survival rate of animals after surgical removal of the adrenal glands was
80%.
The
body
weights were
measured
semi-
152 TABLE 1
Effects of adrenalectomy and dexamethasone on hippocampal and striatal dynorphin A (1-8) content Values are means_+ S.E.M, of 7 rats.
Region
Treatment
Tissue content (pmol / g tissue) 7 days
30days
60 days
Hippocampus
CONT ADX ADX + DEXA
17.4 _+1.0 14.2 _+ 1.0 * * 15.3_+ 1.1
25.1 _+ 1.7 14.1 _+1.5 * * 19.4_+2.5
21.9 _+0.5 9.3 _+0.8 * * 19.5_+ 1.1
Striatum
CONT ADX ADX + DEXA
31.6 +_2.0 34.7 _+3.1 35.7 _+3.1
31.4 _+ 1.7 23.1 _+ 1.3 * 32.6 _+0.8
32.8 _+ 1.4 27,5 + 1.5 * 36.3 _+2.0
* P < 0.05; ** P < 0.001 compared to controls.
hippocampal DYN A(1-8) were decreased by 58%. However, at this time point, there was no further decrease of DYN in the striatum. Throughout all survival intervals, dexamethasone replacement consistently prevented the decrease of DYN A(1-8) in ADX rats (Table I). The levels of ME in the hippocampus and striatum were also determined using the same tissue extracts used for DYN A(1-8) RIA. The ME levels of ADX animals were comparable to the CONT animals (Table II). (ANOVA) Statistical analysis indicated there were no significant differences among the three groups (Ta' ble II). The fluctuation in the actual amount of DYN and enkephalin peptides in the three control groups in Tables I and II is due to the different set of experimental assays. Immunohistochemical examination of hippocampi from ADX rats indicated that the decrease in DYN A(1-8) was most obvious in the dentate gyrus and mossy fibers 90 days after adrenalectomy. We also detected a substantial decrease in DYN A(1-8) after 60 days (Fig. 1B). In 22 out of the 24 ADX subjects studied chosen from all three time points (30, 60 and 90 days), thionin staining showed the dentate granule cell to be morphologically intact (Fig. 1E, from 60 day animals), while immunostaining revealed a significant depletion of DYN A(1-8). In two of the animals,
monthly throughout the entire course of the study. The loss in body weight after ADX was time-dependent and most evident at 90 days post-surgery (30 days, 90%; 60 days, 84%; and 90 days, 76% of control body weight). In the group of ADX + DEXA treated animals, their body weight was reduced even further by the end of day 90 after adrenalectomy (30 days, 70%; 60 days, 69%; and 90 days, 56% of control). Serum corticosterone levels of ADX rats were measured with a standard corticosterone RIA and were found to be extremely low, averaging about 3% of the serum corticosterone level of control animals (CONT, 286 + 58 ng/ml; ADX from 1, 2, 3 month rats, < 8.6 + 5.2 ng/ml, n = 16).
Adrenalectomy selectively reduced DYN A(1-8) immunoreactivity in the hippocampus RIA measurement demonstrated that adrenalectomy selectively reduced hippocampal DYN A(1-8) in a time-dependent manner (Table I). Seven days after ADX, a statistically significant decrease (18%) of DYN was observed in the hippocampus. The decrement in hippocampal DYN was even more evident at day 30 (44%). A modest decrease of DYN A(1-8) level (26%) in the striatum was also observed at day 30. Two months after ADX, the effects on hippocampal DYN A(1-8) were even more pronounced: the levels of TABLE II
Effects of adrenalectomy and dexamethasone on hippocampal and striatal [MetS]-enekephalin content Values are means_+S.E.M, of 7 rats.
Region
Treatment
Hippocampus
CONT ADX ADX+DEXA
Striatum
CONT ADX ADX+DEXA
Tissue content (pmol / g tissue) 7 days
30 days
105 -+ 90.6+ 92.4_+ 2070 2560 2470
5.2 5.3 5.2
_+ 190 _+160 _+ 120
121_+ 101_+ 102_+
60 days 9.2 6.8 7.7
2070_+ 120 1790_+ 96 2450_+ 160
147_+ 135± 144+
3.3 4.t 2.9
2230_+ 114 2370_+223 2510_+ 156
153
o
I0
Fig. 1. Immunoreactivi~ for dynorphin A(1-8) (DYN) in the hippocampus 60 days after adrenalectomy (ADX), ~ animals .'.~,m each time-point (30, 60 and 90 days) of study were subjected to immunohistochemical studies. Results from three ADX Ilrottps were qualitatively similar, except the decrease of DYN staining in the 30 day groups was not as robust as in the 60 and 90 day grOUl~ Therefore, only results from the 60 day group are illustrated as an example. Coronal sections of the dorsal hippocampus showing DYlq in the granule cell mossy fiber pathway. A marked decrease in DYlq from conUrol (A) can be observed in an ADX rat after 60 days (B). Immediate dexamethasone (DEXA) supplement attenuates the loss of DYN in ADX rats (C). High magnification of thionin staining in dentate granule layers (D-F) shows the intactness of dentate granule cells in the hippocampus of ADX (E) as well as the ADX + DEXA (F) rats.
h o w e v e r , t h e r e w a s a p a r t i a l loss o f d e n t a t e g r a n u l e cells t h r o u g h o u t t h e d e n t a t e l a y e r s a f t e r A D X in a d d i t i o n to t h e d e c r e a s e o f D Y N A ( 1 - 8 ) i m m u n o r e a c t i v i t y . I n e a c h o f t h e s e cases, m e a s u r i n g t h e l e n g t h a n d t h e
w i d t h o f t h e d e n t a t e g r a n u l e cell l a y e r s h o w e d t h a t a p p r o x i m a t e l y 7 0 % o f cells r e m a i n e d . B o t h o f t h e s e animals were from the same experiment and same time p o i n t (60 days). W i t h D E X A s u p p l e m e n t a t i o n in t h e
154 drinking water, DYN A(1-8) peptide depletion was substantially diminished (Fig. 1C,F, from 60 day animals). In agreement with the RIA findings, we did not observe any significant changes in ME in the hippocampus or striatum either 30, 60 and 90 days after ADX. Histochemical examination of the striatum of ADX subjects revealed no apparent difference in number of perikarya or in DYN A(1-8) (data not shown).
