Rij rat model of absence epilepsy

Experimental Neurology 289 (2017) 103–116

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Research Paper

Disinhibition of the intergeniculate leaflet network in the WAG/Rij rat model of absence epilepsy Lukasz Chrobok a, Katarzyna Palus a, Jagoda Stanislawa Jeczmien-Lazur a, Anna Chrzanowska a, Mariusz Kepczynski b, Marian Henryk Lewandowski a,⁎ a b

Department of Neurophysiology and Chronobiology, Institute of Zoology, Jagiellonian University in Krakow, Krakow, Poland Faculty of Chemistry, Jagiellonian University in Krakow, Krakow, Poland

a r t i c l e

i n f o

Article history: Received 25 August 2016 Received in revised form 21 December 2016 Accepted 25 December 2016 Available online 30 December 2016 Keywords: Intergeniculate leaflet Absence epilepsy Miniature inhibitory postsynaptic currents GABA Infra-slow oscillations GFAP

a b s t r a c t The intergeniculate leaflet (IGL) of the thalamus is a retinorecipient structure implicated in orchestrating circadian rhythmicity. The IGL network is highly GABAergic and consists mainly of neuropeptide Y-synthesising and enkephalinergic neurons. A high density of GFAP-immunoreactive astrocytes has been observed in the IGL, with a probable function in guarding neuronal inhibition. Interestingly, putatively enkephalinergic IGL neurons generate action potentials with an infra-slow oscillatory (ISO) pattern in vivo in urethane anesthetised Wistar rats, under light-on conditions only. Absence epilepsy (AE) is a disease characterised by spike-wave discharges present in the encephalogram, directly caused by hypersynchronous thalamo-cortical oscillations. Many pathologies connected with the arousal system, such as abnormalities in sleep architecture and an insufficient brain sleep-promoting system accompany the epileptic phenotype. We hypothesise that disturbances in the function of biological clock structures, controlling this rhythmic physiological process, may be responsible for the observed pathomechanism. To test this hypothesis, we performed an in vitro patch-clamp study on WAG/Rij rats, a well-validated genetic model of AE, in order to assess dampened GABAergic synaptic transmission in the IGL expressed as a lower IPSC amplitude and reduced sIPSC frequency. Moreover, our in vivo extracellular recordings showed higher firing rate of ISO IGL neurons with an abnormal reaction to a change in constant illumination (maintenance of rhythmic neuronal activity in darkness) in the AE model. Additional immunohistochemical experiments indicated astrogliosis in the area of the IGL, which may partially underlie the observed changes in inhibition. Altogether, the data presented here show for the first time the disinhibition of IGL neurons in a model of AE, thereby proposing the possible involvement of circadian-related brain structures in the epileptic phenotype. © 2017 Elsevier Inc. All rights reserved.

1. Introduction The intergeniculate leaflet (IGL) is a small thalamic nucleus interlaid between the dorsal and ventral parts of the lateral geniculate complex (DLG and VLG, respectively; Hickey and Spear, 1976; Moore and Card, 1994). Its only known function is to collect and integrate cues arising Abbreviations: AP-5, 2-amino-5-phosphonopentanoic acid; CNQX, 6-cyano-7nitroquinoxaline-2,3-dione; DLG, dorsal part of the lateral geniculate complex; ENK, enkephalin; GABA, γ-amino butyric acid; GFAP, glial fibrillary acidic protein; IA, A-type potassium current; IGL, intergeniculate leaflet; ISO, infra-slow oscillations; IT, T-type calcium current; mIPSC, miniature inhibitory postsynaptic currents; NPY, neuropeptide Y; OPN, olivary pretectal nucleus; SCN, suprachiasmatic nucleus; sIPSC, spontaneous inhibitory postsynaptic currents; SWD, spike-wave discharges; TTX, tetrodotoxin; VLG, ventral part of the lateral geniculate complex; VLPO, ventrolateral preoptic area. ⁎ Corresponding author. E-mail addresses: [email protected] (L. Chrobok), [email protected] (K. Palus), [email protected] (J.S. Jeczmien-Lazur), [email protected] (A. Chrzanowska), [email protected] (M. Kepczynski), [email protected] (M.H. Lewandowski).

http://dx.doi.org/10.1016/j.expneurol.2016.12.014 0014-4886/© 2017 Elsevier Inc. All rights reserved.

from the retina and non-specific brain systems. The efferents of the IGL primarily reach the suprachiasmatic nuclei (SCN), which is the location of the master biological clock; therefore, the structure is considered to be implicated in the modulation of circadian rhythmicity (Harrington and Rusak, 1986; Harrington, 1997; Morin, 2012). In the rat and hamster, both the IGL and SCN can be readily identified in glial fibrillary acidic protein (GFAP) immunostained sections (Morin et al., 1989). IGL neurons can be divided into two major subpopulations, both expressing γ-amino butyric acid (GABA), but differing in terms of synthesised neuropeptides (Moore, 1989; Moore and Speh, 1993; Lima et al., 2012). The first subpopulation is known to produce neuropeptide Y (NPY) and to send their axons to the SCN (Card and Moore, 1989; Glass et al., 2010). NPY-synthesising cells firing tonic action potentials and are non-responsive, inhibited or show complex reactions to light (Zhang and Rusak, 1989; Takatsuji et al., 1991; Thankachan and Rusak, 2005). Recently, we have shown that these IGL neurons can be distinguished during in vitro patch-clamp recording by their marked A-type potassium current (IA; Chrobok et al., 2016). Moreover, NPY can serve as a

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marker of IGL borders, because it is not synthesised in the adjacent nuclei in the rat (Moore and Card, 1994; Harrington, 1997). The second neuronal subpopulation (representing the majority of IGL cells) expresses the opioid neuropeptide enkephalin (ENK) and connects contralaterally located IGLs (Mantyh and Kemp, 1983; Mikkelsen, 1992; Harrington, 1997). As shown previously, under in vivo conditions with urethane anaesthesia, putatively enkephalinergic neurons are able to express a highly specific firing pattern, called infra-slow oscillations (ISO), which are observed under constant illumination only. ISO can be characterised by a rhythmically alternating activity phase (called the intraburst), where neurons generate action potentials in a high frequency fashion, and a subsequent silent phase (extraburst). The period of these oscillations is about 2 min (b0.01 Hz; Lewandowski et al., 2000, 2002; Lewandowski and Błasiak, 2004; Blasiak and Lewandowski, 2013). ISO can be found in not only the IGL, but also while recording various retinorecipient brain structures such as the SCN, olivary pretectal nucleus (OPN), DLG or VLG (Miller and Fuller, 1992; Albrecht and Gabriel, 1994; Albrecht et al., 1998; Filippov and Frolov, 2005; Szkudlarek et al., 2008). We have also shown that putatively enkephalinergic IGL neurons can be differentiated by T-type calcium conductance (IT; Chrobok et al., 2016). Recent experiments performed by our group show that ENK is employed as the main inner IGL neurotransmitter and that enkephalinergic, not NPY-synthesising neurons provide network inhibition (Palus et al., 2017). Absence epilepsy is a neurological disease characterised by the presence of spike-wave discharges (SWD) in the encephalogram accompanied by behavioural arrest. WAG/Rij rats, used in this study, are a well-known model of absence epilepsy, sharing most of the genetic and behavioural disturbances observed in human patients (Epps and Weinshenker, 2013). Among the direct causes of SWD in absence epilepsy, mutations in T-type calcium channel genes (Crunelli and Leresche, 2002) and impaired GABA-dependent inhibition can be listed (Staak and Pape, 2001; Li et al., 2006, 2007; Liu et al., 2007; Brockhaus and Pape, 2011). Additionally, it has been shown that the GABA transporter GAT-1, expressed exclusively in thalamic astrocytes, is crucial for the genesis of seizures (De Biasi et al., 1998; Cope et al., 2009). Despite the fact that SWD are generated in the hypersynchronous thalamo-cortical loop, this type of epilepsy is connected with retinal pathologies (described in WAG/Rij rats only; Lai et al., 1975; O'Steen and Donnelly, 1982), sleep-promoting system insufficiencies (Halasz, 1991; Suntsova et al., 2009) and glial cell impairment (Akin et al., 2011; Sitnikova et al., 2011), with SWD showing circadian rhythmicity (van Luijtelaar and Coenen, 1988; Smyk et al., 2011, 2012). The sleeppromoting brain circuits are reliant on the ventrolateral preoptic area (VLPO; Szymusiak et al., 1998; Gaus et al., 2002), and in an extensive study by Suntsova et al. (2009), the pathological function of this critical hypothalamic area was shown in WAG/Rij rats. In our previous study carried out on single IGL neurons, we showed a decrease in the amplitude of IT in the brain slices obtained from young WAG/Rij rats in comparison with control Wistar rats (Chrobok et al., 2016). We hypothesised that the disturbances in biological clock structures, such as the IGL or SCN, which maintain brain circadian inhibition, may underlie one of the causes of the VLPO insufficiencies and therefore contribute to sleep architecture changes in absence epilepsy (Chou et al., 2002; Saint-Mleux et al., 2007; Suntsova et al., 2009). The aim of the present study was to evaluate possible disturbances the GABAergic IGL network function. First, we focused on the characterisation of GABAergic synaptic transmission in single, immunohistochemically identified IGL neurons by miniature and spontaneous inhibitory postsynaptic current (mIPSC/sIPSC) recordings using an in vitro patchclamp technique and performed a comparison between age-matched presymptomatic WAG/Rij and Wistar rats. By the sustained application of GABA (100 μM) and picrotoxin (100 μM), we were also able to describe the evoked and tonic GABAergic currents. Next, we studied the eventual disturbances in IGL oscillatory neuron physiology performing single-unit extracellular in vivo recordings on adult Wistar and WAG/

