kappa opioid control of seizures produced by a virus in an animal

doi:10.1093/brain/awl008 Brain (2006), 129, 642–654

Kappa opioid control of seizures producedby a virus in an animal model

Marylou V. Solbrig,1,2 Russell Adrian,1 Janie Baratta,1 Julie C. Lauterborn3 and George F. Koob4

1Department of Neurology, 2Department of Pharmacology, 3Department of Anatomy and Neurobiology,University of California-Irvine, Irvine and 4Molecular and Integrative Neurosciences Department,The Scripps Research Institute, La Jolla, CA, USA

Correspondence to: Marylou V. Solbrig, Department of Neurology, 3226 Gillespie Neuroscience Research Building,University of California-Irvine, Irvine, CA 92697-4292, USAE-mail: [email protected]

Epilepsy remains a major medical problem of unknown aetiology. Potentially, viruses can be environmentaltriggers for development of seizures in genetically vulnerable individuals. An estimated half of encephalitispatients experience seizures and�4% develop status epilepticus. Epilepsy vulnerability has been associatedwitha dynorphin promoter region polymorphism or low dynorphin expression genotype, in man. In animals, thedynorphin system in the hippocampus is known to regulate excitability. The present study was designed to testthe hypothesis that reduced dynorphin expression in the dentate gyrus of hippocampus due to periadolescentvirus exposure leads to epileptic responses. Encephalitis produced by the neurotropic Borna disease virus in therat caused epileptic responses and dynorphin to disappear via dentate granule cell loss, failed neurogenesis andpoor survival of new neurons. Kappa opioid (dynorphin) agonists prevented the behavioural and electroence-phalographic seizures produced by convulsant compounds, and these effects were associated with an absence ofdynorphin from the dentate gyrus granule cell layer and upregulation of enkephalin in CA1 interneurons, thusreproducing a neurochemical marker of epilepsy, namely low dynorphin tone. A key role for kappa opioids inanticonvulsant protection provides a framework for exploration of viral and other insults that increase seizurevulnerability and may provide insights into potential interventions for treatment of epilepsy.

Keywords: seizure; encephalitis; Borna disease virus; hippocampus; dynorphin

Abbreviations: BDNF = brain derived neurotrophic factor; BDV = Borna disease virus; BrdU = bromodeoxyuridine;DCX = doublecortin; GFAP = glial fibrillary acidic protein; IR = immunoreactivity; KOR = kappa opioid receptor;NLX = naloxone; nor-BNI = nor-binaltorphimine; NeuN = neuronal nuclei

Received August 8, 2005. Revised November 9, 2005. Accepted December 15, 2005. Advance Access publication January 6, 2006

IntroductionSeizures and status epilepticus increase encephalitis morbidity

and mortality. Despite advances in diagnostic techniques, no

causal agent is identified in the majority of encephalitis cases

(Koskiniemi et al., 2001; Glaser et al., 2003), which limits

agent-specific therapies. The opioid system in the hippocam-

pus is known to regulate excitability (Henriksen et al., 1982;

Wagner et al., 1993; Weisskopf et al., 1993; Madamba et al.,

1999). Dynorphin distribution and regulation are consistent

with endogenous anticonvulsant function in experimental

models (Kanamatsu et al., 1986; Tortella, 1988; Gall et al.,

1990; Douglass et al., 1991; Hong et al., 1992; Romualdi et al.,

1995; Simonato and Romualdi, 1996; Pierce et al., 1999) and a

low dynorphin expression genotype in man is associated with

epilepsy vulnerability (Stogmann et al., 2002; Gambardella

et al., 2003). Recent hypotheses have also addressed the

role of dynorphin in viral seizures (Solbrig and Koob, 2004).

In man, Borna disease virus (BDV) has been associated

with hippocampal sclerosis (de la Torre et al., 1996;

Czygan et al., 1999) and neuropsychiatric disorders (Lipkin

et al., 2001; Ikuta et al., 2002). In rats, BDV causes a persistent

infection with time-based neurological sequelae (Narayan

et al., 1983; Solbrig et al., 1994), one of which has an epileptic

phenotype (Solbrig and Koob, 2004; Solbrig et al., 2005).

Given dynorphin expression can be an important deter-

minant of seizure vulnerability, we tested the hypothesis

that reduced dynorphin expression in the dentate gyrus

of hippocampus owing to periadolescent virus exposure

leads to epileptic responses. Using neuropharmacological,

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anatomical, electrophysiological and biochemical/metabolic

techniques to study BDV-infected rats, we find imbalance

in hippocampal opioid systems causes seizures and a kappa

opioid controls seizures. Besides illustrating viral pathways to

disease, the work provides a framework for exploration of

viral and other insults that increase seizure vulnerability.

Materials and methodsAnimalsSubjects were male Lewis rats (Charles River Labs, Wilmington, MA,

USA) group housed on a 12 h light–dark cycle with ad libitum

access to food and water. All experimental procedures were

performed in compliance with institutional (University of

California-Irvine Institutional Animal Care and Use Committee;

Animal Welfare Assurance no. A3416-01) and National Institutes

of Health guidelines.

Infection of animalsAnaesthesia was induced with inhaled methoxyflurane administered

in a closed bell jar containing gauze soaked with anaesthetic to

achieve 0.22% minimal alveolar concentration (Committee on

Pain and Distress in Laboratory Animals, ILAR, 1992). Under

methoxyflurane anaesthesia, 4-week-old males were infected intra-

cerebrally with BDV (BD rats) by injection of 1.6 · 104 tissue culture

infectious dose units, strain He/80-1, or sham infected with sterile

phosphate buffered saline (PBS) (NL rats) (Solbrig et al., 1994).

Neuropharmacological testing was 6 weeks after infection (Solbrig

et al., 1994). Additional behavioural, morphological and histological

analyses were at different times after infection, as indicated.

