Epileptogenic spikes and seizures but not high voltage spindles are induced by local frontal cortical application of γ-hydroxybutyrate

91

Epilepsy Research, 15 (1993) 91-99 0920-121 l/93/$06.00

EPIRES

0

1993 Elsevier Science Publishers

B.V. All rights reserved

00569

Epileptogenic spikes and seizures but not high voltage spindles are induced by local frontal cortical application of y-hydroxybutyrate

Jurij BrankaEk*,

Hannele

Lahtinen,

Esa Koivisto

and Paavo J. Riekkinen

Department of Neurology, University of Kuopio, Kuopio, Finland (Received

6 December

1992; revision

Key words: High voltage

Combining

the methods

hydroxybutyrate to induce rhythmic (HVS, 69

of microdialysis

(GHB) on frontal during

jerks and epileptic discharges

(seizures)

but did not induce

occurring

spikes invading for the generation

HVS the amplitude

the contralateral

we have examined

generalized

application (~2

behavior

cortex

the effect of unilaterally,’

petit mal epilepsy.

of GHB induced

HVS or spike and waves,

epileptogenic

intracortically

as reported

after

cortex frequently

triggered

and terminated

discharges

(seizures)

Generalized, bilaterally synchronous high voltage spindles (HVS) or spike and wave (SW) activity in the electroencephalogram (EEG) of freely moving rats are regarded as animal models of petit ma1 Recently, the genetic predetermination of frontal cortical spindle rhythms was suggested in laboratory rats of the Wistar strain52. yI.

Correspondence to: Jurij BrankaEk, Ph.D., Department of Physiology II, Heinrich-Heine-University, Moorenstr. 5, D4000 Dusseldorf, Germany. Tel.: (49211) 31 12793; Fax: (49211) 3114231; E-mail: [email protected]. * Present address: Department of Physiology II, HeinrichHeine-University, Moorenstr. 5, D-4000 Diisseldorf, Germany.

high voltage

Hz) behaviorally

and contraversive

y-

is known spindles

systemic

application.

probe was suppressed

after local intracortical

accompanied

movements

towards

In the group

by

the left

of rats with

by GHB and GHB-induced

local HVS. The results point to different induced

applied

GABA metabolite,

(HVS rats), while others never had any HVS.

spikes (co.5

convulsions

of the HVS on the side of the microdialysis

of HVS and spikes and epileptic

Without

in most of the animals

Hz) with behavioral

Introduction

epilepsy9,28*3075

Frontal

1993)

GHB, spontaneous

of animals

spontaneous

Microdialysis;

12 February

resembling

In those both groups hindlimb

y-Hydroxybutyrate;

1993; accepted

endogenous

awake and immobile

intracortical

IO February

EEG activity in freely moving rats. GHB, a natural

occasional

myoclonic

spindles;

and EEG recording,

cortical

spike and wave activity,

Hz) were observed

received

application

neural mechanisms of GHB.

Hydroxybutyrate (GHB), a natural endogenous GABA metabolite5334 and putative neurotransmitter49’50, is known to induce rhythmic spike and wave activity after systemic injections in rats, resembling generalized petit ma1 7, ’ 8.4’. GHB was also shown to induce HVS-like rhythmic spike and wave discharges after systemic injection in a rat strain without spontaneous HIS”. In spite of recent progress investigating the origin of HVS-like thalamo-cortical oscillations especially at the level of the thalamus8.43P45, the role of cortical and corticothalamic pathways as a possible trigger of the thalamo-cortical oscillation is still undetermined. The present experiments were designed in order to test the hypothesis that not only thalamic, but also cortical and cortico-thalamic circuitries are epilepsy’,’

92

involved

in triggering

or generation

of HVS and

SW. The aim of this study was to investigate

the

bine local drug application analysis of somatostatin-like

and neurochemical immunoreactivity25.

The probe

perfused

effect of unilateral, intracortical application of GHB in both HVS rats and rats without HVS.

cial cerebrospinal

Portions

ing of (mM) NaCl

of this work have been published

in ab-

stract form6.24.

was continuously fluid (aCSF), (138)

with artifi-

a solution

NaHC03

consist-

(1 l), KC1 (5)

Material and methods

KH2P04 (1) CaC12 (1) MgC12 ( 1)26 and 0.2% bovine serum albumin (BSA) (pH adjusted to 7.4 by gassing with 95:5% 02:C02). Perfusion with aCSF