Adrenalectomy reduced the abundance of PDYN mRNA in the hippocampus To investigate the possibility that the depletion of DYN A(1-8) peptide in the hippocampus after adrenalectomy is due to a decrease in biosynthesis, measurement of mRNA-encoding PDYN by Northern blot analysis and in situ hybridization was performed. In two month ADX animals, northern blot analysis showed that the PDYN mRNA was selectively reduced in the hippocampus of ADX animals, while PDYN mRNA in the frontal cortex and striatum remained unchanged (HP 49.0% + 4.0; ST 94.9% + 6.9 FC 96.4% + 3.1) (Fig. 2). The PDYN mRNA levels in the ADX + DEXA treated hippocampus were not different from the CONT group (HP 86.7% _+8.1; ST 96.7% + 5.2; FC 96.2% ± 6.8) (Fig. 2). This experiment has been replicated three times. Fig. 3A shows the in situ hybridization signal for PDYN mRNA in the dentate granule cell layer of hippocampus in CONT rats. This signal was greatly reduced in cases when animals were ADX for 90 days or 60 days (Fig. 3B). The degree of signal reduction
CONT
~
r
ADX
w
-
F
-
-
-
. . . . . . . . . . . . .
Effects of ADX on Dyn m R N A Level FC
/
ST
\
/
HC
\
CON]" ADX+DEXAADX CONT ADX+ DEXA ADX
/
\
CONT ADX+ DEXA ADX
.,,,v-- 2.4 kb
60 Day Treatment Fig. 2. Northern blot analysis of the effects of ADX on PDYN mRNA in the rat frontal cortex (FC), striatum (ST) and hippocampus (HC). The gels are from sham-operatered rats (CONT), adrenalectomized rats (ADX) and adrenalectomized rats with dexamethasone replacement (ADX+ DEXA) for 60 days. Note the marked reduction in PDYN mRNA level in the hippocampus of ADX rats. This experiment was replicated twice.
Fig. 3. in situ hybridization of the dentate granule cells from shamoperated rats (A; CONT), adrenalectomized rats (B; ADX) and adrenalectomized rats with dexamethasone replacement (C; ADX + DEXA) animals. Samples are from the 60 day treatment group. ADX greatly reduced the mRNA signal and supplementation with DEXA reversed the effect of ADX on PDYN mRNA. Prodynorphin mRNA is illustrated in dark-field autoradiograms of coronal sections through the dorsal hippocampus.
was less apparent at 30-days after ADX (data not shown), In 9 out of the 12 ADX animals, the thionin counterstain showed that cellular integrity was pre-
155 served in the same dentate gyri that showed a reduction in the PDYN mRNA. In 3 animals, however, there was a reduced number of cells in the dentate granule layer (but not in the CA1 or CA3 fields of the hippocampus). These animals (2 from 60 day ADX and, 1 from 90 day ADX) showed a greater decrease in PDYN mRNA. These three animals also came from the set of experiments in which we previously found 2 animals with cell loss in immunocytochemical staining. In agreement with the changes in DYN A(1-8) in the immunocytoehemical data, ADX-induced depletion of PDYN mRNA throughout 30, 60, and 90 days was substantially prevented by DEXA replacement. In 9 of the ADX + DEXA-treated rats, PDYN mRNA (Fig. 3C) was slightly higher than that observed in the CONT animals (Fig. 3A). No loss of cells in the dentate gyms, CA1, CA2, or CA3 regions was found in these DEXA replaced ADX rats. The proenkephalin mRNA is constitutively low in the hippocampus. ADX did not seem to reduce the message that is normally detected on the dentate granule cells in control animals. DISCUSSION The present study demonstrated that adrenalectomy causes a time-dependent decrease in the steady-state levels of DYN A(1-8) and the abundance of PDYN mRNA in granule cells of the dentate gyms. DEXA replacement was able to largely reverse the effects of ADX, suggesting that glucocorticoids are essential for the functional expression of the PDYN gene in the hippocampus. Regulation of the expression of opioid peptides by glucocorticoids has been documented both in cell culturelS'E7'ESand in rat brain 2'17. Chao et al. 3 reported that ADX decreased the abundance of proenkephalin mRNA in the striatum but not in the hippocampus. The status of DYN A(1-8) was not assayed in this prior reseach. In our study, we observed that ADX caused a profound decrease in DYN A(1-8) in the hippocampus and to a much lesser extent in the striaturn. In the striatum, the maximum attenuation of DYN A(1-8) occurred at 30 days after ADX, whereas at 60 days post-ADX, the loss of DYN A(1-8) in this region was less pronounced. In comparison, the percent loss of DYN A(1-8) 30 days post-ADX was significantly less in the striatum than in the hippocampus (17% vs. 44%, respectively). This is interesting given the fact that there is also a high concentration of glucocorticoid receptors in the striatum. This in addition, suggests that PDYN biosynthesis in the striatum is not as glucocorticoid-dependent as in the hippocam-
pus and that other factors play an important role in sustaining PDYN synthesis in this brain region after A D X 2. In contrast to the robust decrease in DYN A(1-8) content, ME content (Table II) and proenkephalin mRNA 2 remained unchanged in the hippocampus after ADX. Immunocytoehemical studies revealed that the intensity of immunostaining of ME in both the perforant path and the mossy fibers were similar between CONT and ADX animals (data not shown). These results show the differential sensitivity of these two opioid peptides in the hippocampus to the removal of glucocorticoids. It has been reported that ADX causes the degeneration of dentate granule cellsTM. Thus, it is possible that the decrease in DYN A (1-8) content in our study is due to the loss of dentate granule cells. However, there are two observations which argue against this possibility: (i) There was a dissociation between the degree of granule cell loss and the extent of DYN A(1-8) decrease in most of the ADX rats. More than 80% of ADX rats which exhibited either no apparent or only a minor loss of granule cells invariably showed a severe decrease in DYN A(1-8) immunostaining in the mossy fiber pathway (Fig. 1). Of the 5 out of 36 animals that showed granule cell death in our 60-days and 90-days time-course study, the loss was most pronounced at 90-days post-ADX. As reported earlier by Gould et al. 7, we also found that most of the major cell death appeared in the suprapyramidal layer of the dentate gyrus while the CA3, CA2 and CA1 fields remained intact. Only in these individual animals could the