DrugsDrugs used were the general opioid antagonist naloxone (NLX)

(1 mg/kg s.c.) (Sigma, St Louis, MO, USA), the kappa opioid

receptor (KOR) agonist U-50488 (trans–(6)-3,4–dichloro-N-

methyl-N–[2-1(-pyrrolidinyl)cyclohexyl] benzeneacetamide) (10,

20, 40 mg/kg s.c.) (Sigma) (Tortella et al., 1986), morphine sulphate

(5 mg/kg s.c.) (NIDA, Bethesda, MD, USA), nor-binaltorphimine

(nor-BNI) (10 mg/kg s.c.) (Tocris, Ellisville, MO, USA) dissolved

in saline. Naloxone dose dependently produces seizures in BD rats

(Solbrig et al., 1996) and 1 mg/kg s.c. is always convulsant in

BD animals. A 10 mg/kg s.c. nor-BNI is a mid-range dose that

modifies analgesic or hyperthermic responding (Endoh et al.,

1992). Morphine 5 mg/kg s.c. is an analgesic dose that initiates

the development of dependence (Britton et al., 1985; Schulteis

et al., 1997). U-50488 40 mg/kg s.c. is the lowest dose reported to

fully protect against supramaximal electroshock seizures (Tortella

et al., 1986).

ElectroencephalographyBD and NL rats were anaesthetized with ketamine + xylazine

(87 mg/kg + 13 mg/kg, i.p.) (Western Medical Supply, Arcadia,

CA, USA). Stainless steel screw electrodes (Plastics One, Roanoke,

VA, USA) were implanted in the cranium over the right and left

retrosplenial cortices (overlaying hippocampus), fixed to the skull

with dental cement. Ground electrodes were placed in the frontal

bones over the prefrontal cortex. EEG signals were recorded con-

tinuously for up to 3 h after injections of naloxone (1 mg/kg s.c.),

nor-BNI (10 mg/kg s.c.) and morphine (5 mg/kg s.c.). EEG signals

were obtained from unrestrained rats using commutators with

flexible recording cables (Plastics One, Roanoke, VA, USA), and

amplified and displayed in a Grass Polygraph (Model P511) using

manufacturer’s settings, data acquisition (PolyVIEW/XL) and

analysis software (Astro-Med, Inc./Grass Telefactor, W. Warwick,

RI, USA). After surgery, animals were allowed to recover for

1 week before study. Seizures were characterized by rhythmic

spike or sharp wave discharges on the EEG tracings, with amplitudes

at least two times higher than baseline, and accompanied by

epileptic-like behaviours (staring spells, behaviour arrest, twitches,

chewing, wet dog shakes, clonus, rearing with loss of balance)

(Racine, 1972). Neuropharmacological experimental groups con-

tained 4–8 BD rats and age-matched controls. EEGs also were

recorded from younger animals where indicated.

Data analysisNumbers of animals observed with epileptic behaviours were ana-

lysed using the Information Statistic for non-parametric indepen-

dent samples (Kullback, 1968). Observations of epileptic-like

behaviours were verified with recorded EEGs.

HistochemistryThe hippocampal formation was chosen for morphological study,

based on localization of seizures to hippocampal areas (Solbrig

et al., 2005) and tropism of virus to this region (Narayan et al.,

1983). Dynorphin and enkephalin immunoreactivity (IR) in hippo-

campal formation were examined 6 weeks after infection, processing

free-floating 50 mm frozen sections as described (Solbrig et al., 2005)

with primary antibodies to dynorphin A (1–17) (1:500, Serotec,

Oxford, UK) or met5-enkephalin (1:500, Chemicon, Temecula,

CA, USA) (n = 4 per group, BD or NL). Antiserum against

dynorphin A(1–17) has <0.001% cross reactivity to Dyn A(1–8),

leu5-enkephalin, a-neo-endorphin and Dyn B (1-13). Met5-enkepha-

lin antibody has 5.8% cross reactivity to leu5-enkephalin and <0.1%

to other endorphins.

Next, viral effects on dynorphin expression in hippocampal for-

mation were examined, measuring transcript levels in hippocampus

and tracking development and maturation of granule cells, the only

dynorphin-expressing cells of hippocampal formation.

Northern blot analysesEffect of BDV infection on preprodynorphin (PPD) transcription in

hippocampus was examined by northern hybridization. An aliquot

of 15 mg total RNA from hippocampal tissue of BD rats 6 weeks after

infection, extracted in Tri-Reagent (Molecular Research Centre Inc,

Cincinnati, OH, USA) was size-fractionated in 2.2 M for-

maldehyde/1% agarose gels, transferred to nylon membranes, UV

crosslinked and hybridized to random primed 32PDNA fragments

generated from cloned DNA representing dynorphin sequence

(Civelli et al., 1985) with GAPD transcripts as control for RNA

loaded. Autoradiographic signals were quantified by phosphorima-

ging (Storm 840 Phosphorimager; Molecular Dynamics, Sunnyvale,

CA, USA) (n = 8 per group).

To measure viral effects on hippocampal cell proliferation, bro-

modeoxyuridine (BrdU) labelling was performed. To identify cell

types exhibiting BrdU or BDV immunostaining, double-label studies

were performed. To characterize viral effects on development of

neurochemical phenotype, double label studies were performed.

Kappa opioid control of viral seizures Brain (2006), 129, 642–654 643



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BrdU injectionsBrdU (Sigma) dissolved in saline was administered to rats at 5 weeks

of age (1 week after BD infection) at a dose of 50 mg/kg i.p. ·3(3 times on day 0) to label dividing cells (Kuhn et al., 1996). To

evaluate the effect of BDV on cell proliferation, rats were sacrificed

the following day. To evaluate effects of BDV on survival of newly

born cells, rats were sacrificed 1, 2.5 or 4 weeks after last BrdU

injection (n = 4 per experimental group). Brains of all rats were

processed immunohistochemically for combined BrdU and markers

of several cell types using peroxidase or fluorescent methods to assess

phenotype of newly born cells (Kuhn et al., 1996).

