Ž .Brain Research 745 1997 248–256

Research report

Amygdala-kindled seizures increase the expression of corticotropin-releasingŽ .factor CRF and CRF-binding protein in GABAergic interneurons of the

dentate hilus

Mark A. Smith a,), Susan R.B. Weiss a, Richard L. Berry b, Li-Xin Zhang a, Mike Clark a,Gwen Massenburg a, Robert M. Post a

a Biological Psychiatry Branch, National Institute of Mental Health, 10r3N212, Bethesda, MD 20892, USAb Department of Pharmacology, Uniformed SerÕices UniÕersity of the Health Sciences, Bethesda, MD, USA

Accepted 17 September 1996

Abstract

Ž .Kindling, a model of temporal lobe epilepsy, induces a number of neuropeptides including corticotropin-releasing factor CRF . CRFitself can produce limbic seizures which resemble kindling in some aspects. However, tolerance to the convulsant effects of CRF develops

Ž .rapidly. Hypothetically, this could be explained should seizures also induce the CRF-binding protein CRF-BP , which has beenpostulated to restrict the actions of CRF. Therefore, in the present study, we used in situ hybridization to examine the effects ofamygdala-kindled seizures on the mRNA levels of CRF and CRF-BP. Kindled seizures markedly elevated CRF and CRF-BP in thedentate gyrus of rats. CRF and CRF-BP were induced almost exclusively in GABAergic interneurons of the dentate hilus. The CRF andCRF-BP interneurons also expressed neuropeptide Y but not cholecystokinin. CRF appeared to have an excitatory role in the dentategyrus as it decreased the afterhyperpolarization of dentate granule neurons. These results suggest that CRF may contribute to thedevelopment of amygdala kindling. However, the compensatory induction of CRF-BP may serve to limit the excitatory effects of CRF inthe dentate gyrus.

Keywords: Corticotropin-releasing factor; CRF-binding protein; Hippocampus; Kindling; Co-expression; Neuropeptide Y; Cholecystokinin

1. Introduction

Ž .Corticotropin-releasing factor CRF is a neuropeptideimportant in the endocrine and behavioral regulation of thestress response. Additionally, CRF may act as an excita-tory neurotransmitter or neuromodulator in the central

Ž .nervous system CNS . Central administration of CRFincreases neuronal firing, particularly in the locus coeruleusw x34 and the hippocampus where it inhibits the slow after-

Ž . w xhyperpolarization AHP 1,28 . Moreover, ovine CRF,Ž .when injected intracerebroventricularly i.c.v. into ro-

dents, produces tonic clonic seizures after a 3–7 hourw xdelay 11 . Epileptiform activity begins in the amygdala

and then spreads to the dorsal hippocampus and cerebralw xcortex 3 .

The unusually long latency to produce seizures led us to

) Corresponding author. Present address: DuPont Merck, ExperimentalStation, E400r4448, PO Box 80400, Wilmington, DE 19880, USA. Fax:

Ž .q1 302 695-3730.

investigate whether i.c.v. CRF was producing a kindling-like stimulation of the limbic system that eventually re-sulted in full-blown motor seizures as suggested by Ehlers.Indeed, we found that pretreatment with ovine CRF accel-erated the development of subsequent electrical kindling of

w xthe amygdala 36 . However, the role of CRF in limbickindling is complicated by several observations. First, incontrast to its facilitation of electrical kindling, daily ad-ministration of CRF quickly produced tolerance to its own

w xconvulsant effects 36 . Likewise, prior electrical kindlingof the amygdala prevented seizures in response to subse-quent CRF administration. And unlike ovine CRF, admin-istration of rat CRF does not consistently produce general-ized seizures, and the epileptiform activity is confined to

w xthe dorsal hippocampus 20,21 .Some of the seemingly paradoxical effects of CRF

administration might be explained by the rather complexphysiology of CRF, whose actions are regulated not onlyby multiple receptors, but also by a non-receptor CRF-bi-

Ž . w xnding protein CRF-BP 25 . CRF-BP mRNA is widely

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 96 01157-2

( )M. A. Smith et al.rBrain Research 745 1997 248–256 249

distributed in the CNS including the amygdala and hip-w xpocampal formation 26 . The role of CRF-BP is not clear,

but it may serve to limit the action of CRF. Interestingly,ovine CRF binds to the rat CRF receptor but not to theCRF-binding protein.

