Long-lasting effects of feline amygdala kindling on monoamines, seizures and sleep

Brain Research 892 (2001) 147–165 www.elsevier.com / locate / bres

Research report

Long-lasting effects of feline amygdala kindling on monoamines, seizures and sleep Margaret N. Shouse a

a,b ,

*, Richard J. Staba a,b , S. Ferhaan Saquib a , Paul R. Farber a

Department of Veterans Affairs, Greater Los Angeles Health Care System (151 A3), Sepulveda, CA 91343, USA b Department of Neurobiology, Center for Health Sciences, UCLA, Los Angeles, CA 90063, USA Accepted 21 November 2000

Abstract This report describes the relationship between monoamines, sleep and seizures before and 1-month after amygdala kindling in young cats (,1 year old; n58; six female and two male). Concentrations (fmoles of norepinephrine or NE, dopamine or DA and serotonin or 5-HT) were quantified in consecutive, 5-min microdialysis samples (2 ml / min infusion rate) from amygdala and locus ceruleus complex (LC) during four, 6–8-h polygraphic recordings before (n52) and 1 month post-kindling (n52); 5-min recording epochs were temporally adjusted to correspond to dialysate samples and differentiated according to dominant sleep or waking state (lasting $80% of 5-min epoch) and degree of spontaneous seizure activity (number and duration of focal versus generalized spikes and spike trains and behavioral seizure correlates). Post-kindling records in each cat were divided into two groups (n51 record each) based on higher or lower spontaneous EEG and behavioral seizure activity and compared to pre-kindling records. We found: (1) before and after kindling, NE and 5-HT but not DA concentrations were significantly lower in sleep than waking at both sites; (2) after kindling, each cat showed cyclic patterns, as follows: (a) higher NE, 5-HT and DA concentrations accompanied increased seizure activity with delayed sleep onset latency and increased sleep fragmentation (reduced sleep state percentages, number of epochs and / or epoch duration) in one recording versus (b) lower monoaminergic concentrations accompanied reduced seizure activity, rapid sleep onset and reduced sleep disruption in the other recording. The alternating, post-kindling pattern suggested ‘rebound’ effects which could explain some controversies in the literature about chronic effects of kindling on monoamines and sleep–waking state patterns.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Epilepsy: basic mechanisms Keywords: Monoamine; Developmental amygdala kindling; Sleep; Microdialysis; Cat

1. Introduction Considerable literature links monoamines to the regulation of electroencephalographic (EEG) and behavioral components of sleep and arousal as well to epileptogenesis. For example, spontaneous, extracellularly recorded discharge rates of putative norepinephrine (NE) and serotonin (5-hydroxytryptamine or 5-HT) containing neurons in the pontine tegmentum reach a peak during alert waking, decline during slow-wave sleep (SWS), the feline equivalent of NREM sleep in humans [82], and are virtually silent during REM sleep (e.g., Refs. *Corresponding author. 2242 S. Bentley Ave. [ 4, Los Angeles, CA 90064-1940, USA. Tel. / fax: 11-310-473-3128. E-mail address: [email protected] (M.N. Shouse).

[30,31,47,48]). These cells are reciprocally connected with adjacent pontine as well as forebrain structures, including the amygdala [3,4,34,44–46,68,69], which also participate in the generation of physiological and behavioral sleep and arousal components [35]. In contrast, discharge rates of dopamine (DA) containing neurons — which originate from substantia nigra and ventral tegmentum of midbrain — do not change across the sleep–wake cycle [30], even though these cells project to similar pontine and forebrain sites implicated in EEG and behavioral activation [13,34,35,41,44,48,55,59,76]. Seizure disorders are characterized by abnormal EEG and behavioral activation, evidenced by increased frequency and / or amplitude of neuronal discharges, often associated with increased seizure-related behavioral activity (e.g., Refs. [53,76]). The role of monoamines in

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03265-0

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epileptogenesis and in recurrent seizure activity is also well documented. Spontaneous and experimentally induced deficiencies in NE, DA and / or 5-HT have been implicated in the onset and perpetuation of many seizure disorders [1,2,7,14,15,49,50,52,58,60,64,74,89], including the amygdala kindling model of temporal lobe epilepsy (TLE [2,14,15,49,52,60,74,89]). Conversely, many experimental procedures designed to increase monoaminergic activity have proven antiepileptic properties [4–6,10,11,16,43, 60,64,74,84,88]. Seizure disorders are also thought to reciprocally affect monoamines as well as sleep–waking states [12,22, 26,28,70,73,75,77,78]. Persistent changes have been reported after kindling, which is characterized by transsynaptic effects on distal structures and related functions (e.g., Refs. [14,22,49,53]). Short-term post-kindling changes are usually consistent and characterized by increased monoaminergic concentrations during seizures [14,17,20,38,42,43,71,84] followed by depletion associated with delayed REM onset #24 h post-ictal (e.g., Ref. [71]). However, findings of long-term post-kindling changes in either monoamines or sleep are inconsistent [14,49,73]. Some authors report rebound increases in monoamines and / or sleep [12,17,13,14,20,42,51,73,74], some report chronic suppression [22,73], and some report no long-term changes at all [14,28,49]. Several factors could contribute to these discrepancies. Examples are differences in kindling paradigm, species and age of animals as well as severity of seizure disorder, duration and / or timing of post-kindling follow-up. This study focused on severity of seizure disorder as a factor in long-term changes in monoamine concentrations and sleep parameters. Some information about duration and timing of post-kindling follow-up is also provided. The protocol employed microdialysis to measure concentrations of NE, DA and 5-HT as a function of polygraphic recordings obtained before and 1 month after kindling in our developmental feline TLE epilepsy model (e.g., Refs. [72–78]). This kindling procedure employs electrical stimulation of amygdala in pre-adolescent cats. Typically, about 60% of pre-adolescent cats develop spontaneous, stage 6 or generalized tonic–clonic convulsions (GTCs) when kindled to stage 6 [72]. Nearly all manifest some degree of recurrent seizure activity [75], with peaks in spontaneous seizure discharge generalization during EEG synchronized states of waking and sleep (e.g., Ref. [76]). The kindling paradigm used here involved kindling to stage 3, which is the initial seizure discharge generalization stage [72,73,78]. The objective was to determine whether spontaneous seizure activity would develop after this modified kindling procedure and, if so, to detect interactions with sleep–waking states and monoamine concentrations. There were two collection sites: (1) the amygdala, which receives terminals from brainstem areas containing monoaminergic cells thought to affect focal and general-

ized seizure discharge as well as EEG and behavioral arousal [3,13,44–46]; and (2) the pontine tegmentum, in the vicinity of the locus ceruleus complex (LC), where NE-containing cell bodies predominate (e.g., Ref. [79]). LC is also innervated by DA and 5-HT cells involved in seizure and arousal events [42–44,61–63,68,69,82] and is reciprocally connected with amygdala [3,14,46].