Histological proceduresA one-in-six series of sections from control and BD animals surviving

1 day, 1, 2.5 and 4 weeks after the injection of BrdU were processed

for BrdU (1:400 for DAB, Chemicon; 1:100 for fluorescence, Accu-

rate, Westbury, NY, USA) and several cell type markers—neuronal

nuclei (NeuN) (1:1000, Chemicon) anti-NeuN for mature neurons,

glial fibrillary acidic protein (GFAP) (1:2000, Dako, Glostrup,

Denmark) for astroglia, OX42 (1:500, Serotec) for microglia and

mouse monoclonal antibody (38/15 H76) against BDV nucleo-

protein (1:500, gift of L. Stitz). Other primary antibodies

used were dynorphin A (Serotec 1:500), met-enkephalin (1:500,

Chemicon), MASH1 (mammalian achaete-scute homologue)

(1:250, Chemicon) and doublecortin (DCX) (1:500, Chemicon).

BrdU labelling required the following pretreatment: DNA denatura-

tion (50% formamide in 2· SSC, 65oC, 2 h), acidification (2N HCl

37oC, 30 min), rinse (0.1 M boric acid pH 8.5, 10 min) to reduce

reactive aldehydes, and membrane permeabilization (0.25% Triton,

3% normal horse serum in PBS, 1 h). Because of reports of BrdU

promoting neuronal apoptosis (Sekerkova et al., 2004), histology was

evaluated in additional animals age matched to neuropharmacology

animal subjects. MASH1 labelling required wash in 0.3% H2O2 in

methanol for 15 min, PBS washes and overnight incubation in 6%

normal goat serum with 0.2% Tween-20. Other antibodies used

incubations in 3% NGS and 0.2% Triton X-100, except dynorphin

A and met-enkephalin, which excluded Triton-X. Antigens were

visualized with Alexa-488 or Alexa-546 secondary antibodies

(1:1000, Molecular Probes, Carlsbad, CA, USA), or biotinylated sec-

ondary antibodies (Vector, Burlingame, CA, USA) processed by ABC

histochemical method (Hsu et al., 1981) and developed with 3,30-diaminobenzidine. Control sections were processed with omission

of primary antisera. Nissl substance was stained by Cresyl violet.

Timm staining to label zinc in mossy fibre synaptic vesicles and

evaluate mossy fibre sprouting was performed on a separate

group of BD animals 6 weeks after infection and age-matched NL

uninfected animals according to the procedure of Vaidya et al.

(1999). Brightfield and fluorescence signals were detected with

a Nikon E800 epifluorescence microscope equipped with Spot2

CCD Digital Camera and Spot and Spot Advanced imaging soft-

ware. Photomicrograph images were transferred into Photoshop and

assembled using Adobe Photoshop CS.

BrdU quantificationSections of 50 mm thickness were collected on a freezing microtome

through the entire rostrocaudal hippocampal formation with every

sixth section slide mounted for BrdU counting. Slides were examined

for BrdU-positive cells in the granule cell layer of dentate gyrus. BrdU

positive cells within the subgranular zone that were within 2-cell

body widths of the granule cell layer were considered part of the

granule cell layer (Kuhn et al., 1996). All BrdU-positive cells within

the granule cell layer, regardless of size or shape, were counted under

·400 magnification throughout the rostral-caudal extent of the gran-

ule cell layer. Total numbers of BrdU-stained cells in the granule cell

layer were multiplied by six and reported as total numbers of cells

in the granule layer. Raw data for cell counts were statistically ana-

lysed using Student’s t-tests (P < 0.05). To control for differences

in bioavailability or cell uptake of BrdU between uninfected and BD

rats, BrdU-labelled cells in corpus callosum were counted in selected

sections of age-matched NL and BD rats. The subventricular pro-

liferative zone was not used as a control because animals were

infected by intracerebroventricular (ICV) inoculation.

Analysis of phenotypeA one-in-six series of sections from control and BD animals surviving

1 day, 1 week, 4 weeks after injection of BrdU were double-labelled

for NeuN, GFAP, BDV, OX42, dynorphin A ormet-enkephalin using

fluorescent methods. At least 25 BrdU cells in the granule cell layer

per rat were examined for colocalization and analysed using Spot

Advanced Image Analysis or Bio-Rad MRC-600 laser confocal

microscope. Percentages of colabelled BrdU-positive cells were deter-

mined by manual counts of digitally recorded images. Differences in

treatment groups, set in 2 · 2 tables, were assessed by Chi-square

analysis.

Other chemistriesHost soluble factors with regulatory actions on neurogenesis or

dynorphin were assessed.

Coticosterone levelsCirculating corticosterone from whole blood collected at 6 p.m. from

BD rats 6 weeks after infection and age-matched controls, were

assayed by radioimmunoassay (ICN-rat corticosterone Immuno-

chem Double Antibody 125I RIA kit, Linco Diagnostic Services,

St Louis, MO, USA) with the sensitivity or detection limit approxi-

mately 5.7 ng/ml. Duplicate samples were analysed and expressed as

ng/ml. Group differences were analysed by Student’s t-tests with

significance set at P < 0.05 (n = 8–10 per experimental group).

BDNF (brain-derived neurotrophic factor)analysesBD rats from separate groups of animals, 6 weeks after infection

and age-matched controls, were euthanized by inhaled methoxy-

fluorane, administered in a closed bell jar containing gauze soaked

with anaesthetic to achieve 0.22% minimal alveolar concentration,

followed by decapitation. Brains were rapidly removed, cut into

coronal sections by razor blades using an ice-cold aluminium

alloy mould, and the entire hippocampal formation (dentate

gyrus and CA regions) dissected from these sections. Tissue was

manually homogenized in lysis buffer (137 mM NaCl, 20 mM

Tris, 10% glycerol, 1 mM PMSF, 10 mg/ml aprotinin, 1 mg/ml

leupeptin, 0.5 mM Na vanadate, 1% NP-40), as described

(Lauterborn et al., 2000). Total BDNF protein content for each

sample was measured using the BDNF Emax Immunassay System

(Promega, Madison, WI, USA) with absorbance at 450 nm deter-

mined using a plate reader. Data analysis—differences between BD

and NL groups in BDNF were analysed by Student’s t-tests with

significance set at P < 0.05 (n = 8 per group).