In a previous study, we found that amygdala-kindledw xseizures induced CRF in the hilus of the dentate gyrus 32 .

The hilus, or polymorphic layer, of the dentate gyruscontains a heterogeneous population of interneurons, someof which express GABA and serve to inhibit granuleneurons. These hilar interneurons can be further subdi-

Žvided according to the various neuropeptides neuropeptide.Y, somatostatin, cholecystokinin etc. that are co-expressed

Ž w x .with GABA see 24 for review . Which of the many hilarinterneuron subtypes express CRF is unknown. Moreover,the functional role of CRF in the dentate gyrus has notbeen described. Therefore, the purpose of the presentinvestigation was three-fold: first to determine what effect,if any, CRF might have on the electrophysiology of den-tate granule neurons; second, to characterize neurochemi-cally the hilar interneurons in which CRF is expressed; andthird, to investigate the effects of kindling on the expres-sion of CRF binding protein with the hope of sheddinglight on the apparent cross-tolerance between kindled andCRF-induced seizures and the differing convulsant proper-ties of ovine and rat CRF.

2. Materials and methods

2.1. Amygdala kindling

ŽMale Sprague-Dawley rats Taconic Farms, German-.town, NY weighing approximately 300 g were sterotaxi-

cally implanted with a bipolar platinum-iridium electrodeŽinto the left amygdala 5.7 mm posterior, 4.5 mm lateral

.and 2 mm ventral to intra-aural zero . One week followingsurgery, rats received once daily stimulation with 800 mAof current at 60 Hz for 1 s, or were handled in an identical

Ž .manner but did not receive current sham controls . Ratswere stimulated until they had multiple generalized seizuresŽ .means17.0"2.9, range 6–23 . The kindled and shamkindled rats were sacrificed either 24 h or 4 days after the

Ž .last seizure ns6 ratsrgroup . Because the results fromthe two sham groups were virtually identical, the data fromthe two sham groups were combined.

2.2. Electrophysiological recordings from hippocampalslices

Intracellular recording from dentate granule neuronsw xwas performed as previously described 6,27 . Hippocam-

Ž .pal slices 400 mm thick were obtained from normal 100g male Sprague-Dawley rats. Slices were maintained at358C in an interface recording chamber. Dentate granuleneurons with resting membrane potentials more negative

than y60 mV were used for study. The cells werevoltage-clamped at y57 mV for the duration of recording.Stimulation pulses were 0.5 nA for 500 ms. After baseline

Ždata were gathered, 150 nM ratrhuman CRF Bachem,.Torrance, CA was added to the artificial CSF perfusate

for 10 min, and the effects on electrical stimulation wererecorded.

2.3. Riboprobes

Antisense cRNA probes were transcribed from appro-priate linearized plasmids using T7, Sp6 or T3 poly-

Žmerases according to the manufacturer’s instructions Am-.bion, Austin, TX . The cRNA probe for rat CRF was

transcribed from a 1 kb cDNA insert in pGEM 4 contain-Žing the full length coding region of rat CRF kindly.provided by Dr. K. Mayo, Northwestern University . The

specificity of the CRF riboprobe has been previouslyw xdescribed 13 . For the CRF-binding protein riboprobe, we

utilized a pBS II SK plasmid containing a Pst I fragmentŽcoding for the first 600 bp of the cDNA designated

.59–10 kindly provided and characterized by Dr. W.W.Ž . w xVale and colleagues Salk Institute 25,26 . For glutamate

decarboxylase, we used a pBS plasmid containing a 2.7 kbcDNA for rat glutamic acid decarboxylase kindly provided

Ž .by Dr. D.I. Gottlieb Washington Univ., St. Louis, MOw x38 . We linearized the plasmid with PÕuII which made a1. 1 kb transcript. For neuropeptide Y, we used a 511 bpfragment from the coding region of rat NPY inserted into

ŽpBS generously provided by Dr. S. Sabol NHLBI,. w xBethesda, MD 12 . The NPY cDNA was linearized with

FspI to make a riboprobe about 500 bp in size. The fulllength 535 bp cholecystokinin riboprobe was labeled and

w xcharacterized as described 41 . We found that in situhybridization using sense copies of the above cDNAs ascontrol probes did not produce specific signals.