2. Methods

2.1. Surgery Aseptic stereotaxic neurosurgery was performed on eight pre-adolescent cats (six females and two males), aged 4.5–5.5 months, average weight 2.1 kg [29]. Animals were pre-anesthetized with an admixture of ketamine hydrochloride (100 mg / ml) and acepromazine maleate (10 mg / kg) administered i.m. prior to insertion of an i.v. catheter into the cephalic or femoral vein. Deep anesthesia was induced and maintained by barbiturate (0.1–0.2 mg per i.v. injection of 50 mg / ml sodium pentobarbital) administered to effect in four cats. The other four cats were intubated, and deep anesthesia was maintained by 2% isofluorane. Surface and depth electrodes (n540 per cat) were implanted to record sleep–wake state patterns and seizure discharge propagation. Stereotaxic coordinates were extrapolated from the atlases of Rose and Goodfellow [66] for kittens, and Snider and Niemer [81] for adult cats, as previously reported (e.g., Refs. [73–75,77,78]). Jeweler’s screws were threaded into the bone, bilaterally, over frontal (anterior or A24, lateral or L4, L8), posterolateral (A1, posterior or P1, L1) and occipital (P4, L4, L6) cortices to record cortical EEGs and also over the orbit in the frontal sinus to register eye movements (electrooculograms or EOG). Stainless steel wires were inserted into the nuchal musculature to record tone (electromyogram or EMG). Depth electrodes with 1-mm deinsulated tips were inserted bilaterally to record EEG activity in entorhinal cortex (A13, L13, horizontal or H-6), the lateral geniculate nucleus (LGN; A10, L10, H12.3), amygdala (A10, L10, H-4) and LC (P3, L2.5, H-2.5). Bipolar strut electrodes were implanted into entorhinal cortex; all other depth electrodes were tripolar, concentrically arranged leads. For microdialysis, 18-gauge stainless steel guide cannulae were situated in the middle of concentrically arranged recording electrodes 5 mm above target sites in amygdala and pons. Smaller gauge microdialysis probes (polyimide tubing with 2 mm length, Eicom DM-22 membrane) were constructed to measure monoamine concentrations at amygdala and LC recording sites.

2.2. Experimental protocol There were three main experimental phases: pre-kindling baseline, kindling, and post-kindling follow-up.

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2.2.1. Pre-kindling baseline After a minimum 2-week post-operative recovery, animals were adapted to the recording chamber for $24 h before baseline 6–8-h polygraphic recordings of sleep– waking state physiology. Preliminary baseline recordings were obtained on two consecutive days prior to bilateral probe insertion in amygdala and LC. Sleep–waking state parameters were continuously recorded 8–24 h post-insertion just before each dialysate sample collection (left hemisphere only). Dialysate samples were collected during two, 6–8-h polygraphic recordings when cats were 5.0–6.0 months old. 2.2.2. Kindling Two weeks to 1 month later, the 6–6.5-month-old subjects underwent an interim, 1-day kindling experiment using the same probe insertion, polygraphic recording and microdialysis protocol as in initial baseline, with two exceptions. Post-insertion collection was limited to a 3-h period, and seizures were induced by electrical stimulation of left amygdala [78]. Each stimulus consisted of a 1-s train of 60 Hz, biphasic square waves of 1 ms duration. Prior to this second phase of the experiment, the animals had no additional probe insertions, nor had they received kindling stimulation. The 3-h collection protocol (12, 5-min samples each) was interrupted only to establish initial focal or generalized afterdischarge (AD) thresholds, as follows:

1. (1) Baseline: 12, 5-min samples were collected while cats were monitored for sleep–waking state only (1 h). 2. (2) Initial focal AD threshold determination: focal AD refers to local EEG seizure discharge in the stimulated (left) amygdala associated with staring (kindling stage 1 at this stimulation site) and eye-lid twitching (kindling stage 2). Amplitude of the first stimulus was set at 100 mA; stimulus amplitude was incremented by 100 mA every 60 s until the first focal AD was evoked. This is similar to the initial threshold procedure used at this stimulation site in previous studies (e.g., Ref. [74]). A mean of 1164 stimulations with a 1-min interstimulus interval or ISI was required to evoke the first focal AD in the eight cats. All stimuli were delivered in drowsy waking or SWS states. 3. (3) Focal AD with microdialysis: 12, 5-min dialysis samples were collected in relation to stimuli applied during spontaneous sleep or waking states. Focal AD was elicited at a rate of one AD per 5-min sample (1 h). Seizures were evoked using a 1-min ISI beginning 100 mA below threshold established within the previous 5-min collection period. This threshold procedure is the same as in previous work examining postkindling thresholds at this amygdala stimulation site (e.g., Ref. [74]). The procedure was adopted here in

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order to induce one stage 1 or 2 seizure per 5-min sample (the number of stimulations ranged from one to three per sample). Stimulations began 2-min into each collection to account for a 2-min dead time, which reflects the delay between the probe tip and the outlet in the collection vial. All stimuli were delivered in drowsy waking or SWS states. 4. (4) Initial generalized AD threshold determination: the procedure was the same as for initial focal AD determination except that the end-point was eliciting the first generalized AD. Generalized AD is defined by EEG seizure discharge in kindled amygdala, with spread to other recording sites. Behavioral seizure accompaniment is equivalent to a mild stage 3 seizure at this amygdala stimulation site and consists of lipsmacking usually with salivation, head nodding (clonus) and / or limb clonus. A mean of 1567 stimulations with a 1-min ISI was required to achieve the first generalized AD in the eight cats. All stimuli were delivered during drowsy waking or SWS states. 5. (5) Generalized AD with microdialysis: 12, 5-min dialysis samples were collected in relation to spontaneous sleep or waking states, and generalized AD was elicited at a rate of once per 5-min sample (1 h). Otherwise, the procedure as well as number or stimulations (n51–3) needed to elicit generalized AD was the same as for focal AD. Except for three, 5-min epochs in which stimuli were delivered during alert waking, all other stimuli were delivered during drowsy waking and SWS states. The objective of employing this 1-day kindling paradigm, as opposed to the usual practice of evoking AD once per day (e.g., Ref. [72]), was to elicit and to differentiate focal amygdala seizures from mild generalized seizures. All cats were kindled to the same focal or generalized seizure stage criterion. Compressing the kindling protocol to 1 day was intended to achieve kindling while the cats remained in an adolescent age range and to avoid multiple probe insertions. Confining the kindling progression to stage 3 was intended to minimize seizurerelated mobility, which can alter microdialysis measures independently of spontaneous or evoked EEG seizure discharge [8,86]. It should be noted that there is no kindling rate in this paradigm. There was only number of stimulations to first focal or generalized AD in the initial threshold determinations, followed by repetitive focal or generalized AD elicitation during spontaneous sleep or waking state during microdialysis.

2.2.3. Post-kindling follow-up One month elapsed after the interim kindling experiment, and the 7–7.5-month-old subjects underwent the same protocol as before kindling, with two exceptions. Polygraphic recordings were not initiated until commence-

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ment of dialysate collection, and both post-kindling recordings lasted 8 h. Overall, cats had a total of five separate probe insertions from the beginning to the end of the entire protocol. Preliminary results have been reported elsewhere [77,78]. The present report focuses mostly on a comparison of the first two 6–8-h pre-kindling records to the two 8-h records obtained 1 month after kindling. Cats were under continuous visual and polygraphic surveillance for sleep–waking state and seizure activity during dialysate collection. Five-minute samples were obtained using infusion of artificial CSF. Infusion rate (2 ml / min), and collection was regulated by a CMA 100 microinjection pump (Carnegie Medicin). Artificial CSF (Harvard) consisted of mM concentrations of 150 Na 1 , 3.0 K 1 , 1.4 Ca 21 , 0.8 Mg 21 , 155 Cl 2 , and 1 Na 2 HPO 4 , pH 7.5. Each 5-min (10 ml) sample was collected in vials containing 5 ml preservative (pH 3.5), frozen in dry ice and stored in a 2808C freezer.