Sixteen adult 4-6 month old male Wistar rats (250-350 g) were anesthetized with chloral hydrate (360 mg/kg, i.p.) and mounted into a stereotaxic instrument with bregma and lambda at the same height. Guide cannulas for microdialysis probes (Carnegie Medicine AB, Sweden) were implanted in 13 rats unilaterally into the outer layer of the right parietal cortex with a 20” angle from horizontal and a 5” angle from sagittal level with coordinates A = 6.0, L = 3.6 and V = 2.8 relative to bregma. In three rats guides were inserted unilaterally near the hippocampus (A = 6.5, L = 4.5, V = 4.0) the reticular nucleus of thalamus (A = 3.0, L = 3.5, V = 4.0) or the ventrolateral thalamus (A = 2.5, L = 4.5, V = 4.0). Stainless steel screws for epidural recordings of neocortical EEG were threaded into tapped holes over the frontal neocortex (A = 2.0, L = 2.5). Two additional stainless steel screws were placed over the cerebellum. They served as indifferent and ground electrodes, respectively. The fixation of guide cannulas and electro-

had no effect on the frontal cortical EEG. GHB (Sigma) was added to the CSF solution in two concentrations: 10 and 20 mg/ml. The flow rate (3 yl/min) of the perfusate was controlled by means of a microinfusion pump (Carnegie Medicine AB). According to control measurements made by Carnegie Medicin laboratories’0 with the same type of membrane and a flow rate of 2 PI/ min, a recovery rate of 25% was found for GABA’“, a molecule of similar size compared to GHB. With a flow rate of 3 &min, chosen in order to reduce the sampling period and still allowing a measurable recovery of somatostatin, the relative membrane recovery for GHB will be less than 25%. A membrane recovery of less than 25% for GHB means that less than 25% of the GHB concentration inside the microdialysis probe will reach the brain surrounding the probe. After a balancing time (at least 30 min), the experiment consisted of four 40 min perfusion sessions with (1) aCSF solution, (2) aCSF + 10 mg/ml GHB, (3) aCSF + 20 mg/ml GHB and (4) with aCSF solution again

des was done with dental acrylate. After at least 5 days recovery from surgery, the animals were habituated to the recording box (40 x 40 x 40 cm). The rat’s movement was monitored using a small magnetoinductive device fixed to the occipital part of the skull. The EEG was recorded bilaterally from the frontal cortex, amplified, filtered (l-100 Hz), sampled (200 Hz) and stored together with the animal’s movement recordings on hard disk. Control recordings of the EEG were made during a 30 min period of immobile and awake behavior. The dummy of the guide cannula was removed and a microdialysis probe (membrane projecting the guide: 0.5 x 4 mm; molecular cut off below 20 000 Da; for diffusion kinetics see Amberg and Lindefors’) inserted via the cannula into the frontal cortex, hippocampus or thalamus. Microdialysis was used in order to com-

(‘washing’). Simultaneously, two 20 min episodes of EEG were recorded and stored together with the animal’s movement for off-line analysis. The frequency, duration and latency of epileptogenic spikes, epileptic discharges (seizures) and spontaneous HVS were measured by means of a software package (GlobalLab, Datatranslation). During each of these sessions fractions were collected in order to measure the somatostatin-like immunoreactivity (SLI) in the perfusate. The results have been reported elsewherez5. The mean values and standard errors were calculated. Statistical analysis was performed using the non-parametric Wilcoxon tests for paired differences and for two samples. When data collection was finished, the animals were deeply anesthetized and perfused through the

93

left ventricle with 0.9% saline and then with 4% formaldehyde in 0.1 M phosphate buffer. Coronal sections of 25 pm were cut, mounted on slides and stained with hematoxylin-eosin. The localization of the microdialysis probes was verified by light microscopy. Results Prior to the installation of the microdialysis probe control recordings of the EEG of each of the 16 rats during 30 min periods of immobile and awake behavior were analyzed. Spontaneous HVS (intra-HVS frequency of 6-9 Hz) with a mean frequency of occurrence of 3.4 + 0.8/min and a mean total duration of 9.4 + 2.6 min (31.4 + 8.6% of the control recording period) were observed during awake and immobile behavior in 12 of the animals (HVS rats), while four rats (non-HVS rats) never had HVS. Fig. 1 illustrates the effect of unilateral microdialysis perfusion of the frontal cortex with a single dose of 10 mg GHB/ml aCSF (reaching the brain at 0 min) on the frontal cortical EEG of both hemispheres during 60 min in rat MO7 with spontaneous high voltage spindles. The presence of the dialysis probe itself and 20 min perfusion with aCSF had a negligible effect on the EEG and are

I

I 10

0

20

Fig. 1. The frontal microdialysis

30

cortical

40

EEG during

(A) with 10 mg GHB/ml

50

unilateral

[mill]

50

intracortical

aCSF (perfusion

from 0

to 40 min) and aCSF alone (after 40 min) in a rat with spontaneous

high voltage

desynchronized bars)

spindles

and of epileptic

(seizures:

(M07).