2.4. Riboprobe in situ hybridization.

Ž .Frozen brain sections 15 mm at the level of the dorsalhippocampus were cut on a cryostat and mounted ontogelatin-coated slides. In situ hybridization using riboprobes

w35 x w xlabeled with S UTP was performed as described 30,37 .Briefly, the sections were fixed in 4% paraformaldehyde,dehydrated in increasing concentrations of ethanol, anddelipidated in chloroform. For each slide containing twobrain sections, a saturating amount of 35S-labeled ribo-

Ž 6 .probe 1–2=10 cpm was added to 50 ml hybridizationŽ .buffer containing 20 mM Tris-HCl pH 7.4 , 50% for-

Ž .mamide, 300 mM NaCl, 1 mM EDTA pH 8 , 1=

Denhardt’s, 250 mgrml yeast tRNA, 250 mgrml totalRNA, 10 mgrml salmon sperm DNA, 10% dextran sul-

Ž .fate, 100 mM dithiothreitol DTT , 0.1% SDS, and 0.1%sodium thiosulfate. Hybridization took place overnight at548C. The next day the sections were rinsed in 4=SSC,treated with 20 mgrml RNAse A, rinsed in decreasing

( )M. A. Smith et al.rBrain Research 745 1997 248–256250

Fig. 1. Effects of kindling on CRF and CRF-BP mRNAs in the dentate gyrus of rats. Autoradiographs are from sham or kindled rats through the level ofŽ .the dorsal hippocampus. Note increased CRF and CRF-BP expression in the hilar area of the dentate gyrus dg . Actual width of pictured brain slice is 1.2

cm.

concentrations of SSC at room temperature and finallywashed in 0.1=SSC for 1 h at 658C. The slides wereapposed to Kodak Biomax MR film for approximately 3

Ž . Ž .days CRF-BP or 7 days CRF . Some sections wereŽ .dipped in photographic emulsion Kodak NTB-2 and de-

veloped after 4 weeks in Kodak D19 developer.

2.5. Double labeling in situ hybridization

To examine the co-expression of two different mRNAspecies in the same cell, we hybridized simultaneouslywith a digoxigenin-labeled riboprobe and a second ribo-

33 w xprobe labeled with P-UTP as previously described 30 .Digoxigenin labeling with digoxigenin-UTP was carried

Žout according to the manufacturer’s instructions Genius.Kit, Boerhinger Mannheim . The digoxigenin-labeled probe

Žwas visualized by preincubating the slides in 100 mM.Tris, 150 mM NaCl, pH 7.5 containing 3% normal goat

serum and 0.3% Triton X-100 for 30 min at room tempera-ture. Then rabbit anti-digoxigenin antibody conjugated with

Ž . Ž .alkaline phosphatase Boerhinger Mannheim 1:500 wasadded and the slides incubated for 3–4 h at room tempera-ture. The slides were washed and nitroblue tetrazolium and5-bromo-4-chloro-3-indolyl phosphate used to produce ablue-black reaction product in the cells where the digoxi-genin-labeled probe had hybridized. Slides were dipped in

Ž .photographic emulsion Ilford K.5D and developed after4–6 weeks in Kodak D19 developer. The number of cells

Žin which hybridization of the digoxigenin probe indicated.by diffuse reaction product in the cytoplasm and radioac-

Žtive probe indicated by small dark silver grains over the.cell occurred was assessed by observation at 400=

Ž . Ž .Fig. 2. CRF-BP mRNA in dentate gyrus from sham A and kindled B rat. Darkfield photomicrographs show increased CRF-BP mRNA expression 24 hŽ .after the last seizure in the dentate hilus from a kindled rat. Note increase in expression in individual hilar neurons indicated by arrows and the absence of

Ž .expression in the granule layer gl . Bars100 mm.

( )M. A. Smith et al.rBrain Research 745 1997 248–256 251

Fig. 3. Effects of kindling of CRF and CRF-BP mRNA in the dentatehilus 1 or 4 days after the last seizure. Average levels of mRNA are

ŽŽ . .expressed as dpmrmg =area through a section of the dentate gyrus.Levels are significantly different from sham at ) P -0.01, or ) ) P -

0.001.

magnification in 2–4 sections of the dorsal hippocampusŽ .from each animal ns4–8 animalsrgroup .