2.3. Data analysis 2.3.1. Dialysates Dialysate samples from amygdala and LC were analyzed for the presence of three monoamines in chromatograms. The appearance of peaks at retention times of |2.7, 4.9 and 11.1 min was considered positive identification of NE, DA and 5-HT, respectively [77,78]. Monoaminergic concentrations were calculated by measuring area under the peak and calibrated to standards obtained at the beginning, middle and end of each polygraphic / microdialysis recording. Peak amplitude measurements of standards were linear between 0.1 and 10 pg / ml injected. Dialysates were analyzed by an ESA Coulochem II Model electrochemical detection HPLC system, an ESA model 5011 analytic cell with dual porous graphite electrodes (the first electrode was set at 2100 mV and the second at 1220 mV), a guard cell set at 1350 mV, an ESA MD-150 C-18 column, and a commercial mobile phase for monoamines (ESA-MD-IM). Flow rate across the column was 0.6 ml / min. Sample concentrations were temporally adjusted to polygraphic recordings to account for the 3-min ‘dead time’, which reflects the recovery delay between membrane tip to and tube outlet in the collection vial (versus 2-min during the interim kindling experiment). Dialysate concentrations were differentiated according to sleep versus waking state parameters and also as a function of spontaneous focal versus generalized seizure discharge during each 5-min epoch. Because mobility can alter microdialysis measurements (e.g., Refs. [8,86]), epochs containing repetitive, excess mobility (e.g., walking, grooming, eating lasting $2-min) were deleted. All deleted epochs occurred during alert waking, when spontaneous seizure activity was infrequent. Spontaneous seizure activity was mild and, as during the interim kindling experiment, consisted of staring, eye-lid twitches, salivation / lip-smack-

ing, head clonus (head nodding) or limb clonus. These seizures never lasted more than 1.5 min and were not a criterion for sample exclusion. The percent of samples excluded for excess mobility was comparable in prekindling and 1-month post-kindling records and comprised #3% of alert waking epochs per record. In vivo samples were adjusted to reflect in vitro recovery measured prior to sample collection and in 50% of the cases after the conclusion of each pre-kindling and 1 month post-kindling recording / collection. Relative recovery rates were 12.860.1% for NE, 12.560.8% for DA and 12.060.9% for 5-HT before amygdala insertion and 136 0.8% for NE, 12.26 0.9% for DA and 11.96 0.7% prior to LC insertion. Recovery rates determined after collections were #1% lower than pre-insertion at both sites.

2.3.2. Sleep–waking state parameters Four states were identified, as previously described and illustrated [18,73,75,77]. These were alert waking (W1), drowsy waking (W2), SWS and REM sleep. Wakefulness was differentiated into alert waking, defined by low-voltage fast EEG, irregular high-voltage fast eye movements and increased tone (EMG amplitude) when compared to drowsy waking with its higher voltage background EEG ($50 mV in over 50% if 1-min epochs with #2 sleep spindles). Sleep was differentiated into SWS which, when compared to drowsiness, contains longer trains of background EEG (.50% of 1-min epochs with .50 mV per 1-min epoch), .2 sleep spindles per min, slower eye movements and reduced tone) and REM sleep (which is defined by tonic components, including low voltage, fast EEG and loss of tone plus phasic components including rapid eye movements, ponto-geniculo-occipital (PGO) spikes and sometimes muscle twitches). These states seldom lasted for the entire 5-min interval corresponding to the microdialysis sample. For this reason we used dominant state, defined as the state occupying $80% of the sample time, as the criterion for correlating sleep–waking state to monoaminergic sample concentrations. We previously reported highly positive correlations (r5$0.9, P,0.01) between percent of sleep or wake state and sample concentrations in each cat during 6–8-h records before kindling [77]. Correlations in 1-month postkindling findings were .0.93, P,0.01 per cat. Variability in post-insertion delay to collection times (8 h in four cats; 24 h in four cats) can affect extracellular chemical concentrations [8,85,86] but was excluded because of low inter-subject variability. The percent time, number of epochs and epoch duration (min) lasting $1 min, as well as the number of epochs lasting $80% of 5-min collection periods, were calculated for each state in each pre- and post-kindling recording. SWS and REM sleep onset latencies (min from beginning of recording / sample collection) were also determined.

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2.3.3. Seizure activity measures Each 5-min epoch was also assessed for spontaneous seizure activity. EEG seizure indices included: the number of focal or isolated spikes (,70 ms, $100 mV; .1-s interspike interval) in left amygdala or LC, the incidence and duration of spike trains ($3 spikes with ,1-s interspike interval) in left amygdala only (focal) and in left amygdala and LC simultaneously (generalized). We targeted these two structures because they were the dialysis sites. Amygdala and LC discharges were assessed separately as well as jointly because cats kindled as preadolescents tend to develop spontaneous multifocal epilepsy [72,75]. In this study, we detected focal spikes in amygdala and LC separately, but there were no spike trains seen in LC independently of kindled amygdala, hence the designation ‘generalized’. We also quantified the number and duration of behavioral seizure manifestations. As specified above, there were only five behavioral seizure manifestations, which collectively lasted a maximum of 1.5 min per 5-min sample and typically much less. 2.4. Statistical analysis [39] Each cat showed the same trends for each variable throughout the experiment. Accordingly, per capita (per cat) means were used for statistical analysis. A combination of analysis of variance (ANOVA) paradigms with repeated measures and / or non-repeated measures was employed. Post-hoc comparisons were Newman–Keuls tests for dependent (repeated measures) or independent (non-repeated measures) measures. Pearson product moment correlations and multiple regression were also used, as detailed in the results section; a -level was set at 0.05.

2.5. Histology Cats were sacrificed with an overdose of sodium pentobarbital (5 cc of 50 mg / ml nembutal) and perfused with 10% formaldehyde in physiological saline to verify stereotaxic placements. Histology was based on 30-mm frozen coronal sections stained with thionine.

3. Results

3.1. Histology Fig. 1 illustrates coronal sections through insertion, infused and / or recorded sites in amygdala (A) and pons where LC cells originate (B). Amygdala and LC placements are reconstructed for perfused (left) side only [9,81]. Illustrative histological sections of bilateral placements are shown on the right. Findings indicate accurate placements. There were no apparent macroanatomical differences between insertion only and perfused versus contralateral

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insertion but non-perfused sites. This is consistent with our preliminary report on six of the eight subjects [77].

3.2. Pre- versus 1 -month post-kindling findings Simple repeated measures analysis of variance statistical evaluation compared pre- versus post-kindling per cat means for each seizure, sleep–waking state and monoaminergic concentration variable in the two 6–8-h recordings obtained before kindling versus the two 8-h records. There were no statistically significant differences (F range51.1–2.3, df51,7, P.0.1) except for seizure parameters. None of the cats showed spontaneous seizure activity in records obtained before or during kindling trials. However, all cats exhibited spontaneous seizure activity recorded 1 month after the interim kindling experiment. When compared to the 6–8-h pre-kindling baseline records and to kindling trials, each cat displayed differing degrees of spontaneous seizure activity in the two recordings obtained 1 month after kindling. Specifically, each cat displayed spontaneous seizure activity which was higher in one post-kindling seizure record and lower in the other. Four of the cats showed higher seizure activity in the first post-kindling record than the second, and four cats showed the opposite patterns. Accordingly, all post-kindling records were evaluated for seizure, sleep–waking state and monoaminergic concentrations based upon each cat’s 1month post-kindling record of high versus low spontaneous seizure activity, regardless of the order in which higher versus lower spontaneous seizure occurred.