Rij rats. Additional immunohistochemical studies were performed to identify possible GFAP-immunoreactive astrocyte changes in the IGL of epileptic rats. 2. Material and methods 2.1. Animals Animals were maintained and used in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and Polish law. Experimental protocols were approved by the Ethics Committee of the Jagiellonian University in Krakow. Our experiments were designed to reduce stress, suffering and the number of animals used in the study. Animals were bred at the Institute of Zoology, Jagiellonian University under a 12:12 light-dark cycle (lights on at 08:00 am) with food and water available ad libitum. Constant environmental conditions (temperature of 23 °C, 67% humidity) were provided. 2.2. Electrophysiology in vitro 2.2.1. Brain tissue preparation All in vitro experiments were performed on brain slices containing IGL obtained by a procedure described previously in detail (Chrobok et al., 2016; Palus et al., 2015). In short, male Wistar (n = 25) and WAG/Rij (n = 24) rats in two age groups (14/15 and 21/22 days old) were deeply anaesthetised with isoflurane (2 ml/kg body weight, Baxter) and decapitated between 9:00 and 10:00 am. The brain was quickly removed from the skull and immersed in ice-cold normal artificial cerebrospinal fluid (ACSF), saturated with carbogen (95% O2, 5% CO2), composed of (in mM): 125 NaCl, 25 NaHCO3, 3 KCl, 1.2 NaH2PO4, 2 CaCl2, 2 MgCl2 and 10 glucose. Thalamic coronal slices (250 μm thick) were cut using a vibroslicer (Leica VT1000S, Heidelberg, Germany). Then, sections containing IGLs were transferred to the preincubation chamber for 30 min at 32 °C, then another 60 min at room temperature, and finally placed in the recording chamber at 30 °C. 2.2.2. Voltage clamp recordings All recordings were performed in the whole-cell configuration obtained by applying negative pressure from an Ez-gSEAL100B Pressure Controller (Neo Biosystem, San Jose, USA). Recording electrodes were placed above the IGL under visual microscopic control. Single IGL neurons were identified under 40× magnifying objective with a Zeiss Examiner microscope fitted with infrared differential interference contrast (Göttingen, Germany). The recorded signal was amplified by a SC 05LX (NPI, Tamm, Germany) amplifier, low-pass filtered at 2 kHz and digitised at 20 kHz. Spike2 and Signal (Cambridge Electronic Design Inc., Cambridge, UK) software were used for the recordings. Additionally, at the beginning of each recording, voltage clamp depolarising steps (15 steps, 200 ms duration, 700 ms interval) were given in 3 mV increments (first step −80 mV) from the holding potential of −115 mV, in order to classify IGL neurons (Chrobok et al., 2016). A liquid junction potential of approximately − 15 mV was added to the measured membrane potential. Experiments were carried out in voltage clamp mode, with patch pipettes filled with a high Cl− intrapipette solution containing (in mM): potassium gluconate 16, KCl 129, HEPES 10, MgCl2 2, Na2ATP 4, Na3GTP 0.4, EGTA 1 and 0.05% biocytin (pH = 7.4 adjusted with 5 M KOH; osmolality ~ 300 mOsmol/kg) in order to obtain a symmetrical concentration of Cl− across the membrane. Recordings of inhibitory postsynaptic currents (IPSC) and applications of GABA (100 μM) and picrotoxin (100 μM) were performed at a holding voltage of −90 mV. 2.2.3. Statistics and data analysis The analysis of IPSC frequency, amplitude, rise time and decay constant was conducted with commercially available Mini Analysis software (Synaptosoft Inc., Decatur, GA). This program detects and

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measures spontaneous synaptic events according to the amplitude, rate of rise, duration and area under the curve (fc). Synaptic events were detected manually with an amplitude threshold of 3 pA and area threshold of 50 fc. Analysis of whole-cell GABA-evoked current amplitude was performed in Spike2 (CED, Cambridge, UK). The membrane time constant (τ) was determined by fitting to a single exponential. Membrane capacitance (Cm) was calculated according to the equation Cm = τ / R. Current density was calculated by dividing the current amplitude by Cm and expressed in pA/pF. All in vitro data statistics were analysed using GraphPad Prism (GraphPad Software, Inc. USA) software. The Mann-Whitney test and Student's unpaired t-test were used and p b 0.05 was considered as significant. Data are represented as the mean value ± SEM. 2.2.4. Immunohistochemical verification As previously described (Chrobok et al., 2016; Palus et al., 2015), an immunolabelling protocol was used to visualise NPY-immunoreactivity within the IGL in order to verify the localisation of the recorded cell. Briefly, primary rabbit NPY antisera (1:8000; Sigma-Aldrich, Schnelldorf, Germany) and secondary anti-rabbit AlexaFluor 647-conjugated antisera (1:300; Jackson ImmunoResearch, West Grove, PA, USA) were used to identify NPY-positive neurons. During the electrophysiological experiments, IGL neurons were filled with biocytin (0.05%); therefore, incubation with Cy3-conjugated ExtrAvidin (Sigma-Aldrich, Schnelldorf, Germany) during the immunolabelling protocol allowed us to visualise the recorded neurons. Brain slices were viewed using an A1-Si Nikon Inc. (Japan) confocal laser scanning system built on an inverted microscope Nikon Ti-E (Japan). The system was equipped with two detection channels with 488 and 638 nm diode lasers for excitation. The images were stored on a hard disk and further analysed with NIS-Elements (Nikon, Japan) software. Only those neurons which were identified in the area of NPY-positive cell bodies were considered IGL neurons. 2.2.5. Reagents Tetrodotoxin citrate (TTX, Tocris, Bristol, UK), 6-cyano-7nitroquinoxaline-2,3-dione (CNQX, Tocris, Bristol, UK), 2-amino-5phosphonopentanoic acid (AP-5, Tocris, Bristol, UK) and bicuculline methiodide (Bic, Tocris, Bristol, UK) were stored as a stock solution (100× concentrated, dissolved in 0.9% NaCl) at −20 °C. GABA (SigmaAldrich, Schnelldorf, Germany), 100× concentrated in 0.9% NaCl, was stored at 4 °C. On the day of the experiment, each stock solution was dissolved in freshly prepared ACSF in order to obtain working solutions: GABA (100 μM), TTX (0.5 μM), CNQX (10 μM), AP-5 (40 μM) and Bic (20 μM). All drugs were delivered by bath perfusion and approximately 200 s was needed for the substance to reach the recording chamber via the tubing system. In order to induce GABA-evoked tonic whole-cell currents, 10–15 ml of GABA working solution was applied. 2.3. Electrophysiology in vivo 2.3.1. Extracellular recordings Data were obtained from 16 male Wistar (290–350 g) and WAG/Rij rats (215–330 g). At the beginning of each experiment, rats were anaesthetised with an intraperitoneal injection of urethane (1.5 g/kg; Sigma-Aldrich, Schnelldorf, Germany). An additional dose of urethane (10-20% of the initial dosage) was administered in cases when withdrawal and ocular reflexes indicated that the animal had not yet reached the state of deep anaesthesia. Core body temperature and electrocardiography were monitored throughout the experiment. Anaesthetised rats were held in a stereotaxic frame (Advanced Stereotaxic Instruments, Warren, Michigan, USA) via ear canals and incisor bars. Stereotactic points (bregma and lambda) were always located at the same level. The skull was exposed by a midline scalp incision and a craniotomy was performed directly above the IGL (4.0 mm lateral, 4.5–4.8 mm posterior, bregma as reference). Recording borosilicate

