Adrenalectomy attenuates kainic acid-elicited increases of messenger RNAs for neurotrophins and their receptors in the rat brain

0306-4522/93 $6.00 + 0.00 Pergamon Press Ltd 0 1993 IBRO

Neuroscience Vol. 54, No. 4, pp. 90%922, 1993

Printed in Great Britain

ADRENALECTOMY ATTENUATES KAINIC ACID-ELICITED INCREASES OF MESSENGER RNAs FOR NEUROTROPHINS AND THEIR RECEPTORS IN THE RAT BRAIN G. BARBANY* and H. PERSSON Department of Medical Chemistry, Laboratory of Molecular Neurobiology, Karolinska Institute, Box 60400, S-10401 Stockholm, Sweden Abstract-Treatment with excitotoxin kainic acid is known to increase the level of messenger RNAs for nerve growth factor and brain-derived neurotrophic factor in the brain. In this study we have used quantitative in situ hybridization to analyse the effect of glucocorticoids on kainic acid-induced increase of nerve growth factor and brain-derived neurotrophic factor messenger RNA in the rat brain. In adrenalectomized animals, the kainic acid-mediated increase of brain-derived neurotrophic factor messenger RNA in the hippocampus and the cerebral cortex was reduced by 50% compared to sham-operated animals. The increase of nerve growth factor messenger RNA elicited by kainic acid in the dentate gyrus was almost completely abolished in adrenalectomized animals. No significant change was seen in c-fos messenger RNA in the hippocampus of adrenalectomized rat after kainic acid injection compared to sham-operated kainic acid-treated rats, while a three-fold reduction was seen in the cerebral cortex. Dexamethasone injection prior to kainic acid administration potentiated the kainic acid-induced increase of nerve growth factor messenger RNA in the dentate gyrus and the piriform cortex. In contrast,

dexamethasone pretreatment did not potentiate the kainic acid-mediated increase of brain-derived neurotrophic factor messenger RNA. We also examined the effect of adrenalectomy and kainic acid injection on tropomyosin receptor kinase B and C messenger RNA, encoding essential components of high-affinity receptor for brain-derived neurotrophic factor/neurotrophin-4 and neurotrophin-3, respectively. Following adrenalectomy no change of tropomyosin receptor kinase B or C messenger RNA was detected in any of the brain regions studied compared to sham-operated animals. The injection of kainic acid caused four-fold and two-fold increases of tropomyosin receptor kinase B messenger RNA in the dentate gyms and cerebral cortex, respectively, but no change in tropomyosin receptor kinase C messenger RNA in any of these regions. In adrenalectomized animals receiving kainic acid, the level of tropomyosin receptor kinase B messenger RNA was decreased both in the dentate gyrus and cerebral cortex as compared to sham animals treated with kainic acid. Taken together, the data suggest that excitotoxins and glucocorticoids both influence expression of brain-derived neurotrophic factor and nerve growth factor messenger RNA in the brain, but by two different mechanisms, where the effect of excitotoxin-evoked seizures is modulated by glucocorticoids.

and maintenance of neurons in the vertebrate nervous system depend on neurotrophic factors. The prototype and best characterized of these factors is nerve growth factor (NGF), which supports peripheral sympathetic and neural crest-derived sensory neurons (reviewed in Refs 42 and 60). In the CNS, NGF supports basal forebrain choline& neurons.‘2*20,39,61 NGF belongs to a family of structurally related proteins, collectively known as the neurotrophins, which apart from NGF includes brain-derived neurotrophic factor (BDNF),6,4’ neurotrophin-3 13,23,3’.32,46*H and neurotrophin-4,‘8,27 also known as neurotrophin-5.’ NGF, BDNF and neurotrophin-3 are all expressed in neurons in the brain, and the highest levels of mRNAs for all three factors in the rat brain are found

in the hippocampus.3J3*‘4~22*s3 The more recently isolated neurotrophin4 is also expressed in the brain, though at lower levels than the other three neurotrophins. 7,26In the CNS, BDNF has been shown to support the survival of retinal ganglion cells,29 basal forebrain cholinergic neurons’ and mesencephalic dopaminergic neurons25.3*in cell culture. Much less is known about the neurotrophic roles of neurotrophin3 and neurotrophin-4 in the brain, though both factors have recently been shown to stimulate the survival of embryonic locus coeruleus neurons in culture.15 The neurotrophins exert their effect via a highaffinity receptor where a protein tyrosine kinase constitutes an essential component of the receptor.48 The product of the tropomyosin receptor kinase (trk) proto-oncogene, a transmembrane protein of mol. wt 140,000 (pl40”‘) is a functional receptor for NGF.33x36 pl40”’ is a member of a tyrosine kinase family of transmembrane receptors which also includes the products of the trkB (p14SrkB) and

Development

*To whom correspondence should be addressed. BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor; trk, tropomyosin receptor kinase.