disappearance of dentate granule cells have contributed to the greater reduction in the diminished levels of DYN A(1-8) in the mossy fibers and PDYN mRNA in the cell bodies. (ii) The failure of ADX to alter the content of ME (Table 2) and the immunostaining of ME in the mossy fibers as mentioned before, and the status of proenkephalin mRNA 3 suggests that the robust decrease of DYN A(1-8) in the hippocampus was not the consequence of extensive degeneration of dentate granule cells. The decreased content of DYN A(1-8) in the hippocampus of ADX rats was accompanied by a similar degree of reduction in PDYN mRNA levels. Northern blot analysis revealed a 50% reduction of PDYN mRNA in the hippocampus, but not in the striatum or frontal cortex. The In situ hybridization study also confirms these findings. The results by both methods suggest that the ADX-elicited decrease in DYN A(1-8) content is due to a reduction in the biosynthesis of this peptide. The decrease of both DYN A(1-8) and its mRNA in ADX rats was reversed by DEXA supplement, suggesting that glucocorticoid receptors are in-
156 volved in the r e g u l a t i o n of P D Y N expression in the d e n t a t e gyrus. P r e l i m i n a r y results from o u r l a b o r a t o r y s h o w e d that daily injections o f D E X A or i m p l a n t a t i o n o f D E X A p e l l e t s into rats p r o d u c e d i n c r e a s e s in the h i p p o c a m p a l c o n t e n t o f D Y N A ( 1 - 8 ) (Thai et al., u n p u b l i s h e d observation). S i m i l a r t r e a t m e n t also ind u c e d significant i n c r e a s e s in h i p p o c a m p a l a n d striatal D Y N p e p t i d e s in day 27 rats which suggest strongly for a physiological role t h a t g l u c o c o r t i c o i d s have in D Y N p e p t i d e regulation. T h e essential role o f glucocorticoids for t h e n o r m a l expression of h i p p o c a m p a l P D Y N seen in this study was f u r t h e r s u p p o r t e d by in vitro d e n t a t e g r a n u l e cell c u l t u r e s which s h o w e d t h a t glucocorticoids w e r e r e q u i r e d in the c u l t u r e m e d i u m in o r d e r to p r o d u c e d e t e c t i b l e levels of D Y N A ( 1 - 8 ) (X. He, N I E H S , R e s e a r c h T r i a n g l e Park, NC, U S A , p e r sonal c o m m u n i c a t i o n ) . T h e exact m e c h a n i s m s u n d e r l y i n g the r e g u l a t i o n of D Y N e x p r e s s i o n by g l u c o c o r t i c o i d s in t h e h i p p o c a m p u s is not clear. It has b e e n d e m o n s t r a t e d in cell c u l t u r e systems t h a t g l u c o c o r t i c o i d s serve as permissive factors in r e g u l a t i n g the e x p r e s s i o n of the p r o e n k e p h a l i n g e n e ~5'28. G l u c o c o r t i c o i d s m a y affect t h e e x p r e s s i o n of t h e D Y N g e n e directly o r indirectly by c h a n g i n g the function of o t h e r systems. T h e p r o t r a c t e d t i m e course for the d e c r e a s e o f the h i p p o c a m p a l D Y N c o n t e n t a f t e r A D X suggests t h a t t h e effect o f g l u c o c o r t i c o i d s m a y be indirectly m e d i a t e d by s o m e growth factor m e c h a n i s m s . In s u p p o r t o f this idea, A l o e ~ r e p o r t e d t h a t A D X d e c r e a s e d nerve growth f a c t o r in rat hippocampus. In s u m m a r y , e n d o g e n o u s g l u c o c o r t i c o i d s a r e essential for t h e l o n g - t e r m r e g u l a t i o n of t h e s t e a d y - s t a t e e x p r e s s i o n o f t h e P D Y N g e n e in the h i p p o c a m p u s . Excess g l u c o c o r t i o i d s e l e v a t e s D Y N p e p t i d e s in the h i p p o c a m p u s a n d r e m o v a l of this s t e r o i d h o r m o n e d e p l e t e s D Y N p e p t i d e s a n d its m R N A . T h e ability of g l u c o c o r t i c o i d s to positively r e g u l a t e t h e e x p r e s s i o n of g e n e s coding for o p i o i d p e p t i d e s suggests the i m p o r t a n c e o f t h e i n t e r a c t i o n b e t w e e n b o t h systems in m o d u lating the synaptic t r a n s m i s s i o n in the h i p p o c a m p a l circuitry. Acknowlegements. We thank Dr. David Milthorn (University of North
Carolina) for providing the prodynorphin oligo-probe and Dr. James Douglas (University of Oregon) for the dynorphin cRNA probe. REFERENCES 1 Aloe, L., Adrenalectomy decreases nerve growth factor in young adult rat hippocampus, Proc. Natl. Acad. Sci. USA, 86 (1989) 5636-5640. 2 Chao, H.M., Choo, P.M. and McEwen, B.S., Glucocorticoid and mineralocortieoid receptor mRNA expression in the rat rain, Neuro-endocrinology, 50 (1989) 365-371. 3 Chao, H. and McEwen, B., Glucocorticoid regulation of prepro-
enkephalin messenger ribonucleic acid in the rat striatum, kmdocrinology, 126 (1990) 3124-3130.
4 Chavkin, C., Shoemaker, W., McGinty, J., Bayon, A. and Bloom. F., Characterization of the prodynorphin and proenkephalin neuropeptide systems in rat hippocampus, .L Neurosci.. 5 (1985) 808-816. 5 Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. 6 Foster, L. and Dunn, R., Single antibody technique for radio-immunoassay of cortisol in unextracted serum or plasma, Clin. Chem., 20 (1974) 365-368. 7 Gould, E., Wooley, C.S. and McEwen, B.S., Short-term glucocorticoid manipulations affect neuronal morphology and survival in the adult dentate gyrus, Neuroscience, 37 (1990) 367-375. 8 Henriksen, S.J., Weisner, J.B. and Chouvet, G., Opioids in the hippocampus: progress obtained from in vivo electrophysiological analyses. In J. McGinty and D. Friedman (Eds.), Opioid in the Hippocampus, NIDA Research Monograph 82, 1988, pp. 67-93. 9 Jiang, H.K., Owyang, V., Hong J.-S. and GaUagher, M., Elevated dynorphin in the hippocampal formation of aged rats: relation to cognitive iml6airment on a spatial learning task, Proc. Natl. Acad. Sci. USA, 86 (1989) 2948-2951. 10 Kanamatsu, T., Obie, J., Grimes, L., McGinty, J.F., Yoshikawa, K., Sabol, S. and Hong, J., Kainic acid alters the metabolism of metS-enkephalin and the level of dynorphin A in the rat hippocampus, J. Neurosci., 6 (1986) 3094-3102. 11 Lee, P.H.K., Grimes, L. and Hong, J.-S., Glucocorticoids Potentiate Kainic acid-induced seizures and wet dog shakes, Brain Res., 480 (1989) 322-325. 12 Lothman, E.W., Bertram, E.H. III and Stringer, J.L., Functional anatomy of hippocampal seizures, Prog. Neurobiol., 37 (1991) 1-82.