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glass micropipettes were prepared on a horizontal puller (Sutter Instruments, CO P-97, USA) and filled with 4% Chicago Sky Blue (Sigma Aldrich, Schnelldorf, Germany) dissolved in 2 M NaCl. Extracellular neural signals were amplified using preamplifier and CyberAmp 380 amplifier (Axon Instruments, U.S.A., final amplification 10,000×; filtration 300–3000 Hz). To record signals on the computer, the CED Micro mkII interface and Spike2 software (Cambridge Electronic Design Inc., Cambridge, UK) were used. To determine the recording site, an electrical current of 25 mA was ejected from the recording micropipette. 2.3.2. Experimental protocols At the beginning of each registration, the recording electrode was located within the borders of the IGL. To trace changes in the neuronal activity of the IGL, a firing rate histogram (1 s bins) was generated on-line. Only neurons with the characteristic infra-slow oscillatory (ISO) pattern were qualified to perform the protocol, which consisted of 15 min periods of light and darkness. 2.3.3. Histology At the end of each experiment, rats were perfused transcardially with 0.1 M buffered physiological saline (PBS) followed by 4% paraformaldehyde in PBS (pH 7.4). The brain was removed and post-fixed in 4% paraformaldehyde for 12 h. The following day, brains were sectioned at 100 μm slices on a vibroslicer (Leica VT1000S, Heidelberg, Germany). Locations of the recording site marks were established using a light microscope and stereotaxic atlas (Paxinos and Watson, 2007). 2.3.4. Data analysis All single units were sorted manually using Spike2 (Cambridge Electronic Design Inc., Cambridge, UK) principal components analysis. The rhythmicity and spectral content of the recordings were assessed by Fast Fourier Transforms (FFTs) and autocorrelation analysis, conducted in Statistica (StatSoft, Inc., Tulsa, USA). Analysis of intra- and extrabursts was performed with the use of a custom-made MatLab script (MathWorks, USA). Periods, mean frequencies and intra-/extraburst parameters of ISO activity units were statistically compared with GraphPad Prism (GraphPad Software, San Diego, CA, USA). Wilcoxon, Mann-Whitney and Student's unpaired t-tests were applied. 2.4. GFAP immunohistochemistry and data analysis In order to perform immunostaining of GFAP, three Wistar and three WAG/Rij rats (age-matched, 4 months old) were perfused transcardially, as described previously (sections: 2.3.4, Electrophysiology in vivo, Histology). After the perfusion procedure, brains were sectioned into 50 μm coronal slices containing IGLs and submitted to the immunostaining procedure. At first, slices were rinsed in PBS and subsequently placed in fresh PBS containing 0.6% Triton-X100 (Sigma-Aldrich, Schnelldorf, Germany) and 10% normal donkey serum (NDS, Jackson ImmunoResearch, West Grove, PA, USA) at room temperature. After 30 min, slices were washed with PBS and incubated with goat GFAP antisera (1:200, Santa Cruz Biotechnology Inc., CA, USA) and rabbit NPY antisera (1:8000, Sigma-Aldrich, Schnelldorf, Germany) in PBS containing 2% NDS and 0.3% Triton-X100 overnight. Next, slices were rinsed again in PBS and incubated with anti-rabbit AlexaFluor 647 and anti-goat AlexaFluor 488-conjugated antisera (both 1:300; Jackson ImmunoResearch, West Grove, PA, USA) overnight. At the end of the procedure, slices were rinsed again in PBS and mounted on the glass slides with Fluoroshield™ (Sigma-Aldrich, Schnelldorf, Germany). Slices were viewed under a confocal microscope, as described previously (sections: 2.2.4, Electrophysiology in vitro, Immunohistochemical verification). To assess the features of astrogliosis, the mean grey value and area fraction of GFAP-immunopositive cells were obtained using an image processing system (Image J2; v. 2.0.0-rc41/1.50d, NIH, USA). Mean grey value measurements were evaluated as the sum of the grey values

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of all the pixels in the selected region of interest (ROI) covered by the mask and divided by the number of pixels. The area fraction was defined as the percentage of pixels in the ROI. Both measurements were calculated for thresholded pixels (values set at 70–255). The ROI corresponds to the circle placed in the area of the IGL; data from three circles were averaged and analysed. 3. Results 3.1. Amplitude of miniature GABAergic synaptic currents is lower in the IGL of presymptomatic WAG/Rij rats At first, miniature inhibitory postsynaptic currents (mIPSCs) were examined in slices derived from 14/15 day old Wistar and WAG/Rij rats. In total, we recorded 75 IGL neurons in both rat strains, i.e. Wistar (n = 40) and WAG/Rij (n = 35), using the patch-clamp technique. During the first experiments, the presence of mIPSC was verified by bicuculline (20 μM) application, which caused the disappearance of all recorded events (Fig. 1A). To check for any possible differences in GABAergic synaptic transmission, we compared mIPSC parameters between WAG/Rij and Wistar rats. The mean mIPSC amplitude was statistically lower in WAG/Rij rats in comparison to Wistar rats (64.95 pA ± 1.78 pA, n = 18 vs. 80.24 pA ± 4.25 pA, n = 23, p = 0.0054, Student's unpaired t-test, Fig. 1B). The mean mIPSC frequency did not differ significantly between the two rat strains (0.87 Hz ± 0.08 Hz, n = 18 vs. 0.95 Hz ± 0.10 Hz, n = 23, WAG/Rij and Wistar rats, respectively, p = 0.56, Student's unpaired t-test, Fig. 1C). Both results were confirmed by comparing data from the respective cumulative distribution histograms of mIPSC amplitudes and frequencies (Fig. 1B,C). We also evaluated the possible differences in mIPSC kinetics; both the rise time and the decay constant were found to be not significantly different between the two rat strains (rise time: 1.73 ms ± 0.05 ms, n = 18 vs. 1.70 ms ± 0.06 ms, n = 23; decay constant: 4.93 s ± 0.38 s, n = 18 vs. 4.88 s ± 0.40 s, n = 23, WAG/Rij and Wistar, respectively, prise time = 0.68, pdecay const = 0.94, Student's unpaired t-test, Fig. 1F). The second group of neurons examined (n = 34) were obtained from 21/ 22 day old rats. Similarly, we observed a significant difference in the mIPSC amplitude (90.61 pA ± 6.92 pA, n = 17 vs. 72.96 pA ± 4.44 pA, n = 17, Wistar and WAG/Rij rats, respectively, p = 0.039, Student's unpaired t-test, Fig. 1D), but not in the frequency (0.87 Hz ± 0.13 Hz, n = 17 vs. 0.79 Hz ± 0.08 Hz, n = 17, Wistar and WAG/Rij rats, respectively, p = 0.94, Mann-Whitney test, Fig. 1E). Again, both results were confirmed in cumulative plots (Fig. 1D, E). As in the 14/15 day old group, we did not observe any changes in the rise time (1.56 ms ± 0.04 ms, n = 17 vs. 1.69 ms ± 0.07 ms, n = 17, Wistar and WAG/Rij rats, respectively, p = 0.098, Mann-Whitney test) or the decay constant (3.53 s ± 0.18 s, n = 17 vs. 4.01 s ± 0.28 s, n = 17, Wistar and WAG/Rij rats, respectively, p = 0.27, Mann-Whitney test, Fig. 1H) between the two rat strains. In all parameters presented above, no differences between IT- and IA-expressing IGL neurons were noted (data not shown). 3.2. Spontaneous GABAergic synaptic transmission in the IGL is dampened in presymptomatic WAG/Rij rats In order to verify whether the changes observed while studying the miniature synaptic transmission can be seen in spontaneous inhibitory postsynaptic currents (sIPSC), additional recordings in the standard ACSF (without TTX) were carried out on 8 Wistar and 10 WAG/Rij rats. As in case of mIPSC, the sIPSC amplitude was lower in WAG/Rij comparing to Wistar rats, what was significant for 14/15 day old (73.88 pA ± 6.38 pA, n = 17 vs. 59.52 pA ± 3.46 pA, n = 23, Wistar and WAG/Rij, respectively, p = 0.041, Student's unpaired t-test, Fig. 2A) and 21/22 day old group (82.07 pA ± 8.22 pA, n = 13 vs. 62.05 pA ± 3.65 pA, n = 12, Wistar and WAG/Rij, p = 0.041, Student's unpaired t-test, Fig. 2C). Interestingly, the sIPSC frequency was heavily