2.6. Data analysis

Autoradiograms were analyzed by a ‘blind’ observerusing image analysis software developed by Wayne Ras-

w xband at NIH as described 31 . The optical density wascorrected for film non-linearity using 14C standards andconverted to dpmrmg wet tissue. Measurements weretaken from 4–6 sections from each animal. Data areexpressed as the mean"standard error of the meanŽ .S.E.M. . Differences between groups were tested statisti-

Ž .cally by analysis of variance ANOVA followed by thepost hoc Tukey-Kramer comparison for multiple groupsusing SuperANOVA software.

3. Results

3.1. Effects of kindled seizures on CRF and CRF-BP

Using in situ hybridization, we examined the effects ofamygdala-kindled seizures on the expression of CRF and

Fig. 4. Correlation between CRF and CRF-BP mRNA levels in theŽŽ .dentate gyrus. Individual levels of mRNA are expressed as dpmrmg =

.area through a section of the dentate gyrus. Data derived from anexperiment in which rats were electrically stimulated before or after

Ž .carbamazepine contingent tolerance which induced CRF and CRF-BPw xmRNAs to varying degrees 35 .

CRF-binding protein in the rat brain. As previously re-w xported 32 , kindled seizures dramatically increased CRF

Ž .mRNA expression in the dentate hilus Fig. 1 . And again,kindled seizures did not alter CRF mRNA levels in thehippocampal pyramidal layers, cerebral cortex, or centralnucleus of the amygdala. In the present study, we alsoexamined the effects of kindling on the expression of thebinding protein for CRF. In unstimulated animals, CRF-BPmRNA was present in scattered neurons of the hippocam-pal formation but was not expressed in granule cells orpyramidal layers of the hippocampus. Basal expression ofCRF-BP appeared to be higher than that of CRF. Yet, in asimilar fashion to that of CRF, amygdala kindling in-creased CRF-BP mRNA levels specifically in neurons of

Ž .the dentate hilus Figs. 1 and 2 . It appeared that thenumber of neurons expressing CRF-BP increased as well

Ž .as the level of expression per cell after kindling Fig. 2 . Incontrast, kindled seizures did not alter the expression ofCRF-BP expression in the cerebral cortex, amygdala orelsewhere in the brain that we observed.

CRF and CRF-BP mRNA levels were elevated 24 hŽ .after the last seizure Fig. 3 . Similar results were observed

in a second kindling experiment in which rats were sacri-

Table 1Co-expression of CRF mRNA in dentate hilar interneurons containing GABA, NPY or CCK mRNAs in kindled rats

Co- No. of cells expressing No. of cells No. of cells expressing % of co-transmitter % of CRF cellstransmitter co-transmitter only expressing both co-transmitter cells also expressing also expressing

CRF only and CRF CRF co-transmitter

GABA 819 6 119 12.7% 95.2%NPY 52 4 77 59.7% 95.1%CCK 128 198 5 3.8% 2.5%

( )M. A. Smith et al.rBrain Research 745 1997 248–256252

Ž .ficed 4 h after the last seizure results not shown . Overall,the regulation of CRF and CRF-BP during kindled seizureswas very similar as evidenced by a strong correlation

Ž .between CRF and CRF-BP mRNA levels Fig. 4 . How-ever, while CRF levels had returned to baseline by 4 dafter the last seizure, we found that CRF-BP expression

Ž .was still significantly elevated at that time Fig. 3 .

3.2. Co-expression of CRF and CRF-BP in GABAergicinterneurons

By hybridizing simultaneously with 33 P- and digoxi-genin-labeled probes, we determined what other neuro-transmitters were contained in the hilar neurons that ex-pressed CRF and CRF-BP after kindling. CRF and CRF-BP

w33 x Ž . Ž .Fig. 5. Co-expression of CRF or CRF-BP with GAD, NPY or CCK. Hybridization of P UTP riboprobes specific for CRF A,B,C or CRF-BP D,E,F isŽ . Ž . Ž .indicated by small dark silver grains. Hybridization of digoxigenin-labeled GAD A,D , NPY B,E and CCK mRNAs C,F are indicated by the diffuse