Abbreviations:

909

910


RAKHAVV

trkC (~145”~‘) genes. ~145”~” and ~145”~’ have recently been shown to be essential components of functional high-affinity receptors for BDNF and neurotrophin-3. respectively.4”~47~59p14SrkH is also a functional receptor for neurotrophin-4.‘.” ” Chronic seizures produced by a unilateral electrolytic lesion of the dentate gyrus hilus lead to increased levels of NGF and BDNF mRNAs in the hippocampus. “Z Administration of the excitotoxin kainic acid also causes a marked but transient increase of NGF and BDNF mRNAs in the hippocampus and neocortex.4.‘h.h’ Moreover, seizures induced by electrical kindling stimulations in the hippocampus lead to a marked and transient increase of NGF and BDNF mRNAs in the dentate gyrus and the parietal and piriform cortices.” Increases of NGF and BDNF mRNAs have also been demonstrated in dentate gyrus granule cells following cerebral ischemia and insulin-induced hypoglycemic coma.45 The same brain insults also lead to marked but transient increased of trkB mRNA and protein in the hippocampus. whereas no change has been seen for trk and trkC mRNAs.‘” Taken together, these data have led to the hypothesis that neurotrophins and their receptors could provide and facilitate a local trophic support within the hippocampus that protects against ncuronal necrosis after brain insults. Glucocorticoids have also been implicated in ncuronal death. High levels of glucocorticoids administered experimentally or induced by stress arc toxic to hippocampal pyramidal CCIIS~~and have been shown to exacerbate neuronal insults.‘” The mechanism of glucocorticoid neurotoxicity is not clear. though scvera] Factors have been implicated. such as impairment of glucose uptake” and enhancement of glutamatergic signals.’ Another possibility is decreases in the expression and synthesis of ncurotrophins which could lead to impaired survival of ncurans. In support of this hypothesis. it has recently been shown that dexamethasone produces a short term increase in neurotrophin mRNA expression followed by a lasting decrease both in the hippocampus and the cerebral cortex.c.J2 In the present study. we have analysed changes in mRNA expression for neurotrophins and their high-affinity receptors in adrenalcctomized animals after kainic acid injections. WC further analysed the effect of the synthetic glucocorticoid dexamethasone on the kainic acid-elicited increase in BDNF and NGF mRNAs.

EXPERIMENTAL PROCEDURES

Male Sprague-Dawley rats (I go-200 g) were purchased from Alab (Sweden), and housed under standard light conditions receiving water and lab-chow ud lihitwn. Adrenalectomy (n = 12) and sham operation (laparotomy; n = 12) were performed under pcntobarbital anesthesia. Following adrenalectomy, drinking water was supplemented with 3% saline to compensate for the loss of salt. Three days

