Distribution of Fos-positive neurons in cortical and subcortical structures after picrotoxin-induced convulsions varies with seizure type

BRAIN RESEARCH Brain Research 683 (1995) 73-87

ELSEVIER

Research report

Distribution of Fos-positive neurons in cortical and subcortical structures after picrotoxin-induced convulsions varies with seizure type John O. Willoughby *, Lorraine Mackenzie, Andrei Medvedev, Jennifer J. Hiscock Centre for Neuroscience and Department of Medicine, Flinders University and Medical Centre, PO Box 2100, Adelaide, SA 5001, Australia Accepted 7 March 1995

Abstract The distribution of Fos protein was mapped in rat brain following a single non-focal convulsive seizure. Single seizures were induced with intravenous picrotoxin in unhandled animals housed in isolation. Different convulsive behaviours occurred unpredictably. The least severe seizures were predominantly localised to the face, head and forelimbs, without loss of posture control (restricted seizures). The most extensive seizures affected all limbs and trunk, sometimes with falling (generalised seizures). There was a correlation between seizure behaviour and distribution of Fos induction. After restricted seizures, Fos was induced at highest levels in neocortex and piriform cortex and was prominent in entorhinal cortex, caudal-ventral caudate-putamen and amygdala. Regions of thalamus were consistently and lightly labelled, but Fos induction did not occur in hippocampus. After generalised seizures, there was Fos induction in cortex but less than after restricted seizures and, in three of four animals, also in dentate gyms, hippocampus and subiculum. There was occasional or variable labelling of thalamus, basolateral amygdala and caudate-putamen. One animal with generalised seizures showed no hippocampal Fos induction. The findings indicate that picrotoxin induces seizures with at least two different patterns of neuronal involvement. The cortex, part of the caudate-putamen, amygdala and thalamus are involved in restricted seizures while the hippocampus, cortex and thalamus are involved in generalised seizures. The results do not support the view that generalised seizures are a progression from restricted forms. Cortical Fos involvement is entirely consistent with the participation of cortex in non-focal epilepsy. In these non-focal seizures, the dentate-hippocampus may be a source of excitation to cortex in the generalised group while the cortex appears to be the predominant site of excitation in the restricted group.

Keywords: Picrotoxin; Convulsion; Fos; Immunohistochemistry; Cortex; Thalamus; Caudate-putamen; Hippocampus; Rat

I. Introduction This study was undertaken to identify neurons active during a single non-focal convulsive seizure in order to reveal the populations of neurons that participate in such seizures and, ultimately, to characterise the essential populations. Picrotoxin, an antagonist at the y-aminobutyric acid ( G A B A ) - A receptor-gated chloride channel, as well as the related agent pentylenetetrazol, and bicuculline, induce convulsions when administered systemically or intracerebrally [10,13,17,19,22,28]. In a primate form of generalised epilepsy, impaired G A B A binding in cortex and hippocampus has been demonstrated [33]. W e have ini-

* Corresponding author. Fax: (61) (8) 204-5458. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved

SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 3 6 6 - 5

tially chosen to study convulsions caused by impaired inhibition, namely, those induced by picrotoxin. Current hypotheses about the process of generalised epilepsy attribute roles to structures that have strong connectivity with the cortex, and there is evidence that thalamus [21] and hippocampus [33], respectively, are important in feline penicillin epilepsy and in baboon with apparently spontaneous grand mal epilepsy. The possible participation of other subcortical regions is undefined. Convulsions of various types transiently induce the stereotyped appearance of a series of immediate early gene proteins such as Fos, which is one of the first to be expressed. The m R N A s and proteins are immunohistochemically detectable in cerebral cortex and limbic system for specific periods after a convulsion [5,8,10,13,17,19,22,28,29]. These studies provide evidence that Fos protein is induced in response to strong synaptic activation. It can also be induced by other treatments, for

74

J.O. Willoughby et aL /Brain Research 683 (1995) 73-87

example hormones, neurotransmitters, injury and growth factors [14,27]. Fos immunohistochemistry has proven useful in defining nerve pathways activated by specific physiological processes [14,25,31]. Numerous studies have shown that the regional patterns of Fos mRNA or Fos protein expression are grossly similar following multiple seizures induced by kainic acid, picrotoxin, electro-shock and forebrain injections of convulsant agents and have revealed dramatic involvement of dentate gyrus in some aspect of generalised convulsions [5,8,10,17,19,22,28,29]. Audiogenic seizures cause a completely different pattern of Fos expression, predominantly in brainstem structures [30] similar to Fos expression after seizures induced by application of bicuculline to the inferior colliculus [19]. However, many of these studies have been carried out in handled animals and after prolonged periods of sustained or repetitive seizure activity (commonly 15 min or more, terminated by diazepam, for example) so that the relationship between a particular seizure and Fos expression cannot be precisely defined. Furthermore, handling itself induces Fos in cortical neurons [35]. We have therefore defined the cortical and subcortical distributions of Fos caused by a single convulsion induced by an intravenous infusion of picrotoxin in the isolated, unrestrained rat.

welded to the steel screws and soldered to a female integrated circuit socket, all of which were embedded in dental cement. Recordings were made via a FET amplifier [4], which was incorporated into the male connector by which the EEG leads were attached to the rat's head. Monopolar recordings were made between the cortical electrodes and the indifferent electrode using a Beckman EEG polygraph or a MacLab. Half-amplitude cut-off frequencies were at 1 and 70 Hz. Animals were allowed to recover for at least 5 days and then placed in cages inside isolation recording chambers to acclimatise for 3 days. The venous line and EEG lead were connected the day before infusion so that animals were studied in isolation, without handling or restraint. Video-monitoring was also undertaken.

2.3. Infusions Intravenous infusions were made using a Model 341B Sage Pump delivering 0.1 ml per min. Picrotoxin 1 m g / m l (Sigma, St Louis, USA) was dissolved in 10% dimethylsulfoxide (Ajax Chemicals, Sydney, Australia) in distilled water and heparin (David Bull Labs, Mulgrave, Australia) 30 units per ml. The infusate was administered for 1 min every 3 - 4 rain, and was continued until convulsions were induced or control conditions were fulfilled. All infusions were commenced between 08:30 and 09:00 h.

2. Materials and methods

2.4. Pentobarbitone clamp

2.1. Animals

Some animals were quiescent while others were aroused after seizures and even though the picrotoxin infusion had ceased, a second seizure occurred approximately every 1 in 8 animals. To prevent post-seizure arousal and more behavioural seizures occurring, pentobarbitone (Boehringer Ingelheim, Artarmon, Australia) was infused via the intravenous catheter immediately following the first convulsive event at a sufficient rate to keep the animal lightly anaesthetised for 1.5 h, after which it was deeply anaesthetised and perfused for immunohistochemical studies. Preliminary determinations indicated that pentobarbitone did not alter Fos-expression in the areas we had selected for study. Pentobarbitone did induce Fos in the area of the nucleus basalis, central amygdaloid nucleus, Islands of Calleja and, possibly, habenula, which we did not otherwise examine (to be described in a separate paper). We refer to this procedure as 'pentobarbitone clamp'.

