Optical imaging reveals reduced seizure spread and propagation velocities in aged rat brain in vitro

Neurobiology of Aging 24 (2003) 345–353

Optical imaging reveals reduced seizure spread and propagation velocities in aged rat brain in vitro M. Holtkamp a,∗ , K. Buchheim a , H. Siegmund b , H. Meierkord a b

a Neurologische Klinik, Universitätsklinikum Charité, Schumannstr. 20/21, 10117 Berlin, Germany Johannes-Müller-Institut für Physiologie, Universitätsklinikum Charité, Humboldt-Universität Berlin, Berlin, Germany

Received 14 March 2002; received in revised form 3 June 2002; accepted 21 June 2002

Abstract Old age is the most common time for patients to develop epileptic seizures, and due to their frequent unusual clinical presentation the diagnosis of epilepsy is often delayed in the elderly. It is as yet unknown if pronounced alterations in the plastic properties of aging nervous tissue contribute to these phenomena. We employed a non-lesional in vitro epilepsy model to study seizure susceptibility, spread pattern, and propagation velocities in combined hippocampal-entorhinal cortex slices of aged rats and controls using electrophysiological methods and imaging of intrinsic optical signals. In aged animals we saw a less extensive spread of seizure-like events into areas adjacent to the region of onset of activity and a decreased spread velocity in various anatomical regions. In addition, both the activity-dependent shrinkage of the extracellular space (ECS)-volume and the extracellular K+ concentration were significantly reduced compared to controls. The results of this study are consistent with the clinical observation that epileptic seizures in the elderly have a reduced tendency to spread. In addition, our data suggest that in the absence of structural lesions seizure susceptibility in the aging brain is not increased. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Aging; Epilepsy; Low-Mg2+ -model; Seizure-spread; Propagation velocity; Extracellular space-volume; Extracellular K+ concentration; Intrinsic optical signal

1. Introduction Epilepsy is a disease with common onset in the extremes of life. In senescence the incidence of epileptic seizures and epilepsy is highest, even exceeding that seen in childhood [64]. There is evidence that in infants and young children the increased risk of epilepsy—at least in part—is due to decreased inhibitory and increased excitatory mechanisms seen in discrete periods of brain development [18,22,30,61,70]. In contrast, the mechanisms involved in seizure susceptibility and expression in the elderly are largely unclear. There is little doubt that the high incidence of cerebral structural lesions (i.e. vascular, infectious, neoplastic, neurodegenerative, traumatic), metabolic disturbances and the potentially pro-convulsive co-medication play important roles [13,23]. It is also well known that there are pronounced alterations in the plastic properties of the aging brain in the absence of structural lesions [49,56,63]. However, the effects that such changes in brain plasticity may have on seizure suscepti∗ Corresponding author. Tel.: +49-30-450-560102; fax: +49-30-450-560932. E-mail address: [email protected] (M. Holtkamp).

bility, regions of onset, spread patterns and seizure spread velocities are poorly understood. Experimental studies have revealed controversial results concerning seizure susceptibility and aging. Various in vivo studies suggest both, an increased [12,26,32,34] and a decreased [17,33,52] susceptibility with aging. Also, several in vitro studies have been carried out evaluating neuronal excitability of old tissue in stimulation experiments. Again, increases [5,38] and decreases [14,53,55] of neuronal excitability with aging have been reported. Therefore, the current experiments were carried out in an attempt to clarify such contradictory results and to better understand how age-related differences in susceptibility to seizure activity affect the spatiotemporal pattern of discharge onset, seizure spread and velocities. In view of a number of unusual clinical presentations of seizures in the elderly [19] we hypothesized that the spatiotemporal behavior of epileptiform discharges in aged tissue may help to explain such unusual features. To this end we used the combined hippocampal-entorhinal cortex slice preparation in vitro for electrophysiological investigations and imaging of intrinsic optical signals (IOS). The latter approach makes use of the activity-dependent changes in the optical tissue