644 Brain (2006), 129, 642–654 M. V. Solbrig et al.



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ResultsSeizures in BD ratsICV infection of 4-week-old periadolescent Lewis rats is an

established model of encephalitis (Narayan et al., 1983). To

assess effects of infection on electroencephalographic tracings,

serial EEGs of BD rats were recorded over the course of infec-

tion in freely moving rats from screw electrodes overlaying the

hippocampus. After 3 weeks of infection, the earliest EEG

changes were frequent sharp waves. Disorganized EEG pat-

terns, with complex polyphasic waves containing an admix-

ture of frequencies, were obtained 4 weeks after infection from

BD rats showing startle myoclonus and hyperactivity. More

encephalopathic EEGs with burst suppression patterns—high

voltage bursts of slow waves intermingled with sharp transi-

ents or spikes against depressed background—were obtained

6 weeks after infection from BD rats showing stereotypical

behaviours, rearing, falling or wild running. Furthermore,

within another 2–4 weeks, repetitive sharp waves on EEG

were captured, time-locked to spontaneous seizures as

periods of behavioural arrest, staring spells, automatisms

such as lip smacking, blinking, head nodding or retrocollis

(dorsal head and neck extensions), falls or clonic limb move-

ments (Fig. 1 lower tracings). Epileptiform activity was not

observed in NL uninfected rats (Fig. 1 upper tracings).

A kappa opioid blocks seizuresThe general opiate antagonist NLX is a dose-dependent con-

vulsant in BD rats (Solbrig et al., 1996), producing periodic

sharp waves on hippocampal EEGs together with staring

spells, behaviour arrest, eye blinks or clonic movements,

and c-fos elevations in hippocampus and amygdala (Solbrig

et al., 2005). To further evaluate a role for opioids in brain

excitability, the effect of a kappa opioid on seizures induced by

naloxone was examined. NLX (1 mg/kg s.c.) rapidly and con-

sistently caused seizures with a transition from background

Fig. 1 Spontaneous epileptiform activity in BD rats. Representative EEGs recorded from hippocampal leads from NL uninfected rats (top)and BD rats (middle and lower tracings) during the course of infection (animal ages 7, 8, 10 and 12 weeks). Baseline activity from freelymoving NL rats characterized by low amplitude desynchronized activity have little variation, in contrast to progression from increasedsharp waves (7 weeks), to complex polyphasic waves (8 weeks) and high amplitude bursts (10 weeks) in BD rats during the course ofinfection. A spontaneous seizure with myoclonus occurs when background burst activity transitions to continuous spike or sharpwave discharges (BD rat, 12 weeks).




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burst activity to rhythmic spike or sharp wave discharges

within 5 min of drug administration (Fig. 2A). Administra-

tion of the selective KOR agonist U-50488 (40 mg/kg s.c.)

10 min before NLX (1 mg/kg s.c.) prevented NLX-induced

seizures in all BD rats tested (BD NLX 8/8 seizures versus BD

U50488 + NLX 0/8 seizures; 2I = 22.18 df = 1, P < 0.001, n = 8

per group) (Fig. 2A, lower tracing). Breakthrough seizures, as

isolated EEG spikes and runs of sharp waves were recorded

from BD animals receiving lower doses of U-50488 (10 and

20 mg/kg s.c.) (not pictured).

To confirm a role of kappa opioids in protecting the

brain from overstimulation, behavioural effects of the kappa

opioid antagonist nor-BNI were examined. Administration of

the KOR antagonist nor-BNI (10 mg/kg s.c.) had convulsant

effects, producing recurrent hippocampal spike or sharp wave

discharges accompanied by staring spells, lip trembling

or smacking, rhythmic trunk flexions or hindlimb extensor

spasms. Ictal episodes occurred 45 min to 2 h after drug

administration (BD nor-BNI 7/7 seizures versus NL

nor-BNI 0/4 seizures; 2I = 9.87, df = 1, P < 0.01, n = 4–7

per group), consistent with the peak KOR blocking effect

(Endoh et al., 1992) (Fig. 2B).

To evaluate a mu opioid receptor component to the sei-

zures, BD rat behaviours were probed using the mu opioid

receptor agonist morphine. Morphine (5 mg/kg s.c.) induced

epileptic behaviours: staring spells or myoclonic jerks of trunk

or forepaws time-locked to periodic spike or wave discharges

on EEG (BD morphine 5/5 seizures versus NL morphine

0/5 seizures 2I = 13.86, df = 1, P < 0.001, n = 5 per group).

Ictal episodes, beginning 15–20min after drug administration,

were frequent at 30 min (Fig. 2C), approximating the kinetics

of binding and receptor-induced internalization of opioids

with their receptors in mammalian cells (Gaudriault et al.,

1997).

Specificity of the KOR system for anticonvulsant effects was

assessed by U-50488 treatment prior to morphine. Adminis-

tration of the KOR agonist U-50488 (40 mg/kg s.c.) to BD rats

10 min before morphine (5 mg/kg s.c.) prevented seizures in

all rats tested (BD U50488 + morphine 0/5 seizures versus

BD morphine 8/8 seizures, 2I = 17.32, df = 1, P < 0.001,

n = 5–8 per group), but the KOR agonist-morphine combi-

nation depressed both consciousness and EEG tracings

(Fig. 2C, lower tracing).