3.3. Seizure variables Fig. 2 illustrates onset of the longest electroclinical spontaneous focal (A) and generalized (B) seizure in the cat who showed the longest stimulus-evoked seizures during kindling. Spontaneous EEG seizure discharge patterns as well as the type, timing and duration of EEG and behavioral seizure activity resembles that seen after stimulus-evoked focal versus generalized seizures during kindling. Focal EEG seizure discharge trains originated in kindled (left) amygdala; generalized seizure discharge spread from amygdala to ipsilateral entorhinal cortex and then to ipsilateral LC and / or thalamus (LGN lead) before propagation to contralateral recording sites. Spontaneous behavioral seizure correlates were mild, even in this cat. Behavioral seizure correlates consisted of staring and eye twitching associated with focal amygdala EEG seizure discharge equivalent to stage 1–2 kindled seizures evoked from this stimulation site. Salivation / lip-smacking with head clonus (head nodding) and / or limb clonus was associated with EEG seizure discharge propagation equivalent to stage 3 kindled seizures. Generalized seizure activity was more prevalent in slow-wave sleep (SWS) than drowsy waking, as was the case during the interim

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Fig. 1. Insertion / infusion probe and EEG recording sites in left amygdala and LC in eight cats. Left: Reconstructions of tips based on Snider and Niemer [81] for amygdala versus Berman [9] for LC. Note that P2 in the Berman atlas corresponds to P3 in Snider and Niemer. Right: bilateral coronal sections from one cat. Abbreviations: IC, inferior colliculus; BC, brachium conjunctivum; BP, brachium pontis.

kindling study when all but three seizures were evoked during these two sleep or waking states. A main difference between spontaneous and stimulusevoked seizures was the mean incidence and duration of seizures during 5-min dialysis collection epochs. During kindling, only one focal or generalized seizure was evoked per 5-min dialysis collection epoch; the mean number of behavioral seizure events (range, 1–5) during focal AD plus behavioral seizures (n51.66 0.3) lasted 2.46 2.3 s. Generalized AD plus behavioral seizures (n53.860.4) lasted 6.164.2 s. Table 1 summarizes the 1-month postkindling findings for these and other seizure parameters. The incidence of spontaneous focal and generalized seizures per 5-min collection was higher (all .1 per 5-min epoch), but average seizure duration was usually briefer than during stimulus-evoked trials regardless of increased or decreased spontaneous seizure activity. Simple repeated measures ANOVA comparing indices of

spontaneous behavioral seizure activity in 1-month postkindling records revealed several significant intra-subject differences between records with increased versus decreased seizure activity (F range514–18.2, df. 1,7, P, 0.01). It should be noted that the S.D.s reflect inter-subject variability, which is not reflected in the error term for repeated statistical measures analysis. Rather, the error term reflects intra-subject variability; this difference explains how statistical significance can be achieved even when S.D.s overlap the means. Another difference between 5-min epochs containing stimulus-evoked versus spontaneous seizures recorded 1 month after kindling is that spontaneous seizures were registered in all four sleep–waking states. Fig. 3 illustrates the sleep–waking state distribution of spontaneous seizure activity in the same cat depicted in Fig. 2 during the two 1-month post-kindling recordings (a: decreased seizure activity; b: increased seizure activity). Except for the

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Fig. 2. Spontaneous seizures in the cat with the longest seizures during and after kindling. These were the longest spontaneous seizures recorded in this cat. Onset, EEG seizure discharge generalization pattern, if any, and behavioral seizure correlates are depicted during (A) focal seizures and (B) generalized seizures. Some form of arousal (e.g., eye movements) preceded and followed seizure onset in both cases. The focal seizure occurred during drowsy wakefulness; the generalized seizure began in slow-wave sleep. Abbreviations: L, left; R, right; MC, motor cortex; EOG, electrooculogram; amy, amygdala; ecx, entorhinal cortex; LGN, lateral geniculate nucleus of thalamus; LC, locus ceruleus; EMG, electromyogram. Recording speed510 mm / s. (→) Onset of stage 1 seizure (stare), stage 2 seizure (eye-twitching), stage 3 seizure (lip-smacking / salivation, head clonus and forelimb clonus). (→*) Onset of limb clonus.

difference between recordings in overall quantity of spontaneous seizure activity, the sleep–wake state distribution of focal and generalized seizure discharge was similar during increased versus decreased seizures in postkindling observations. Seizure activity was less prominent

in states characterized by asynchronous cell discharge patterns, notably alert waking and REM sleep, as evidenced by highly localized isolated spikes in amygdala and / or LC, than in states characterized by synchronous neuronal discharges, including drowsy waking and SWS,

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Table 1 Means6S.D.s for the number and duration of focal versus generalized spontaneous seizures 1-month after kindling in eight cats a (A) ↓ Seizure activity

No. of EEG and behavioral seizures / 5-min epoch EEG and behavioral seizure duration (s / seizure) No. of behavioral manifestations (n55) / seizure) Duration of specific behavioral seizure manifestations (s / seizure) Stage 1–2 Stare Eye twitch Stage 3 Salivation / lip-smack Limb clonus Head clonus

(B) ↑ Seizure activity

Focal seizures

Generalized seizures

Focal seizures

Generalized seizures

1.360.7

2.261.2

2.961.3*

3.161.4†

2.460.4

3.060.7

3.660.6*

4.560.9†

1.960.7

2.460.2

2.760.3*

3.060.4†

0.460.2 2.060.5

0.560.2 2.560.3

0.660.2 3.060.4*

0.560.3 3.860.6†

2.160.3 2.160.4 2.060.5

3.960.7† 4.060.9† 3.960.5†

a

Findings are sorted as a function of (↓) (A) or (↑) (B) seizure activity (n51, 8 h record per cat each). Findings are averaged across four sleep–waking states, and number of EEG and behavioral seizures corresponds to number of spike trains in 5-min collection epochs (see Table 2). All other measures of seizure indices are averaged per seizure in each 5-min epoch for each cat. *F5P,0.01, focal seizure parameter significantly higher during (↑) versus (↓) seizure activity; †F5P,0.01, generalized seizure parameter significantly higher during (↑) versus (↓) seizure activity.

when focal and generalized seizure discharge trains were most prevalent. Table 2 summarizes the population data for sleep– waking state parameters and verifies the trends seen in Fig. 3. The cat illustrated in Figs. 2 and 3 had more spontaneous seizure activity in the first recording (B) than the second (B), but, as mentioned above, cats were evenly divided in that four showed more seizure activity in the first than the second recording and four showed the opposite trend. For this reason, means differentiating each cat’s records with increased versus decreased seizure activity were combined regardless of order. Statistical analysis consisted of two-way repeated measures analysis of variance, also known as an A3B3S design in which A5high versus low seizure records (n52 repeated measures), B5sleep or waking states (n54 repeated measures) and S5per capita means (n58 cats) applied to each seizure variable separately. Focal and generalized seizure parameters in the increased seizure activity recording was significantly higher in all states than during the recordings with decreased seizure activity (between seizure conditions: F range5 30.2–40.7, df51,7, P,0.01; between state: F range5 13.5–14.3, df53,21, P,0.01). Nevertheless, focal and generalized seizure activity was higher in drowsy waking and SWS than during alert waking or REM in both recordings. All post hoc tests (dependent or paired comparisons) were significant at the 0.05–0.01 level except for the incidence of focal discharges and duration of spike

trains in alert waking versus REM in the low seizure activity recording.