Male inbred Sprague-Dawley rats, known not to spontaneously express spike and wave discharges [34], weighing 400-600 g, were housed in individual cages at an ambient temperature of 22°C with a 12:12 h light/dark cycle. Animals were given unrestricted access to food and water. We analysed 14 animals, 6 controls and 8 with seizures (2 groups of 4).

2.2. Electroencephalography (EEG) Animals were prepared with a jugular venous catheter and with cortical electrodes for EEG, primarily to determine if electrographic seizures might occur in the absence of motor convulsions. We followed the method of implantation of recording electrodes as described by Buszaki and others [4], except that fewer electrodes were implanted. Animals were anaesthetised with pentobarbitone 60 m g / k g intraperitoneally. Electrodes consisted of stainless steel screws inserted through the skull but not penetrating dura and positioned as follows: an indifferent electrode anteriorly over the frontal sinus, 3 pairs of electrodes 2.5 mm from the midline and 2 mm anterior and 2 and 6 mm posterior to bregma and an earth electrode in the occipital bone overlying the cerebellum. Fine wires were micro-

2.5. Immunohistochemistry Fos immunohistochemistry was undertaken 90 min after the seizure or control conditions were completed. This is within the time period in which Fos can be identified or is maximal in neurons that are known to express Fos [7,10]. Animals were deeply anaesthetised with pentobarbitone and perfused via the ascending aorta with 1% sodium

75

3.0. Willoughby et al. / Brain Research 683 (1995) 73-87

Table 1 Antibodies Species and Antigen

Dilution

Immunopositivity for:

Supplier

Rabbit Fos (#PC05) Mouse parvalbumina Rabbit calretinin Mouse calbindin ~ Goat anti-rabbit Donkey anti-mouse

1:1000 ]:10,000 1:10,000 1:5000 1:200 1:200

Fos reticular thalamicnucleus central medial thalamicnucleus rabbit primary mouse primary

Oncogene Science, Uniondale,USA Sigma, St. Louis, USA Swant, Bellinzona,Switzerland Sigma, St Louis, USA Vector, Burlingham,USA Jackson Immunoresearch,Philadelphia,USA

Monoclonal antibody.

nitrite in 0.01 M phosphate buffer followed by 1 litre of fixative (phosphate-buffered formaldehyde 4%, picric acid 15%, pH 7.4). After microwave post-fixation to 68 ° C, brains were cut with a Vibratome at 33 /xm and the sections were then processed for Fos immunohistochemistry using Fos antibody (Table 1) and the streptavidinbiotin-horseradish peroxidase complex technique (Amersham, UK). Sections were rinsed in 50% ethanol and incubated successively in 20% normal horse serum (NHS), primary Fos antibody in 1% NHS in Trismabuffered saline and 0.01% sodium azide for 2.5 days and then secondary antirabbit antibody (Table 1). The secondary antibody was localised using nickel enhanced diaminobenzidine with glucose oxidase to generate peroxide and yielded a dark blue or black reaction product [18]. Different sections were sequentially processed with a second primary antibody to allow definition of regions within the thalamus. The antibodies and dilutions are listed in Table 1. These incubations were carried out overnight at room temperature with 1% NHS in 0.1 M phosphate buffer. The secondary antibodies were localised using the unenhanced diaminobenzidine method with glucose oxi-

dase to generate peroxide and yielded a brown reaction product [18]. Thus Fos-labelled nuclei were distinguishable from neuronal-marker-immunopositiveperikarya by colour and location. The specificity of the Fos antibody was tested by preincubating the antiserum with synthetic M protein [25] at 2 - 2 0 / ~ g / m l for 24 h before application to the tissue. All nuclear staining was abolished. Sections processed without Fos or other primary antibodies but subject to all second antibody and diaminobenzidine reaction procedures, also revealed no staining of nuclei or cell bodies. 2.6. Quantitation

Fos immunoreactivity was observed in cell nuclei in several cortical [36] and subcortical [23] regions. Counts of all darkly or lightly labelled immunoreactive nuclei were undertaken at 1 0 0 - 2 0 0 × magnification on an Olympus BH-2 Microscope using the Magellan morphometry programme [12] on a computer interfaced to the microscope via a drawing tube. Table 2 lists the areas selected for counts of Fos-immunopositive neurons and the areas are

Table 2 Areas selected for Fos-positivecell counts Region

AP level a

Cells counted

Units

10.2 8.2 7.2 1.96 1.96

200/xm strip, pia to corpus callosum 200 txm strip, pia to corpus callosum 200/xm strip, pia to dorsal endopiriformarea 200/xm strip, pia to corpus callosum variable area, cellular layer

n n n n n/mm 2

5.7 5.7 5.7

200/xm strip, granularlayer 200/xm strip, pyramidallayer 200 /xm strip, pyramidallayer

n n n

7.2

200 /xm strip, corpus callosumto int. capsule

n/mm 2

7.2 7.2 7.2

area definedby calretininlabel area defined by parvalbuminlabel variable area enclosed by reticularnucleus

n/ram 2 n n/ram 2

5.7 5.7

200 × 400/xm strip, perpendicularto optic tract 200 tzm strip, parallel to optic tract, through maximumdiameterof basolateral amygdala

n n

Cortex:

Frontal Frl cortex h Parietal Parl cortex b Piriform cortex Entorhinalcortex Subiculum Hippocampus: Dentate gyms CA 3 CA 1 Basal ganglia: Caudate-putamen Thalamus: Central medial n. Reticular n. Ventral lateral n. Amygdala: Medial amygdala. Basolateral amygdala

Coronal level anterior to the interauralline [23]. b AS defined in Zilles [36].

76

J.O. Willoughby et al. / Brain Research 683 (1995) 73-87

2.7. Statistical analysis

IiJ Fig. 1. Output of Magellan program indicating the areas in which Fos positive cells were quantitated (described in Table 2): frontal Frl (a); parietal Parl (b); piriform (c); subiculum (d); entorhinal (e); central medial thalamic nucleus (f); ventral lateral thalamic nucleus (g); reticular thalamic nucleus (h); caudate putamen (i); CA1 (j); dentate gyrus (k); CA3 (1); medial amygdala (m); basolateral amygdala (n).

shown in Fig. 1. The areas chosen were from important subregions within each major structure; specifically for cortex: motor, somatosensory, olfactory and integrative cortices; for hippocampal structures: the dentate gyrus, CA1, CA3 and the outflow (subiculum); for thalamus: areas projecting purely to cortex (ventral lateral), to both cortical and subcortical areas (central medial) and purely to other thalamic regions (reticular) [15]; for amygdala, the regions having separate hypothalamic projections [24]. A small area in caudate-putamen was also counted. Each region was counted in three sections that were not necessarily adjacent or ipsilateral. Preliminary counting revealed that the mean of three counts was within 5% of the mean after counting homologous areas in more than 6 sections. No corrections were made for nuclear size, cell size, or for cells crossing the edge of the section. Fos-immunopositive counts in strips were expressed as mean and standard error of counts and in areas as counts per mm 2 (Table 2).