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properties that are intrinsic to the tissue itself [20,27]. We have previously demonstrated in brain slices from adult and juvenile rats that intrinsic optical imaging is an excellent tool to map spatiotemporal patterns of seizure propagation [10,46,70]. The low-Mg2+ -model represents an acute experimental model which is frequently used in epilepsy research [68]. The discharges in this model mainly result from unblocking of the N-methyl-d-aspartate (NMDA) receptor [48]. Omitting Mg2+ from the artificial cerebrospinal fluid (ACSF) produces short recurrent discharges in the hippocampus, prolonged seizure-like events and late recurrent discharges in the entorhinal cortex and neighboring structures [10,48,68]. The seizure-like events are characterized extracellularly by long lasting negative field potential shifts superimposed by fast action potentials that match the initial tonic-like and then clonic-like electrographic activity [3]. Using IOSs we have analyzed the onset, spread pattern and velocity of the seizure-like events. In addition, we have tried to gain some insight into the mechanisms associated

with spread patterns in aged tissue by analyzing parameters that are well known to be involved in other age groups: activity-induced extracellular space (ECS)-volume changes and alterations in extracellular K+ concentrations [15].

2. Methods 2.1. Combined hippocampal-entorhinal cortex slice preparation The experiments were performed on combined hippocampal-entorhinal cortex slices that were prepared as previously described [10,70]. A schematic drawing of such a brain slice is shown in Fig. 1A. Male Wistar rats aged 12–14 weeks, in the following, termed adult rats or controls, and rats aged 24–26 months, in the following, termed old rats, were decapitated under deep ether anesthesia. The brains were rapidly removed and washed with cold ACSF.

Fig. 1. (A) Schematic drawing of the combined entorhinal cortex-hippocampus slice preparation used in the current experiments. The relevant anatomical structures, which are present in both age groups, are outlined. TE: temporal cortex; PC: perirhinal cortex; lEC/mEC: lateral/medial entorhinal cortex; Sb: subiculum; CA1/3: cornu ammonis area 1/3; DG: dentate gyrus. (B) Electrographic and optical features during a seizure-like event of a control animal. Time courses of the extracellular field potential (f.p.), extracellular potassium concentration [K+ ]0 and change in light transmittance (T/T) as measured in the lEC. (C) Evolution in time and space of the optical signal. In the original video image a bipolar stimulation electrode is placed in the stratum radiatum of CA3 (Schaffer collateral) and a recording electrode can be identified in the stratum pyramidale of CA1. Prior to omission of Mg2+ , the viability of the slice is evaluated by evoking field EPSPs in the CA1 (for details see Section 2). Another recording electrode is placed in the lEC. During illumination from below, myelin-rich regions such as the alveus appear dark, whereas regions in which neurons are densely packed such as CA1 and three appear bright. A series of eight pseudocolored subtraction images (for detail see Section 2) shows the evolution of the optical features associated with the same seizure-like event that is analyzed in (B). While there is only optical noise in the first pseudocolored image (5 s prior to seizure onset), initial optical changes are seen in the lateral entorhinal cortex in the next image (0 s, onset). There is a bidirectional spread towards the temporal cortex (which is reached after 9 s) and the subiculum 5 s after the onset. At the electrographic offset of the seizure-like event in the lEC maximal changes in light transmittance of 1.1% are found here and in the adjacent PC. The decline of the optical signal change takes approximately 28 s after electrographic offset of the seizure-like event before baseline is reached again.

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Brain slices (400 ␮m) containing the temporal cortex, the perirhinal cortex, the lateral and medial entorhinal cortex, the subiculum, the dentate gyrus, and the ventral hippocampus were cut in nearly horizontal plane using a Vibratome (752 M Vibroslice; Campden Instruments Ltd., Loughborough, UK). Slices were stored at 35 ◦ C in ACSF which contained 124.0 NaCl, 3.0 KCl, 2.0 MgSO4 , 2.0 CaCl2 , 1.25 NaH2 PO4 , 26.0 NaHCO3 , and 10.0 glucose in mM (pH 7.4), oxygenated with 95% O2 , and 5% CO2 under interface conditions. Slices were individually transferred to an interface-recording chamber, placed on a transparent membrane (0.4 ␮m Millicell culture plate inserts; Millipore, Bredford, MA, USA), illuminated from below, and continuously perfused (1.5–2 ml/min) with prewarmed (35 ◦ C) and carbonated ACSF. Warmed, humified 95% O2 , and 5% CO2 gas mixture was directed over the surface of the slices. Epileptiform activity was induced by omitting Mg2+ from the ACSF.