EEG (white bars), discharges

The periods of epileptic

of normal

or

spikes (hatched

with myoclonic

convulsions

black bar) are shown at the top (see also Fig. 2). The

lower row shows the frontal microdialysis

cortical

probe (B). Vertical

EEG contralateral bar: I mV (positive

to the up).

therefore not shown in the figure. After a latency of 10 min, the spontaneous HVS and synchronized EEG waves were suppressed and epileptogenic spikes appeared on the side of microdialysis (A), instantly invading the contralateral cortex (B). At 18 min bursts of spikes and epileptic discharges appeared. The behavioral manifestations of this type of epileptic discharge were contralateral myoclonic convulsions and contraversive head movements towards the rat’s hindlimb occasionally followed by licking of the contralateral hindlimb. Epileptic spikes were frequently but not always accompanied by shortlasting jerks or twitches. At 40 min GHB was replaced by aCSF. Five minutes later the epileptic discharges with myoclonic convulsions disappeared and the low frequency epileptic spikes returned. After about 30 min a normal EEG pattern with spontaneous HVS reappeared on both hemispheres. During the time of GHB perfusion, the amplitudes of spikes and epileptic discharges rose slowly and declined only after replacing the GHB by aCSF. Similar effects were found in all of the 13 rats with a cortical probe (Fig. 2). After 10 mg/ml GHB (at 0 min in Fig. 2) nine of 13 rats exhibited epileptic spikes with a mean latency of 17.9 f 2.2 min but no seizures. In those nine rats 20 mg GHB/ml aCSF was used and epileptic discharges (seizures) resulted with a mean latency of 9.3 f 1.5 min. The epileptic discharges were accompanied by myoclonic convulsions and contraversive head movements, all toward the animal’s hindlimb, and occasionally followed by licking. The remaining four rats already showed seizures after .lO mg/ml GHB. No further dose was used in these rats except in rat MO6 for 4 min. Two of these rats displayed mainly epileptic discharges with myoclonic convulsions and almost no spikes. After washing with aCSF the epileptic discharges disappeared with a mean latency of 9.9 + 2.6 min (N = 12) and epileptic spikes reappeared in nine of 12 rats (mean latency: 15.5 f 3.3 min). Finally, 31.0 + 2.4 min after replacing GHB with aCSF, desynchronized and normal spontaneous EEG returned. In two rats, the experiments could not be completed due to mechanical damage of the microdialysis probe. No significant differences were found between

94

GAMMA-HYDROXY6~TYRATE RATS MO2 7

____-----.--1 '-=----i v

" v

MO9 -

.

v

Ml3 -

I MO3 p MO7 p

.

---.

Ml1 -

SEIZURES

-.

MiO*M06*.

-9

MOl*-

-

0

v

20

.

40

60

7

80 100 120 140

TIME Iminl c=l norm. EEG

m

SPIKES

V 20 mQ/ml OH5 Fig. 2. The effect of GHB rats and three normal

leptic spikes;

black

seizures (for animal mg GHB/ml

cortical

EEG; hatched

bars: periods

bars:

periods

bars: periods

with epileptic

aCSF starting

from 0 min. White triangles:

perfusion

with aCSF

alone

aCSF;

(‘washing’);

due to mechanical

black

triangles:

question damage

of

with epi-

discharges

MO7 see also Fig. I). The perfusion

with 20 mg GHB/ml

SPIKBS

EEG in 10 HVS

HVS (*). White

perfusion

sions not completed

SEIZURES

WASHING (aCSF)

on frontal

rats without

or desynchronized

V

m

and

with IO start of start

marks:

of ses-

of the microdia-

lysis membrane.