When the assumptions for analysis of variance were met, means were compared using one way analysis of variance, otherwise a non-parametric mood median test was used (Minitab Statistical Software, Pennsylvania, USA). Regression analysis was used to test relationships between Fos-counts and dosage of picrotoxin. A significance level of 0.05 was used throughout.

2.8. Nissl stains To obtain an estimate of the proportion of cells demonstrating Fos-positivity after seizures, the coverslips were removed from one rat in each seizure category and stained using cresyl violet. Such staining revealed only nuclei in sections that had already been immunohistochemically processed. Nuclei were then matched to those already counted for Fos and previously unstained nuclei, that were not obviously microglia or endothelial cells or dark evenly stained nuclei (likely to be glia), were also counted. Total cells counted by this method provided an estimate of total neuronal cell number in any region.

2.9. Fos-positive cell distribution in cortical laminae After all cortical regions had been mapped for Fos-positive cells, the data comprising coordinates of the borders of the cortical strips and the cells counted within these borders, were reanalysed as follows. The distances of cells from the pial border, taking into account its natural curvature, were calculated as well as the depth of the cortex from pia to corpus callosum. The strip was then divided into 20 bins of equal size and the numbers of cells within each bin were counted and plotted. Distributions of cells were obtained in absolute numbers, as well as for selected animals, in relative units, namely, percentage of Fos-positive cells to estimated neuronal number within each bin in each strip. By averaging over the homologous cortical areas in each category of seizure, a comparison was made separately for each bin between the seizure groups using unpaired t-test.

3. Results

3.1. Behaviour Administration of picrotoxin at a rate of 0.1 m g / m i n for 1 min every 3 - 4 rain induced an initial arousal then a

Fig. 2. Low-power photomicrographs representative of the cortical areas in which Fos-positive cells were quantitated in controls (Con), in animals with restricted seizures (R) and generalised seizures (G). The sections have been single-labelled for Fos to show its distribution clear of other markers. Differences in Fos induction between restricted and generalised seizures are not obvious. Frontal Frl = A; Parietal Parl = B; Piriform = C; Entorhinal = D. Bar = 300 um.

J.O. Willoughby et al. / Brain Research 683 (1995) 73-87

Con

77

G

R

A

B

C

ja,

D

°~'~

78

J.O.

Willoughby

et al. / Brain

Research

683

(1995)

73-87

R

G

A

°',~"

r °

B z,~;.

.

-

~

"

,

°

•

• . .

.

....:

°° . t o

.

C :°

-

•

.

°,~,

r.

..

.'..

D 3.'-

i' ..~2 •

, °h •

"

.5

,

. I

79

J.O. Willoughby et al. / Brain Research 683 (1995) 73-87

prolonged reduction in motor activities followed by myoclonic jerks, followed finally by convulsions. All electrical seizures were accompanied by behavioural seizures. W e observed a range of convulsive responses: (i) tonic or clonic events mainly confined to the head or to the head and forelimbs, occasionally with rearing, but with the animal maintaining its stance (defined herein as a restricted seizure), (ii) tonic or clonic events involving the head and all limbs associated, sometimes with loss of postural control (defined herein as a generalised seizure). Involvement of the trunk and hindlimbs, therefore, distinguished a restricted from a generalised seizure. Generalised seizures commenced in the same way as restricted seizures with head and forelimb clonic activity and this was immediately followed by involvement of the trunk and hindlimbs. Restricted seizures occurred more frequently than generalised seizures, and increasing the infusion rate to 0.1 m g / m i n for 1 min every 3 min instead of every 4 min did not alter this. W e were unable to predict how severe the seizure would be in terms of dose of picrotoxin (Fig. 4), or duration or distribution of motor activity. The first four animals fulfilling behavioural criteria for each experimental group were utilised. 3.2. E l e c t r o e n c e p h a l o g r a p h y

In animals undergoing vehicle infusion (n = 2) or the 0.6 m g / k g dose of picrotoxin (n = 1), the E E G did not reveal any electrographic discharges. In those infused with a dose of 1.2 m g / k g a convulsion did not always eventuate (n = 1) or it was prevented by pentobarbitone clamp (n = 2). These animals exhibited myoclonic jerks of head and sometimes upper limbs which were associated with short runs of high-voltage spikes on E E G as previously described for G A B A antagonists [1]. W e have not observed electrical seizures without behavioural seizures. There was a wider range in duration of generalised seizures (18 to 73 s) than restricted seizures (22 to 30 s). Seizures occurred unpredictably at doses of picrotoxin ranging between 0.9 m g / k g and 2.0 m g / k g . The EEG during seizure initially revealed bilateral complex activity consisting of spikes and waves which evolved after 5 - 1 0 s into regular synchronous high-voltage spikes separated by slow waves or electrically flat periods. There were no EEG features that enabled differentiation between generalised and restricted seizures. 3.3. F o s expression

Vehicle-infused animals (n = 2) and those receiving 0.6 m g / k g (n = 1) or 1.2 m g / k g (n = 3) doses of picrotoxin

800

c -i 0

RS

600

El.

,,=-

IIGS

~Qs

400

k-

Z

]~as

GSl

.J 200

0

1t" Ii

C(2} 0

C 0.5

C(3) 1.0

1.5

2.0

2.5

Picrotoxin dose {mg/kg)

Fig. 4. Picrotoxin-Fos cell count dose-response relationship in Frontal Frl (motor) cortex. Each point is the mean and S.E.M. of counts in three separate sections through Frl in each rat. Induction of Fos can be seen to be entirely dependent on the occurrence of seizures. Animals with generalised seizures (defined in text) had lower Fos counts than animals with restricted seizures (see also Fig. 5). C, control; RS, restricted seizure; GS, generalised seizure. without convulsions exhibited a few scattered Fos-positive neurons in cortex (Fig. 2), caudate-putamen, thalamus, hippocampus or amygdala (not shown), and scattered Fospositive neurons in many hypothalamic and some pontomesencephalic nuclei. Thus, there was no Fos induction in these six animals which constituted the control, no convulsion, group (all graphs). Fos was expressed in many forebrain regions after single convulsions as illustrated in the low-power photomicrographs in Figs. 2 and 3. Of special note is that different animals exhibited different patterns of Fos induction. In generalised seizures (n = 4), the main feature was induction in cortex (4 of 4) and hippocampal structures (3 of 4). In all restricted seizures (n = 4), the main findings were of Fos induction in cortex, basolateral amygdala and caudate-putamen but not hippocampal structures. There was no relationship between Fos counts and electrographic seizure durations (data not shown). Quantitation was undertaken to enable statistical comparison between the two groups. Considering primary motor ( F r l ) cortex in detail, there were different Fos-counts depending on seizure type (Fig. 4, and see below). The seizure threshold ranged from 0.9 to 2.0 m g / k g picrotoxin and there was no clear relationship between dose and seizure type. Similar Fos c o u n t / p i c r o t o x i n relationships were obtained for all brain regions examined and, as for frontal F r l cortex, there was discontinuous expression of Fos, dependent on the occurrence of a seizure. The one animal in the generalised seizure group that failed to exhibit hippocampal Fos-induc-

Fig. 3. Low-power photomicrographs representative of the appearances in hippocampus (A), subiculum (B), caudate putamen (C) and amygdala (D) in animals with restricted seizures (R) and generalised seizures (G). Differences in Fos expression can be seen in regions indicated by arrowheads. Bar in B and C = 200 ~m; Bar in D = 415 /xm, also refers to A.