Encephalitis induces dynorphinloss in hippocampusTo assess neuropathological effects, BD rat brains were

examined. BD rats 6 weeks after infection showed an

atrophic, disorganized hippocampal formation with laminar

disruption and apparent increased density of cells in

hilus (Fig. 3A). The granule layer was preserved as a rim of

NeuN-positive cells (Fig. 3B). A proportion of surviving

neurons were BDV positive, recognized by BD nucleoprotein

antibody (Fig. 3A, top right).

Coincident with granule cell loss, mossy fibre labelling was

not seen in BD rats. Timm staining was attenuated in the hilus

and granule cell layer, and absent from the molecular layer

(Fig. 3C). In Fig. 3D and E, neither dynorphin nor enkephalin

labelling in cell bodies is discernible in BD rats.

Encephalitis induces enkephalinincrease in CA1In contrast to the dentate gyrus, met-enkephalin immuno-

staining was faint in cell bodies that were sparsely scattered

in stratum radiatum of hippocampal field CA1 of NL rats

(Fig. 3F) but prominent in CA1 interneurons of BD rats

(Fig. 3F arrows). Cell bodies labelled in CA1 were: (regio

superior and inferior) stratum pyramidale cells that extend

apical dendrites into stratum radiatum of hippocampal field

CA1, stellate and bipolar stratum radiatum neurons with

a dendritic process radiating to pyramidal layer and another

to hippocampal fissure. The cells correspond in localization

and morphology with previously described populations of

enkephalin-like immunoreactive interneurons of the stra-

tum pyramidale, stratum radiatum, and stratum radiatum–

stratum lacunosum-moleculare interface (Gall et al., 1981).

BD rat met-enkephalin-like IR cells also immunostained with

BDV nucleoprotein antibody (Fig. 3F, right).

Failed neurogenesis and survival ofnew neurons contribute to dynorphinloss in hippocampal formationTo study the developmental aspect of dynorphin expression

in hippocampal formation, the development and maturation

of granule cells, the only dynorphin-expressing cells of the

Fig. 2 Pharmacological analysis of epileptic phenotype with EEG recordings from hippocampal leads from freely moving BD and NL rats.(A) In BD rats, NLX (1 mg/kg s.c.) administration elicits rhythmic (2 per second) high amplitude spike or sharp wave discharges anddisplays of behaviour arrest, blinks or head nods. This seizure, recorded 15 min after drug administration, is typical of patterns recordedfrom 5–45 min after NLX. U-50488 treated animals have no seizures. Pretreatment with the selective KOR agonist U-50488 (40 mg/kg s.c.)before a single dose of NLX (1 mg/kg s.c.), prevents clinical and electrographic seizures (n = 8 per group). In NL animals, NLXadministration elicits increased numbers of sharp waves, but no seizures. U-50488 and U-50488-NLX treated NL animals have noepileptiform activity (n = 8 per group) Representative EEGs recorded 15 min after drug administration are shown in A. (B) In BD rats,nor-BNI (10 mg/kg s.c.) administration elicits rhythmic sharp wave discharges and synchronous clonic trunk movements in the 45–120 mininterval after drug administration. Nor-BNI treated NL animals have no seizures (n = 4–7 per group). Representative EEGs recorded 45 minafter drug administration are shown in B. (C) In BD rats, morphine (5 mg/kg s.c.) administration elicits seizures: staring spells and blinkstime locked to rhythmic sharp wave discharges recorded 15–45 min after morphine. Pretreatment with U-50488 (40 mg/kg s.c.) before asingle dose of morphine (5 mg/kg s.c.) prevents clinical and electrographic seizures. In NL animals, morphine elicits increased EEG activity,but morphine- and U-50488-morphine-treated NL animals have no seizures (n = 5–8 per group). Representative EEGs recorded30 min after drug administration are shown in C. (D) EEGs of vehicle-treated animals are shown for comparison.




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hippocampal formation (McGinty et al., 1983) were examined

following injections of BrdU (50 mg/kg i.p. ·3) on day 7

of infection. BD and control rats were given BrdU to label

dividing cells and sacrificed at selected time points to track

proliferating cells. Differences in numbers and distribution of

proliferating cells were apparent at all time points. BD rats

showed fewer BrdU-positive cells in the granule cell layer

at 1 day (BD 6974 6 330 versus NL 12511 6 672, P <




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0.01) as well as fewer BrdU-positive cells at all survival times

following BrdU injection (at 1 week BD 4282 6 332 versus

NL 77746 1099, P < 0.05; at 2.5 weeks, BD 16576 482 versus

NL 38926 370, P < 0.05; at 4 weeks, BD 13566 105 versus NL

3719 6 279, P < 0.01) (Fig. 4A).

Granule proliferative layers were disrupted or displaced by

neovascular areas, concentric circular areas of BrdU positive

cells along the subgranular zone (Fig. 4B, arrow). Diffuse

background staining seen at 2.5 and 4 weeks was attributed

to increased blood brain barrier permeability and penetration

of brain parenchyma by non-specific IgGs (Fig. 4B). There

were no apparent differences in bioavailability or cell uptake

of BrdU between uninfected and BD rats. BrdU-labelled cell

counts in corpus callosum did not differ across groups at

early survival times (data not shown).

Next, viral effects on new neuron survival were character-

ized. To track cell phenotypes present in the BrdU-labelled

cell population of the granular zone, sections were immuno-

stained for cell markers. Proportions of each indicated

phenotype were calculated in marker/BrdU double-positive

cells (Fig. 4C). Quantification of NeuN, BrdU double-positive

cells showed that NeuN was expressed in 82% of BrdU cells

in NL rats and that NeuN was expressed in 47% of BrdU

cells of BD rats at Day 1. With NeuN-labelled cells account-

ing for only 9% of the surviving BrdU cells in BD rats at

4 weeks versus 86% in NL controls, levels of NeuN/BrdU

cells after 4 weeks were significantly lower in BD rats (x2 =

19.795, P < 0.0001). At the later, 4-week time point, BrdU

cells were perivascular, not neural, and not recognized by

BDV antibody. Thus, BD animals lost more new neurons

than controls. BrdU cells showed neither dynorphin A IR,

a marker for mature granule cells, nor met-enkephalin IR.