and t-1. PEKSSOY

atier surgery. six adrenaletomized

an~t 4tx sham anutrai\ were injected i.p. with 6 mg/kg kainic acid. while the reman; ing animals were injected with saline and killed 2 h after the injection. In a different set of experiments the animals \\erc injected with dexamethasone (5 mg!kg: ,J -- 6). kainic acid (6 mg/kg; n -= 6) or both (n = IO). Dexamethasone w:ti injected 2 h 01 = 6) and 24 h (it = 4) prior, lo kainic acid. ‘fhc animals were killed by CO2 inhatation
Tissue sections (14 /cm) through the hlppocampus ucrc cut on a Lcitz cryostat and thawed onto poly-t.-lysmrcoated slides (50 pgg/ml). After fixation in 4% paraformaldehydc for 30min. the sections wcrc dehydrated in :t graded ethanol series including a 5 min incubation in chlorof(>rm Hybridization was performed in 50%” formamide. 4 .~ sodium chloride sodium citrate buffer (I x:sodium chioride sodium citrate buffer is 150 mM Ma<‘l. 15 mM sodium citrate, pH 7.0). I x Denhardt’s solution. 10% dexti-dn ~ulfate. 0.25 mg/ml yeast tRNA, 0.5 mgimi sheared salmon sperm DNA, t % N-lauroyl-sarcosine. 0.02 M -Na:PO! (pH 7.0) and 0.05 M dithriothreitol with 5 r; LO”c.p.m ml of the respective probe. The sections uere hybridized fc11 IS h in a humidified chamber with 0.1 ml of hybndizatlon cocktail/slide. They were subsequently rmsed. washed four times (IS min each) at 42 C in 0.1 ‘+ sodium chloride bodium citrate buffer. air dried and exposed to Hyperfilm [Imax (Amersham. U.K.). The sections were dipped il: Kodak NTB-2 photoemulsion (diluted I : I in water). exposed for five to six weeks at 4 C. developed. fixed and counterstained with Cresyl Violet. To detect BDNF-specific mRNA. :L _X)-mer ohgonuclcotide complementary to nucleotides bFO.690 of the riot BDNF mRNA was used. For NGF-spicific mRNA. .I 50-mer oligonuclcotide complementar! to nucleotides X69 91X of the rat NGF was used. Thrsc oligonucleotidcz+ arc the same as those used prcviousiy.” .Fhe trk6 all and full length probes are complementary to nucleotidcs 1314 1361 and 1260~ 1307. respectively. in the rat trkB mRNA sequence of Middlemas et lrl.” For trkC’. a 50-mer oiigonucleotide complementary to nucieotides 1186 1235 m the rat trkC sequence of Merlio r~f &‘I was used. These probes arc characterized in detail. Including their hybridirLItion specificit). in Ernfors (J/ II/.“’ zlnd Mcrlio (‘I ul.” (‘-foa-specitic mRNA was detected ualng a 48-mer oligonucleotide complementary to nucteotides 543 591 in the rat c-fos sequence of C&ran er t/l.’ ‘TIr oligonucleotides were labeled at the 3’-end with a-t%l4TP using terminal deoxyribonucleotide transferase (iB1: New Haven. CT) to ;t specific activity of approximately itPc.p.m.;~g and purified on a Nensorb column (Du Pant, Wilmington, DE) prior to use. The specificity of the hybridizatloii was assessed 1)) competing out the signal of the f’5S_F1ATP-labeicd probe hq adding a IOO-fold excess of the same ut&be?cd oligonucleotidc to the hybridization cocktail.

The autoradiograms were analyscd tijth a Mtcrocomputcr Imaging Device (Imaging Research Inc., Canada). Optical density values were determined and conv&ed to relative levels of radioactivity using autoradiographic 14Cmicro scales as external standards exposed to the same film. Two tissue sections from each brain were quantified and six measurements of each 340 pm: were rakcn in ea& anatom ical section. Mean values were calculated from six animals in each experimental group. Data are presented as mean + S.E.M. Statislical analyxh were performed using ANOVA followed by the Fischer PL.SD test.

ovulation

of mRNAs for ~euro~op~ns

and their receptors

Fig. I. Expression of BDNF mRNA in the brain of adrenalectomized rats after kainic acid treatment. Prints of autoradiograms from sham animals receiving saline, sham animals plus kainic acid, adrenalectomized animals plus kainic acid and adrenalectomhcd animals plus saline. Note the attenuation of the kainic acid-mediated increase in BDNF mRNA in dentate gyrus, CA1 region of the hippocampus, neocortex and p&form cortex after a~enal~tomy. Bright-field micrographs of auto~~o~ph~ emulsions from the granule ce% in the dentate gurus and piriform cortex from the same brains. Scale bar for the prints is 2.3 mm and is the Sam@for all other prints. Scale bar for the bright-tield micrographs is 20 pm and is the same far all other bright-field photographs. adx, adrenalectomized; dg, dentate gyrus; KA, kainic acid; pyr, pyramidal cell layer; pir, piriform cortex.

911

Fig. 2. Expression of NGF mRNA in the brain of adrenalectomized rats after kainic acid trwtment. Prints of a~tor~~o~ram~ from sham animals retxiving saline, sham animals phB kninic-asid. adrwtalectomiZwl an&x& pfus b&&c acid and ~dr~~~~~~o~d animals p&s saline. Note tke &most w@ete ~~~~~e~~~ of the kitinic acid-mediated increase is NCF mRMA in the dentate gyms. ~~gh~-~e~d r&rograpbs df autoradiographic emulsions from the granule cells in the dentate gyus and from the piriform cortex from the same brains. Scale bars and abbreviations as in Fig. I.