J.O. Willoughby et al. / Brain Research 683 (1995) 73-87

8(I

800 -IFRONTAL Frl

.00

i

600

.oo

Ill

[]

300

P,.,FO.M

--

(4),.

.~

q-

° o

200

0

100

Q,.

o

o NONE

RS GS SEIZURE TYPE

NONE

RS SEIZURE TYPE

GS

Fig, 5. Histograms of Fos-positive cell counts (4-S.E.M.) in cortex, (a) neocortex, (b) paleocortcx. Fos counts in both frontal and parietal cortices after restricted seizures are significantly higher than after generalised seizures. For piriform and entorhinal cortices, the differences in Fos counts between restricted and generalised seizures are not significant. NONE, control; RS, rcstricted seizure; GS, generalised seizure; , significantly different from controls (mood median test).

(el Piriform Cortex

(a) Frontal Frl Cortex 60

el

e2o4

•

5

•

6

70

•

1

•

~

3

•

•

60

50

¢n

0

0

to

40

0

40

0

3o

¢/)

0

5O

30

0

20

1.1.

20

10

PIA

~

,

,

.

i

1

.

.

.

.

i

5

.

.

.

.

10

i

.

.

.

.

15

CO

i

PIA

(b) Parietal Par1 Cortex

80

ole

70

3

e4

5

•

•

=

= ~

6

.

.

.

.

5

.

.

.

.

10

.

.

.

.

~,CC

15

20

{d) Entorhinal Cortex

40

•

•

1

2

•

•

3

e4e

5

•

6e

3O

6O

'~

so

0 (.3

4O

0 0

3O

0

~q 0 U-

1

20

2O

U. 10

2O 10 0

PIA

'

1

5

'

i

.

.

.

.

10

i

.

.

.

.

15

1

CC

PIA

CC

1

20

DEPTH

D E P T H B E L O W PIA (bins) •

Generalised

5

seizure

0

Restricted

10

15

20

B E L O W PIA (bins)

seizure

Fig. 6. Distributions of cell counts ( + S.E.M.) according to depth bclow pia, indicating the laminar distribution of activated neurons after restricted (n = 4) and generalised seizures (n = 4). The bin means tend to be lower for generalised than for restricted seizures, even in piriform and entorhinal cortices where the overall counts did not significantly differ. The positions of the layers in cortex are indicated by numbers from l - 6 . These estimates were derived from Zilles for neocortex [36], Haberly and Price for piriform [11] and Swanson, Kohler and Bjorklund for entorhinal cortex [32]. CC, corpus callosum; * significantly different (unpaired t-test).

J.O. Willoughby et al. / Brain Research 683 (1995) 73-87

tion (see below), exhibited cortical Fos induction. Fos counts in this and all other regions are therefore presented as bar graphs. In neocortical regions, Frl and Parl, quantitation revealed that Fos-induction was consistently higher in animals that exhibited restricted seizures (n = 4) than in animals with generalised seizures (n = 4) (Fig. 5a). In all animals the absolute Fos counts were highest in layers 2 - 4 (Fig. 6a and b). At all levels (depths) of cortex, Fos counts were greater in restricted seizures relative to generalised seizures in keeping with the total Fos counts (Fig. 6a and b). In an individual animal in each seizure group, Fos induction occurred in approximately 80% of cells after a restricted seizure and in 60% after a generalised seizure (Table 3). The Fos- relative to neuronal counts for individual animals at different cortical depths are shown in Fig. 7 and indicate that the variation in laminar distribution of Fos was related to differences in underlying cell densities. In neocortex, approximately 80% of cells in all bins in an animal with a restricted seizure were Fos positive. After a generalised seizure there were fewer cells at all levels, especially in layers 4 to 6 (Fig. 7). In piriform and entorhinal cortices, total Fos counts were not significantly different in the two seizure types (Fig. 5b). The laminar distribution of Fos-positive neurons revealed higher counts at all depths in restricted seizures compared with generalised seizures, with some significant differences (Fig. 6c and d). In an individual animal in each seizure group, Fos induction occurred in a higher proportion of cells after a restricted seizure than after a generalised seizure and in entorhinal cortex, there was a reduced proportion of Fos-positive cells compared to other cortical regions in both seizure types (Table 3). The Fos counts relative to neuronal counts for individual animals are shown in Fig. 7 and again indicated that the variation in laminar distribution of Fos was related to differences in underlying cell densities, except after generalised seizures in entorhinal superficial layer 2 and layer 6 where relatively fewer cells were Fos positive. In subiculum, there was a definite but low level of Fos-positivity in control animals. Striking increases in Fos-positive neurons occurred only after generalised seizures in 3 of 4 animals (Figs. 3 and 8). There was a marked increase in Fos-positivity in dentate gyrus in 3 of 4 animals with generalised seizures (Figs. 3 and 9), but no change from controls after restricted seizures. Similarly, counts in CA3 and CA1 were elevated in 3 of 4 animals only after generalised seizures (Figs. 3 and 9). Fos was induced in nearly all dentate granule cells throughout the dentate gyms (Table 3), but in CA1, even more pyramidal cells were Fos-positive in the ventral part of the hippocampus than in the dorsal area we quantitated. The only animal with a generalised seizure without dentate and hippocampal Fos expression had a short duration convulsion (25 s). The bulk of the caudate-putamen remained Fos-nega-

81

tive. In the caudal-ventral part of this structure, however, there was consistent Fos-induction after restricted seizures and in only one animal with a generalised seizure (Figs. 3 and 10). Up to 60% of cells in a strip through this region were Fos-positive after restricted seizures (Table 3). In amygdala, Fos-positive cell counts were increased in medial amygdala after both restricted and generalised seizures (Figs. 3 and 11). Note that only a small proportion of cells in this structure are Fos-positive (Table 3). In basolateral amygdala, Fos induction was consistently seen after restricted seizures, while only 1 of 4 animals with generalised seizures exhibited Fos induction (an animal in which hippocampal induction of Fos also occurred). Some 70% of cells in a strip through basolateral amygdala were Fos-positive (Table 3) in restricted seizure. In thalamic nuclei there were increases in Fos-positivity after both seizure types (Fig. 12). However, in reticular nucleus only 4% of cells expressed Fos after a restricted seizure (Table 3), a consistent finding, whereas only 2 of 4 animals exhibited increases after a generalised seizure, but to much higher levels (20%). In central medial nucleus there were Fos-positive cells in control animals, so that the increase with seizures was approximately 2-3-fold with an additional 10-20% of cells expressing Fos (Table 3). Fos-labelling was always pale in ventral lateral thalamus, unlike all other regions, and definite labelling could be identified in 2 of 4 animals with generalised seizures and there were very low numbers of Fos-positive neurons in 2 of 4 animals with restricted seizures. Approximately 4 0 50% of ventral lateral thalamic neurons express Fos after single seizures (Table 3).