Microglia became the predominant new cell type. A total of

82% of BrdU-positive cells co-label with the microglial

marker OX42 at the 4-week time point (Fig. 4C), and

OX42-stained cells flood the dentate gyrus and perivascular

areas (Fig. 4D).

BDV is an infection of CNS neurons and glia (Gosztonyi

and Ludwig, 1995) and successfully establishes infection

in neonatal, young and mature rodent brains. This is reflected

in BrdU + proliferating cells being co-labelled by BDV

antibody early in infection in our model. To begin to char-

acterize the early neural lineage cells that are recognized

and infected by BDV, double IHC was performed using

antibodies to BDV and Mash-1, a beta helix-loop-helix

transcription factor expressed in neuronal progenitors

(Ross et al., 2003). Proliferating cells committed to a neural

fate were infected and expressed BDV nucleoprotein one

week after infection (Fig. 5B). However, later in infection,

neuroblasts or DCX positive cells (Gleeson et al., 1999;

Brown et al., 2003) could not be recognized (Fig. 5D). Clearly,

BDV infection is damaging to developing neurons. At which

stage virus is toxic to developing neurons will be the subject

of further study.

To study actual dynorphin expression in hippocampal for-

mation, the effect of BDV infection on dynorphin mRNA in

hippocampal formation was examined by northern blotting.

PPD transcripts in BD rat hippocampus were decreased but

not absent (BD 1165 6 800 versus NL 15255 + 1037, mean

optical density, arbitrary units, t(1,14) = 10.749, P < 0.0001,

n = 8 per group). Low corticosterone (Thai et al., 1992) or

elevated BDNF (Croll et al., 1994) can downregulate dynor-

phin at the level of transcription. To examine whether host

soluble factors could be linked to decreased transcription or

stability of a dynorphin precursor mRNA in surviving dentate

granule cells, serum corticosterone and hippocampal BDNF

levels were measured. No group differences in BDNF levels in

hippocampal formation were observed [BD 1.064 6 0.122

versus NL 1.011 6 0.125 pg/100 mg, t(1,15) = 0.299, P =

0.7689 n = 8–9 per group]. Corticosterone levels were not

reduced, but instead were increased in BD rats [BD 282.00 6

30.027 versus NL 212.20 6 11.563 ng/ml, t(1,16) = 2.354,

P < 0.05, n = 8–10 per group], to suggest a host role in

inhibition of neurogenesis (Cameron and Gould, 1994).

DiscussionThe present study supports the hypothesis of a critical role of

opioids in the maintenance of a balanced tone of activity

in hippocampal circuits related to seizure induction, and a

key role for the KOR in anticonvulsant protection in viral

seizures in rodents. Our results show a rodent model of

viral encephalitis based on BDV reproduces a functional

neurochemical change, low dynorphin tone, important in

epilepsy vulnerability. Genotypes producing low dynorphin

tone are associated with temporal lobe epilepsy and febrile

seizures in man (Stogmann et al., 2002; Gambardella et al.,

2003). BD rats lack hippocampal dynorphin owing to

Fig. 3 Viral-induced opioid pathology in coronal sections through hippocampus. (A) Nissl-stained sections show contraction ofhippocampus with laminar disruption and nerve cell loss in BD rats. Some dentate granule cells are labelled by BDV antibody (arrow,top right). (B) Robust NeuN IR in dentate layers, hilus and CA3 in NL rats contrasts with residual granule layer preserved as rim ofNeuN-positive cells in BD rat (right). (C) Higher magnification shows Timm staining of hilus, portions of granule cell layer andmolecular layer near tip of dentate gyrus in NL rats. Such staining is attenuated in BD rats, and absent from supragranular regions(right). (D) A network of dynorphin A-like IR cells and the processes of granule cells are visible in the NL rat but absent in the BD rat.(E) The overlapping dentate network of met-enkephalin-like IR cells and processes in the NL rat is reduced in the BD rat to scatteredIR particles through granule and subgranule layers. (F) In CA1, met-enkephalin-like IR interneurons are faint in NL rat, but they aremuch more intensely stained in the BD rat (arrows). Met-enkephalin-like IR cells also expressed BDV nucleoprotein antigen (arrows).Scale bars = 200 mm (A, B and F: photographed at 100·; C, D and E: photographed at 200·). NL, normal uninfected, BD, Borna diseaserat, g, granule cell, hf, hippocampal fissure, aBD, anti-BD, BDV nucleoprotein antibody.




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depopulation of the granule layer and loss of competence of

surviving granule cells to express dynorphin. Mechanisms of

dentate gyrus dynorphin depletion as well as upregulation of

enkephalin elsewhere in the hippocampus are demonstrated.

The non-selective opioid receptor antagonist NLX has

been shown to produce seizures in BD rats which map to

hippocampal and limbic circuits (Solbrig et al., 1996, 2005).

Their seizures have a periodicity of 1–2 per second, within

the dynamic range for frequency-dependent facilitation of

hippocampus (Nadler, 2003). In this study, administration

of the kappa agonist U-50488 controls NLX seizures, while the

selective kappa antagonist nor-BNI reproduces the convulsive

phenotype. NLX seizures, with latencies of <5 min, were

distinct from morphine seizures, with onsets 15–20 min

after drug administration, and were frequent at 30 min,

matching the internalization kinetics of binding and

receptor-induced internalization of opioids with their

receptors (Gaudriault et al., 1997).

Specificity of kappa effect also is supported by the finding

that the kappa agonist U-50488 blocks convulsant effects of

morphine (5 mg/kg s.c.) in BD rats. The results suggest that

a resting, non-seizing hippocampus requires balanced kappa

and mu opioid tone. Once endogenous opioid systems are

destabilized, seizures result.