Fig. 3. Spontaneous duced

epileptic

spikes (~0.5

IiVS rats (N = 10) and those without HVS (N = 3). Also, no significant correlations could be found among the HVS rats between the frequency of occurrence or total duration of the spontaneous HVS and the rat’s susceptibility to GHB. Clear differences between the intra-HVS frequency of spontaneous HVS (~6 Hz) and the intraburst frequency of the GHB-provoked epileptic discharges (~2 Hz) or the frequency of epileptic spikes (~0.5 Hz) are shown in a representative example from one and the same rat in Fig. 3. High voltage spindles, epileptic discharges and epileptic spikes are shown with the same amplitude calibration and time scales. It is evident that the intraburst frequency of the GHB-induced epileptic discharges is more than three times lower than the

high voltage

discharges

spindles

and seizures

Hz) from one representative

the same amplitude

calibration

and corresponding

(vertical

(>6

Hz), GHB-in-

( < 2 Hz) and epileptic HVS rat shown

with

bars: 1 mV, positive up)

time scales (20 s and 500 ms).

intra-HVS frequency of the spontaneous high voltage spindles. In three of the 10 HVS rats, high voltage spindles occurred spontaneously during intervals between epileptic spikes in the frontal cortex, contralateral to the probe side, whereas on the side of microdialysis the amplitude of HVS was strongly diminished (Fig. 4). The amplitudes of the GHB-induced epileptic spikes and epileptic discharges were similar on both the side of microdialysis and the contralateral frontal cortex. Epileptic spikes provoked by GHB in the perfused side of the cortex instantly invaded the con-

95

RlGHT

FRONTAL

CORTEX

lMlCRODlALVSlS

WITH

ture of spikes and spike-triggered HVS appeared. High voltage spindles were never observed after

GHE)

local frontal cortical microdialysis with GHB rats spontaneously not exhibiting HVS. Local

LEFT

FRONTAL

CORTEX

(CONTRALATERAL

/

I

t

application

of

10 or 20 mg/ml

GHB into the right and reticular thalamic

hippocampus, ventrolateral nuclei had no effect on the

frontal

or the behavior

cortical

EEG

of the rat.

The frequency or duration of HVS was not changed in HVS rats, nor were HVS induced in the one tested rat without spontaneous HVS with a microdialysis probe in the ventrolateral thalamus.

TO MICRODIALYSIS)

HYS

control

in

Discussion

Fig. 4. HVS but not epileptic discharges on the side of the microdialysis HVS and epileptic

discharges

(lower row). Vertical

(seizures) are suppressed

probe (upper appear

row) whereas

on the contralateral

bar: 1 mV; horizontal

both side

bar: 10 s.

tralateral cortex, triggered and terminated local HVS (Fig. 5). On the contralateral cortex a mix-

GHB

PROVOKED

SPIKES TRIGGER HIGH VOIZAGE SPINDLES

RIGHT FPONUL

LRPT PRONUL

Fig. 5. Suppression termination

of HVS on the side of the microdialysis

of HVS in the contralateral

The main results of the present study are the findings that unilateral, local intracortical application of GHB induces epileptogenic spikes and epileptic discharges with myoclonic convulsions (seizures) and suppresses the amplitude of the spontaneous EEG waves, including HVS. GHB-induced spikes and epileptic discharges and the suppression of the spontaneous EEG resembled the effect

CORTRX (Ylcrodlal~lr

CORTRX (ContraMoral

probe (upper

cortex (lower row, right column).

with

GIU)

to Ylcrodialfrlr)

row, right and left columns) Vertical

min (left column).

bar:

1 mV: horizontal

and spike-evoked

triggering

bars: 2 s (right column)

and and 1

96

of topical neocortex16

application of penicillin to or the effect of systemic application

penicillin’ 5,‘9 rather voltage spindles.

than

the

spontaneous

the of high

Both the genetically predetermined spontaneous HVS 28,5’s2 and the HVS-like SW activity induced by systemic i.p. injections of 25&375 mg/kg GHB are recognized as animal models of petit ma1 epilepsy','7,'8.'9. A similar origin of both rhythmic activities was recently suggested”. Our results point to different neural mechanisms for the generation of HVS and the induction of spikes and epileptic discharges after local intracortical application of GHB. Contrary to the systemic injections of GHB, local intracortical application revealed clear differences between spontaneous HVS and GHB-induced spikes and epileptic discharges in frequency, amplitude and origin. A similar spiking, starting with single spikes at lower serum concentrations of GHB and bursts of spikes with intervening periods of desynchronization and occasional myoclonic jerks at higher concentrations, was described in cats during systemic GHB administration36. EEG slowing with serum concentrations of 150 pg/ml GHB and myoclonic seizures with concentrations above 500 pg/ml were reported in monkeyss7. Similar EEG abnormalities were described after systemic injection in adult and immature rats. Systemic administration of GHB below 400 mg/kg provoked spike and wave discharges resembling somewhat the HVS starting from 14 day old animals. In rats under 14 days of age, concentrations above 400 mg/kg GHB showed EEG voltage suppression, spikes and bursts of spikes. Lower concentrations had no effect on EEG3s. Snead38 suggested different mechanisms for the ‘early hypersynchrony’ (HVS-like spike and wave discharges) and the voltage suppression with spiking. The anti(petit mal)epileptic drug ethosuximide had no effect on the voltage suppression or spiking in rats under 14 days of age but was effective against the early hypersynchrony produced by GHB in animals after the third postnatal week3s. Intracortical administration of GHB in the present study seems to resemble the EEG abnormalities shown in postnatal rats. Similar to the lower dose systemic injections in immature rats the HVS-like

spike

and

experiments

wave

discharges

in the early part

and in the later part

were

absent

of the microdialysis

served EEG voltage depression spiking as was found by Snead with higher dose systemic

in our

of the microdialysis we ob-

accompanied in immature

injections38.