Ž . Ž .black reaction product. Note that some GAD cells co-express CRF A or CRF-BP D as indicated by the closed arrows. Also note that CRF isŽ .co-expressed with NPY B . CRF-BP is co-expressed with some NPY neurons as indicated by the closed arrow, but other neurons apparently express

Ž .CRF-BP but not NPY as indicated by the open arrow pointing towards a cluster of grains in the absence of digoxigenin labeling E . There was noŽ .co-expression of CRF C with CCK as indicated by the open arrows pointing to a cluster of grains without digoxigenin. Likewise, CRF-BP did notŽ . Ž .co-localize with CCK F , where the open arrow points to a large cluster of grains which are slightly over-exposed in the absence of underlying reaction

Ž .product compare to D .

( )M. A. Smith et al.rBrain Research 745 1997 248–256 253

Table 2Co-expression of CRF-BP mRNA in dentate hilar interneurons containing GABA, NPY or CCK mRNAs in kindled rats

Co- No. of cells No. of cells No. of cells expressing % of co-transmitter % of CRF-BP cellstransmitter expressing co- expressing both co-transmitter cells also expressing also expressing

transmitter only CRF-BP only and CRF-BP CRF-BP co-transmitter

GABA 456 14 172 27.4% 92.4%NPY 163 29 192 54.1% 86.9%CCK 111 321 0 0% 0%

Table 3Effects of kindling on the percentage of GABAergic hilar interneuronsco-expressing CRF or CRF-BP mRNAs

Ž . Ž .GAD GADq % GADq %total CRF CRF-BP

Ž . Ž .Sham 144"19 1.5".5 1.1 14.7"3 10.4b aŽ . Ž .Kindled 157"22 19.8"5 12.6 43.0"6 27.1

Data represent the average number of neurons per hippocampal sectionexpressing the particular mRNA or combination of mRNA species. Thenumber of cells expressing the mRNA is greater in the kindled groupcompared to the sham at a P -0.01 or b P -0.001.

were almost exclusively expressed in GABAergic neuronsas evidenced by the presence of glutamate decarboxylaseŽ .GAD mRNA. Most of the CRF and CRF-BP neurons

Ž .also expressed neuropeptide Y NPY mRNA. In contrastthere was virtually no co-expression of cholecystokinin

Ž .with CRF or CRF-BP see Fig. 5 and Tables 1 and 2 .Compared to non-stimulated sham animals, kindling

significantly increased the proportion of GAD-expressingŽneurons in which CRF and CRF-BP were induced Table

Table 4Effects of kindling on the percentage of NPY hilar interneurons co-ex-pressing CRF or CRF-BP mRNAs

Ž . Ž .NPY NPYq % NPYq %total CRF CRF-BP

Ž . Ž .Sham 31"5.4 2.2".5 7.1 11.8"4.2 38.4a a aŽ . Ž .Kindled 60"13 15.4"3.6 25.3 32.0"4.1 52.4

Data represent the average number of neurons per hippocampal sectionexpressing the particular mRNA or combination of mRNA species. Thenumber of cells expressing the mRNA is greater in the kindled groupcompared to the sham at a P -0.01.

Fig. 6. Co-expression of CRF and CRF-BP in the dentate hilus. Photomi-33 Žcrograph of P-labeled CRF hybridization indicated by small black

. Žsilver grains and digoxigenin-labeled CRF-BP indicated by dark, diffuse.reaction product . Closed arrow indicates a neuron expressing both CRF

and CRF-BP mRNAs. Open arrow points to a cluster of grains notoverlying reaction product, thus indicating a neuron which expressedCRF but not CRF-BP. Bars10 mm.

. w x3 . Consistent with our previous study 32 , the number ofGAD mRNA-positive neurons in the hilus did not changeafter kindling. The proportion of NPY neurons expressingCRF and CRF-BP also increased after kindling, eventaking into consideration the increased number of hilar

Ž .neurons expressing NPY Table 4 .It was also determined that co-expression of CRF with

Ž .CRF-BP occurred in the dentate hilus Fig. 6 . Approxi-mately 50% of CRF neurons in the dentate hilus alsoexpressed CRF-BP mRNA, while 32% of the total numberof CRF-BP neurons co-expressed CRF following kindlingŽ .Table 5 .