ionophore I cocktail A 60031 Fluka; tetraethylammonium– potassium ion exchanger cocktail 477317 Corning. One electrode was placed in layer II or III of the lateral entorhinal cortex, and the other initially in the stratum pyramidale of the hippocampus (cornu ammonis, area 1: CA1). Bipolar stimulating electrodes were placed in the stratum radiatum and the extracellular field response was recorded from the stratum pyramidale of CA1. Only those slices were accepted for further recording in which the population spike recorded in the stratum pyramidale of CA1 revealed amplitudes larger than 2 mV. After test stimuli had proved the viability of the slices, this recording electrode was transferred from area CA1 to the medial entorhinal cortex. In the current study only spontaneously occurring seizure-like events induced by Mg2+ -free ACSF lasting more than 10 s were analyzed. Data were recorded on chart recorder and on computer hard disc. For acquisition and analysis of the electrophysiological data a special software (pClamp 6.0.3; Axon Instruments, Foster City, CA, USA) was used.

2.2. Ion-sensitive microelectrodes

2.3. Optical imaging

The recordings were started after more than 1 h of equilibration of the slices in normal ACSF. Two ion-selective microelectrodes were used to record extracellular field potentials and to determine changes in extracellular potassium concentrations, in some of the experiments one of the electrodes was used to measure the activity-induced changes in extracellular tetraethylammonium chloride (TEA+ ) concentrations. Changes in TEA+ concentrations reflect changes of the ECS-volume since TEA+ added to the ACSF in concentrations of 2 mM is largely restricted to the extracellular compartment [51], although uptake by glial cells does take place to a steady-state intracellular level [4]. To translate the recorded potential values (mV) of potassium and TEA+ in concentration values (mM) we used a modified Nernst equation:

The slices were transilluminated from below using unfiltered light from a halogen lamp supplied by a stabilized voltage-regulated power supply (KL 1500; Schott, Wiesbaden, Germany). Homogeneous illumination was achieved by a curved glass fiber (Ø 8 mm) lead directly underneath the slice below the transparent floor of the interface chamber. The slices were viewed from above with an upright binocular microscope (MS 5; Leica, Bensheim, Germany) through a 2.5× objective. Video images recorded by a CCD camera (8 bit; Sanyo, Osaka, Japan) were obtained with a monocular phototube (Leica). During the experiments the images were stored on videotape using a S-VHS video-recorder (AG-6720A; Panasonic, Osaka, Japan) which allowed further off-line analyzes. Some experiments were analyzed on-line using the algorithm described below. The complete video signals were converted at a 12.5 MHz ratio into 480 lines by 640 pixel per line employing a frame-grabber board (DT2855; Data Translation, Marlboro, MA, USA) and in-house software. The 8-bit information of each converted pixel represents 1 of 256 gray levels. The first image used for the analysis was captured 5–10 s before the electrical onset of the seizure-like event and the last image at least 45 s after the end of the discharges. The aim was to examine changes of the intrinsic optical signal; therefore, the first image in a series served as control which was subtracted from each of the following images. These subtracted images disclosed areas in the slice where light transmittance changed over time. Pseudocolored images were calculated by subtracting the control image from subsequently acquired images and assigning a color lookup table to the relative pixel values. The series of subtraction images typically contained 150 images with an interimage interval of 350 ms. To graph data from specific slice areas, free-hand regions of interest (ROIs) were placed over

log[Ion]1 =

EM + log[Ion]0 , (s × v)

where EM indicates the recorded potential, s denotes the electrode slope obtained calibration, v is the valence of the specific ion, [Ion]0 denotes the ion concentration at rest, and [Ion]1 is the ion concentration during activity. Relative changes in ECS-volume were calculated using the formula suggested by Dietzel et al. [15]:
 [TEA+ ]0 Shrinkage of ECS (%) = 1 − × 100, [TEA+ ]1 where [TEA+ ]0 indicates the TEA+ signal in mM before activity and [TEA+ ]1 indicates the maximal TEA+ signal in mM during activity. Ion-selective micro-electrodes were prepared and calibrated using the method described by Lux and Neher [41]. The following resins were used: potassium–potassium