13 McEwen, B. and Gould, E., Adrenal Steroid Influences on the Survival of Hippocampal neurons, Biochem. Pharmacol., 40 (1990) 2393-2402. 14 McGinty, J.F., Henriksen, S.J., Goldstein, A., Terenius, L. and Bloom, F.E., Dynorphin is contained within hippocampal mossy fibers: immunochemical alterations after kainic acid administration and cotchicine-induced neurotoxicity, Proc. Natl. Acad. Sci. USA, 80 (1983) 589-593. 15 Naranjo, J.R., Moccheni, I., Schwartz, J.P. and Costa, E., Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells, Proc. Natl. Acad. Sci. USA, 83 (1986) 1513-1517. 16 Reul, J. and de Kloet, R., Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation, Endocrinology, 117 (1985) 2505-2511. 17 Rossier, J., French, E., Gros, C., Minick, S., Guiltemin, R, and Bloom, F.E., Adrenalectomy, dexamethasone or stress alters opioid peptide levels in rat anterior pituitary but not intermediate lobe or brain, Life Sci., 25 (1979) 2105-2112. 18 Sapolsky, R., A mechanism for glucocorticoid toxicity in the hippocampus: increase neuronal vulnerabibtity to metabolic insults, J. Neurosci., 5 (1985) 1228-1232. 19 Sar, M., Application of avidin-biotin complex technique for the localization of estrodial receptor in target tissue using monoclonal antibodies. In G.R. Bullock and P. Petrusz (Eds.), Technique in Immunocytochemistry, VoL 3, Academic Press, New York, 1985, pp. 33-54. 20 Seroogy, K., Bayliss, D., Szymeczek, H6kfett, T. and Millhorn, D., Developmental transience of neuropeptide gene expression in rat brainstem as determined by in situ hybridization. In W.E. Stumpf and H.F. Solomom (Eds.), Using Autoradiography and Correlati~,e Imaging In Vitro and In Viuo, Academic press, Orlando, in press. 21 Stovitor, R., Valiquette, G., Abrams, G., Ronk, E., Sollas, A., Paul, L. and Neubort, S., Selective loss of hippocampal granule cells in the mature rat brain after adrenalectomy, Science, 243 (1989) 535-538. 22 Thai, L., Hong, J.-S. and Gallagher, M., Perforant path deafferentationalters hippocampal dynorphin and enkephalin content in rats, Soc. Neurosci. Abstr., 17 (1991) 395.
157 23 Tertian, D.M., Hernandez, P.G., Rea, M.A. and Peters, R.I., ATP Release, Adenosine Formation, and modulation of dynorphin and glutamic acid release by adenosine adnalogues in rat hippocampal mossy fiber synaptosomes, J. Neurochem., 53 (1989) 1390-1399. 24 Winer, B.J., Statistical Principles in Experimental Design, McGraw-Hill, New York, 1963. 25 Xie, C.-W., Mitchell, C.L. and Hong, J.-S., Perforant path stimulation differentially alters prodynorphin mRNA and prodynorphin mRNA levels in the entorhinal cortex-hippocampal region, Mol. Brain Res., 7 (1990) 199-205. 26 Xie, C.W., MeGinty, J., Lee, P., Mitchell, C. and Hong, J.-S., A glutamate antagonist blocks perforant reduction of dynorphin peptide and prodynorphin mRNA levels in rat hippocampus, Brain Res., 562 (1991) 243-250.
27 Yoshikawa, IC and Sabol, S.L., Expression of the enkephalin precursor gene in C6 glioma cells: regulation by /]-adrenergic agonists and glucocorticoids, Mol. Brain Res., 1 (1986) 75-83. 28 Yoshikawa, K. and Sabol, S.L., Glucocorticoids and cyclic-AMP synergistically regulate the abundance of preproenkephalin messenger RNA in neuroblastoma×glioma hybrid cells, Biochem. Biophys. Res. Commun., 139 (1986) 1-10. 29 Zhang, W.Q., Mundy, W., Thai, L., Hudson, P., Gallagher, M., Tilson, H. and Hong, J.-S., Decreased glutamate release correlates with elevated dynorphin content in the hippocampus of aged rats with spatial learning deficits, H~ppocampus, 1 (1991) 391-397.
150
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BRESM 70489
Regulation of prodynorphin gene expression in the hippocampus by glucocorticoids Linda Thai
a,b Paul H.K.
Lee "'*, Jack Ho a, Harold Suh a and John S. Hong
~
a Laboratory of Molecular and IntegratiL,e Neuroscience, National Institutes ofEnt, ironmental Health Sciences, Research Triangle Park. NC 27709 (USA) and t, Curriculum in Neurobiology, Unit:ersity of North Carolina, Chapel Hill. NC 27514 (USAi (Accepted 26 May 1992)
Key words." Adrenalectomy; Dexamethasone; Hippocampus; Dynorphin; Enkephalin; Gene regulation; Opioid peptide; Steroid hormone
The regulation of prodynorphin gene expression by glucocorticoids in the hippocampus was examined in rats that were adrenalectomized (ADX) either 7, 30, 60 and 90 days prior to sacrifice. Peptide levels in the hippocampus of ADX rats were determined by radioimmunoassay and immunocytochemistry. Prodynorphin (PDYN) mRNA was measured by Northern blot analysis and in situ hybridization. A time-dependent decrease in dynorphin A(I-8) (DYN) levels in the hippocampus (18% at 7 days; 44% at 30 days; 58% at 60 days) of ADX rats was found, which was accompanied by a comparable decrease in the abundance of PDYN mRNA. An in situ hybridization analysis revealed that both the number of positively hybridized cells and the number of silver grains per cell were decreased in the dentate gyrus after ADX. The administration of dexamethasone after surgery reversed the peptide and m R N A attenuation induced by ADX. ADX had no effect on the expression of proenkephalin mRNA or [MetS]-enkephalin immunoreactivity in the hippocampus. Examination of thionin-counterstained tissue showed that the dentate granule cell layer was intact. The decrement of DYN expression in this system is proposed to have resulted from the removal of glucocorticoid input and not dentate granule cell loss. This study provides the strong evidence for a differential susceptibility of these two opioid peptides in the hippocampus to the removal of glucocorticoids. In addition, these data provide support for a potentially selective, glucocorticoidpermissive component in PDYN gene expression.