3.4. Other experimental effects on Fos expression In addition to the effects of pentobarbitone referred to above, after pentobarbitone without a seizure, small Fospositive cell nuclei were observed in the hilus of the dentate gyrus of the hippocampus. These cells did not double label with any neuronal marker, but were clearly positive for glial fibrillary acidic protein, indicating that they were glia. In animals that had convulsive seizures, a small number (less than 10%) of small and medium-sized nuclei in cortex were localised in cells that labelled with GFAP. Most of these cells were in layer 1 and were excluded from the Fos counts in this study. Glial expression of Fos in this experimental model is being presented separately (Hiscock, J.J., Mackenzie, L. and Willoughby, J.O., unpublished).

4. Discussion

Physiological stimulation and pharmacological manipulations can cause Fos induction in a distribution that is specific to the stimulus and consistent with the known function of the neurons as determined by other methods

J.O. Willoughby et al. / Brain Research 683 (1995) 73-87

82

RESTRICTED

SEIZURE

Frontal F r l

GENERALISED

Cortex

Frontal F r l

100

.o g M

.o~

70

SEIZURE [ 100

,0

[ ,o

-,o

40 ~

[,o~

60

•' i

50

Cortex

io 40

5O

2°°o~',°O~o.,o

40 3O 20

Ii°~

10 0

PIA

.

.

.

.

.

1

.

.

.

.

.

5

.

.

.

.

,

10

Parietal

.

.

.

15

Par1

.

,

CC

0

PIA 1

20

5

10

15

20

Parietal Par1 Cortex

Cortex

100

- 100 120

80 ~

120'

- 80

m

~ I00

~°

~ioo.

o

0

N 6o

,,6 40

80. ') 6 0 "

20 u-

2O

40.

~

0

~ 20. b-

,o 2o PIA

O" PIA ff 1

CC 1

10

5

15

20

.

.

.

.

.

.

.

.

.

5

Piriform Cortex

.

.

.

.

10

Piriform

, . . . . 15

T CC 20

Cortex

100

100

F]-.o

100 -~ c

~

i

so ~o

80

60'

~ 4o~

--

~

L,°~

o

L0

~

,o z

:o,O

40

d (n 20

~ 20O"

80

PIA

.

.

.

.

1

.

.

.

.

.

.

5

.

.

'

.

.

.

J

%

.

~P'~

.

15

Entorhinal ~

.

10

CC

0

O

PIA

20

CC 1

5

Cortex "

)

10

Entorhinal

L

15

20

Cortex

100

t lOO

ao ~

80 ~t

so

,°~

I

6O 4w

~o

=c 50 o ~ 4o

.J ~ 3o Z 2o ,,4 ¢~ 10 0

PIA

T -

, • , . . . . S

, . . . . 10

, . . . . 15

4o z

,o

ul

~ ~ ~o

~ CC 20

0

2o o

PIA

,

~ 5

DEPTH BELOW PIA (bins)

CC 10

15

DEPTH BELOW PIA (bins) • FOS

o NISSL

•

FOSINISSL (X)

20

J.O. Willoughby et al. / Brain Research 683 (1995) 73-87

Restricted seizure Fos +

Nissl

Nissl

DENTATE {4)

0

Generalised seizure % Fos Fos +

[•

200

Table 3 Proportions of cells Fos-positive (+_S.E.M.) from single animals with either restricted or generalised seizure

83

L)

CA 3

150

w

% Fos

[~

CA 1

0

c)

Cortex: Frl Par1 Piriform Entorhinal

551+76 738+8 288+_17 356+_11

665+97 899+_30 354+_15 520+_10

81+1 82+_2 82+_1 68+_3

376+21 466+_49 213+_25 210+_4

634+43 857+_57 288+_29 477+_25

59+2 57_+2 74_+1 44+_2

0 0 0

--

0 0 0

151+27 167+27 54_+3 82+5 11_+2 98_+4

93+_1 66+-3 11+-1

Hippocampus: Dentate CA3 CA1

-

100

o

0 a.

I

50

0 U_

(6)

(4)

NONE

RS

Basal ganglia: Caudate Amygdala: BL Medial

322+-73 541+-75 59+_6 17+_7

568+_36

3_+1

174+_28 251+_15 69+_8 4_+2 19+_3 231+_26 8_+1 14+_3

170_+38 210+_11

3+_1 4+_3

GS

SEIZURE TYPE Fig. 9. Histograms of Fos-positive cell counts ( + S.E.M.) in hippocampal structures. NONE, control; RS, restricted seizure; GS, generalised seizure; *, significantly different from restricted seizure ar, d control groups (mood median test).

Thalamus: CM RT VL

98+8 38+_1 61_+15

463+_23 21+_1 115+_3 195+_3 37-+1 955+_75 4-+0 249-+25 1010-+31 20_+1 126_+16 47+_7 77+_25 187+_42 43+_7

BL, Basolateral; CM, central medial; RT, reticular; VL, ventral lateral.

[3,14,31]. Thus, mapping the distribution of Fos after convulsive seizures could reveal some of the overall pattern of neuronal activation associated with the seizure. In some situations, however, not all neurons likely to be involved in physiological functions can be demonstrated to induce Fos with a presumably relevant stimulus, thus it seems probable that some neurons do not express Fos 1500

- - 1500

(4)

(3)

E E

0

when activated [31] or with a seizure, the process does not provide a critical stimulus for Fos induction [26]. In the present study, a significant proportion of cells expressed Fos in all brain regions we examined. We have therefore accepted that the distribution of Fos reflects, at least in part, the pattern of neurons activated during seizures. Furthermore, in structures where Fos is induced we have assumed that the number of Fos-positive nuclei is correlated in some unknown way with strength of stimulus to that structure. Evidence that number of labelled cells and intensity of labelling are related to stimulus duration has

1000

1000

5oo

500 (4)

(61

9

14)

14"1

NONE

RS

Type

° GS

of seizure

Fig. 8. Histograms of Fos-positive cell counts ( _ S.E.M.) in subiculum. NONE, control; RS, restricted seizure; GS, generalised seizure; , significantly different from restricted seizure and control groups (mood median test).