A role for hippocampal opioid systems in seizure vulner-

ability is in agreement with previous studies of animal

models of seizure risk following in utero morphine exposure

(Schindler et al., 2004). In male rats prenatally exposed to

morphine, epileptiform activity is more easily induced in

entorhinal cortex (Velisek et al., 2000) and systemic naloxone

increases seizure susceptibility (Schindler et al., 2004). In utero

morphine exposure reduces dynorphin-derived peptides in

CA3 and increases mu opioid receptors in dentate gyrus,

CA3, CA1 in adult male rats, as well as decreasing pro-

enkephalin mRNA/met-enkephalin peptide and increasing

prodynorphin mRNA/dynorphin B peptide in granule

layer of dentate gyrus. Naloxone seizures, measured as

decreased latency to bicuculline seizures after naloxone

administration, are associated with reduction in dynorphin

peptides in CA3 in these animals (Schindler et al., 2004). After

prenatal morphine exposure, ovariectomy and hormone

replacement, female adult rats have similarly increased

MOR binding in CA3 and increased susceptibility to flurothyl

seizures (Slamberova et al., 2003).

In our model, NLX is convulsant by blocking KOR tone

from an already weakened or attenuated dynorphin system,

and morphine is convulsant by enhancing mu over kappa

tone. However, one cannot exclude convergent down-

stream or ion channel responses to NLX and morphine as

explanation for similarities in convulsant effects.

The chemical neuroanatomy of BD rat hippocampus is

consistent with the neuropharmacological results. Dynorphin

IR was absent in the dentate granule layer, and enkephalin IR

increased in CA1 of BD rats. Owing to retention of a dentate

layer of NeuN positive cells, loss of mature granule cells can-

not fully account for dynorphin loss. BrdU studies revealed

that the virus interfered with granule cell development and

survival, such that young neurons were lost too early to repo-

pulate the granule layer with mature dynorphin-positive

phenotypes. The dentate granule layer was sporadically

infected, thus limiting the direct role of virus in transmitter

downregulation (Hans et al., 2001). Hippocampal BDNF

levels were unchanged in BD rats and serum corticosterone

levels were increased, and such changes have not been

reported to downregulate dynorphin (Thai et al., 1992;

Croll et al., 1994). Effects at gene-expression level are not

excluded, but direct causes through viral interference or

host soluble factors were not found. However, BD rats show

early EEGs with high levels of bursting waves, and seizures

have been shown to deplete hippocampal dynorphin at the

mRNA and peptide levels (Xie et al., 1989; Douglass et al.,

1991, reviewed in Simonato and Romualdi, 1996). Thus, a

different host factor, subclinical seizures, could exhaust

dynorphin from surviving granule cells, triggering an evolu-

tion to clinical seizures. These results add to previous studies

of viral and immunological determinants of virulence, which

have already demonstrated depopulation of the granule layer

resulting from inflammatory/immune-based neuronal loss

(Gosztonyi and Ludwig, 1995; Planz and Stitz, 1999). The

chief damaging elements are cell loss, failure of neurogenesis,

depletion of dynorphin with seizures and inability to restore

dynorphin to meet demand. No doubt cell loss is important,

but it is multiple factors, acting together, that expose key

sites of vulnerability in the brain’s carefully regulated control

of excitability. One could speculate based on the present study

that a significant contribution to overall dynorphin pathology

is depletion of dynorphin with seizures.

The classic hippocampal circuit is a trisynaptic circuit

utilizing glutamatergic neurotransmission. At each step,

excitatory tone is modulated by a diverse group of inhibitory

and excitatory neurons (Freund and Buzsaki, 1996). In

normal brain, the dentate granule cells serve as a high-

resistance gate or filter, inhibiting propagation of hypersyn-

chronous discharges from entorhinal cortex to hippocampus.

Gating depends on several factors, including release of

inhibitory neurotransmitters, such as dynorphin and others,

and structural integrity. BDV-induced loss of the neuro-

pharmacological and structural gate, as shown in this study

by failed neurogenesis and infection of young neurons,

may prove important in initiation and spread of temporal

lobe seizures. Dynorphin or kappa opioid presynaptic

inhibitory effects in hippocampus through Shaker type potas-

sium channels on mossy fibres (Simmons and Chavkin, 1996)

are presumed lost to the BD rats. However, dynorphin also

decreases neuronal activity via post-synaptic actions on

potassium M channels of principal CA3 and CA1 hippo-

campal neurons (Moore et al., 1994; Madamba et al.,

1999). It is the anticonvulsant actions of KOR agonists on

CA3 or CA1 principal neurons, neurons that survive in

BD viral infection, that mediate the pharmacological

effects observed in this study, which may be considered

therapeutically relevant.




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Fig. 4 Viral effects on neurogenesis, survival and phenotype. (A) Numbers of BrdU-labelled cells in granule layer of hippocampus atvarious time points after BrdU injection. BDV infection decreases numbers of BrdU labelled cells within granule cell layer and subgranulezone 1 day, 1, 2.5 and 4 weeks after BrdU injection, relative to normal uninfected rats. *P < 0.05, **P < 0.01 values are mean (6 SEM)cell counts per animal for entire hippocampus (n = 4 per group for each time point). (B) Distribution of BrdU-labelled cells. Coronalsections through the hippocampus of NL and BD rats show proliferating cells stained by antibodies against BrdU at 1 day, 1, 2.5 and 4 weeksafter BrdU injection. Arrow indicates area of inflammatory angiogenesis interrupting granule layer proliferation in BD rat. (C) Percentage ofBrdU positive cells that colabelled with NeuN, GFAP, BDV antibody or OX42, measures neurons, glia, infected cells and microglia,respectively, among proliferating cells. During BD, there is a progression of BrdU-positive phenotypes, from NeuN and GFAP earlyin disease, to OX42 late in disease. Roughly 50% of BrdU-labelled cells are NeuN-positive at day 1, 82% of BrdU cells are OX42immunoreactive at 4 weeks. BrdU-labelled cells were scored within granule cell layer and subgranule zone. NeuN, neuronal nuclei, GFAP,glial fibrillary acidic protein, OX42, monocyte microglia marker. (D) OX42 immunofluorescence staining shows the microglial response toBD infection 4 weeks after BrdU injection. The dentate gyrus, hilus, fissure and perivascular areas of BD rat hippocampus are heavily stained(lower panel) compared with NL rat hippocampus. Scale bars = 200 mm.