dampened in WAG/Rij rats, especially in younger animals (14/15: 3.80 Hz ± 0.67 Hz, n = 17 vs. 1.12 Hz ± 0.13 Hz, n = 23; 21/22: 1.72 Hz ± 0.38 Hz, n = 13 vs. 0.93 Hz ± 0.15 Hz, n = 12, Wistar and WAG/Rij, p14/15 = 0.0001, p21/22 = 0.034, Mann-Whitney test, Fig. 2B,D). All of the differences were confirmed by comparing data from the respective cumulative distribution histograms (Fig. 2A-D). In case of sIPSC kinetics, IGL neurons derived from 14/15 day old WAG/Rij rats were characterised by longer decay constant (5.51 s ± 0.34 s, n = 17 vs. 7.12 s ± 0.48 s, n = 23, Wistar and WAG/Rij, p = 0.014, Student's unpaired t-test, Fig. 2E) with no differences in rise time (2.22 s ± 0.11 s, n = 17 vs. 2.15 s ± 0.10 s, n = 23, Wistar and WAG/ Rij, p = 0.67, Student's unpaired t-test, Fig. 2E). This differences were lacking in 21/22 day old group (rise time: 1.65 s ± 0.04 s, n = 13 vs. 1.69 s ± 0.08 s, n = 12; decay constant: 3.41 s ± 0.14 s, n = 13 vs. 3.66 s ± 0.28 s, n = 12, Wistar and WAG/Rij, prise = 0.59, pdecay = 0.43, Student's unpaired t-test, Fig. 2G). 3.3. Tonic GABAergic currents in IGL are not disturbed in WAG/Rij rats In the next set of experiments, we checked whether the GABAevoked whole-cell currents differed between WAG/Rij and Wistar rats in the recorded IGL neurons. In total, we recorded 54 IGL neurons on which GABA (100 μM; 10–15 ml) was applied tonically in order to evoke a stable tonic current (Fig. 3A). Similarly to IPSC recordings, 14/ 15 and 21/22 day old Wistar and WAG/Rij rat groups were examined. No significant differences in the current density were noted (14/15: 45.77 pA/pF ± 9.54 pA/pF, n = 11 vs. 60.69 pA/pF ± 18.01 pA/pF, n = 13; 21/22: 93.68 pA/pF ± 20.20 pA/pF, n = 15 vs. 108.70 pA/pF ± 14.34 pA/pF, n = 15, Wistar and WAG/Rij, respectively, p14/15 = 0.718, p21/22 = 0.231, Mann-Whitney test, Fig. 3B). In order to examine possible differences in tonic GABAergic currents between IGL neurons derived from 8 Wistar and 10 WAG/Rij rats, we applied picrotoxin (100 μM; 15–20 ml) to block GABAA conductance, that was elicited as an outward whole-cell current (Fig. 3C). No differences in the current density between rat strains were found, neither in 14/15 day old (3.45 pA/pF ± 1.06 pA/pF, n = 10 vs. 4.09 pA/pF ± 0.88 pA/pF, Wistar and WAG/Rij, p = 0.31, Mann-Whitney test, Fig. 3D), nor in 21/22 day old group (3.58 pA/pF ± 0.97 pA/pF, n = 6 vs. 3.34 pA/pF ± 0.54 pA/pF, Wistar and WAG/Rij, p = 0.94, Mann-Whitney test, Fig. 3D). 3.4. Age-dependent changes in IGL inhibition Although beyond the main scope of this study, interesting developmental observations can be performed with the comparison between two age groups in both Wistar and WAG/Rij rats. There were no significant changes in mIPSC amplitude between 14/15 and 21/22 day old rats of both strains (Wistar: 80.24 pA ± 4.07 pA, n = 23 vs. 64.95 pA ± 1.7 pA, n = 18; WAG/Rij: 90.61 pA ± 6.5 pA vs. 72.96 pA ± 4.19 pA, 14/15 and 21/22, pWistar = 0.19, pWAG/Rij = 0.10, Student's unpaired ttest, Fig. 4A). The mIPSC frequency did not change with age neither in Wistar (0.94 Hz ± 0.10 Hz, n = 23 vs. 0.87 Hz ± 0.14 Hz, n = 17, 14/ 15 and 21/22 day old, respectively, p = 0.43, Mann-Whitney test, Fig. 4B) nor in WAG/Rij rats (0.87 Hz ± 0.08 Hz, n = 18 vs. 0.79 Hz ± 0.09 Hz, n = 17, 14/15 and 21/22 day old, respectively, p = 0.51, Student's unpaired t-test, Fig. 4B). However, mIPSC kinetics (both the rise time and decay constant) were shortened in older animals, that was significant in Wistar rats (rise time: 1.70 ms ± 0.06 ms, n = 23 vs. 1.56 ms ± 0.04 ms, n = 17; decay constant: 4.88 s ± 0.40 s, n = 23 vs. 3.53 s ± 0.19 s, n = 17, 14/15 and 21/22 day old, respectively, prise = 0.045, Mann-Whitney test, pdecay = 0.0097, Student's unpaired t-test, Fig. 4C,D). In case of WAG/Rij rats, the only difference in mIPSC kinetics was noted for decay constant (4.93 s ± 0.38 s, n = 18 vs. 3.80 s ± 0.22 s, n = 17, 14/15 and 21/22, p = 0.027, Mann Whitney test, Fig. 4D), but not for rise time (1.73 s ± 0.05 s, n = 18 vs. 1.69 s ± 0.07 s, n = 17, 14/15 and 21/22, p = 0.42, Mann Whitney test, Fig. 4C).

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Fig. 1. Abnormalities in IGL miniature synaptic GABAergic transmission in WAG/Rij rats. (A) Raw mIPSC recording (holding potential = −90 mV; TTX 0.5 μM, CNQX 10 μM, AP-5 40 μM) of an IGL neuron in Wistar and WAG/Rij rats. Downward deflections were found to be mIPSC as they vanished in the presence of bicuculline (20 μM). (B) In the 14/15 day old group, the mIPSC amplitude was lower in WAG/Rij than in Wistar rats, reflected by the change in the cumulative distribution and mean amplitude value (**, p = 0.005, Student's unpaired ttest). (C) No changes in mIPSC frequency were noted. Similar changes were measured in 21/22 day old rats, i.e. the mIPSC amplitude was lower in WAG/Rij rats (D; *, p = 0.039, Student's unpaired t-test), with no differences in frequency of recorded events (E). (F, H) No changes in mIPSC kinetics were found, neither in 14/15 nor in 21/22 day old rats. (G, I) Averaged mIPSC recorded from IGL neurons in Wistar and WAG/Rij rats are presented for both 14/15 and 21/22 day old rats, respectively. Cumulative probability histograms were constructed for each individual cell and averaged, subsequently. All data were analysed based on 200 s recording traces, regardless the number of events. At all bars, curves and traces, grey indicates Wistar and red indicates WAG/Rij rats.