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3.1. Influence of TEA+ on electrophysiological and optical data

subiculum, medial and lateral entorhinal cortex, perirhinal cortex, and temporal cortex. The average digital intensities were sampled, stored at ASCII files, and graphed using Excel 5.0 (Microsoft). The figures and plots illustrate relative differences in light transmittance (T). The magnitude of the intensity difference (T) was expressed as a percentage of the digital intensity of the control image in a series. Maximum changes in light intensity (T/T) typically ranged from 0.5 to 2.7% during epileptiform activity, whereas noise usually was below 0.05%. Thus, the signal-to-noise ratio ranged from 10 to 54. We have previously shown the synchronous electrographic onset and the light transmittance changes (for details and methods see [10,70]). To analyze propagation of a single seizure-like event in time and space subsequently captured images were compared. Corresponding pixels in consecutive images exceeding a previously chosen threshold of light intensity change (10% of maximum) clearly above the optical noise were marked. By this approach a circumscribed area of intrinsic optical signal change associated with epileptiform activity could be displayed. Changes in this area from one image to another exhibited specific spread patterns such as area of onset, direction and extent of propagation, and velocity of propagation. The latter was determined by dividing the distance of the wavefront between subsequent images by the interimage interval.

In the group of old animals we evaluated the effects of TEA+ on the electrophysiological and optical parameters. In the first step we compared the results of the experiments with TEA+ to those without the substance, to discriminate a possible pharmacological effect of TEA+ . In the presence of TEA+ there was no significant difference in the latency to the onset of the first seizure-like event (TEA+ : 28 ± 3.6 min versus naive: 55 ± 12.6 min; P = 0.08). There was also no significant effect on the duration of seizure-like events (23.6 ± 2 s versus 23 ± 1 s; P = 0.8) or on the amplitude of the DC shift (1.2 ± 0.06 mV versus 1.44 ± 0.1 mV; P = 0.17). Velocities of spread of epileptiform activity, region of onset, direction, and extent of propagation were not influenced by TEA+ . However, in the presence of TEA+ the potassium shift was significantly higher (10.4±0.3 mM versus 8.4 ± 0.3 mM; P < 0.001) and the amplitude of the intrinsic optical signal associated with seizure-like events was significantly lower (0.8 ± 0.05% versus 1.8 ± 0.1; P < 0.001). Therefore, we excluded the changes of potassium and amplitude of the intrinsic optical signal in the presence of TEA+ from further analysis. The following data refer to all experiments in which TEA+ did not affect the results.

2.4. Data analysis and presentation

3.2. Electrophysiological features

All data are expressed as mean values ± standard error of the mean (S.E.M.). ANOVA/Fishers PLSD test was used for statistical analysis, differences between measurements were considered significant with P < 0.05. A graphic program (CorelDraw 10.0) was used to prepare images and graphs.

Following the wash-out of Mg2+ the first seizure-like events in the adult control group appeared after 35 ± 3.5 min (n = 5) and there was a similar latency in the old rats (n = 8) of 41.6 ± 7.8 min (P = 0.53). Comparing duration of seizure-like events in controls (23.9 ± 1.56 s) to old rats (23.1 ± 1.1 s) we saw no significant difference (P = 0.69). Old animals revealed significantly lower amplitudes of DC shifts (1.4 ± 0.1 mV versus 2.3 ± 0.1 mV; P < 0.001) and significantly lower potassium shifts (8.4 ± 0.3 mM versus 10.5±0.48 mM; P < 0.001). Basic electrophysiological parameters over the time course of an exemplary seizure-like event for a control and an old animal are shown in Figs. 1B and 2A. Table 1 summarizes the basic electrophysiological features of the two groups.

3. Results Sixty-four seizure-like events in eight combined hippocampus-entorhinal cortex slices from five old rats and 59 seizure-like events in six slices from five adult rats (control group) were investigated. In four slices of three old animals the ECS-marker TEA+ was added to the ACSF.

Table 1 Basic parameters of seizure-like events in brain slices of adult and old rats Duration (s)

[K+ ]0 (mM)

f.p. (mV)

Change of IOS (%)

Adult rats SLE (n)

23.9 ± 1.56 59

10.5 ± 0.48 39

2.3 ± 0.1 59

1.5 ± 0.04 54

Old rats SLE (n)

23.1 ± 1.1 64

8.4 ± 0.3 44

1.4 ± 0.1 64

1.8 ± 0.1 43

Significance (P)

=0.69

<0.001

<0.001

<0.01

[K+ ]0 , maximal extracellular potassium-concentration; f.p., extracellular field potential; IOS, intrinsic optical signal; s, second; mM, millimol; mV, millivolt; SLE, seizure-like event; n, number.