3.4. Sleep–waking state data Tables 3 and 4 show differences between the two 6–8-h pre-kindling, which were averaged across state (n52 records per cat), and compared to the two, 8-h postkindling sleep–waking state parameters as a function of increased versus decreased seizure discharge (n51 record per cat). Table 3 shows results from records evaluated for states lasting $1 min. Table 4 shows the number of 5-min epochs containing $80% of each state, converted to percentages of total to equate for differences in length of pre-kindling versus 1-month post-kindling records. Statistical analysis consisted of a simple repeated measures ANOVA in which means for each variable in the eight cats were assessed separately as a function of the three records (F range55.0–6.8, df52,14, P5,0.01). Post-hoc comparisons revealed several differences when pre-kindling records were compared to 1-month post-kindling records. The most pronounced differences were between post-kindling records with increased versus decreased seizure activity. The main findings were:

1. (1) Post-kindling records with decreased seizure activity exhibited lower percentages of waking and

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Fig. 3. Illustrative samples of polygraphic recordings during four sleep or waking states obtained during after kindling as a function of (↓) seizure activity (A; left) versus (↑) seizure activity (B; right) in one cat. Each polygraphic sample lasted 30. All recordings shown are from the left (infused) side. Mcx, motor cortex; EOG, electrooculogram; Lamy, left amygdala; LLC, left locus ceruleus; EMG, electromyogram. Recording speed510 mm / s. (→) Focal spikes in left amygdala or LC; (—) spike trains in left amygdala and / or LC.

higher percentages of sleep than during pre-kindling baseline (P,0.05–0.01). Post-kindling reductions in waking percentages were associated with reduced number of epochs (P,0.05). Increased SWS time was associated with rapid onset latency (P,0.001) and fewer epochs (P,0.01) of longer duration (P,0.01), whereas increased REM sleep time was associated with rapid onset latency (P,0.01) and with more frequent epochs (P,0.05) of normal duration. 2. (2) The opposite trends were found in post-kindling records with increased seizure activity. In these records, cats displayed increased waking versus reduced sleep percentages when compared to pre-kindling results and post-kindling records with reduced seizure activity. Waking and SWS percentages were associated

with changes in number and duration of epochs as well as delayed SWS onset latency. Reduced incidence of REM sleep epochs and delayed REM onset accounted for REM sleep suppression (versus pre-kindling, P, 0.05; versus post-kindling records with reduced seizure activity, P,0.01). These findings suggest a cyclic pattern in which decreased post-kindling seizure activity promoted sleep, whereas increased seizure activity delayed sleep onset and fragmented sleep structure. Table 5 and Fig. 4 confirm and extend this observation. Table 5 shows the percentage of spike-wave trains which disrupted ongoing sleep states. Focal and particularly generalized seizure trains more frequently disrupted

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Table 2 Three indices of EEG seizure activity 1-month after kindling a Post-kindling (↓) Seizure activity

(↑) Seizure activity

Before kindling

Post-kindling (↓) Seizure activity

()↑ Seizure activity

Alert waking (W1) Mean % time Mean no. of epochs Mean epoch duration (min)

20.163 12.163 8.065

11.864† 7.062† 8.161

27.163* 18.462* 7.262

Drowsy waking (W2) Mean % time Mean no. of epochs Mean epoch duration (min)

30.964 14.066 10.665

25.265† 10.163† 11.964

44.962* 24.162* 8.963

5.161.3*† 3.361.4*† 3.561.4*†

SWS Mean % time Mean no. of epochs Mean epoch duration (min)

33.165 20.163 7.966

43.565* 15.262† 13.762*

19.962† 25.363* 3.861†

5.961.2*† 3.861.2*† 4.161.3*†

REM Mean % time Mean no. of epochs Mean epoch duration (min)

15.963 11.561 6.164

19.564* 14.762* 6.463

8.162† 6.060.5† 6.562

SWS Onset latency (min)

25.167

10.068†

13.069†

REM Onset latency (min)

63.9615

30.269†

93.5612*

Alert waking ( W1) Amygdala Mean no. of spikes Mean no. of spike trains Mean train duration (s)

1.460.4 1.260.2 1.560.7

3.161.3* 2.161.2* 3.861.5*

Amygdala and locus ceruleus Mean no. of spikes Mean no. of spike trains Mean train duration (s)

1.560.3 1.360.2 1.760.4

3.061.2* 2.261.1* 3.961.2*

Drowsy waking ( W2) Amygdala Mean no. of spikes Mean no. of spike trains Mean train duration (s)

2.560.2† 2.360.2† 2.860.3†

Amygdala and locus ceruleus Mean no. of spikes Mean no. of spike trains Mean train duration (s)

3.160.3† 2.660.1† 2.860.2†

SWS Amygdala Mean no. of spikes Mean no. of spike trains Mean train duration (s)

3.960.4† 3.660.2† 3.860.2†

Table 3 Sleep–waking state parameters (means6S.D.s in eight cats) in 6–8-h records before and in 8-h records 1 month after kindling as a function of (↑) or (↓) spontaneous seizure activity a

6.861.3*† 5.661.1*† 4.561.1*†

a

Amygdala and locus ceruleus Mean no. of spikes Mean no. of spike trains Mean train duration (s)

4.360.4† 4.760.3† 3.960.2†

8.061.5*† 5.961.7*† 8.262.3*†

REM Amygdala Mean no. of spikes Mean no. of spike trains Mean train duration (s)

1.560.2 0.560.1†† 1.660.1

1.960.3*†† 0.960.2*†† 2.760.2* Ii

Amygdala and locus ceruleus Mean no. of spikes Mean no. of spike trains Mean train duration (s)

1.260.1†† 0.160.2†† 1.260.1††

1.960.2*†† 0.260.2*†† 1.960.1*††

a

Means6S.D.s for eight cats are shown as a function of: (a) (↓) or (↑) spontaneous seizure activity, including isolated spikes per mm (,70 ms, $100 mV with $1-s interspike intervals), number of spike trains per 5-min epoch ($3 spikes / s) and duration of spike trains (s); (b) recording site (amygdala versus amygdala and locus ceruleus); and (c) onset during sleep or waking state. Note: spike trains correspond to EEG and behavioral seizure correlates in amygdala alone (focal) and both amygdala and locus ceruleus (generalized). *P,0.05 higher than (↓) seizure activity condition; †P,0.05 higher than alert waking and REM; ††P,0.05 lower than alert waking.