Regulation of mRNAs for neurotrophins and their receptors

913

A similar attenuation of the kainic acid-mediated increase of BDNF mRNA was seen in the frontoEflects of kainic acid injections on neurotrophin parietal and piriform co&es. The level of BDNF messenger RNA expression in adrenalectomized rats mRNA in adrenalectomized animals was reduced by 25% in both hippocampus and the cortical The effects on the expression of mRNA for neuroregions compared to sham-operated controls. Thus, a clear attenuation was seen irrespective of whether trophins in the adult rat brain following an i.p. the kainic acid-mediated increase was compared to injection of the excitotoxin kainic acid were analysed by quantitative in situ hybridization in normal vs the levels in sham-operated or adrenalectomized adrenalectomized animals. As shown previously,4*‘6s63 animals. The increase of NGF mRNA in the dentate gyrus elicited by kainic acid was ahnost com2 h after injection of kainic acid, nine-fold and fourfold increases were found in the mRNA levels for pletely abolished in adrenalectomized animals BDNF and NGF, respectively, in the dentate (Figs 2, 3). For both NGF and BDNF, the kainic acid-mediated increase was due to a larger number gyrus, compared to sham-operated control animals of neurons expressing detectable amounts of either (Figs l-3). BDNF mRNA was also increased threeof the two mRNAs, as well as an increased intenfold in the CA1 region but only slightly in the CA3 sity of labeling over individual neurons. The increases region of the hippocampus. Treatment of adrenalin the number of labeled cells and the labeling ectomized animals with kainic acid elicited a 4.5fold intensity over individual cells were both reduced in increase of BDNF mRNA in the dentate gyrus and a I.8-fold increase in the CA1 region (Figs 1, 3). adrenalectomized animals (Figs 1, 2). RESULTS

1 ooo-

BDNF mRNA

000 -

0

r

5 ti c

600-

.

SHAMcSALlNE

0

SHAM + KAINIC

hJ

ADREtW_ECTCMY+ KAINIC

fl

ADRENALECTCX4Y + !MJNE

0

F

‘-

9

400-

1

2oo-

II_

I

I3

DENTATE GYRUS

400

I=.

1 ..

300

-

200

-

ob!ll3

*Ilk9

CA1

OOQ,

CA3

FP-CORTEX

OOBS

PIWORM CORTEX

NGF mRNA

.i?

5 + F = 4

. .

J1oo-

1

b

ih

DENTATE GYRUS

PIRIFORM CORTEX

Fig. 3. Autoradiograms were quantified using a microcomputer imaging device. Means are calculated from six animals and presented as mean f S.E.M. Statistical analysis was performed using ANOVA followed by the Fischer PLSD test. Asterisks indicate statistically significant differences between kainic acid treatment of normal and adrenalectomized animals (**P < 0.001). CAL and CA3, regions CA1 and CA3 of the hippocampus; FP-CORTEX, frontoparietal cortex.

-,-,.-

.._ -,

Regulation of mRNAs for neurotrophins and their receptors The e$ect of kainic acid on c-fos messenger RNA expression in adrenalectomized animals

Following kainic acid injection, c-fos mRNA has been shown to increase dramatically in the brain, preceding the increase in NGF and BDNF mRNAs.4 The level of c-fos mRNA was analysed in adrenalectomized vs sham-operated animals after kainic acid injection using quantitative in situ hybridization. The level of c-fos mRNA was dramatically increased in the hippocampus, cingulate and piriform cortices and throughout the cerebral cortex 2 h after kainic acid injection (Fig. 4). In adrenalectomized animals the level of c-fos mRNA after kainic acid injection was elevated to the same extent in the hippocampus, as compared to sham-operated controls. However, in the cerebral and piriform cortices, the level of c-fos mRNA after kainic acid injection was reduced three-fold and two-fold, respectively, in adrenalectomized animals compared to kainic acid injection in sham-operated controls (Fig. 5).