INTRODUCTION Within the hippocampal formation, dentate granule ceils serve to restrict or amplify signals that originate in extrahippocampal sites and propagate into the hippocampus proper 12. It is also known that dynorphin (DYN), an endogenous opioid peptide, is synthesized by dentate granule cells, and is stored in and released by their axons, the mossy fibers 1'4'14'23. The expression of DYN has been demonstrated to be negatively regulated by glutamatergic transmission. For example, during the normal process of aging, when afferent perforant path fibers are sparse and glutamatergic synaptic activity is lessened, DYN immunoreactivity in the mossy fiber axon terminals has been shown to be increased 9'29. This peptide up-regulation can also be demonstrated by lesioning the perforant path input to the dentate granule cells 22. In contrast, stimulation of
the perforant path causes an increase in glutamate release and suppresses the expression of prodynorphin (PDYN) mRNA 25. Furthermore, perforant path stimulation-induced down-regulation of PDYN mRNA was prevented by pretreatment with a glutamate receptor blocker, y-D-glutamylglycine 26. Thus, DYN regulation in the hippocampus has consistently been shown to be modulated by glutamatergic transmission. The hippocampus appears to be the principal neural target for glucocorticoids because it contains a high concentration of both type I and type II corticosteroid receptors 16. Glucocorticoids, secreted by the adrenal cortex under chronic physiological stress, are capable of damaging or destroying hippocampal pyramidal neurons by sensitizing them to various metabolic insults 1~. Glucocorticoid-induced damage to the hippocampal pyramidal cells is similar to the cell loss during aging, yet the mechanisms underlying the neurotoxic effect of
Correspondence: J.S. Hong, Laboratory of Molecular and Integrative Neuroscience, National Institute of Environmental Health Science, Research Triangle Park, NC 27709, USA. Fax: (1) (919) 541-0841. * Present address: Division of Cell Biology, Burroughs Wetlcome Co., Research Triangle Park, NC 27709, USA.
151 this steroid hormone
have not been well character-
i z e d TM. I n c o n t r a s t , d e n t a t e g r a n u l e cells a r e r e s i s t e n t to the toxic effects of glucocorticoids. Recent literature has suggested that glucocorticoids might even be essential f o r t h e s u r v i v a l o f t h e d e n t a t e g r a n u l e cells t3'21. Because of the potent effects of glucocorticoids on the hippocampus, we examined the effect of ADX the expression of PDYN
and proenkephalin
on
g e n e s , in
the rat hippocampus. Here we report that ADX selectively d e c r e a s e s t h e l e v e l s o f D Y N a n d its m R N A
immunoreactivity
in t h e h i p p o c a m p u s . F u r t h e r m o r e ,
this
d e c r e a s e c a n b e b l o c k e d by t h e s y n t h e t i c g l u c o c o r t i coid, d e x a m e t h a s o n e . MATERIALS AND METHODS
Animals Male, 10- to 12-week-old Fischer-344 rats (Charles River, Raleigh, NC) weighing 280-300 g were used. These animals were housed 3 per cage in a room maintained at 21 :t:2°C on a 12:12 h light-dark cycle.
Treatment of animals The animals were randomly divided into 3 groups: sham-operated (CONT), adrenalectomized (ADX) and adrenalectomized plus dexamethasone replacement (ADX+DEXA). The sham-operation or adrenalectomy was performed through a bilateral dorsal incision under methoxyflurane (Metofane, Pitman-Moore, Washington Crossing, NJ) anesthesia as described elsewhere Ix. After surgery, ADX rats received 0.9% saline in addition to 4% sucrose in their drinking water and A D X + D E X A rats received 1 m g / l of dexamethasone (Sigma Chemical Company, St. Louis. MO), a synthetic glucocorticoid, in saline. The completeness of ADX was verified by determining the serum concentration of corticosterone by radioimmunoassay (RIA).
Radioimmunoassays The tissue levels of [MetS]-enkephalin (ME) and DYN A(1-8) were determined by RIA as described in detail elsewhere 1°. Briefly, animals were decapitated at 4 different time points: 7, 30, 60, or 90 days post surgery and assayed at different time. Brain regions were immediately dissected and frozen on dry-ice. Tissue was homogenized in 2 M acetic acid at 4°C. Homogenates were heated in boiling water for 5 min and centrifuged at 15,000× g for 20 rain. Supernatants were lyophilized to dryness. The residues were reconstituted in RIA buffer and incubated either with, 125I-labeled DYN or 125I-labeled ME, together with, the corresponding antiserum for 18 to 24 h at 4°C. The antiserum against DYN A(1-8) had the following cross-reactivities: DYN A(1-13), 0.02%; DYN A(1-17), 0.01%; ME, 0.01%. The cross-reactivities of the ME antiserum with other opioid peptides were: DYN A(1-8), < 0.06%; [LeuS]-enkephalin 0.47%; and ~-endorphin, 0.2%. Blood samples from the trunk were taken immediately after decapitation. The standard method of corticosterone assays is described elsewhere 6. Briefly, serum were separated by centrifugation (9,681× g). Samples from the CONT group were diluted 50-fold prior to assay. The cortisol antibody was purchased from Radioassay Systems Laboratories, Inc. (Carson, CA).
Immunocytochemistry DYN and ME immunostaining were measured by immunocytochemistry (ICC) in CONT, ADX and ADX + DEXA rats (30, 60 and 90 days after surgery). Eight animals/group were used for this experiment. Rats were anesthetized with 4% chloral hydrate and perfused transcardially with 30 ml of 1% paraformaldehyde per 100 g
body weight before fizing with 70 ml of 4% paraformaldebyde per 100 g body weight at 4°C. Brains were then removed and post-fixed in 4% paraformaldehyde for 24 h. Forty-~m-thick vibratome sections were then incubated, free-fioating, in 2% normal goat serum, followed by incubation with either the DYN A(1-8) or ME antiserum. The avidin-biotin immunoperoxidase method with 3,Y-diaminobenzidine tetrahydrochloride, as the chromagen was used 19. Sections were then mounted onto gelatin-coated slides and coverslipped with Permount.
Northern blot analysis Total RNA was extracted from the hippocampus, striatum and frontal cortex using the guanidinium thiocyanate procedure described in detail elsewhere 5. This extract was further purified by repeated water extractions followed by ethanol precipitation, air dried, and dissolved in water. The content of total RNA was quantified by measuring absorbance at 260 nm. For blot analysis, total RNA was denatured with dimethyl sulfoxide, electrophoresed on a 1.5% agarose gel containing 6% formaldehyde, and transfered to a nylon membrane. A 32p-labeled PDYN cRNA probe was used for PDYN mRNA measurement as described 25. The blot was incubated with a prebybridization buffer at 60~C for 12-15 h. The [32pkRNA probe was then added to the incubation mixture and hybridized with the blots at 60°C for 24 h. After hybridization, the blots were washed, air-dried and exposed to Kodak XAR-5 film at -70°C. Autoradiograms were quantified by scanning densitometry.