SWS by leading to awakening in recordings with increased seizure activity than in recordings with decreased seizure activity. This finding was statistically significant, using simple repeated ANOVAs (F range59.0–11.7, df51,7, P,0.001). Spikes or spike trains rarely disrupted REM in records containing either high or low seizure activity, and

Scoring is based on states lasting $1 mm (see Table 4 for corresponding 5-min epochs). *P,0.05–0.01 higher than pre-kindling baseline; †P,0.05–0.01 lower than pre-kindling baseline.

there were no statistically significant differences between post-kindling records in the percentage of disruptive focal or propagated seizure trains during REM (F5P.0.1). Fig. Table 4 Means6S.D.s (n58 cats), converted to percent of 100, for number of 5-min epochs containing $80% of sleep or waking states corresponding to dialysate samples obtained during 6–8-h records before kindling and 8-h records 1 month after kindling a Before kindling

Post-kindling (↓) Seizure activity

(↑) Seizure activity

Alert waking (W1) Mean no. of epochs

3462

2863†

4464*

Drowsy waking (W2) Mean no. of epochs

2862

2262†

3263*

SWS Mean no. of epochs

2563

3162*

1863†

REM Mean no. of epochs

1362

1961*

661†

a

Post-kindling records are sorted as a function of (↑) or (↓) spontaneous seizure activity (n51 record each per cat). *P,0.05–0.01 higher than pre-kindling baseline; †P,0.05–0.01 lower than pre-kindling baseline.

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Table 5 Percentage of spike-wave trains discharge disrupting sleep state in eight cats (mean6S.D.) as a function of 5-min epochs during 8-h records (n51 each) containing (↓) or (↑) seizure activity Post-kindling (↓) Seizure activity

(↑) Seizure activity

SWS Amygdala Amygdala and locus ceruleus

11.264 20.965

23.965‡ 82.364‡

REM Amygdala Amygdala and locus ceruleus

3.162 8.563

5.362 7.864

‡F5P,0.001 from (↓) percentage of seizure trains disrupting sleep.

4 illustrates these points in the form of hypnograms constructed for the cat portrayed in Figs. 2 and 3.

3.5. Monoamine concentrations Fig. 5 shows the population data for amygdala (A) and LC (B). Means6S.D.s of concentrations of each monoamine per cat per state were combined during pre-kindling baseline records (top) for comparison with means6S.D.s obtained in post-kindling records containing increased (middle) versus decreased higher (bottom) seizure activity. Findings for each monoamine are expressed in fmoles / 10ml sample. Results were adjusted for in vitro probe recovery which was about 13% for each monoamine at both sites throughout the experiment, as quantified in the data analysis section (see Section 2). Statistical analysis consisted of two-way analyses of variance (ANOVAs) with repeated measures on one factor, also known as an A(B3S) design. In ANOVA 1, concentrations of each monoamine were examined separately in which A5the non-repeated measure (n52 infusion sites), B5repeated measures obtained during pre-kindling baseline, post-kindling records with increased seizure activity versus post-kindling records with decreased seizure activity (n53), and S5mean concentration per cat (n58). In ANOVA 2, the three monoamines were examined in each experimental condition separately as a function of collection site and sleep or waking state. In this analysis, A5the non-repeated measure (n52 infusion sites), B5sleep or waking state (n54) and S5mean concentration per cat (n58). In ANOVA 3, the three monoamines were compared to each other as a function of experimental conditions at each site separately. In this analysis, A5the non-repeated measure (n53 transmitters), B5the repeated measure (n53 experimental conditions) and S5mean concentration per cat (n58). Data in Fig. 5 generated three main findings:

Fig. 4. Hypnograms illustrating sleep patterns in an 8-h record obtained before kindling (A), during the post-kindling record with (↓) seizure activity (B) and during the post-kindling record with (↑) seizure activity (C) in the cat portrayed in Figs 2 and 3. Seizure discharge occurred only in post-kindling records. GSTs, generalized seizure trains, which did not interrupt on-going sleep (empty circle) or awakened the cat from sleep (filled circle). A higher percentage of GSTs disrupted on-going sleep in the post-kindling record with (↑) seizure activity (bottom) than in the post-kindling record with (↓) seizure activity (middle). Sleep–wake state abbreviations as in Fig. 3.

1. (1) Concentrations of each monoamine significantly differed between both 1-month post-kindling seizure conditions when compared to pre-kindling baseline (ANOVA 1, repeated measures factor, df52,28, NE: F521.8, P,0.001; DA: F532.2, P,0. 001; and 5HT: F524.3, P,0.001) and at both sites (ANOVA 1, non-repeated measure, df51,14, F range511.7–12.2, P,0.01). Post-hoc paired multiple comparisons revealed significantly higher concentrations of each monoamine during records with increased seizure activity versus lower concentrations during records with decreased seizure activity. When compared to pre-kindling baseline:

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Fig. 5. Mean6S.D. concentrations (fmol / 10 ml sample) of norepinephrine (NE), serotonin (5-HT) and dopamine (DA) in amygdala (A; left) and pons (B; right) in eight cats. Results are shown as a function of four sleep or waking states before kindling (top), in post-kindling records with (↓) seizure activity (middle) and post-kindling records with (↑) seizure activity (bottom). W-1, alert waking; W-2, drowsy waking; SWS, slow-wave-sleep; REM, rapid-eye-movement sleep.

M.N. Shouse et al. / Brain Research 892 (2001) 147 – 165

(a) NE concentrations in amygdala declined by 43.3% during decreased seizure activity (P,0.01) and elevated by 51.6% during increased seizure activity (P,0.01). LC concentrations declined by 35.3% during decreased seizure activity (P,0.01) and increased by 19% during increased seizure activity (P,0.05). (b) DA concentrations in amygdala declined by 52.4% during decreased seizure activity (P,0.01) and increased by 92.8% during increased seizure activity (P,0.01). LC concentrations reduced by 40% during decreased seizure activity (P,0.01) but increased by 115% during increased seizure activity (P,0.01). (c) 5-HT concentrations in amygdala declined by 45.5% during decreased seizure activity (P,0.01) and increased by 45.4% during increased seizure activity (P,0.01). LC concentrations reduced by 36.2% during decreased seizure activity (P,0.01) and increased by 41.9% during increased seizure activity (P,0.01). 2. (2) Comparisons between sleep–waking states (ANOVA 2, repeated measure, df53,42) showed that concentrations of NE (F514.6, P,0.01) and 5-HT (F57.1, P,0.01) declined from waking to SWS, including transitions into REM, and reached a minimum during stable REM sleep, whereas DA concentrations did not change across the sleep–wake cycle (F51.1, P.0.1). State-dependent patterns for NE and 5-HT as well absence of n state-dependent DA changes were detected before and 1 month after kindling at both infusion sites (ANOVA 2, non-repeated measure, df51,14, F range511.7–12.2, P, 0.01). State-dependent findings were the same for each transmitter at each site and in each experimental condition Pre-kindling versus 1 month post-kindling means therefore were collapsed for each cat in each state to generate the following percentages. When compared to alert waking: (a) NE concentrations in amygdala declined by 19.2% in drowsiness, 42.7% in SWS and 72.1% during REM. Pontine NE concentrations declined by 26.2% in drowsiness, 39.8% in SWS and 72% during REM (P,0.05–0.01 in all comparisons). (b) 5-HT concentrations in amygdala declined by 20.2% in drowsiness, 49.6% in SWS and 76.4% during REM. Pontine 5-HT concentrations declined by 18.7% in drowsiness, 39.9% in SWS and 67.7% during REM (P,0.05–0.01 in all comparisons). (c) DA concentrations never changed by more than 1% at either site and in any state (P.0.1 in all comparisons). 3. (3) Comparisons between transmitters revealed higher concentrations of NE and 5-HT than DA at both sites (ANOVA 3, non-repeated measures factor, df52,14, amygdala, F513.5; pons, F518.9, P,0.01) and in all

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three experimental conditions (repeated measures factor, df52,42, amygdala, F515.1; pons, F519.2, P, 0.01). Post-hoc comparisons verified the non-repeated measures main effect in all three experimental conditions (P,0.05–0.01) and the repeated measures main effect in all states (P,0.05–0.01) except REM in the amygdala. Higher concentrations of NE and 5-HT were detected in LC versus amygdala, whereas DA concentrations were higher in amygdala than in LC regardless of the three experimental conditions (P,0.01).