915

(Fig. 6), whereas no statistically significant changes were detected in the CA3 and CA1 regions (Fig. 7). In adrenalectomized animals receiving kainic acid, the level of trkB all mRNA in the dentate gyrus was significantly decreased (P < 0.01) compared to sham animals after kainic acid injection (Figs 6, 7). In the piriform cortex, the increase of trkB all mRNA was approximately two-fold after kainic acid injection, and adrenalectomy abolished this increase almost completely (Fig. 7). In the dentate gyrus, trkB full-length mRNA increased 1.5fold after kainic acid treatment and this increase was completely abolished in adrenalectomized animals. No significant increase of trkB full-length mRNA was detected in the areberal cortex after kainic acid treatment (data not shown). The effect of kainic acid on trkC mRNA expression was analysed in the cerebral cortex and the hippocampus. In these two brain regions, no significant change in mRNA levels for trkC was detected after kainic acid injection, nor after adrenalectomy or the combined treatment (Fig. 8).

Eflects of kainic acid on tropomyosin receptor kinase B and C messenger RNA expression

Next, we analysed the effect of adrenalectomy and kainic acid injection on trkB and trkC mRNA expression. Two different probes were used to detect trkB mRNA: one which detects all trkB mRNA, i.e. encoding either truncated or full length trkB receptors, and a second which detects trkB transcripts encoding only the full length receptor. Following adrenalectomy, no significant change in the level of trkB or trkC mRNA was detected in any of the brain regions examined. The injection of kainic acid caused a four-fold increase in the level of trkB all mRNA in the dentate gyrus

1000

-

800

-

c-fos

z

P ‘$ :

600

-

400

-

200

-

The effect of systemic dexamethasone injection 2 h prior to kainic acid injection on neurotrophin mRNA expression was analysed. Dexamethasone administered 2 h prior to kainic acid potentiated the increase in NGF mRNA significantly (P < 0.001) in both the dentate gyrus and the piriform cortex (Fig. 9). This potentiation was also seen when kainic acid was administered 24 h after dexamethasone had been injected (Fig. 10). The level of BDNF mRNA in the hippocampus or cerebral cortex did not differ

mRNA

x f

E

Effect of dexamethasone and kainic acid on neurotrophin messenger RNA expression in the brain

q

SHAM + KAHIC

19

ADRENALECTOMY + KAINIC

..

DENTATE GYRUS

(

CA1

II

FP-CORTEX

PIRIFORM CORTEX

Fig. 5. Quantification of c-fos mRNA using a microcomputer imaging device. Means are calculated from six animals and presented as mean f S.E.M. Statistical analysis was performed using ANOVA followed by the Fischer PLSD test. Asterisks indicate statistically significant differences between kainic acid treatment of normal and adrenalectomized animals (*P < 0.01; **P < 0.001). Abbreviations as in Fig. 3.

Fig. 6. Expression of tskR all mRNA in the brain of adrenalectomized rats after kainic acict twatmcnt. Prints of autoradiograms From sham animals receiving saline. sham animals plus kainic acid. adrenaiectomized animals plus kainic acid and adrenalectomized animals plus saline. Bright-fieId micrographs ot autoradiographic emulsions from the granule cells in the dentate gyrus and from the piroform cortex from the same brains. Abbreviations and scale bars as in Fig. I.

signi~cantly between animals receiving both treatments (with a 2-h interval between them) or oniy kainic acid. fn the piriform cortex, a I .4-fold increase of BDNF mRNA was seen in dexamethasone plius kainic acid-treated animals compared to treatment

with kainic acid alone (Fig, 9). fn contrast. when kainic acid was administered 24 ir after dexamethasane injection, the increase in 5XXGF mRN.4 was sign~tica~tly attenuated in both the dentate gyrus and piriform cortex compared to animals receiving kainic

~~~ati~nof mRFJAs

for ~u~~p~~s

and their receptors

917

**

trkB all mRNA *

q WXM+SALINE q SHAM * KAINIC h3

q

DENTATE GYRUS

CA3

CA1

ADRENALECTOMY+KAINlC ADRMCTOMY

*

+ SALINE

FP-CORTEX

PIRIFORM CmTEX

Fig. 7. Autoradiograms after trkB all mRNA hybridization quantified using a microcomputer imaging devise. Means are calculated fram six animals and presented as mean f S.E.M. Statistical analysis was performed using ANOVA followed by the Fischer PLSD test. Asterisks indicate statistically significant differences between kainic acid treatment of normal and adrenalectomixed animals (‘8’ -C0.01; **P -~0.001). Abbreviations as in Fig. 3.

acid alone (Fig. 10). ~~t~~t~e~~

of the animals with

dexamethasone did not aiter the kainic acid response of mRNA encoding either trkB all or full-length receptors in any of the brain regions examined (data not shown).