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Fig. 2. (A) Electrographic and optical features during a seizure-like event of an old animal. Time courses of the extracellular field potential (f.p.), extracellular potassium-concentration [K+ ]0 , change in light transmittance (T/T), and TEA+ as measured in the lEC. The TEA+ -signal reveals a shrinkage of the extracellular space (ECS)-volume of about 5%. (B) In the original video image of the combined entorhinal cortex-hippocampus slice preparation of an old animal a bipolar stimulation electrode in the stratum radiatum of CA3 and a recording electrode in the lEC can be identified. At seizure onset (0 s, onset) an increase in light transmittance occurs in the mEC, which slowly spreads towards the temporal cortex. We never saw invasion of the subiculum in slices of the aged animals. The pseudocolored control image taken 5 s before seizure onset and the subtraction image 40 s after seizure offset (40 s, post) display optical noise only.

3.3. Changes in the extracellular space-(ECS) volume We studied the changes in ECS-volume in 10 seizure-like events from two slices of two old animals using TEA+ -sensitive microelectrodes. The concentration of this ECS-marker increased during seizure activity from the baseline of 2 mM TEA+ concentration to 2.11 ± 0.01 mM. This indicates a shrinkage of the ECS-volume during the seizure-like activity of 5 ± 0.2% (Fig. 2A). 3.4. Optical features During seizure-like events in each case an increase in light transmittance amounting to a plateau was seen followed by a gradual decline to baseline. Examples of the evolution of the optical signal in time and space of a control and an aged animal are shown in Figs. 1C and 2B. The amplitude of the change in light transmittance was significantly higher in the aged animals (1.8 ± 0.1% versus 1.5 ± 0.04%; P < 0.01) (Table 1). 3.5. Regions of onset and spread patterns We looked for the anatomical regions in which the first changes in light transmittance were seen and identified these as regions of onset. In adult animals all seizure-like events

started in the EC, with 37% in the medial (mEC) and 63% in lateral EC (lEC). In the old rats 49.2% of seizure-like events started in mEC, 43.1% in lEC, and 7.7% in TE. In a further step we analyzed the spread patterns of each seizure-like event. From the regions of onset the wavefront propagated bidirectionally towards the subiculum and the temporal cortex. In cases with onset in the temporal cortex propagation was monodirectional towards the subiculum. In the control group only, the seizure-like events regularly invaded the subiculum which was never observed in old animals (for details see Fig. 3 A.1 and A.2). 3.6. Spread velocities Ranking of spread velocity in the different regions was equal in both, control and old animals, from lowest to highest: perirhinal cortex, entorhinal cortex, and temporal cortex. In the adult animals velocity in the subiculum was fastest (1.34 ± 0.1 mm/s). Comparison of speed in the two age-groups revealed significantly lower velocities in the old animals in the entorhinal (0.5 ± 0.03 mm/s versus 0.81 ± 0.04 mm/s; P < 0.001) and in the perirhinal cortex (0.3 ± 0.03 mm/s versus 0.6 ± 0.06 mm/s; P < 0.001). Velocity in the temporal cortex was lower in the old animals as well (0.87 ± 0.11 mm/s versus 0.95 ± 0.08 mm/s) but difference did not reach a significant level (P = 0.53) (Fig. 3B).

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4. Discussion

Fig. 3. (A) Schematic drawings of the combined entorhinal cortexhippocampus slice preparation in the two age groups with regions of onset and spread patterns. For anatomical regions see Fig. 1A. The pacemaker regions are indicated as black stars. The numbers in stars reflect the total number of seizure-like events generated in the actual pacemaker region. The black lines, numbers and arrows show the route and direction of spread as well as the numbers of seizure-like events which ceased in a defined region. Note that in control slices (A.1.) invasion of subiculum frequently occurred, whereas this region never was invaded in old animals (A.2.). (B) Velocity of spread in different regions in the two groups. In slices of old animals the spread velocities are slower in all regions, which was significant for the entorhinal and perirhinal cortex (EC and PC). TE: temporal cortex; and Sb: subiculum.