The comparison of sIPSC between two age groups revealed similar changes to those observed in case of mIPSC. The amplitude of sIPSC did not vary significantly between younger and older animals, neither in Wistar (73.88 pA ± 6.38 pA, n = 17 vs. 82.07 pA ± 8.22 pA, n = 13, 14/15 and 21/22 days old, respectively, p = 0.43, Student's unpaired t-test, Fig. 4E) nor in WAG/Rij rats (59.52 pA ± 3.46 pA, n = 17 vs. 62.05 pA ± 3.65 pA, n = 13, 14/15 and 21/22 days old, p = 0.64, Student's unpaired t-test, Fig. 4E). However, sIPSC frequency was lower in older Wistar rats (3.79 Hz ± 0.67 Hz, n = 17 vs. 1.72 Hz ± 0.38 Hz, n = 13, 14/15 and 21/22 day old, p = 0.022, Mann-Whitney

test, Fig. 4F), what was not observed in case of WAG/Rij rats (1.12 Hz ± 0.13 Hz, n = 23 vs. 0.93 Hz ± 0.15 Hz, n = 12, 14/15 and 21/22 day old, p = 0.65, Student's unpaired t-test, Fig. 4F). As in case of mIPSC, the sIPSC kinetics were distinctly shortened in 21/22 day old rats in comparison to 14/15 day old group. This was true for both Wistar (rise time: 2.22 ms ± 0.11 ms, n = 17 vs. 1.65 ms ± 0.04 ms, n = 13; decay constant: 5.50 s ± 0.34 s, n = 17 vs. 3.41 s ± 0.14 s, n = 13, 14/15 and 21/22 day old, prise = 0.0001, pdecay b 0.0001, Student's unpaired t-test, Fig. 4G,H) and WAG/Rij rats (rise time: 2.15 ms ± 0.10 ms, n = 23 vs. 1.69 ms ± 0.08 ms, n = 12; decay constant:

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Fig. 2. Dampened spontaneous synaptic GABAergic transmission in IGL of WAG/Rij rats. (A) The sIPSC amplitude was lower in the 14/15 day old WAG/Rij than in Wistar rats, reflected by the change in the cumulative distribution and mean amplitude value (*, p = 0.041, Student's unpaired t-test). (B) The sIPSC frequency was also lower in WAG/Rij rats, what was shown as the change in the cumulative distribution (higher inter-event intervals) and mean frequency value (***, p = 0.0001, Mann-Whitney test). (C, D) Similar changes were measured in 21/ 22 day old rats, i.e. the sIPSC amplitude was lower in WAG/Rij rats (*, p = 0.041, Student's unpaired t-test; C), together with lower sIPSC frequency (*, p = 0.034, Mann-Whitney test; D). (E, G) The only significant difference in sIPSC kinetics was found between decay constants in 14/15 day old rats (*, p = 0.015, Student's unpaired t-test). (F, H) Averaged sIPSC recorded from IGL neurons in Wistar and WAG/Rij rats are presented for both 14/15 and 21/22 day old rats, respectively. Cumulative probability histograms were constructed for each individual cell and averaged, subsequently. All data were analysed based on 200 s recording traces, regardless the number of events. At all bars, curves and traces, grey indicates Wistar and red indicates WAG/Rij rats.

7.12 s ± 0.48 s, n = 23 vs. 3.66 s ± 0.28 s, n = 12, 14/15 and 21/22 day old, prise = 0.0037, pdecay b 0.0001, Student's unpaired t-test, Fig. 4G,H). Finally, we compared GABA-evoked whole-cell current density, which was significantly increased in the 21/22 day old group in both Wistar (45.77 pA/pF ± 9.53 pA/pF, n = 11 vs. 93.68 pA/pF ± 20.20 pA/pF, n = 15, 14/15 and 21/22 days old, respectively, p = 0.047, Mann-Whitney test, Fig. 3B) and WAG/Rij rats (60.69 pA/pF ± 18.01 pA/pF, n = 13 vs. 108.70 pA/pF ± 14.34 pA/pF, n = 15, 14/15 and 21/22 days old, respectively, p = 0.007, Mann-Whitney test, Fig. 3B). In the case of tonic outward current evoked by picrotoxin application, no age related changes were noted when current density was compared, both in Wistar (3.45 pA/pF ± 1.06 pA/pF, n = 10 vs. 3.58 pA/pF ± 0.97 pA/pF, n = 6, 14/15 and 21/22, p = 0.71, Mann-Whitney test, Fig. 3D) and WAG/Rij rats (4.09 pA/pF ± 0.88 pA/pF, n = 10 vs. 3.34 pA/pF ± 0.54 pA/pF, n = 9, 14/15 and 21/22, p = 0.49, Student's unpaired t-test, Fig. 3D). 3.5. IGL neurons exhibit ISO even in darkness in the absence epilepsy model During our in vivo experiments, the infra-slow oscillatory (ISO) activity was extracellularly recorded from the IGL, as shown previously in adult Wistar rats (Lewandowski et al., 2000, 2002; Lewandowski

and Błasiak, 2004; Blasiak and Lewandowski, 2013). In Wistar rats, ISO activity occurred in photopic conditions in all recorded IGL neurons (n = 10) and diminished completely in darkness in seven cases. For the remaining three cells, a residual activity pattern resembling the beginning and the end of intrabursts was retained. Therefore, for WAG/Rij rats, two distinct ambient lighting conditions were chosen: light-on and light-off, which refer to strong photopic (~900 lx) and scotopic (~1 lx) conditions. Surprisingly, the rhythmic pattern was maintained in WAG/Rij rats during light-off conditions (n = 8). The ISO activity recorded within the IGL in two rat strains is shown in Fig. 5. In both Wistar and WAG/Rij rats, oscillatory activity was detected by the presence of secondary peaks in the autocorrelation function (Fig. 5B, G) and bimodal frequency distributions (Fig. 5C, H). The application of FFT algorithms indicated the period of the firing pattern, which was close to 2 min (Fig. 5D, I). In Wistar rats, a significant decrease was noted in the mean firing rate under light-off conditions (4.07 Hz ± 0.71 Hz vs. 0.37 Hz ± 0.18 Hz, n = 10, p = 0.002, Wilcoxon test, Fig. 5E). Parallel analysis performed in WAG/Rij rats showed no significant differences between the two lighting conditions (11.04 Hz ± 3.47 Hz vs. 7.64 Hz ± 2.5 Hz, n = 8, light-on and light-off, respectively, p = 0.17, Student's paired t-test, data not shown). In the next step, we examined the possible differences between ISO characteristics under light-on and light-off conditions in WAG/Rij

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Fig. 3. Whole-cell GABAergic currents in the IGL. (A) Raw recording traces (holding potential = −90 mV; TTX 0.5 μM, CNQX 10 μM, AP-5 40 μM) obtained from IGL neurons in Wistar and WAG/Rij rats showing whole-cell currents evoked by GABA (100 μM, blue bars) application. (B) The bar graph shows no significant differences in current density between two rat strains in both 14/15 and 21/22 day old groups. The whole-cell current density evoked by tonic GABA application was significantly higher in older animals (Wistar: *, p = 0.047, Mann-Whitney test; WR: **, p = 0.007, Mann-Whitney test). (C) Raw recording traces showing tonic outward currents elicited by picrotoxin (100 μM, grey bars) application on IGL neurons. (D) No significant differences in current density were noted neither between two rat strains nor age groups. Grey indicates Wistar and red indicates WAG/Rij rats. In younger animals, both colours are light, whereas darker colours indicate older rats.

rats. The ISO periods obtained for each neuron under two lighting conditions by means of FFT were compared, but no notable differences were found (117.5 s ± 13.95 s, n = 10 vs. 106.3 s ± 14.26 s, n = 8, light-on and light-off, respectively, p = 0.25, Wilcoxon test, Fig. 5J). The duration of the intra-/extraburst phases was another useful parameter to describe rhythmic neuronal IGL activity. The mean length of the intraburst was shorter in darkness (59.70 s ± 10.48 s, n = 10 vs. 42.20 s ± 8.56 s, n = 8, light-on and light-off, respectively, p = 0.026, Student's unpaired t-test, Fig. 5K), but the mean length of the extraburst did not change when changes in the lightening surroundings occurred (63.40 s ± 10.12 s, n = 10 vs. 63.31 s ± 5.27 s, n = 8, light-on and light-off, respectively, p = 0.38, Wilcoxon test, Fig. 5K). To complete the ISO analysis in WAG/Rij rats, the global frequency within the intra- and extraburst phases was calculated. Neither the intra- nor extraburst global firing rate showed evident differences between light-on and light-off conditions (intraburst: 17.08 Hz ± 4.85 Hz vs. 14.42 Hz ± 4.80 Hz, n = 8, p = 0.43, Student's unpaired t-test, Fig. 5L; extraburst: 5.01 Hz ± 1.76 Hz vs. 4.07 Hz ± 1.83 Hz, n = 8, light-on and light-off, respectively, p = 0.38, Wilcoxon test, Fig. 5L). 3.6. IGL oscillatory neurons in WAG/Rij rats display higher neuronal activity in vivo Next, we set out to determine whether the oscillations vary between strains. In order to address this issue, periods of rhythmic patterns recorded under photopic conditions (~900 lx) were compared between Wistar and WAG/Rij rats. Unexpectedly, the period of ISO activity did not differ between groups (125 s ± 6 s, n = 10 vs. 117.5 s ± 13.95 s, n = 8, Wistar and WAG/Rij rats, respectively, p = 0.17, Mann-Whitney test, Fig. 6A). The difference in the mean duration of the activity phase was at the limit of statistical significance; a tendency toward a reduced intraburst length in WAG/Rij rats was noted (81.67 Hz ± 5.49 Hz, n = 10 vs. 59.70 s ± 10.48 s, n = 8, Wistar and WAG/Rij rats, respectively, p = 0.06, Student's unpaired t-test, Fig. 6B). Moreover, mean extraburst length was significantly shorter in Wistar rats in comparison to the