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Beyond the dentate gate, interneurons showing increased

enkephalin IR, which co-localized with BDV IR in inter-

neurons of stratum radiatum and stratum pyramidale

of CA1, are positioned to influence mu receptors over a

wide distribution and strengthen hippocampal excitation.

In rodents, mu opioid receptors are on a neurochemically

heterogeneous subset of hippocampal interneurons, most

frequently on interneurons that are specialized to inhibit

pyramidal cells (Blasco-Ibanez et al., 1998). In this distri-

bution, interneurons use mu opioids to limit their own

activity as well as that of their targets (Drake and Milner,

2002; Drake et al., 2002). Thus, there is a net excitation,

either by enkephalin upregulation arising as a direct effect

of virus (Solbrig et al., 2002), or by overstimulation of

stratum radiatum interneurons. Prominent enkephalin stain-

ing in BD rats may signify recurrent seizures, with CA1

enkephalin interneurons activated in feedforward manner

by Schaffer collaterals, using the hippocampal intrinsic

excitatory circuit of CA3 projections to CA1. Prominent

enkephalin staining may also signify more interconnected

networks, with reorganization of CA1 associational pathways

into more excitatory networks (Esclapez et al., 1999; Lehmann

et al., 2001).

Thus, viral infection, by producing opioid system

destabilizations, induces the same limbic opioid changes as

would be anticipated during frequent hippocampal seizures

(i.e. dentate dynorphin depletion by perforant path activity

and CA1 enkephalin interneuron stimulation by feedforward

Schaffer collateral excitatory activity). These opioid changes

promote and sustain a proconvulsive state. The Borna model

illustrates circumstances that deplete dynorphin from the

granule cell layer and upregulate enkephalin elsewhere in

the hippocampus. The BD rat overlaps with other epilepsy

models with dynamic neuropeptide profiles that have estab-

lished a role of dynorphin deficits in seizures. However, the

BD model goes on to demarcate an increased, excitatory

enkephalin network in CA1 and supports an additional

role for CA1 enkephalin upregulation in seizure vulnerability.

The model may apply to any condition with granule cell loss,

silencing or failed neurogenesis and enhancement or redirec-

tion of excitation to CA1. Thus, the model may generalize to

other epilepsies where dynorphin depletion due to recurrent

seizures and enkephalin upregulation in CA1 interneurons

due to repetitive hippocampal circuit stimulation occurs.

The epileptic syndrome produced by the BD rat is greatly

simplified at the neuropharmacological level by the observa-

tion that restoration of dynorphin tone can prevent seizures.

Normally, KOR receptors are found on mossy fibre terminals,

principal neurons, perforant pathway and supramamillary

afferents (reviewed in Solbrig and Koob, 2004). The dynor-

phin system, hypothesized to be a neuromodulatory homeo-

static system, may be released on demand by excessive stimuli.

KOR activation in hippocampus achieves effects desirable

in anticonvulsants. The KOR effects include opening K+

channels (Simmons and Chavkin, 1996) or closing Ca2+

channels (Rusin et al., 1997), thereby controlling pre-synaptic

transmitter release, increasing M type K+ currents to stabilize

membranes and post-synaptically silence excitatory neuro-

transmission (Madamba et al., 1999), or acting on classes

of interneurons (Racz and Halasy, 2002) to desynchronize

the gamma aminobutyric acid inhibitory network. Due to

preservation of some of these actions in BD rats, a single

pharmacological manipulation, the use of a drug with narrow

(KOR) specificity, appears to overcome themix of neurophar-

macological and lesion effects produced in BD rats.

A dominant role for dynorphin, trumping other trans-

mitters, in defending against encephalitic seizures, may be

an important part of the normal neuroadaptive response

of the brain to overstimulation. A more radical view pos-

sibly important for the pathogenesis of seizures is that the

phenotype is determined by the most prevalent break

with homeostasis, which in this case is by dynorphin, an

inhibitory/modulatory neurotransmitter, and such a view

suggests the feasibility of exploring the use of kappa opioid

agonists in refractory seizures.

AcknowledgementsThe authors thank Lothar Stitz for the gift of BDV nucleo-

protein antibody, Chris Gall for helpful discussions, Steve

Henriksen and his lab members for advice on EEG recording,

and Mike Arends for editorial assistance. This work was

supported by National Institutes of Health grant NS042307

from the National Institute of Neurological Disorders and

Fig. 5 Viral effects on proliferating, progenitor and newly bornneurons. (A and B) Double immunohistochemical staining forMash-1 (red) and BDV (green) shows Mash-1 labelled neuralprogenitors in NL uninfected rat hippocampus (A) and Mash-1IR cells also labelled by BDV antibody (B) 1 week after infection.(C and D) DCX IR bodies and filamentous processes of newneurons are present in dentate layers and hilus of NL rats.(C) Arrows indicate young neurons in granule layer withperpendicular processes extending towards the molecular layer.In BD rats 5 weeks after infection, there are disorganized,amorphous clumps of DCX-immunopositive material (D). aBD,anti-BD, BDV nucleoprotein antibody, DCX, doublecortin, g,granule cell layer, h, hilus, m, molecular layer. Scale bars = 50 mm.Photographed at 200·.




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Stroke to MVS and UC Discovery BIO 03-10408 and Cortex

Pharmaceuticals CP-360222 to JCL.

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