3.6. Interaction between seizure, sleep and monoamine concentrations Fig. 6 contrasts sleep–waking state time with changes in monoaminergic concentrations in 1-month post-kindling records with increased versus decreased seizure activity. Shown are percent changes from pre-kindling baseline in these variables after kindling (top5amygdala, bottom5 LC). Findings are also separated according to the four cats with reduced seizure discharge in the first versus second recording (left) and the four cats showing the opposite order (right). As described earlier, differences were statistically significant from baseline, indicating increased monoamines and reduced sleep time in records with increased seizure activity, whereas reduced monoamines were associated with increased sleep in records with decreased seizure activity. There were no significant differences as a function of order of recording (P.0.1). Pearson product moment correlations verified that records with the highest degree of seizure activity were associated with the highest concentrations of monoamines and lowest sleep time, whereas records with the least spontaneous seizure activity contained lowest monoaminergic concentrations as well as highest sleep percentages, regardless of whether the first or second recording contained more seizure activity (r range50.7–0.78, df56, P,0.05). Further, concentrations of monoamine correlated both individually (r range50.73–0.75, df56, P,0.05) and collectively (multiple r range50.88–0.92) with various seizure variables, whereas only concentrations of NE (r5 0.8, P,0.05) and 5-HT (r50.79, P,0.05) correlated with total sleep time (multiple r50.86). Thus, regardless of the type of statistical analysis, we found that (1) increases in all three monoamines predicted increased seizure activity, whereas (2) only increased NE and 5-HT predicted increased sleep disruption.

4. Discussion Results confirm and extend previous work about monoaminergic regulation of EEG and behavioral arousal events

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M.N. Shouse et al. / Brain Research 892 (2001) 147 – 165

Fig. 6. Per capita means6S.D. percent change from pre-kindling baseline (0) in eight cats during two, 8-h post-kindling recordings obtained on consecutive days (first recording, filled bars; second recording, unfilled bars). Four cats exhibited (↓) seizure activity in the first recording followed by (↑) seizure activity in the second recording (left), and four showed (↑) seizure activity in the first recording followed by (↓) seizure activity in the second recording (right). Findings for NE, 5-HT and DA are shown for amygdala (top) and pons (bottom). Percent change in TST (total sleep time) is also shown to emphasize the fact that increased monoaminergic concentrations accompanied increased seizure activity and reduced sleep time, whereas reduced monoaminergic concentrations accompanied reduced seizure activity and increased sleep time regardless of the order of recordings.

as they relate to sleep–waking state patterns, epilepsy and their interaction. Specifically, (1) intra-subject comparisons revealed higher concentrations of NE and 5-HT in pons than amygdala, whereas DA concentrations are higher in amygdala than LC both before and after kindling; (2) inter-site comparisons showed that NE and 5-HT were higher in amygdala in all states except REM, whereas NE and 5-HT levels in LC were higher than DA in all sleep–waking states both before and after kindling; (3) only NE and 5-HT declined from waking to sleep at both collection sites, whereas DA levels did not change as a function of sleep–waking state at either site, again before and after kindling; (4) post-kindling monoaminergic and sleep–waking state parameters differed from pre-kindling values only when post-kindling records were differentiated

based on severity of seizure disorder. Specifically, each kindled subject displayed a cyclic pattern in which increased spontaneous seizure activity was associated with increased extracellular concentrations of monoamines and sleep disruption, whereas decreased spontaneous seizure activity was associated with reduced monoaminergic concentrations and reduced sleep disruption when compared to pre-kindling baseline. Technical considerations complicate interpretation (e.g., Refs. [8,80,85,86]). For example, we cannot exclude differences in probe recovery rate that may have occurred during sample collection. However, relative in vitro recovery percentages were similar prior to insertion and at the conclusion of studies at both collection sites, and in vivo monoaminergic findings were stable over the collec-

M.N. Shouse et al. / Brain Research 892 (2001) 147 – 165

tion period relative to standards. We cannot eliminate the role of differences in probe placement in spite of similarities in probe tip location at both sites based upon available histology. We did not have an unkindled control group with which to compare monoamines and sleep– wake state parameters over a comparable time frame. Studies assessing transmitter concentrations over a 7-day post-insertion period found linear reductions over time [65]. Our study involved five probe insertions and collections, but insertions and dialysate collections were spread out over time [85], and results revealed no linear reduction in monoamine concentrations before, during or after kindling. Repetitive insertions and sampling may have contributed to the suppression of monoamines seen 1 month post-kindling during reduced spontaneous seizure activity, but findings were more closely associated with degree of post-kindling seizure activity and sleep-related changes than with the chronology of recordings and dialysate collection sequence. Technical limitations not withstanding, the observed differences could also have a valid anatomical and physiological basis. Higher concentrations of NE in pons versus amygdala may reflect somatodendritic extrusion, which is well-established by in vitro studies of monoamines, including NEcontaining cells which predominate in the LC (e.g., Refs. [16,21,24,56,80]). Higher concentrations of 5-HT in the vicinity of LC versus amygdala could reflect the presence of some 5-HT cell bodies in LC (e.g., Ref. [80]) and / or higher density projections from the dorsal raphe to adjacent regions in the pontine tegmentum and related axonal transmitter release (e.g., Ref. [35]). The presence of lower DA concentrations in LC versus amygdala is consistent with sparser pontine innervation from midbrain (e.g., Ref. [35]). DA and 5-HT concentrations were also lower than NE in amygdala, potentially because we selected a target site at the juncture between the basolateral and central nuclei where NE terminals are thought to predominate [46]. Findings confirm and extend other reports on monoaminergic mechanisms of EEG and behavioral arousal during the sleep–wake cycle [35,37,41,47,48,57,61– 63,68,79], including those employing microdialysis [41,57,61–63,67,87]. EEG desynchronization as well as loss of antigravity muscle tone during REM are thought to result from interactions between localized brain stem cholinergic, glutaminergic and GABAergic neurons, in part as they affect NE and 5-HT containing neurons (e.g., Refs. [37,79]). Cholinergic and GABAergic cells seem to actively suppress discharge rates and chemical release of adjacent NE and 5-HT containing cells during the seizureresistant REM sleep state, characterized by intense EEG activation and profound lower motor neuron inhibition (e.g., Refs. [25,41,57]). Suppression of 5-HT discharge is specifically implicated in the disinhibition of pontine cholinergic cells generating phasic arousal events, particularly PGO spikes (e.g., Refs. [34,41]), which have been