WAG/Rij group (42.89 s ± 3.31 s, n = 10 vs. 63.40 s ± 10.12 s, n = 8, Wistar and WAG/Rij rats, respectively, p = 0.01, Mann-Whitney test, Fig. 6B). Thus, it is worth noting that, in contrast to the Wistar strain, oscillations recorded in WAG/Rij rats were characterised by a short intraburst phase preceded by a long extraburst phase. Also, in these animals, the overall activity was higher both in the light-on and light-off surroundings. To confirm that the ISO patterns between strains were indeed different, the global frequency of high activity and the silent phase was estimated. It was found that the global rate of the intraburst phase was clearly elevated in WAG/Rij rats (5.93 Hz ± 1.03 Hz, n = 10 vs. 17.08 ± 4.85, n = 8, Wistar and WAG/Rij rats, respectively, p = 0.02, Student's unpaired t-test, Fig. 6C). Similarly, the global frequency of the extraburst phase was also apparently increased within this group (0.8 Hz ± 0.26 Hz, n = 10 vs. 5.01 Hz ± 1.76 Hz, n = 8, Wistar and WAG/Rij rats, respectively, p = 0.02, Student's unpaired t-test, Fig. 6C).

3.7. GFAP-positive IGL astrocytes in physiology and absence epilepsy In order to evaluate the accompanying differences in glial morphology, we performed immunohistochemical staining for GFAP-positive astrocytes. As gliosis may underlie changes in neuronal inhibition (Ortinski et al., 2010), the intensity of fluorescence expressed as the mean grey value and the area encompassed by GFAP-positive cells were compared between Wistar and WAG/Rij rats. In total, 25 and 23 slices containing IGLs of the matched anterior-posterior coordinates obtained from three Wistar and three WAG/Rij rats, respectively; these were successfully stained and analysed. The mean grey value was significantly higher in IGL slices obtained from WAG/Rij rats (138.90 ± 1.93, n = 25 vs. 147.20 ± 2.80, n = 23, Wistar and WAG/Rij rats, respectively, p = 0.017, Student's unpaired t-test, Fig. 7). Similarly, the area fraction of GFAP-immunoreactive astrocytes was higher in the absence epilepsy model (9.81% ± 1.59%, n = 25 vs. 19.44% ± 3.76%, n = 23, Wistar and WAG/Rij rats, respectively, p = 0.019, Student's unpaired t-test, Fig. 7). In the whole manuscript, astrogliosis was defined as an increased

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Fig. 4. Age-related differences in GABAergic currents. (A) The amplitude of mIPSC did not significantly differ between 21/22 compared to 14/15 day old rats. This observation did not differ between Wistar and WAG/Rij rats. (B) No differences between age groups were noted in mIPSC frequency. (C, D) mIPSC kinetics were faster in older rats as reflected by the shorter rise time (significant only for Wistar rats: *, p = 0.045, Mann-Whitney test; C) and decay constant (Wistar: **, p = 0.009, Mann-Whitney test; WR: *, p = 0.027, Mann-Whitney test; D). (E) No significant differences in sIPSC amplitude were observed between two age groups in both rat strains. (F) The frequency of sIPSC was significantly higher in younger Wistar rats (*, p = 0.022, Mann-Whitney test) but did not differ in WAG/Rij rats. (G, H) sIPSC kinetics were also faster in older rats — characterised by both shorter rise time (Wistar: ***, p = 0.0001, Student's unpaired t-test; WR: **, p = 0.004, Student's unpaired t-test; G) and decay constant (Wistar: ****, p b 0.0001, Student's unpaired t-test; WR: ****, p b 0.0001, Student's unpaired t-test; H). Grey bars indicate Wistar and red bars indicate WAG/Rij rats. In younger animals, both colours are light, whereas darker colours indicate older rats.

GFAP staining, however, the putatively reactive astrocytes were seen in WAG/Rij rats, what is presented in the bottom panel of Fig. 7. 4. Discussion 4.1. Synaptic and extrasynaptic GABAergic inhibition in physiology and absence epilepsy GABA is the most abundant inhibitory substance in the adult brain as is considered to be the primary neurotransmitter of the circadian timing system (Moore and Speh, 1993). The significance of GABAergic inhibition to IGL physiology has been the subject of previous studies

describing both the electrophysiological effects of GABAergic agents and the types of GABAA receptor subunits (Gao et al., 1995; Harrington, 1997; Palus et al., 2015). The spontaneous activity of GABAergic synapses can be recorded by patch-clamp as a phasic inhibition elicited by inhibitory postsynaptic currents (IPSC). This can be done in two variants: when the neuronal activity in the slice is preserved (spontaneous IPSC) or in the presence of TTX (miniature IPSC). The results provided by this study show significantly reduced phasic GABAergic inhibition upon IGL neurons in presymptomatic WAG/Rij rats in comparison to Wistar rats, expressed as a lower amplitude of both mIPSC and sIPSC. Interestingly, no differences in the frequency nor in the kinetics of mIPSC were noted, which implies postsynaptic

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Fig. 5. Infra-slow oscillatory (ISO) activity in IGL of Wistar and WAG/Rij rats. (A) Exemplary recording (firing histogram, in grey) of a single ISO neuron in light-on and light-off conditions (bin size = 1 s) in Wistar rat. Note the disappearance of ISO activity in darkness. (B) The temporal regularity of spiking is reflected by waves in the autocorrelation function only under light-on conditions. (C) The frequency histogram of the firing frequencies shows the bimodal distribution only in constant light. (D) The period of oscillations under light-on conditions was in the range of two minutes, as assessed by fast Fourier transformation (FFT). (E) In Wistar rats, the mean firing rate was significantly reduced in darkness (**, p = 0.002, Wilcoxon test, n = 10). (F) Analogous recording of a single ISO neuron in WAG/Rij rats under both light conditions (in red). Note the presence of ISO activity even in darkness. The regularity of spiking (G), the bimodal distribution (H) and periodicity (I) persisted under light-off conditions. (J) The mean period of ISO in all oscillatory IGL neurons in WAG/Rij rats was insignificantly shorter after the light was switched off. (K) In darkness, the mean length of the intraburst was shortened (*, p = 0.026, Student's paired t-test, n = 8) with no changes in the mean extraburst length. (L) The mean firing rate was not affected by light conditions in any oscillation phase. In all graphs, orange indicates light-on (sustained lightening ~900 lx) and black indicates light-off conditions (sustained darkness ~1 lx).