In recent years it has become apparent that old age is associated with a high risk to develop epileptic seizures. Indeed, the prevalence and incidence of epilepsy in the elderly now have exceeded those of any other age-group [24]. It is therefore important to improve our understanding of the peculiarities of ictogenesis in aged tissue. A number of reports have indicated that various structural lesions account for the increased seizure susceptibility [13,23]. However, it is as yet unknown which effect changes in brain plasticity associated with old age themselves may exert on ictogenesis. In this study, a combination of electrophysiological and optical imaging methods was used to analyze the spatiotemporal pattern of seizure onset and spread induced by Mg2+ -free solution in hippocampal-entorhinal cortex slices of 2-year-old rats compared to 3-month-old control animals. According to a widely accepted definition we identified “old” or “aged” animals if they had lost approximately half or more of their siblings at the age of 24 months or above [58]. We choose 3-month-old rats as control animals because animals of this age are sexually mature and are commonly used for non-age-dependent in vivo and in vitro studies. The main findings of this study are: seizure-like events in tissue from aged compared to adult animals display more than one region of onset, they show a less extensive spread to adjacent areas and a decreased propagation velocity in distinct anatomical regions. Furthermore, we saw less pronounced activity-induced reductions of the ECS-volume and a smaller increase of the activity-induced extracellular K+ concentration. We used a combination of electrophysiological methods and intrinsic optical imaging to monitor regions of onset, spread patterns and propagation velocities. The important advantage of optical imaging over pure electrophysiology is the ability to provide simultaneous coverage of the whole preparation (see Section 2) with a non-invasive approach. The neurobiological basis of the intrinsic optical signal change is as yet unclear but it is important to note that it is composed of different components in vivo and in vitro. Whereas changes in blood volume or oxygen consumption are important in vivo these factors do not play a major role in the bloodless brain slice preparation in which light scattering appears to be more important [20]. A number of mechanisms may contribute to activity-induced scattering changes. Cytosolic changes such as alteration of its viscosity have been suggested by Hill and Keynes [25]. Since stimulation-induced optical signals could be abolished in the presence of Ca2+ -free ACSF or addition of kynurenic acid, it has been assumed that postsynaptic activation may be important for an optical signal to be provoked [42]. Certainly, changes in ECS-volume also contribute to the signal change but there is no clear cut simple relation between cell swelling (or shrinkage of the ECS-volume) and the direction of the intrinsic optical signal change under interface conditions [9,11,50]. In spite of the poorly understood

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mechanisms underlying the signals, they already represent important tools in neuroscience research [2,20,70]. Regarding the preparation we used in the current experiments it is important to note that in contrast to the isolated hippocampal slice, which is deprived from functionally important adjacent structures such as the entorhinal cortex, the combined preparation as used in the current study allows phenomena such as the initiation and the spread of epileptiform activity to be assessed [10,68]. Analysis of the regions within the slice in which seizure-like events occurred revealed some differences in the two groups. In keeping with previous studies [10,46] seizure-like events in the control group of adult tissue originated solely in the entorhinal cortex. Although the vast majority of seizure-like events in the aged group also originated in the entorhinal cortex, there was a proportion of 8% that arose in the temporal cortex, possibly indicating a tendency towards multifocality in aged tissue. Propagation of epileptiform activity is an important step in the process of secondary generalization involving progressive recruitment of neighboring cells and areas. Unfortunately, there is a paucity of data regarding this phenomenon in aged tissue. Our experiments revealed marked differences in the extent of seizure spread in aged slices and in slices from adult animals. In adult brain control slices the seizure-like events propagated as far as to reach the subiculum but without further invasion of the hippocampus proper. The latter probably indicates that in non-epileptic adult animals the dentate gyrus acts as a seizure filter [10,37]. This is different from immature juvenile tissue in which the critical inhibitory mechanisms have not been established [70] and from chronic epileptic animals where the excessive epileptic discharges lead to an impaired function of the dentate gyrus [6,39]. In contrast, in the aged group seizure spread was less extensive not even exceeding the medial entorhinal cortex and never invading the subiculum. Thus, old age appears to be an important factor as to how and to what extent seizure activity spreads and there is a decreasing tendency of spread from juvenile over adult to senile neuronal tissue. Such reduced extent of seizure spread in the old age group parallels the clinical observation that partial seizures in the elderly have less propensity to spread to adjacent areas and to generalize [13]. The velocities of seizure spread too, represent crucial parameters that participate importantly in the clinical expression of seizures. Again, the underlying mechanisms are largely unclear. It is assumed that synaptic and/or non-synaptic mechanisms play relevant roles depending on the velocity of propagation [16]. There is a fast spread of 100–1500 mm/s in synaptically transmitted interictal discharges [31,60,67]. In the current experiments velocities were slow, not exceeding 1 mm/min in either group. This suggests that synaptic mechanisms are of minor importance. Such a hypothesis is supported by the fact that slow spread is also seen in seizure-like events induced by Ca2+ -free ACSF which propagates in the absence of synaptic trans-