0

Is] NONE

F~ RS SEIZURE

GS TYPE

Fig. 10. Histograms of Fos-positive cell counts (+S.E.M.) in a strip through caudate-putamen. NONE, control; RS, restricted seizure; GS, generalised seizure; *, significantly different from generalised seizure and control groups (mood median test).

Fig. 7. Distributions of cell counts ( + S.E.M.) according to depth below pia in single animals with a restricted seizure and with a generalised seizure together with estimated neuronal number (Nissl) ( + S.E.M.) at the same depths. The relative number of Fos-positive cells is shown above each pair of plots for four cortical regions. In layer 1, few cells are present, producing large variability in the calculated relative Fos-labelling. Counts indicate that the laminar distribution of activated neurons is uniform in comparison to the underlying cell counts, especially for the restricted seizure. There is less relative Fos expression in the most s ut~erfical and the deeper layers in the generalised seizure. CC, corpus callosum. O, Fos counts; O , Nissl count (estimated neuronal number); D, relative Fos/estimated neuronal number.

84

J.O. Willoughby et a l . / Brain Research 683 (1995) 73-87 200

o O

150

(')

100

~'(41

m

M-AMYGDALA

T

[ ~ - ] BL-AMYGDALA

0

(4) ~

50

t

I

(5)

U-

.. RS

NONE

]'_: GS

SEIZURE TYPE Fig. 11. Histograms of Fos-positive cell counts (±S.E.M.) in strips through two regions of amygdala. NONE, control; RS, restricted seizure; GS, generalised seizure; M, medial; BL, basolateral; *, significantly different from controls (mood median test).

tion of the stimulus. Others report the use of diazepam in a similar way [13]. 4.2. Picrotoxin The effect of picrotoxin in inducing Fos was clearly related to the occurrence of a seizure given the discontinuous d o s e - r e s p o n s e relationship between picrotoxin and Fos-induction. Even doses of picrotoxin that were above threshold for other animals, failed to induce Fos unless there was a seizure. This indicates that it is the seizure process that induces Fos rather than any dose-related effects of picrotoxin on neuronal activity. Brain binding sites for p i c r o t o x i n / c o n v u l s a n t s as measured by S-t-butylbicyclophosphorothionate binding, do not consistently correlate with Fos distribution [9], also making a non-convulsant effect on Fos most unlikely. 4.3. Fos distribution

been reported [3]. Because we have no measure of antidromic or other forms of neuronal activation, we have interpreted our findings in relation to normal neuronal connectivity. 4.1. Controls In controls there were few Fos-positive neurons in cortical or subcortical regions and levels were lower than described in other reports. Animals in this study were not handled, unlike all other studies reported, but there were numerous Fos-positive neurons in the hypothalamus and brain stem. With the exception of the region of the basal nucleus, central nucleus of the amygdala and the Islands of Calleja, areas outside the focus of this study, pentobarbitone did not cause Fos induction. For this reason, pentobarbitone clamping may prove widely useful in confining the effects of brief stimuli on Fos-expression to the dura-

1000

RETICULAR N. (counts) CENTROMEDIAL N. (counts/ram2)

8OO

~

VENTROLATERAL N. (counts/ram2)

LL

ca') 600

<'

(4)

400 200

()4.

j

RS

GS

16}

0 NONE

SEIZURE TYPE

Fig. 12. Histograms of Fos-positive cell counts (±S.E.M.) in three thalamic regions. NONE, control; RS, restricted seizure; GS, generalised seizure; *, significantly different from controls (mood median test).

The major finding of this study is that non-focal seizures caused by impaired inhibitory function have at least two different behavioural characteristics that are associated with two patterns of distribution of Fos induction. In mild convulsive events, the animal exhibited head and neck rigidity with forelimb clonic contractions and Fos was induced at highest levels in cortex, caudal-ventral caudate-putamen and basolateral amygdala. In those with generalised convulsive events of head and all limbs, lower levels of Fos induction occurred in cortex and in 3 of 4 animals, there was intense Fos induction in dentate gyrus and to a lesser extent in hippocampus and subiculum, structures in which there was no Fos induction with restricted seizures. Failure to induce Fos in the uninvolved structures in each seizure type is clearly not due to any inability of the ceils to express Fos and indicates that the hippocampus is less activated in restricted seizures than in generalised seizures and conversely, the cortex, amygdala and caudate-putamen are consistently more activated. The finding of an inverse relationship between cortical and dentate-hippocampa| Fos induction provides strong evidence that restricted seizures are not a limited form of generalised seizures and that they are two different kinds of convulsive events, not different stages of a continuous process. Cortical Fos-induction. Consistent with EEG evidence and based on quantitating Fos-positivity in four areas, all areas of cortex are involved in the seizure process. While only motor components of a convulsion are visible, the motor cortex is no more involved than sensory or olfactory cortices. Studies of animals subjected to prolonged seizure activity suggested that every cell in the cortex is likely to be involved in a seizure caused by blockade of inhibition [6,22,25,28]. However, by careful quantitation of Fos after a single seizure, we have demonstrated that different levels of Fos-induction occurred with different seizure types and

J.O. Willoughby et al. / Brain Research 683 (1995) 73-87

that Fos-induction occurred evenly in all laminae throughout the cortex especially in restricted seizures (Figs. 6 and 7). In addition the most severe behavioural seizure was associated with less cortical Fos-induction than with less severe forms of convulsion. With generalised seizures, Fos-induction occurred somewhat less in all laminae, especially in the deeper half of the cortex (Figs. 6a,b and 7). One possible explanation for the widely distributed uniform cortical involvement is that intracortical excitatory connectivity is widespread and its activity is little affected by whatever inhibitory mechanisms remain in the picrotoxin-treated rat. Reduction of Fos-induction in deeper layers might imply the presence of slightly more effective inhibitory circuits in the deeper cortical layers or less effective excitatory inputs. Our findings are consistent with recent electrophysiological and computer modelling evidence [2] that inhibitory mechanisms are slightly more potent in deeper cortical layers. Hippocampal Fos-induction. Fos expression in dentate gyrus, CA3, CA1 and subiculum (Figs. 3, 7 and 8) clearly support the possibility of activation postsynaptically from dentate granule cell to CA3 pyramidal cell to CA1 pyramidal cell to subiculum along known hippocampal projections [32]. Inspection of entorhinal cortex counts (Fig. 5d) revealed that this structure, which is the source of the perforant path to dentate gyrus, was no more activated during a generalised seizure than during a restricted seizure. Furthermore, layer 2 in entorhinal cortex which provides afferents to the perforant path [16], was less activated during a generalised seizure (Fig. 6d). We suggest, therefore, that activation of dentate granule cells during a generalised seizure occurs more as a result of intrinsic dentate-hippocampal mechanisms and less as a result of the cells being driven from entorhinal cortex, and that dentate mechanisms play a crucial role in the early stages of generalised seizure. An inhibitory influence on superficial entorhinal cortex neurons by deeper layer entorhinal neurons has been observed in tissue slice and in vivo electrophysiological studies [16], pointing to a possible physiological mechanism for the apparent lesser involvement of entorhinal layer 2 in generalised seizures. The lack of dentate Fos expression in restricted seizures in which there is intense entorhinal layer 2 involvement, points to the entorhinal-dentate connection as a relative barrier to spread of excitation, also previously described [16]. Hippocampal Fos was not seen in one animal of the generalised seizure group even though its behavioural characteristics and distribution of Fos were otherwise consistent with generalised seizures. In our view this constitutes a limitation of categorising seizures by behaviour alone. The observation indicates that seizures which are behaviourally similar may be produced by different physiological processes. In this single animal, for example, the seizure might have been of cerebrocortical origin or, perhaps, the hippocampus was involved in the seizure though at a level below the threshold for Fos induction.