In situ hybridization histochemistry In situ hybridization histochemistry was performed as described 2°. After decapitation, brains were immediately frozen on powdered dry-ice and the dorsal hippocampus was sectioned coronally at 10 ~ m on a cryostat. Frozen sections were thawed and mounted onto gelatin-coated slides and post-fixed with 4% paraformaldebyde for 10 rain. Brain sections were pretreated with acetic anhydride, delipidated in a graded series of ethanol and chloroform, rebydratod, and dried. A cocktail of two 33 mer synthetic oligodeoxynucleotide probes complementary to bases 618-652 and 691-723, of rat PDYN cDNA were used for hybridization. These two fragments each contains a 64% randomly distributed GC rich region of the PDYN gene. The proenkephalin probe is 39 mer, 56% GC-rich on the transcribing region of the proenkephalin gene (5' AAG, CGC, TAC, CAG, CCA, GAT, GCA, AAG, TCT, CAG, GAA, CTG, CGC, 3'). The probes were labeled with [35S]dATP using terminal deoxynucleotidyl-transferase (Bethesda Research Laboratory, Grand Island, NY), with a specific acitivity of 2x106 cpm/~l. The sections were then hybridized with the ass-labeled probe (5 x 105 cpm/slide) at 370C for 20 h. For post-hybridization washing, the slides were dipped 4 times into 1 × SSC (0.15 M NaC1/0.015 M sodium citrate, pH 7.0) at 60°C, once for 15 rain, and once in 1 × SSC at room temperature for 1 h. The slides were then dried under a vacuum overnight and dipped in Kodak NTB-2 emulsion (2:1 with water) and exposed for 3 weeks before developing in D-19 solution.
Statistics The RIA data for DYN and ME were subjected to a one-way analysis of variance (ANOVA) to test for overall statistical significance. Comparisons between individual groups were made using Fisher's least significant difference test 24. A level of P < 0.05 was considered significant. RESULTS
Effects o f adrenalectomy on body weight and blood corticosterone level R e s u l t s f r o m all h i s t o l o g i c a l s t u d i e s w e r e
pooled
from three separate experiments. The survival rate of animals after surgical removal of the adrenal glands was
80%.
The
body
weights were
measured
semi-
152 TABLE 1
Effects of adrenalectomy and dexamethasone on hippocampal and striatal dynorphin A (1-8) content Values are means_+ S.E.M, of 7 rats.
Region
Treatment
Tissue content (pmol / g tissue) 7 days
30days
60 days
Hippocampus
CONT ADX ADX + DEXA
17.4 _+1.0 14.2 _+ 1.0 * * 15.3_+ 1.1
25.1 _+ 1.7 14.1 _+1.5 * * 19.4_+2.5
21.9 _+0.5 9.3 _+0.8 * * 19.5_+ 1.1
Striatum
CONT ADX ADX + DEXA
31.6 +_2.0 34.7 _+3.1 35.7 _+3.1
31.4 _+ 1.7 23.1 _+ 1.3 * 32.6 _+0.8
32.8 _+ 1.4 27,5 + 1.5 * 36.3 _+2.0
* P < 0.05; ** P < 0.001 compared to controls.
hippocampal DYN A(1-8) were decreased by 58%. However, at this time point, there was no further decrease of DYN in the striatum. Throughout all survival intervals, dexamethasone replacement consistently prevented the decrease of DYN A(1-8) in ADX rats (Table I). The levels of ME in the hippocampus and striatum were also determined using the same tissue extracts used for DYN A(1-8) RIA. The ME levels of ADX animals were comparable to the CONT animals (Table II). (ANOVA) Statistical analysis indicated there were no significant differences among the three groups (Ta' ble II). The fluctuation in the actual amount of DYN and enkephalin peptides in the three control groups in Tables I and II is due to the different set of experimental assays. Immunohistochemical examination of hippocampi from ADX rats indicated that the decrease in DYN A(1-8) was most obvious in the dentate gyrus and mossy fibers 90 days after adrenalectomy. We also detected a substantial decrease in DYN A(1-8) after 60 days (Fig. 1B). In 22 out of the 24 ADX subjects studied chosen from all three time points (30, 60 and 90 days), thionin staining showed the dentate granule cell to be morphologically intact (Fig. 1E, from 60 day animals), while immunostaining revealed a significant depletion of DYN A(1-8). In two of the animals,
monthly throughout the entire course of the study. The loss in body weight after ADX was time-dependent and most evident at 90 days post-surgery (30 days, 90%; 60 days, 84%; and 90 days, 76% of control body weight). In the group of ADX + DEXA treated animals, their body weight was reduced even further by the end of day 90 after adrenalectomy (30 days, 70%; 60 days, 69%; and 90 days, 56% of control). Serum corticosterone levels of ADX rats were measured with a standard corticosterone RIA and were found to be extremely low, averaging about 3% of the serum corticosterone level of control animals (CONT, 286 + 58 ng/ml; ADX from 1, 2, 3 month rats, < 8.6 + 5.2 ng/ml, n = 16).
Adrenalectomy selectively reduced DYN A(1-8) immunoreactivity in the hippocampus RIA measurement demonstrated that adrenalectomy selectively reduced hippocampal DYN A(1-8) in a time-dependent manner (Table I). Seven days after ADX, a statistically significant decrease (18%) of DYN was observed in the hippocampus. The decrement in hippocampal DYN was even more evident at day 30 (44%). A modest decrease of DYN A(1-8) level (26%) in the striatum was also observed at day 30. Two months after ADX, the effects on hippocampal DYN A(1-8) were even more pronounced: the levels of TABLE II
Effects of adrenalectomy and dexamethasone on hippocampal and striatal [MetS]-enekephalin content Values are means_+S.E.M, of 7 rats.