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mission [35]. Comparing our two groups, seizure spread was significantly slower in the entorhinal and perirhinal cortex in the aged slices. It is conceivable that both reduced extent and velocity of spread reflect age-related alterations in synaptic efficiency [43], disrupted local brain circuitry or its faulty repair [29] and/or alterations in activity-induced ECS-volume shrinkage or extracellular K+ concentrations (see below) [28]. Our findings of limited and slow spread of the discharge may well explain a number of clinical peculiarities seen in the presentations of seizures in the elderly. Besides the reduced tendency of partial seizures to generalize there may be very localized EEG discharges [19]. In addition, complex partial seizures may largely consist of staring, an altered mental state or unresponsiveness without evolution of additional features [19]. Such unusual clinical presentations may give rise to diagnostic uncertainties. Indeed, large studies [44,45] have demonstrated that in the elderly epilepsy with recurrent complex partial seizures may go undiagnosed for more than 1 year after the first seizure. The results of the current study indicate that reduced seizure spread and decreased propagation velocities represent important explanatory tissue properties of the aging brain. In order to gain some insight into tissue properties and mechanisms that are involved in the reduced extent of seizure spread and its slow velocity we have compared ECS-volume changes and rises in extracellular K+ concentrations in aged tissue and controls. Both represent seizure discharge-related parameters that are known to be prominently involved in ictogenesis and spread. It is well established that epileptiform activity is associated with a shrinkage of the ECS-volume in vivo [15] and in vitro [21]. Clinically and experimentally, such acute shrinkage in turn has been demonstrated to increase seizure susceptibility and spread [1,59]. The current data, however, demonstrate that the acute activity-related changes in ECS-volume of 5% are considerably less pronounced compared to the 7% we have seen previously in brain slices of adult animals during low-Mg2+ -induced seizure-like events [9]. It is important to note, that the activity-dependent ECS-volume shrinkage reflects the functional changes during seizure-activity, only. It does not account for the ECS-volume fraction itself, which has been shown to be significantly lower in aged rats [62,63]. Thus, it is plausible to assume, that the reduced extent and velocity of seizure spread represent consequences of a wider ECS-volume. The extracellular K+ increases associated with seizurelike events were analyzed using ion-sensitive microelectrodes. It is well established that during electrical stimulation and spontaneous epileptiform activity the extracellular K+ concentration may rise from 3 to 12 mM [21,40]. It has been suggested that increased extracellular K+ accumulation is crucial in the recruitment of neurons in the vicinity of the initial focus and consequent spread of seizure-like events [66]. The mechanisms most importantly involve a reduced K+ driving force [65], an impaired GABAA receptor-mediated

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inhibition [36], and K+ -induced glial cell swelling contributing to ECS-volume shrinkage [69]. Therefore, it was interesting to note that the maximum activity-dependent K+ increases were significantly lower in aged animals compared to controls and this likely represents another important mechanism involved in the reduced extent and velocity of spread. It is unlikely that the smaller activity-dependent K+ increases in aged tissue can be explained by a more efficient K+ uptake by glia since there is evidence for an impairment of glial function during senescence [57,62]. In the current experiments we have employed a standard model of epilepsy in which increased synaptic excitation results mainly from unblocking the NMDA receptor [48]. Could the results of our study have been influenced by the finding that there is a decrease of about 30–40% of the NMDA binding sites in the hippocampus of aged rats [8,47]? Such an assumption is unlikely in view of the fact that there is no significant age-related difference in the ability of Mg2+ to depress the magnitude of the NMDA receptor-mediated EPSP [7,54]. However, it is clearly important to investigate seizure susceptibility of aged brain tissue also using models that are based on other pathophysiological mechanisms. In conclusion, the current study demonstrates that intrinsic optical imaging allows one to analyze spatiotemporal pattern of seizure discharges in aged tissue. The changes in plasticity that occur during the process of aging exert pronounced effects on spread patterns and propagation velocities. The features of restricted seizure spread and reduced propagation velocities may in part be explained by our findings of significantly reduced activity-dependent ECS-volume changes and the less pronounced K+ increases in aged tissue compared to controls.

Acknowledgments We gratefully acknowledge encouragement from U. Heinemann and the excellent technical support from H.J. Gabriel. This study was supported by the DFG grant BU 1331-1 to K.B. and H.M.

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