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Fig. 6. Comparison of infra-slow oscillatory (ISO) activity parameters in IGL between control Wistar rats and the absence epilepsy model — WAG/Rij rats, in light-on conditions (sustained illumination ~ 900 lx). (A) The mean period of oscillations did not differ between strains. (B) ISO activity in WAG/Rij rats was characterised by insignificantly shorter intrabursts (p = 0.067, Student's unpaired t-test, n = 10 and 8, respectively) and significantly longer extrabursts (*, p = 0.012, Mann-Whitney test). (C) ISO neurons in IGL were more active in WAG/Rij rats, as reflected by the higher firing rate in both intra- (*, p = 0.023, Student's unpaired t-test) and extrabursts (*, p = 0.017, Student's unpaired t-test). Grey bars indicate Wistar and red bars indicate WAG/Rij rats.

disturbances in GABAA receptors rather than a reduction in neurotransmitter release, such as differences in the phosphorylation state, channel density, subunit composition or subcellular localisation (Krishek et al., 1994; Browne et al., 2001). In case of sIPSC, a potent reduction of frequency in WAG/Rij rats has been observed, what can further result in insufficient inhibition seen during in vivo experiments, at which activity-dependent GABA release is intact. The differences in IPSC characteristics presented in this study were persistent in both age groups investigated (14/15 and 21/22-days old rats). No differences were noted between putatively NPY-synthesising (IA-expressing) and enkephalinergic (IT-expressing) IGL neurons, that implies a subpopulation-independent disinhibition of the whole IGL network. In other studies, similar impairments in GABAergic inhibition in WAG/Rij rats has been shown in the neocortex (Luhmann et al., 1995), a neuronal site directly involved in SWD generation. Also, injection of bicuculline (a GABAA receptor blocker) into the thalamic reticular nucleus (TRN) was shown to increase SWD activity in Genetic Absence Epilepsy Rats from Strasbourg (GAERS; Aker et al., 2006). A decrease in GABAergic transmission has been shown to increase excitation, a characteristic of epilepsy. However, not only lowered inhibition in the thalamo-cortical loop may govern SWD generation, as enhanced GABAergic inhibition in the hippocampus of WAG/Rij rats has been shown to reduce the SWD number (Tolmacheva and van Luijtelaar, 2007). Taken together, these findings suggest that lowered GABA-dependent inhibition in those structures, consistent with our study, is epileptogenic. On the other hand, a greater amplitude of mIPSC and enhanced single channel chloride conductance have been shown in the intralaminar thalamic nucleus of WAG/Rij rats (Brockhaus and Pape, 2011). Therefore, the statement that GABAergic inhibition in absence epilepsy is disrupted or unbalanced seems to be more appropriate than generally lowered or enhanced. GABAergic inhibition can occur both by the activation of phasic GABAA receptors localised within synapses and persistent stimulation of extrasynaptic GABAA receptors, resulting in tonic suppression of neuronal activity (Mody and Pearce, 2004; Farrant and Nusser, 2005). Enhanced tonic GABAergic inhibition has been recently proposed as the thalamic mechanism required for SWD generation (Cope et al., 2009). The results presented in this study show a slight enhancement of the tonic current elicited by prolonged application of 100 μM GABA onto

IGL neurons in WAG/Rij rats (presented as current density). However, probably due to the relatively high variability of responses, the difference in whole-cell current density did not reach statistical significance. Nevertheless, this tendency is worth noting due to the presence in two age groups (14/15 and 21/22 days old rats). The tonic inward GABAevoked current in our study represented the total current induced by activation of all (synaptic and extrasynaptic) GABAA and GABAB receptors, which are both functionally expressed in the IGL (Palus et al., 2015). Therefore, we cannot extract a specific target underlying the observed differences. Thus, this small enhancement of the total GABA-evoked current may represent a compensatory mechanism opposing the significant reduction in phasic GABAergic currents. Furthermore, the application of picrotoxin, presented in this study, resulted in tonic outward currents, presumably due to the closure of extrasynaptic GABAA receptors. The density of picrotoxin-elicited current did not differ between Wistar and WAG/Rij rats, what may suggest the similar expression of sustained, tonic GABAergic currents in two rat strains. Our patch-clamp in vitro research on presymptomatic WAG/Rij rats was carried out in strict age-matched experimental groups (postnatal day 14/15 and 21/22), since previous reports have shown developmental changes in GABAergic signalling in the absence epilepsy model (Li et al., 2007). The precise mechanism of the age-related changes in GABAA receptor subunit composition remains unknown. However, in another study carried out on intralaminar thalamic neurons, no significant changes in mIPSC parameters from a postnatal age (postnatal days 14–22) were observed (Brockhaus and Pape, 2011). In the present study, we did not observe any significant age-related changes in the mIPSC/sIPSC amplitude, either in Wistar or WAG/Rij rats. As the mIPSC frequency was not altered by age, the sIPSC frequency was lowered in older Wistar rats. The IPSC rise time and decay constant were shortened with age, what was even more distinct for sIPSC. This constitutes an interesting physiological observation, as single IPSCs characterised by faster kinetics are less likely to be averaged and may produce an apparently lower conductance. In contrast, the data show that total inhibition evoked by tonic GABA application increased with age. Summing up, our results suggest that the GABAergic network changes with age in the IGL, becoming more sensitive to GABA application and more selective in receiving phasic inhibition.

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Fig. 7. Astrogliosis in IGL in the absence epilepsy model assessed by GFAP immunoreactivity. (above) Confocal micrographs (single planes) showing GFAP immunoreactivity (in green) in the IGL and neighbouring areas. Note that the IGL can be differentiated from the lateral geniculate complex. White scale bars correspond to 100 μm. (upper right) Astrocyte hypertrophy is indicated by the larger area encompassed by GFAP-immunoreactive processes (*, p = 0.019, Student's unpaired t-test) and greater staining intensity (*, p = 0.017, Student's unpaired ttest). Grey bars indicate Wistar and red bars indicate WAG/Rij rats. (middle panel) Enlargement of the IGL area assessed by the NPY immunoreactivity. From left: GFAP immunoreactive astocytes (in green); NPY immunoreactivity (in magenta); merged signal. White scale bars correspond to 50 μm. (below) Representative high magnifications of GFAP-immunoreactive astrocytes. Note the putatively reactive astrocytes in WAG/Rij rats. White scale bars correspond to 10 μm.

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4.2. Disturbance of the infra-slow oscillatory pattern in the IGL in the absence epilepsy model One of the IGL subpopulations was shown to generate action potentials in the isoperiodic fashion in the extracellular in vivo recordings under urethane anaesthesia. This firing pattern, called infra-slow oscillations (ISO), consists of extra- and intraburst phases of neuronal activity repeated in time with a period close to 2 min (b 0.01 Hz; Lewandowski et al., 2000; Lewandowski and Błasiak, 2004). This kind of activity was found to be expressed by neurons that connect contralaterally located IGLs, which have been shown to be enkephalinergic in the rat (Mikkelsen, 1992; Blasiak and Lewandowski, 2013). Increasing evidence suggests that ISO activity is generated in the retina, since intraocular injections of TTX contralateral to the recording site abolish this rhythmic firing (Lewandowski and Błasiak, 2004). Moreover, the ISO pattern is absent in in vitro slice preparations during both extracellular and patch-clamp recordings (Blasiak and Lewandowski, 2004; Szkudlarek and Raastad, 2007). Not only IGL neurons are known to generate ISO: it has been also shown in other retinorecipient structures such as the SCN, OPN, DLG and VLG (Miller and Fuller, 1992; Albrecht and Gabriel, 1994; Albrecht et al., 1998; Filippov and Frolov, 2005; Szkudlarek et al., 2008). Presumably, due to their occurrence in OPN, ISO are reflected in the pupil diameter (Blasiak et al., 2013). To our great interest, the ISO pattern was modulated by constant light. As this rhythmic firing shares a common generator in ISO-expressing neurons in different visual system structures, we hypothesise that their maintenance or abolishment in darkness depends on the network properties within the ISO-receiving nucleus. For example, the ISO pattern is highly synchronised between the IGL and OPN on the ipsilateral site, as both structures receive oscillatory input from the contralateral retina (Szkudlarek et al., 2008). But, in the case of OPN neurons, the ISO pattern remains in light-off conditions (Szkudlarek et al., 2012), whereas in the IGL, the neuronal activity of oscillatory cells is highly suppressed in darkness and the rhythmicity is lost (Lewandowski et al., 2000; Blasiak and Lewandowski, 2013). As the IGL network, unlike the OPN, is highly GABAergic, we hypothesise that, due to the strong inner inhibition, the excitatory input arising from spontaneous retinal activity (preserved in darkness) is not sufficient to sustain neuronal activity and the ISO pattern in the IGL. This assumption is strengthened by the results presented in this study. As we have generally found that ISO activity is abolished under light-off conditions in the IGL neurons of Wistar rats, in three cases, residual activity resembling the beginning and the end of intraburst was preserved in darkness. The results presented in this study show the occurrence of the ISO pattern in the IGL recorded in adult urethane-anesthetised WAG/Rij rats. Most interestingly, this oscillatory pattern, however changed, persisted under light-off conditions in all IGL neurons recorded. The period of oscillations does not seem to be significantly affected by light, although it was slightly shorter in darkness. The absence of changes in the ISO period between light-on and light-off conditions was also described in the OPN of Wistar rats (Szkudlarek et al., 2012). Interestingly, in our study, the separate analysis of intra- and extrabursts showed that the active phase of ISO in the IGL of WAG/Rij rats was significantly shorter after the light was switched off. The illumination level did not alter the silent phase length, and changed lighting conditions were not able to affect the firing rate, either in the intra- or extraburst phase. The ISO pattern comparison under light-on conditions between the two rat strains shows the differences in both oscillation phase lengths and firing frequencies. In general, IGL neurons in the absence epilepsy model can be characterised by significantly longer extrabursts and a visible shortening of the active phase (p = 0.067, Student's unpaired ttest), with no differences in period length. Due to the fact that the mechanism of ISO is not known, it is impossible to distinguish if the observed pattern abnormalities stem from disturbed retinal function (Lai et al., 1975; O'Steen and Donnelly, 1982) or mechanisms intrinsic to the IGL. Thus, a difference in firing frequency was noted between the two