Region
Treatment
Hippocampus
CONT ADX ADX+DEXA
Striatum
CONT ADX ADX+DEXA
Tissue content (pmol / g tissue) 7 days
30 days
105 -+ 90.6+ 92.4_+ 2070 2560 2470
5.2 5.3 5.2
_+ 190 _+160 _+ 120
121_+ 101_+ 102_+
60 days 9.2 6.8 7.7
2070_+ 120 1790_+ 96 2450_+ 160
147_+ 135± 144+
3.3 4.t 2.9
2230_+ 114 2370_+223 2510_+ 156
153
o
I0
Fig. 1. Immunoreactivi~ for dynorphin A(1-8) (DYN) in the hippocampus 60 days after adrenalectomy (ADX), ~ animals .'.~,m each time-point (30, 60 and 90 days) of study were subjected to immunohistochemical studies. Results from three ADX Ilrottps were qualitatively similar, except the decrease of DYN staining in the 30 day groups was not as robust as in the 60 and 90 day grOUl~ Therefore, only results from the 60 day group are illustrated as an example. Coronal sections of the dorsal hippocampus showing DYlq in the granule cell mossy fiber pathway. A marked decrease in DYlq from conUrol (A) can be observed in an ADX rat after 60 days (B). Immediate dexamethasone (DEXA) supplement attenuates the loss of DYN in ADX rats (C). High magnification of thionin staining in dentate granule layers (D-F) shows the intactness of dentate granule cells in the hippocampus of ADX (E) as well as the ADX + DEXA (F) rats.
h o w e v e r , t h e r e w a s a p a r t i a l loss o f d e n t a t e g r a n u l e cells t h r o u g h o u t t h e d e n t a t e l a y e r s a f t e r A D X in a d d i t i o n to t h e d e c r e a s e o f D Y N A ( 1 - 8 ) i m m u n o r e a c t i v i t y . I n e a c h o f t h e s e cases, m e a s u r i n g t h e l e n g t h a n d t h e
w i d t h o f t h e d e n t a t e g r a n u l e cell l a y e r s h o w e d t h a t a p p r o x i m a t e l y 7 0 % o f cells r e m a i n e d . B o t h o f t h e s e animals were from the same experiment and same time p o i n t (60 days). W i t h D E X A s u p p l e m e n t a t i o n in t h e
154 drinking water, DYN A(1-8) peptide depletion was substantially diminished (Fig. 1C,F, from 60 day animals). In agreement with the RIA findings, we did not observe any significant changes in ME in the hippocampus or striatum either 30, 60 and 90 days after ADX. Histochemical examination of the striatum of ADX subjects revealed no apparent difference in number of perikarya or in DYN A(1-8) (data not shown).
Adrenalectomy reduced the abundance of PDYN mRNA in the hippocampus To investigate the possibility that the depletion of DYN A(1-8) peptide in the hippocampus after adrenalectomy is due to a decrease in biosynthesis, measurement of mRNA-encoding PDYN by Northern blot analysis and in situ hybridization was performed. In two month ADX animals, northern blot analysis showed that the PDYN mRNA was selectively reduced in the hippocampus of ADX animals, while PDYN mRNA in the frontal cortex and striatum remained unchanged (HP 49.0% + 4.0; ST 94.9% + 6.9 FC 96.4% + 3.1) (Fig. 2). The PDYN mRNA levels in the ADX + DEXA treated hippocampus were not different from the CONT group (HP 86.7% _+8.1; ST 96.7% + 5.2; FC 96.2% ± 6.8) (Fig. 2). This experiment has been replicated three times. Fig. 3A shows the in situ hybridization signal for PDYN mRNA in the dentate granule cell layer of hippocampus in CONT rats. This signal was greatly reduced in cases when animals were ADX for 90 days or 60 days (Fig. 3B). The degree of signal reduction
CONT
~
r
ADX
w
-
F
-
-
-
. . . . . . . . . . . . .
Effects of ADX on Dyn m R N A Level FC
/
ST
\
/
HC
\
CON]" ADX+DEXAADX CONT ADX+ DEXA ADX
/
\
CONT ADX+ DEXA ADX
.,,,v-- 2.4 kb
60 Day Treatment Fig. 2. Northern blot analysis of the effects of ADX on PDYN mRNA in the rat frontal cortex (FC), striatum (ST) and hippocampus (HC). The gels are from sham-operatered rats (CONT), adrenalectomized rats (ADX) and adrenalectomized rats with dexamethasone replacement (ADX+ DEXA) for 60 days. Note the marked reduction in PDYN mRNA level in the hippocampus of ADX rats. This experiment was replicated twice.
Fig. 3. in situ hybridization of the dentate granule cells from shamoperated rats (A; CONT), adrenalectomized rats (B; ADX) and adrenalectomized rats with dexamethasone replacement (C; ADX + DEXA) animals. Samples are from the 60 day treatment group. ADX greatly reduced the mRNA signal and supplementation with DEXA reversed the effect of ADX on PDYN mRNA. Prodynorphin mRNA is illustrated in dark-field autoradiograms of coronal sections through the dorsal hippocampus.