~~~~~U~

The results presented in this report show that adrenalectomy attenuates the kainic acid-induced increase of steady-state levels of BDNF mRNA in the

Fig. 8. Expression of trkC mRNA in the brain of adrenalectomixed rats after kainic acid treatment. Prints of autoradiograms from sham animals receiving saline, sham animals plus kainic acid, adrenalectomimd animals plus kainic acid and adrenalectomized animals plus saline. Bright-field micrographs of autoradiographic emulsions from the granttIe eells in the dentate gyrus and from the piriform cortex from the same brains. Abb~~ations and scale bars as in Fig. I.

800

I BDNF mRNA

q

KAiNlC

f!ii

DEX

!?I

DEX

+ KAlNiC

0 DENTATE GYRUS 400

1

FP-OORTEX

PIRIFORM CC%TEX

NGF

DENTATE GYRUS

PIRIFORM

CORTEX

pretreatment with de~rne~k~~e on the k&tic acid-mediated irtor~st?s of NGF and BDNF mRNAs. Aoto~d~o~ms were ~~t~~ ysiq a ~~~~~r ~~~~~.~~~. k&eqs are calctitad from six animal and presenti as ANOVA followed by the F&her PLSD t&t. As kainic &id trc&ent of normal and a&&s I%XWI~~ Fig. 9. The efkx% of ? h

dexamehsoste.

Fig. 10. Rel?tiye amoupts of NGF and BDNF @N#%s. in the dentate gyros after &am&hascme, kd&cxMi&~i# aidd dexame&as=ont p&s k&r& a&d with B 244 ~~~~~~~ are cafcutated from four animals presented as mean +_ S.E.M. Statistical analysis was performed using AirlOVA followed by the Fischer PLSD test. Asterisks indhte statistically significant difE%nces between kainic acid treatment of normal and-animals receiving Wamethasonc pllts kainic acid (**P < 0.001). DEX. dexamethasone,

increase in trkB in the dentate. gyr& and a t&o-foEd the piriform cortex: Ifiowe~% the protX: specific far rnti?As encading Jhe_ fi$t-lea&~ .recep-. tor increased only in the dentate gyrus after kainic tars. Rainic a&l &cited a four-fold

all mRNA increase in

Regulation of mRNAs for n~urotrup~ns

acid treatment. A similar differential response in the increase of trkB all and full-length mRNAs has been seen following kindling induced by electrical stimulations in the hippocampus.@ In adrenalectomized animals, the kainic acid increase of trkB mRNA was significantly attenuated. Both BDNF and trkB mRNAs increase in the granule cells of the dentate gyrus following kindling epileptogenesis, ischemia or insulin-induced hypoglycemic coma, and it has been suggested that the increase of trkB mRNA could be mediated by increased levels of BDNF protein.% Similarly, the reduced increase of trk B mRNA seen in adrenalectomized animals after kainic acid injection could be due to a decreased level of BDNF in adrenalectomized animals treated with kainic acid compared to kainic acid alone, rather than a direct effect of glucocorticoids on trkB mRNA expression. The similar distribution of cells expressing trkB and BDNF in the hippocampus has led to the suggestion of an autocrine or paracrine mode of action, where the same population of cells producing the neurotrophic factors also respond to them via a highaffinity receptor?” Limbic seizures produced by an electrolytic lesion in the dentate gyrus increase NGF and BDNF mRNAs in the ~p~ampus.“.~ Recently, this increase was reported to occur in adrenalectomized animals as well.L7a It would also be of interest to examine the effect of adrenalectomy on increases of neurotrophin mRNA after kindling and ischemia to evaluate if adrenalectomy attenuates these increases, or if this effect is only seen after administration of an excitotoxin. The combined administration of dexamethasone and kainic acid resulted in increased levels of NGF in the granule cells of the dentate gyrus compared to kainic acid only. Dexamethasone has been shown to elicit a biphasic response in NGF mRNA: in the first hours after admi~stration NGF mRNA is transiently increased, followed by a long-lasting decrease.s This could reflect a differential response in different cell populations. In support of this hypothesis, dexamethasone has been shown to increase NGF mRNA in neurons, while a decrease is seen in astrocytes.43 Thus, the increase seen 4 h after dexamethasone might be due to a neuronal response to dexamethasone. The additive effect of dexamethasone and kainic acid on NGF mRNA levels implies that the two treatments elicit increases of NGF mRNA by two different mechanisms. The kainic acid-mediated increase appears to be due to a glutamate release and activation of the excitatory amino acid receptors of the non-N-methyl-D-aspartate subtype,63 while the effect of dexamethasone could be due to the presence of a glucocorticoid receptor responsive element in the NGF promoter.” Surprisingly, when kainic acid was administered 24 h after dexamethasone there was stilt a potentiation of kainic acid increase in NGF mRNA, even though dexamethasone alone produces a decrease in NGF mRNA after 24 h.$ One possible explanation could be increased neuronal excitability