Systemic

by rats in-

jection of GHB (300 mg/kg i.p.) revealed a mixture of HVS and GHB-induced SW activity and HVS were less regular or sinusoidal during GHB compared to control recordings (Brankack, unpublished observations). Depression of the surface negative component in all forms of cortical primary evoked potentials was reported in rats following systemic GHB administration (1 g/kg, i.p.)4. Laborit suggested that GHB acts primarily at the level of the cortex associative pathways. An almost identical pattern of EEG changes as was shown above with GHB has been found after systemic (intramuscular) injection of penicillin in cats, another well known model of petit ma1 epilepsy’5.‘9: generalized bilaterally synchronous 335 Hz spike and wave discharges are seen in the EEG”.“. Furthermore, local cortical application of penicillin (concentrations exceeding 250 IU/ hemisphere) induced a multiple spike pattern with generalized seizure discharges resembling those after local cortical application of 20 mg/ml GHB in our experiments. Local cortical application of weak solutions of penicillin elicited the same cortical electrographic manifestation as with intramuscular injection”. Local application of penicillin to subcortical structures, particularly to the thalamus, was completely incapable of producing any form of epileptic discharge15.16, which is in agreement with our failure (albeit only with two rats) to show any effect on the EEG after local thalamic application of GHB, but is in contrast to recent reports with intrathalamic microinjections of GHB27,4’. The nucleus reticularis thalami was shown to be essential for HVS43-46 and the ventrolateral thalamus participates in frontal cortex generation of ~~~82.4"

Interestingly, GHB-induced spikes sometimes triggered and terminated HVS in the frontal cortex, contralateral to the side of microdialysis. Single transcortical electrical pulse stimulations also trigger and terminate HVS7 as do single pulses in the ventrolateral thalamus22. Epileptic spikes, in-

97

duced by local cortical application of GHB in one hemisphere, invade the contralateral side and may trigger there directly or more likely through the cortico-thalamo-cortical pathways the high voltage spindles, whereas the amplitude of the spontaneous EEG and HVS on the ipsilateral side is diminished or suppressed by the local effect of GHB. On the contralateral side a mixture of spikes, bursts of spikes and spike-triggered HVS can be observed. The suppression of spontaneous HVS and induction of spikes and epileptic discharges during intracortical GHB application clearly demonstrates the different origin of both types of rhythmic EEG activity. The epileptogenic and convulsive actions of GHB similar to that of penicillin seem to be restricted to the neocortex whereas both thalamic and thalamo-cortico-thalamic circuitries may be important for the generation and triggering of spontaneous HVS. Dingledine et al.‘* and Schneiderman have suggested that GABAergic inhibition is blocked by penicillin. The similar effect of intracortically applied GHB suggests that inhibition in the local cortical circuitry is also diminished or blocked by GHB. GHB as a natural endogenous GABA metabolite5334 has been termed a GABA agonist29 but as Snead et a1.42

stated: “GHB is not a GABA agonist because it does not compete for [3H]GABA binding”13,‘4 “nor do GABA, muscimol, or diazepam compete directly for [3H]GHB binding”3q39. “The neurophysiologic effects of GABA differ from those of GHB”20331*32.According to our results GHB rather resembles a GABA antagonist or GABA blocker. The neural mechanisms of the rhythmic SW activity induced by systemic GHB injection and their relationship to spontaneous HVS remains unclear because in our experiments neither intracortical nor intrathalamic local applications of GHB resulted in rhythmic activity, comparable to the SW after systemic injections, but see Liu et a1.27 and Snead et a141. Local effects of GHB on the cholinergic or monoaminergic ascending activating systems in the brainstem or basal forebrain, which modulate thalamo-cortical oscillations2’247*48 should be tested in future experiments. Acknowledgements

We thank A.-L. Gidlund, S. Karhunen and M. Lukkari for expert technical assistance, E. MacDonald, Ph.D. and Ita Walsh for revising the language.

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