85

Sub-cortical Fos induction. Thalamic Fos-induction occurred consistently with both seizure types, except for the ventral lateral nucleus in which labelling was faint and in which there was no Fos-induction in 2 of 4 animals with generalised seizures. These findings suggest that there is involvement of thalamus in both seizure types, although ventral lateral nucleus may not be consistently involved. In basolateral amygdala and caudal-ventral caudate-putamen, consistent Fos-induction occurred with restricted seizures, indicating that these structures and possibly the ventral lateral thalamic nucleus were excited more during restricted than during generalised seizures. Thus Fos levels in cortex, caudate-putamen and amygdala, and possibly ventral lateral thalamic nucleus, are higher in restricted than in generalised seizures. The involvement of subcortical structures in seizures might depend on the degree of their direct participation in the seizure, or on the extent of excitation they receive from the cortex. There are excitatory inputs from cortex to caudate-putamen [20], reticular thalamic nucleus, ventral lateral thalamic nucleus, central medial thalamic nucleus [15] and amygdala [24] that might account for their involvement in the seizure process. Some of these regions have reciprocal excitatory projections to cortex that probably constitute an epileptic substrate [13,15,21]. However, neurons in the caudate-putamen and reticular thalamic nucleus are themselves inhibitory (both GABA synthesising nuclear groups). Even if they play a facilitatory role in synchronising activity in the established phase of the epileptic process they would not participate in feedback activation of the cortex during seizures. These inhibitory neurons appear to be activated as recipients of cortical excitation, rather than themselves producing a seizure discharge. Possible role o f cortex, subcortical structures and dentate-hippocampus. Cortical Fos counts were lower in gen-

eralised than in restricted seizures and in 3 of 4 animals there was concurrent hippocampal-subicular Fos induction in generalised seizures. One possible explanation for this reciprocal relationship is that the structure that first reaches seizure threshold reaches a higher level of activation than if it is 'secondarily' activated by another. This assumes that the cortex is secondarily activated from the hippocampus in generalised seizures and that restricted seizures are the result of a higher level of excitation being generated within cortex by mechanisms additional to any low level excitation of cortex from hippocampus. To the extent that Fos reflects strong neuronal activation, the variable participation in seizures by the amygdala, caudate-putamen and hippocampus, diminishes the individual importance of these structures as essential substrates in this model, although they may well participate at a low level. In the range of seizure types and severities produced by picrotoxin, the cortex and at least one midline nucleus of thalamus appear to be critical structures. In summary, our findings have revealed that single non-focal seizures induced by an antagonist of nervous

86

J.O. Willoughby et al. / Brain Research 683 (1995) 73-87

s y s t e m i n h i b i t i o n , p i c r o t o x i n , are u n p r e d i c t a b l e in r e g a r d to c a u s a l d o s e o f d r u g as w e l l as the n a t u r e a n d s e v e r i t y o f the b e h a v i o u r d u r i n g the seizure. M i l d e r c o n v u l s i o n s w e r e a s s o c i a t e d w i t h d i s t r i b u t i o n s o f F o s i n d u c t i o n that i n d i c a t e a d i f f e r e n t d i s t r i b u t i o n o f i n v o l v e d n e u r o n s f r o m the m o s t s e v e r e f o r m s o f c o n v u l s i o n . A l l a r e a s o f the c e r e b r a l c o r t e x are a c t i v a t e d in c o n v u l s i o n s a n d all cortical l a y e r s are i n v o l v e d . C e r e b r a l cortical a c t i v a t i o n s o m e t i m e s a p p e a r s to b e s e c o n d a r y to d e n t a t e - h i p p o c a m p a l a c t i v a t i o n in the m o s t s e v e r e s e i z u r e s w h i l e s u b c o r t i c a l s t r u c t u r e s participate in less s e v e r e c o n v u l s i o n s , s o m e as a c o n s e q u e n c e o f cortical a c t i v a t i o n .

Acknowledgements S u p p o r t e d b y g r a n t s f r o m the N a t i o n a l H e a l t h a n d M e d i c a l R e s e a r c h C o u n c i l , the A u s t r a l i a n B r a i n F o u n d a tion a n d the F l i n d e r s M e d i c a l C e n t r e R e s e a r c h F o u n d a t i o n .

References [1] Ben-Ari, Y., Tremblay, E., Riche, D., Ghilini, G. and Naquet, R., Electrographic, clinical and pathalogical alterations following systemic administration of kainic acid, bicuculline or pentetrazole: metabolic mapping using the deoxyglucose method with special reference to the pathology of epilepsy, Neuroscience, 6 (1981) 1361-1391. [2] Berman, N.J., Douglas, R.J. and Martin, K.A.C., Gaba-mediated inhibition in the neural networks of visual cortex. In R.R. Mize, R.E. Marc and A.M. Sillito (Eds.), Progress in Brain Research, Vol. 90, Elsevier, Amsterdam, 1992, pp. 443-476. [3] Bullitt, E., Lee, C.L., Light, A.R. and Willcoxon, H., The effect of stimulus duration on noxious-stimulus induced c-fos expression in the rodent spinal cord. Brain Res., 580 (1992) 172-179. [4] Buzsaki, G., Bickford, R.G., Ponomareff, G., Thai, L.J., Mandel, R. and Gage, F.H., Nucleus basalis and thalamic control of neocortical activity in the freely moving rat, 3". Neurosci., 8 (1988) 4007-4026. [5] Cole, A.J., Abu-Shakra, S., Saffen, D.W., Baraban, J.M. and Worley, P.F., Rapid rise in transcription factor mRNAs in rat brain after electroshock-induced seizures, J. Neurochem., 55 (1990) 1920-1927. [6] Dragunow, M. and Robertson, H.A., Generalized seizures induce c-fos protein(s) in mammalian neurons, Neurosci. Lett., 82 (1987) 157-161. [7] Dragunow, M., Yamada, N., Bilkey, D.K. and Lawlor, P., Induction of immediat-early gene proteins in dentate granule cells and somatostatin interneurons after hippocampal seizures, Mol. Brain Res., 13 (1992) 119-126. [8] Duman, R.S., Craig, J.S., Winnston, S.M., Deutch, A.Y. and Hernandez, T.D., Amygdala kindling potentiates seizure-stimulated immediate-early gene expression in rat cerebral cortex, J. Neurochem., 59 (1992) 1753-1760. [9] Edgar, P.P. and Schwartz, R.D., Localization and characterization of 35s-t-butylbicyclophosphorothionate binding in rat brain: An autoradiographic study, J. Neurosci., 10 (1990) 603-612. [10] Gass, P., Herdegen, T., Bravos, R. and Keissling, M., Induction of immediate early gene encoded proteins in the rat hippocampus after bicuculline-induced seizures: Differential expression of krox-24, fos and jun proteins, Neuroscience, 48 (1992) 315-324. [11] Haberly, L.B. and Price, J.L., Association and commissural fiber systems of the olfactory cortex of the rat. I. Systems originating in