and their receptors

919

due to dexamethasone treatment, rendering neurons more susceptible to kainic acid. Supporting this idea, several groups have reported electrophysiological changes in the hippocampus in response to adrenal steroids.9s34 No additive effects of kainic acid and dexamethasone were seen for BDNF in the hippocampus, consistent with the finding that the level of BDNF mRNA is not changed in response to dexamethasone treatment alone after 4 h.5”3 However, when kainic acid was given 24 h after dexamethasane, there was a sign&ant attenuation of the BDNF mRNA response to kainic acid, which is consistent with the decrease in BDNF mRNA seen 24 h after dexamethasone treatment.5 Thus, both adrenalectomy and 24 h dexamethasone treatment attenuate the kainic acid-mediated increase of BDNF mRNA. This unexpected result implies that both reduced and supraphysiological levels of adrenal steroids attenuate the increase of BDNF mRNA. Alternatively, since dexamethasone treatment has been shown to influence a number of neuronal parameters, such as excitability’s and voltage-dependent Ca*’ conductances,35 as well as a number of biochemical parameterQ7 it is possible that the dexamethasoneinduced attenuation of BDNF mRNA was caused indirectly by any of these changes. Taken together, these results suggest that different mechanisms underlie the increases in BDNF and NGF mRNAs seen after kainic acid exposure, which are modulated by dexamethasone in different ways. Recently, a clear dissociation between behavioral seizures and increases in BDNF mRNA after kainic acid in postnatal rats has been reported, suggesting that neuronal activation is not sufficient for up-regulation of BDNF mRNA and that other factors may play a role.” In the ~p~~rnpus, adrenal~tomy si~ificantly attenuated the increase elicited by kainic acid on neurotrophins and trkB mRNAs, but had little effect on c-fos mRNA expression. However, in cerebral cortex, adrenalectomy significantly reduced the increase in c-fos, suggesting that glucocorticoids differentially regulate c-fos mRNA expression in these two brain regions. C-fos belongs to a family of genes that are rapidly and transiently activated by a great variety of stimuli. 52 The c-fos gene encodes a nuclear DNA binding protein which forms heterodimers with other proteins such as c-jun, where the two proteins together constitute the transcription factor API. In fibroblasts, c-fos has been postulated to regulate transcription of the NGF gene by binding to an API site located in the first intron of the NGF gene.” The rapid increase in c-fos mRNA in the brain after kainic acid treatment preceding the increase in BDNF and NGF mRNAs4 is consistent with a similar role for c-fos in the brain. Glucocorticoids have been shown to repress transcription from the collagenase gene by reducing API activity through direct binding to the transcription factor.30,57*61 Our finding that changes in c-fos mRNA in the hippocampus in response to kainic acid and glucocorticoids

G. BAKBANY

920

are not parallel to changes in NGF mRNA levels suggests that glucocorticoids do not attenuate the increase of NGF mRNA by inhibiting transcription of the c-fos gene. Instead, it is possible that glucocorticoids, similar to the repression of transcription from the collagenase gene, inhibit transcription from the NGF gene by direct protein-protein interaction with the API complex.

and k-1.PERSSO~

mRNAs in the hippocampus and piriform cortex. TrkB mRNA expression in these brain regions is increased by kainic acid but not changed in response to altered levels of glucocorticoids. though adrenalectomy reduces the kainic acidmediated increase. Furthermore. WC show that dexamethasone potentiates the increase in NGF mRNA elicited by kainic acid whereas it attenuates the increase in BDNF mRNA. These data imply

CONCLUSION

Our results show that adrenalectomy attenuates the kainic acid-mediated increase of NGF and BDNF

two

different,

partly

isms whereby neurotrophin regulated in the brain.

counteracting,

gene

mechan-

expression

is

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