was less apparent at 30-days after ADX (data not shown), In 9 out of the 12 ADX animals, the thionin counterstain showed that cellular integrity was pre-
155 served in the same dentate gyri that showed a reduction in the PDYN mRNA. In 3 animals, however, there was a reduced number of cells in the dentate granule layer (but not in the CA1 or CA3 fields of the hippocampus). These animals (2 from 60 day ADX and, 1 from 90 day ADX) showed a greater decrease in PDYN mRNA. These three animals also came from the set of experiments in which we previously found 2 animals with cell loss in immunocytochemical staining. In agreement with the changes in DYN A(1-8) in the immunocytoehemical data, ADX-induced depletion of PDYN mRNA throughout 30, 60, and 90 days was substantially prevented by DEXA replacement. In 9 of the ADX + DEXA-treated rats, PDYN mRNA (Fig. 3C) was slightly higher than that observed in the CONT animals (Fig. 3A). No loss of cells in the dentate gyms, CA1, CA2, or CA3 regions was found in these DEXA replaced ADX rats. The proenkephalin mRNA is constitutively low in the hippocampus. ADX did not seem to reduce the message that is normally detected on the dentate granule cells in control animals. DISCUSSION The present study demonstrated that adrenalectomy causes a time-dependent decrease in the steady-state levels of DYN A(1-8) and the abundance of PDYN mRNA in granule cells of the dentate gyms. DEXA replacement was able to largely reverse the effects of ADX, suggesting that glucocorticoids are essential for the functional expression of the PDYN gene in the hippocampus. Regulation of the expression of opioid peptides by glucocorticoids has been documented both in cell culturelS'E7'ESand in rat brain 2'17. Chao et al. 3 reported that ADX decreased the abundance of proenkephalin mRNA in the striatum but not in the hippocampus. The status of DYN A(1-8) was not assayed in this prior reseach. In our study, we observed that ADX caused a profound decrease in DYN A(1-8) in the hippocampus and to a much lesser extent in the striaturn. In the striatum, the maximum attenuation of DYN A(1-8) occurred at 30 days after ADX, whereas at 60 days post-ADX, the loss of DYN A(1-8) in this region was less pronounced. In comparison, the percent loss of DYN A(1-8) 30 days post-ADX was significantly less in the striatum than in the hippocampus (17% vs. 44%, respectively). This is interesting given the fact that there is also a high concentration of glucocorticoid receptors in the striatum. This in addition, suggests that PDYN biosynthesis in the striatum is not as glucocorticoid-dependent as in the hippocam-
pus and that other factors play an important role in sustaining PDYN synthesis in this brain region after A D X 2. In contrast to the robust decrease in DYN A(1-8) content, ME content (Table II) and proenkephalin mRNA 2 remained unchanged in the hippocampus after ADX. Immunocytoehemical studies revealed that the intensity of immunostaining of ME in both the perforant path and the mossy fibers were similar between CONT and ADX animals (data not shown). These results show the differential sensitivity of these two opioid peptides in the hippocampus to the removal of glucocorticoids. It has been reported that ADX causes the degeneration of dentate granule cellsTM. Thus, it is possible that the decrease in DYN A (1-8) content in our study is due to the loss of dentate granule cells. However, there are two observations which argue against this possibility: (i) There was a dissociation between the degree of granule cell loss and the extent of DYN A(1-8) decrease in most of the ADX rats. More than 80% of ADX rats which exhibited either no apparent or only a minor loss of granule cells invariably showed a severe decrease in DYN A(1-8) immunostaining in the mossy fiber pathway (Fig. 1). Of the 5 out of 36 animals that showed granule cell death in our 60-days and 90-days time-course study, the loss was most pronounced at 90-days post-ADX. As reported earlier by Gould et al. 7, we also found that most of the major cell death appeared in the suprapyramidal layer of the dentate gyrus while the CA3, CA2 and CA1 fields remained intact. Only in these individual animals could the disappearance of dentate granule cells have contributed to the greater reduction in the diminished levels of DYN A(1-8) in the mossy fibers and PDYN mRNA in the cell bodies. (ii) The failure of ADX to alter the content of ME (Table 2) and the immunostaining of ME in the mossy fibers as mentioned before, and the status of proenkephalin mRNA 3 suggests that the robust decrease of DYN A(1-8) in the hippocampus was not the consequence of extensive degeneration of dentate granule cells. The decreased content of DYN A(1-8) in the hippocampus of ADX rats was accompanied by a similar degree of reduction in PDYN mRNA levels. Northern blot analysis revealed a 50% reduction of PDYN mRNA in the hippocampus, but not in the striatum or frontal cortex. The In situ hybridization study also confirms these findings. The results by both methods suggest that the ADX-elicited decrease in DYN A(1-8) content is due to a reduction in the biosynthesis of this peptide. The decrease of both DYN A(1-8) and its mRNA in ADX rats was reversed by DEXA supplement, suggesting that glucocorticoid receptors are in-
156 volved in the r e g u l a t i o n of P D Y N expression in the d e n t a t e gyrus. P r e l i m i n a r y results from o u r l a b o r a t o r y s h o w e d that daily injections o f D E X A or i m p l a n t a t i o n o f D E X A p e l l e t s into rats p r o d u c e d i n c r e a s e s in the h i p p o c a m p a l c o n t e n t o f D Y N A ( 1 - 8 ) (Thai et al., u n p u b l i s h e d observation). S i m i l a r t r e a t m e n t also ind u c e d significant i n c r e a s e s in h i p p o c a m p a l a n d striatal D Y N p e p t i d e s in day 27 rats which suggest strongly for a physiological role t h a t g l u c o c o r t i c o i d s have in D Y N p e p t i d e regulation. T h e essential role o f glucocorticoids for t h e n o r m a l expression of h i p p o c a m p a l P D Y N seen in this study was f u r t h e r s u p p o r t e d by in vitro d e n t a t e g r a n u l e cell c u l t u r e s which s h o w e d t h a t glucocorticoids w e r e r e q u i r e d in the c u l t u r e m e d i u m in o r d e r to p r o d u c e d e t e c t i b l e levels of D Y N A ( 1 - 8 ) (X. He, N I E H S , R e s e a r c h T r i a n g l e Park, NC, U S A , p e r sonal c o m m u n i c a t i o n ) . T h e exact m e c h a n i s m s u n d e r l y i n g the r e g u l a t i o n of D Y N e x p r e s s i o n by g l u c o c o r t i c o i d s in t h e h i p p o c a m p u s is not clear. It has b e e n d e m o n s t r a t e d in cell c u l t u r e systems t h a t g l u c o c o r t i c o i d s serve as permissive factors in r e g u l a t i n g the e x p r e s s i o n of the p r o e n k e p h a l i n g e n e ~5'28. G l u c o c o r t i c o i d s m a y affect t h e e x p r e s s i o n of t h e D Y N g e n e directly o r indirectly by c h a n g i n g the function of o t h e r systems. T h e p r o t r a c t e d t i m e course for the d e c r e a s e o f the h i p p o c a m p a l D Y N c o n t e n t a f t e r A D X suggests t h a t t h e effect o f g l u c o c o r t i c o i d s m a y be indirectly m e d i a t e d by s o m e growth factor m e c h a n i s m s . In s u p p o r t o f this idea, A l o e ~ r e p o r t e d t h a t A D X d e c r e a s e d nerve growth f a c t o r in rat hippocampus. In s u m m a r y , e n d o g e n o u s g l u c o c o r t i c o i d s a r e essential for t h e l o n g - t e r m r e g u l a t i o n of t h e s t e a d y - s t a t e e x p r e s s i o n o f t h e P D Y N g e n e in the h i p p o c a m p u s . Excess g l u c o c o r t i o i d s e l e v a t e s D Y N p e p t i d e s in the h i p p o c a m p u s a n d r e m o v a l of this s t e r o i d h o r m o n e d e p l e t e s D Y N p e p t i d e s a n d its m R N A . T h e ability of g l u c o c o r t i c o i d s to positively r e g u l a t e t h e e x p r e s s i o n of g e n e s coding for o p i o i d p e p t i d e s suggests the i m p o r t a n c e o f t h e i n t e r a c t i o n b e t w e e n b o t h systems in m o d u lating the synaptic t r a n s m i s s i o n in the h i p p o c a m p a l circuitry. Acknowlegements. We thank Dr. David Milthorn (University of North
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