161

linked to increased TLE seizure activity in SWS and its transition into REM [75,76]. At other times, Ach, NE and 5-HT containing cells act in concert. Collective increases in electrochemical activity appear to promote tonic behavioral and EEG arousal during the seizure-resistant alert waking state [76] and, conversely, reduced electrochemical activity contributes to lower tonic EEG and behavioral activity during seizure-prone drowsy waking and SWS states [25,35–37,57,68,82,87]. While NE and 5-HT concentrations declined progressively from alert waking to drowsy waking and SWS and reached a nadir in REM, DA concentrations did not change across the sleep–wake cycle. This may account for state-related trends towards overlapping monoamine concentrations, which occurred at both collection sites but was complete only during REM sleep and only in the amygdala. We attribute closer overlap in amygdala than LC to higher relative concentrations of DA in amygdala, which may be due to increased innervation of amygdala than LC, as previously posited [34,45,54]. The basis for negligible changes in cell discharge rates or chemical release of DA in the sleep–wake cycle is unclear. DA regulation is better established for motor than EEG activation since chronic DA depletion is associated with persistent akinesia and / or dyskinesia while EEG changes are transient (e.g., Ref. [35]). Some findings suggest that DA electrochemical activity patterns may be too brief and / or too sustained (e.g., Ref. [35]) to permit correlation with sleep–waking states. Our findings are compatible with the thesis that DA modulation of EEG and motor activity is independent of spontaneous sleep or waking states but not necessarily of seizure activity. Increased seizure activity was accompanied by increased monoaminergic release, consistent with sustained epileptic neuronal discharges with clinically evident behavioral seizure activity [5,17,19,20,27,38,40,42,43,71,84]. Conversely, reduced seizure activity was accompanied by reduced monoaminergic release, consistent with reduced epileptic neuronal discharge and behavioral seizure correlates. Seizure-related change in monoamines occurred in both amygdala and LC even when focal seizure discharge was confined to amygdala. This result is unlike our interim kindling study in which stimulus-evoked seizures were studied over a much more limited time frame but is consistent with previous reports of increased cell discharge in LC after amygdala kindling [33] and elevated forebrain NE levels at microdialysis sites distal to hippocampal kindling [54] in the absence of seizure discharge. Given these findings and the extra-focal changes attributed to the kindling process (e.g., Refs. [14,49]), it may not be surprising that seizure-related changes in monoamines also affected other indices of EEG and behavioral arousal. Increased monoaminergic activity was associated with sleep disruption, while reduced monoaminergic concentrations accompanied improved sleep. Even so, seizures were still more likely to occur in drowsiness and SWS than

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alert waking and stable REM [76], and only NE and 5-HT were correlated with state-specific seizure results. The post-kindling sleep changes seen here differ from previous findings [12,26,28,70], including ours [73]. Many procedural variables have been identified as contributory to the presence, absence and recovery of post-kindling sleep deficits (for a review, see Ref. [73]). The most salient factors here seem to be severity of seizure disorder with respect to fluctuation in post-kindling sleep deficits over time. While subjects in this study were kindled only to stage 3, all exhibited repetitive spontaneous seizures throughout the two 1-month post-kindling recordings. Combining post-kindling data for each cat revealed no differences from pre-kindling baseline. After separating post-kindling records on the basis of severity of seizures, we detected extreme fluctuation in which higher density of seizure activity, particularly generalized seizure discharge, was associated with delayed sleep onset and disrupted ongoing sleep, especially SWS. The opposite pattern was seen in the same animals exhibiting reduced seizure activity either the day before or the day after increased seizure activity. These results are reminiscent of findings by Calvo and Mas [12], who showed that cats kindled as adults displayed significant day-to-day post-ictal variation in REM onset latencies. Periods of post-ictal REM delay alternated with rapid REM onset, and when post-kindling findings were averaged over consecutive days, showed no overall differences from pre-kindling baseline. Similarly, we previously reported that cats kindled as kittens who showed the highest post-kindling REM sleep percentages also showed the least stable, chronic post-kindling REM sleep patterns, including periodic bouts of REM sleep from waking on days of rebound sleep [73,75]. We did not see REM sleep onset from waking in this particular experimental series, but we did detect reduced SWS and REM onset latencies, which can result from extreme sleep deprivation [18,19]. Thus, post-kindling sleep insomnia could be masked by cyclic patterns of sleep loss and sleep rebound. The observed post-kindling changes in monoamines may also shed light on the many conflicting reports detailing short- and long-term changes in monoamines as a factor in kindled epileptogenesis. Some have reported chronic postictal reductions in catecholamines and / or 5-HT (e.g., Refs. [20,22,70]), some have reported no sustained post-kindling change [14,17,49], and some have reported post-kindling increases [6,17,77,78]. Various systemic and focal manipulations used to suppress monoamines, such as 6-OHDA depletion [2,15,50,52], lesions [64], and antagonists [60,74] increase subsequent seizure susceptibility, whereas procedures intended to increase monoaminergic release, such as transplants [4–6,10], agonists and / or uptake inhibitors [7,43,60,84,88] reduce seizure susceptibility. However, effects tend to be more pronounced on kindled epileptogenesis than on the established focus (e.g., Ref. [49]). This, combined with the controversy about long-

term depletion, led some to conclude that monoamines serve a transient role in chronicity of kindled seizure susceptibility and that some other, secondary change — e.g., changes in GABA, acetylcholine, adrenoceptor binding characteristics, etc, rather than transmitter concentrations per se — underlies the permanent increase in seizure susceptibility following kindling in adults and to a greater extent in the young (e.g., Refs. [14,23,32,42,49]). Our post-kindling monoaminergic results — like the sleep deficits — are complicated by the fact that all subjects exhibited spontaneous seizures in varying degrees during post-kindling recordings. The increase in monoaminergic concentrations associated with increased seizure discharge is consistent with microdialysis findings showing increased release of monoamines during seizures in multiple epilepsy models and at diverse collection sites [41,42,88]; in contrast, a suppression of monoamines is associated with reduced seizure activity, at least prior to seizure induction. Our findings complement these by suggesting that excess transmitter release during brief but repetitive periods of seizure discharge may lead to depletion of monoamines. This depletion could create a necessity for restoration of intracellular manufacturing and storage which — in the absence of synaptically driven release — enables return to a more epileptogenic state. In conclusion, because all the animals were epileptic at the time of post-kindling follow-up, our findings do not directly address the controversial issue of whether monoamines contribute to chronic post-kindling seizure or sleep disorders. However, the cyclic alternation of variables does pertain to the concept of ‘rebound’ and may shed light on some apparent discrepancies in the literature. Specifically, post-kindling records with increased seizure activity was affiliated with temporary increases in monoaminergic release, accompanied by EEG and behavioral arousal; these alternated with transient periods of reduced monoamine concentrations and sleep recovery, which could in turn promote seizures and abnormal arousal events in a self-perpetuating fashion.

Acknowledgements Supported by the Department of Veterans Affairs. We thank Drs. K. Tachiki, Paul Dittes, Y. Wada, M. Bier and T. Kodama as well as Mr. J. Langer for technical assistance and the staff of the Sepulveda VA Animal Research Facility for diligent animal care.

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