strains: IGL neurons recorded from WAG/Rij rats can be described as significantly more active during intra- and extrabursts in comparison to those in Wistar rats. We hypothesise that this generally higher activity of IGL neurons in the absence epilepsy model may be the result of insufficient inhibition within the IGL network itself. Our in vivo results are in the agreement with the in vitro experiments presented in this study. The lowered phasic inhibition of IGL neurons observed in presymptomatic WAG/Rij rats could perturb the delicate balance of excitatory and inhibitory synaptic inputs, which may lead to an altered change in firing rate in different constant lighting conditions. Therefore, the data presented here show that IGL neurons in the model of absence epilepsy are pathologically disinhibited, which probably stems from changes on the level of GABAA receptors observed even before seizure onset. The observed phenomenon may result from other disturbances caused by the epileptic phenotype but not explained by our study. According to the literature, a various pathologies have been reported in the WAG/Rij rat retina, which may be the cause of the altered response to light observed at the level of the IGL (Lai et al., 1975; O'Steen and Donnelly, 1982). However, due to the abnormal degeneration of photoreceptors, one would suspect the opposite result, i.e. impaired and not enhanced activity under light-on conditions. The other possibility is abnormal modulation of the IGL firing rate by modulatory brain systems, as previously suggested as one of the causes of SWD (Snead, 1995). 4.3. Gliosis in IGL in absence epilepsy Previous studies have reported an interesting feature of differentiating biological clock structures (IGL and SCN) in the surrounding nuclei, i.e. a high density of GFAP-positive astrocytes (Morin et al., 1989; Harrington, 1997). In our study, we focused on possible changes in IGL astrocytes due to their involvement in neuronal inhibitory transmission. First, the production of GABA in neurons is astrocyte-dependent, and the loss of astrocyte function results in selective dysfunction of GABAergic synapses (Liang et al., 2006; Fricke et al., 2007). Second, gliosis (assessed by enhanced GFAP production) leads to specific impairments in inhibitory and not excitatory transmission, manifested in a lower IPSC amplitude (Ortinski et al., 2010), thus increased expression of glial glutamate transporters was reported in reactive astrocytes (Aronica et al., 2000; Vermeiren et al., 2005). Finally, astrogliosis is also associated with network hyperexcitability in epilepsy (Ang et al., 2006). Our results indicate increased production of GFAP by astrocytes located in the IGL (marked by NPY-immunoreactivity) and their hypertrophy. Based on the rationale presented above, we hypothesise that IGL astrogliosis in the model of absence epilepsy may underlie the observed changes in neuronal electrophysiology. To our knowledge, we are the first to show astrogliosis in biological clock structures in the absence epilepsy model, although impairments in glial cell functioning in other nuclei have been observed before in WAG/Rij rats (Akin et al., 2011; Sitnikova et al., 2011). 4.4. Possible functional significance of IGL GABAergic transmission in absence epilepsy The direct mechanisms of SWD generation are restricted to the hypersynchronous thalamo-cortical loop, but a local imbalance in the functioning of other brain regions may lead to enhancement of the epileptic phenotype (Epps and Weinshenker, 2013). As IGL neurons do not project to the neocortex (Harrington, 1997), direct involvement of the observed abnormalities in the SWD generation seems unlikely. However, insufficiencies in the sleep-promoting system, localised in ventrolateral preoptic area, have been observed in WAG/Rij rats (Suntsova et al., 2009), which reflects the sleep architecture abnormalities seen in epilepsy patients (Baldy-Moulinier, 1992; Maganti et al., 2006). The source of the excessive inhibition upon the VLPO in WAG/Rij rats is still unknown. Moreover, biological clock control over sleep-promoting

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structures is still unclear. Previous studies have shown that the major clock (SCN) does not innervate the VLPO in a direct manner (Chou et al., 2002; Saint-Mleux et al., 2007). It was also shown that, although sparse, there is a direct, unilateral and unidirectional innervation of the VLPO by the IGL (Chou et al., 2002; Morin and Blanchard, 2005). Therefore, we hypothesise that the disinhibition of GABAergic IGL neurons may play a role in the mechanism of sleep-related changes in WAG/Rij rats. As the function of ISO activity in the visual system has not yet been established, it is hard to hypothesise about the role of its altered response to constant light conditions. However, it is known that, under physiological conditions, the activity of ISO expressed by IGL neurons is synchronised with the OPN (Szkudlarek et al., 2008) and probably other visual system structures. Moreover, this characteristic pattern in the IGL was recorded exclusively under light-on conditions in Wistar rats (Lewandowski et al., 2000, 2002; Lewandowski and Błasiak, 2004; Blasiak and Lewandowski, 2013). In WAG/Rij rats, the synchronisation of neuronal activity also takes place under light-off conditions, which resembles the excessive synchronisation observed in other neuronal systems in absence epilepsy (see, e.g. McCormick and Bal, 1997; Crunelli and Leresche, 2002). 5. Conclusions Our study identifies alternations consistent with disinhibition of the IGL network in WAG/Rij rats at different levels. First, we have shown dampened GABAergic synaptic transmission, reflected in a lower IPSC amplitude and sIPSC frequency measured during patch-clamp experiments in vitro. Second, we correlated the observed insufficiencies in phasic inhibition with enhanced activity of ISO neurons in vivo, which were accompanied by an altered reaction to lighting conditions. Finally, immunohistochemical staining showed astrogliosis in the IGL, which has been associated with altered GABAergic transmission at the neuronal site. Together with our previous findings (Chrobok et al., 2016), the data presented in this paper show abnormal functioning of the IGL network in this model of absence epilepsy. Our results are the first to propose the involvement of circadian-related nuclei in the WAG/Rij model of absence epilepsy, with a potential role in circadian disturbances in absence epilepsy. Acknowledgements We wish to thank Dr. P. Orlowska-Feuer for practical training in extracellular single-unit in vivo recording techniques. We also would like to thank Dr. Z Soltys and Dr. J. Kula for help with data analysis. This study was supported by the Ministry of Science and Higher Education project “Diamentowy grant III” 0001/DIA/2014/43, 2014-2017. LC was additionally supported by the Polish National Science Centre doctoral scholarship “Etiuda IV” 2016/20/T/NZ4/00273. The research was carried out using equipment purchased through financial support from the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08). References Aker, R.G., Özyurt, H.B., Yananli, H.R., Çakmak, Y.Ö., Özkaynakçi, A.E., Sehirli, Ü., Saka, E., Çavdar, S., Onat, F.Y., 2006. GABAA receptor mediated transmission in the thalamic reticular nucleus of rats with genetic absence epilepsy shows regional differences: functional implications. Brain Res. 1111, 213–221. Akin, D., Ravizza, T., Maroso, M., Carcak, N., Eryigit, T., Vanzulli, I., Aker, R.G., Vezzani, A., Onat, F.Y., 2011. IL-1β is induced in reactive astrocytes in the somatosensory cortex of rats with genetic absence epilepsy at the onset of spike-and-wave discharges, and contributes to their occurrence. Neurol. Dis. 44, 259–269. Albrecht, D., Gabriel, S., 1994. Very slow oscillations of activity in geniculate neurons of urethane-anaesthetised rats. Neuroreport 5, 1909–1912. Albrecht, D., Royl, G., Kaneoke, Y., 1998. Very slow oscillatory activities in lateral geniculate neurons of freely moving and anaesthetised rats. Neurosci. Res. 32, 209–220.

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