the piriform cortex and adjacent areas, J. Comp. Neurol., 178 (1978) 711-740. [12] Halasz, P. and Tork, I., Imaging of immunohistochemically identified neurons using IBM compatible and Macintosh computers, Neurosci. Lett., Suppl. 34 (1989) 588 [13] Herdegen, T., Sandkuhler, J., Gass, P., Kiessling, M., Bravo, R. and Zimmermann, M., JUN, FOS, KROX, and CREB transcription factor proteins in the rat cortex: basal expression and induction by spreading depression and epileptic seizures, J. Comp. Neurol., 333 (1993) 271-288. [14] Hoffman, G.E., Lee, W., Attardi, B., Yann, V. and Fitzsimmons, M.D., Luteinizing hormone-releasing hormone neurons express c-los antigen after steroid activation, Endocrinology, 126 (1990) 17361741. [15] Jones, E.G., The Thalamus, Plenum Press, New York, 1985. [16] Jones, R.S.G., Entorhinal-hippocampal connections: a speculative view of their function, Trends Neurosci., 16 (1993) 58-64. [17] Lanaud, P., Maggio, R., Gale, K. and Grayson, D.R., Temporal and spatial patterns of expression of c-fos, zif/268, c-jun and jun-B mRNAs in the rat brain following seizures evoked focally from the deep prepiriform cortex, Exp. Neurol., 119 (1993) 20-31. [18] Llewellyn-Smith, I.J., Minson, J.B. and Pilowsky, P.M., Retrograde tracing with cholera toxin B-gold or with immunocytochemically detected cholera toxin B in central nervous system, Methods Neurosci., 8 (1992) 180-201. [19] Maggio, R., Lanaud, P., Grayson, D.R. and Gale, K., Expression of c-los mRNA following seizures evoked from an epileptogenic site in the deep prepiriform cortex: Regional distribution in brain as shown by in situ hybridization, Exp. Neurol., 119 (1993) 11-19. [20] McGeorge, A.J. and Faull, R.L.M., The organisation of the projection from the cerebral cortex to the striatum in the rat, Neuroscience, 29 (1989) 503-537. [21] McLachlan, R.S., Gloor, P. and Avoli, M., Differential participation of some 'Specific' and 'Non-specific' thalamic nuclei in generalized spike and wave discharges of feline generalized penicillin epilepsy, Brain Res., 307 (1984) 277-287. [22] Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping patterns of c-fos expression in the central nervous system after seizure, Science, 237 (1987) 192-197. [23] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. [24] Price, J.L., Russchen, F.T. and Amaral, D.G., The limbic region. II. The amygdaloid complex. In A. Bjorklund, T. Hokfelt and L.W. Swanson (Eds.), Handbook of chemical neuroanatomy. Vol. 5. Integrated systems of the CNS. Part 1, Elsevier, Amsterdam, 1987, pp. 279-388. [25] Sagar, S~M., Sharp, F.R. and Curran, T., Expression of c-los protein in brain: Metabolic mapping at the cellular level, Science, 240 (1988) 1328-1331. [26] Sharp, F.R., Gonzalez, M.F., Sharp, J.W. and Sagar, S.M., c-los expression and (14C) 2-deoxyglucose uptake in the caudal cerebellum of the rat during motor/sensory cortex stimulation, J. Comp. Neurol., 284 (1989) 621-636. [27] Sharp, J.W., Sagar, S.M., Hisanaga, K., Jasper, P. and Sharp, F.R., The NMDA receptor mediates cortical induction of los and los-related antigens following cortical injury, Exp. Neurol., 109 (1990) 323-332. [28] Shehab, S., Coffey, P., Dean, P. and Redgrave, P., Regional expression of fos-like immunoreactivity following seizures induced by pentylenetetrazole and maximal electroshock, Exp. Neurol., 118 (1992) 261-274. [29] Shin, C., McNamara, J.O., Morgan, J.I., Curran, T. and Cohen, D.R., Induction of c-los mRNA expression by after-discharge in the hippocampus of naive and kindled rats, J. Neurochem., 55 (1990) 1050-1055. [30] Snyder-Keller, A.M. and Pierson, M.G., Audiogenic seizures induce

J.O. Willoughby et al. / Brain Research 683 (1995) 73-87 c-fos in a model of developmental epilepsy, Neurosci. Lett., 135 (1992) 108-112. [31] Strassman, A.M. and Vos, B.P., Somatotopic and laminar organization of Fos-like immunoreactivity in the medullary and upper cervical dorsal horn induced by noxious facial stimulation in the rat, J. Comp. Neurol., 331 (1993) 495-516. [32] Swanson, L.W., Kohler, C. and Bjorklund, A., The limbic region. I: The septohippocampal system. In A. Bjorklund, T. Hokfelt and L.M. Swanson (Eds.), Handbook of Chemical Neuroanatomy. VoL 5: Integrated Systems of the CNS, Part 1, Elsevier, 1987, pp. 125-277. [33] Ticku, M.K., Lee, J.C., Murk, S., Mhatre, M.C., Story, J.L., KaganHallet, K., Luther, J.S., MacCluer, J.W., Leland, M.M. and Eidelberg, E., Inhibitory and excitatory amino acid receptors, c-fos ex-

87

pression, and calcium-binding proteins in the brain of baboons (papio hamadryas) that exibit 'spontaneous' grand mal epilepsy, In J. Engel Jr., C. Wasterlain, E.A. Cavalheiro, U. Heinemann and G. Avanzini (Eds.), Molecular Neurobiology of Epilepsy, Elsevier, Amsterdam, London, New York, Tokyo, 1992, pp. 141-149. [34] Willoughby, J.O. and Mackenzie, L., Nonconvulsive electrocorticographic paroxysms (absence epilepsy) in rat strains, Lab. Anim. Sci., 42 (1992) 551-554. [35] Willoughby, J.O., Mackenzie, L., Hiscock, J.J. and Sagar, S., Nonconvulsive spike-wave discharges do not induce Fos in cerebrocortical neurons, Mol. Brain Res., 18 (1993) 178-180. [36] Zilles, K., The Cortex of the Rat, Springer-Verlag, Berlin, 1985.