Table 5Effects of kindling on the co-expression of CRF and CRF-BP mRNAs

CRF only CRF-BP only CRFqCRFBP CRF total CRF-BP total % of CRF with % of CRF-BPCRF-BP with CRF

Sham 1.7"1.5 12.7"0.6 1.3"0.6 3.0"1.0 14"1.0 53"41 9"3.4b b a c c aKindled 8.7"2.1 24"4.5 11.3"4.5 20"3.7 35"4.1 55"12 32"11

Data represent the average number of neurons per hippocampal section expressing the particular mRNA or combination of mRNA species. The number ofcells expressing the mRNA is greater in the kindled group compared to the sham at a P-0.05, b P-0.01, or c P-0.001.

( )M. A. Smith et al.rBrain Research 745 1997 248–256254

Ž .Fig. 7. Effect of CRF on the slow afterhyperpolarization AHP in a non-kindled dentate granule neuron. Intracellular recordings from a single dentategranule neuron stimulated with 0.5 nA current for 500 ms. Baseline recording revealed an AHP of approximately 5 mV. During a 10-min perfusion with150 nM CRF, the AHP decreased to 3 mV. After a 10-min washout period, the AHP returned to 6 mV in amplitude. Results are representative of threesimilar experiments.

3.3. Effects of CRF on the electrophysiology of the dentategyrus

Finally we determined what effect CRF had on dentategyrus electrophysiology in naive rats that had not been

Ž .kindled. CRF 150 nM consistently decreased the slowŽ .afterhyperpolarization AHP in dentate granule neurons

Ž .Fig. 7 . Accomodation was unchanged or slightly in-creased.

4. Discussion

4.1. CRF and CRF-BP co-expression in GABAergic in-terneurons of the dentate hilus

From our initial study on CRF expression during kin-dling, we had tentatively proposed that CRF was beinginduced in dentate hilar interneurons which probably were

w xGABAergic 32 . In the present study, we found thatindeed, not only CRF but also CRF-BP, were inducedalmost exclusively in GABAergic interneurons of the den-tate gyrus by kindling. GABAergic neurons, in addition tothose in the dentate gyrus, may also express CRF. Weobserved in sham animals that many GAD neurons in thecerebral cortex co-expressed CRF or CRF-BP mRNA. Thecoexistence of CRF and GABA has previously been ob-served in the paraventricular nucleus of the hypothalamusŽ . w x w xPVN 23 and in amacrine cells of the retina 40 .

The GABAergic neurons in the dentate hilus can befurther subdivided into groups based on the neuropeptidesthey express. We found that CRF and CRF-BP are co-ex-

Ž .pressed with NPY but not with cholecystokinin CCK . Ina similar fashion, there is very little co-expression of CCK

w xand CRF in the hypothalamic paraventricular nucleus 7 .The hilar neurons that express NPY and CRF probablyexpress additional neuropeptides. NPY interneurons in the

w xhilus usually also contain somatostatin 14 . Using oligo-Ž .nucleotide probes specific for CRF and somatostatin SS ,

we found that 30–50% of SS neurons co-expressed CRFŽ .M.A. Smith, unpublished observation . Because the ma-jority of NPY and SS neurons in the hilus are immunoreac-

w xtive for GAD 15 , it would seem likely that manyGABArNPYrSS neurons in the hilus also express CRFand probably CRF-BP. This conclusion is further sup-ported by the fact that the interneurons that express CRF-immunoreactivity project to the outer portion of the molec-

w xular layer of the dentate gyrus 32 as do NPY and SSw xneurons 2,10,17 where they are thought to terminate

either postsynaptically on granule cells or presynapticallyon perforant path fibers. Thus the CRF interneurons in thehilus are most likely classified as GABAergic polymorphic

Žcells rather than basket cells which often contain CCKand project to the granule neuron cell bodies and inner

. w xmolecular layer 24 .

4.2. Potential releÕance of CRF-BP in limiting the epilep-togenic effects of CRF

The induction of both CRF and CRF-BP mRNA bykindled seizures appeared to be confined to the dentatehilus. We found no evidence of changes in CRF-BP in theamygdala, cortex or elsewhere in response to kindling.Moreover, the induction of CRF-BP in the dentate hiluswas specific in that electroconvulsive seizures also in-creased CRF-BP expression, but immobilization stress did

Ž .not M.A. Smith, unpublished observations .The role of CRF-BP in the regulation of CRF activity is

not clear, but one likely possibility is that CRF-BP limitsthe actions of CRF by preventing CRF from binding to itstransmembrane receptors. This may occur in an autocrine

w xfashion at the site of CRF release, as we and others 26found significant co-expression of CRF and CRF-BPmRNA. However, some hilar neurons clearly expressed

( )M. A. Smith et al.rBrain Research 745 1997 248–256 255

CRF-BP, but not CRF, leading us to speculate that CRF-BPmay also limit the paracrine actions of CRF.

The induction of CRF-BP during kindling may helpexplain our previous observation that electrically kindledseizures prevented the epileptogenic effects of subsequent

w xadministration of CRF 36 . Whether the seizures thatresult from i.c.v. CRF injection also induce CRF-BP isunknown. However, such an induction of CRF-BP mightexplain the rapidly observed tolerance to the convulsant

w xeffects of repeated CRF injection 36 . We cannot rule outthe possibility that down-regulation of CRF receptors mayalso contribute to CRF tolerance. However, in preliminaryexperiments we did not observe a consistent decrease inthe expression of CRF receptor mRNA in the dentate1

gyrus. Very little expression of the CRF receptor occurred1

in dentate granule neurons under baseline conditions. Fu-ture experiments will be necessary to determine if therecently cloned CRF receptor, which may be present in2

w xhigher concentrations in the dentate gyrus 18 , is regulatedby seizure activity.

The binding characteristics of CRF-BP may also helpexplain the observed differences in ovine and rat CRFpro-convulsant effects. Given the fact that ovine and ratCRF bind to the CRF receptor with similar affinities1Ž . w xK s8.3 and 3.3 nM respectively 8 , whereas ovined

CRF has a much lower affinity for the CRF bindingŽ . Ž . w xprotein 250 nM than does rat CRF 0.1 nM 25 , it may

be that CRF-binding protein plays a role in limiting theepileptogenic effects of rat CRF.

4.3. Potential releÕance of CRF in kindled seizures

The functional significance of the induction of CRF ininterneurons of the dentate hilus following kindling is notknown. Whether the excitatory effect of CRF on dentategranule neurons is mediated in a direct or indirect mannerremains to be seen. One possibility is that CRF affectsdentate granule neurons indirectly by stimulating the re-lease of glutamate or GABA as suggested in other brain

w xareas 5,29 . However, we were not able to demonstrate aneffect of CRF on glutamate or GABA release from hip-

Žpocampal slices in vitro M. Clark and M.A. Smith, unpub-.lished observations . Alternatively, CRF may stimulate the

release of another neuromodulator such as norepinephrinew x16 .

Regardless, dentate granule neurons do respond to CRF.I.c.v. injection of CRF can stimulate c-fos expression in

w xthe dentate gyrus 9 . Moreover, in the initial electrophysi-ological studies described here, we found that CRF de-creases the afterhyperpolarization current of dentate gran-ule neurons in a similar fashion to that previously observed

w xin hippocampal pyramidal neurons 1 . Thus CRF mayexcite dentate granule neurons by antagonizing the Ca2q-dependent Kq channel responsible for the slow AHP.

It is of interest that CRF, which appears to be excita-tory, is increased in inhibitory GABAergic neurons during

kindling. The other neuropeptides in the hilar neurons suchas NPY and SS are generally thought to be inhibitoryw x22,39 . It is conceivable that the induction of CRF maypartially counteract the inhibitory effects of GABA, NPYand SS and thereby contribute to the net excitation of thedentate gyrus during kindling.

CRF may also contribute to hippocampal damage in-curred during ischemia or exposure to excitatory aminoacids. A CRF antagonist can attenuate the extent of dam-age in the CA1 pyramidal layer in response to ischemiaw x19 . Likewise the excitatory neuronal damage induced byan NMDA receptor agonist is also reduced by a CRF

w xantagonist 33 . Thus CRF antagonists may have utility inw xpreventing the epileptogenic effects of CRF 4 and have

neuroprotective effects during other insults to the hip-pocampus.

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