Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
J Neurophysiol 112: 2888 –2900, 2014. First published September 10, 2014; doi:10.1152/jn.00406.2014. Regular-spiking cells in the presubiculum are hyperexcitable in a rat model of temporal lobe epilepsy Saad Abbasi and Sanjay S. Kumar Department of Biomedical Sciences, College of Medicine and Program in Neuroscience, Florida State University, Tallahassee, Florida Submitted 29 May 2014; accepted in final form 5 September 2014 TLE; presubiculum; cell classification; hyperexcitability; electrophysiology THE PRESUBICULUM (PrS), DEFINED by Brodmann in the primate brain as area 27, is located between the subiculum and the parasubiculum, and is known to play a critical role in spatial information processing and memory consolidation (Boccara et al. 2010). Additionally, research from animal models of temporal lobe epilepsy (TLE) has implicated the PrS in driving epileptiform activity in neighboring brain structures including the entorhinal cortex (Tolner et al. 2005). Despite its potential to drive parahippocampal circuits, information on the physiological state of its neurons, especially under epileptic conditions, remains greatly limited. Address for reprint requests and other correspondence: S. S. Kumar, Dept. of Biomedical Sciences, College of Medicine, Suite 3300-B, Florida State Univ., 1115 West Call St., Tallahassee, FL 32306-4300 (e-mail: sanjay.kumar @med.fsu.edu). 2888 PrS is cytoarchietectonically distinguishable by a densely packed pyramidal cell layer (L) II that combines with LIII to form the superficial layers, which are separated from the deeper layers (V-VI) by a cell sparse LIV. Input to PrS arises from retrosplenial, cingulate, and visual cortices (Jones and Witter 2007; Kononenko and Witter 2012; van Groen and Wyss 1990a,b; Vogt and Miller 1983), anteroventral and laterodorsal thalamic nuclei (van Groen and Wyss 1990a), and anterior thalamus (Shipley and Sorensen 1975; Yoder et al. 2011). PrS is reciprocally connected with the subiculum (Funahashi and Stewart 1997; Harris et al. 2001; O’Mara et al. 2001) and sends outputs to retrosplenial cortex (Van Groen and Wyss 2003), anteroventral, anterodorsal, and laterodorsal thalamic nuclei (Seki and Zyo 1984; van Groen and Wyss 1990a; Yoder et al. 2011), all fields of hippocampus including the molecular layer of dentate gyrus (Kohler 1985), as well as a prominent bilateral projection to medial entorhinal area (MEA) (Caballero-Bleda and Witter 1994; Honda and Ishizuka 2004). Projections from PrS to MEA terminate in LIII and LI, providing both glutamatergic and ␥-aminobutyric acid (GABA)ergic input (van Haeften et al. 1997), originating predominantly from LIII of PrS (Honda et al. 2011). Projections from PrS to LIII of MEA are of significance to TLE because pyramidal cells in this layer are selectively lost in patients and rodent models of TLE (Du et al. 1993, 1995). Additionally, unilateral ablation of PrS and the adjoining parasubiculum prevents cell loss in MEA, providing a partial neuroprotective effect (Eid et al. 2001). Furthermore, animal model studies have demonstrated that stellate cells in LII of MEA are hyperexcitable in TLE (Bear et al. 1996; Kobayashi et al. 2003; Kumar and Buckmaster 2006; Scharfman et al. 1998) due in part to reduced inhibition mediated by loss of local GABAergic interneurons in LIII (Kumar and Buckmaster 2006). Yet, despite the loss of primary postsynaptic targets in MEA (LIII pyramids), PrS input to MEA is preserved and capable of evoking epileptiform activity specific to this region in epileptic animals (Tolner et al. 2005, 2007). Recently, we characterized PrS neurons into distinct groups using hierarchical cluster analysis based on electrophysiological criteria, establishing baselines for intrinsic properties mediating action potential waveform and synaptic drive in each of the neuron types (Abbasi and Kumar 2013). However, a direct assessment of the physiological state of PrS neurons in animals with TLE has not been previously undertaken and the current study addresses which of these neuronal populations are affected along with the nature of their physiological alterations. Using the well-established pilocarpine model of TLE (Buckmaster 2004), we show that only a subset of neurons from 0022-3077/14 Copyright © 2014 the American Physiological Society www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 Abbasi S, Kumar SS. Regular-spiking cells in the presubiculum are hyperexcitable in a rat model of temporal lobe epilepsy. J Neurophysiol 112: 2888 –2900, 2014. First published September 10, 2014; doi:10.1152/jn.00406.2014.—Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy, characterized by recurrent seizures originating in the temporal lobes. Here, we examine TLErelated changes in the presubiculum (PrS), a less-studied parahippocampal structure that both receives inputs from and projects to regions affected by TLE. We assessed the state of PrS neurons in TLE electrophysiologically to determine which of the previously identified cell types were rendered hyperexcitable in epileptic rats and whether their intrinsic and/or synaptic properties were altered. Cell types were characterized based on action potential discharge profiles followed by unsupervised hierarchical clustering. PrS neurons in epileptic animals could be divided into three major groups comprising of regularspiking (RS), irregular-spiking (IR), and fast-adapting (FA) cells. RS cells, the predominant cell type encountered in PrS, were the only cells that were hyperexcitable in TLE. These neurons were previously identified as sending long-range axonal projections to neighboring structures including medial entorhinal area (MEA), and alterations in intrinsic properties increased their propensity for sustained firing of action potentials. Frequency and amplitude of both spontaneous excitatory and inhibitory synaptic events were reduced. Further analysis of nonaction potential-dependent miniature currents (in tetrodotoxin) indicated that reduction in excitatory drive to these neurons was mediated by decreased activity of excitatory neurons that synapse with RS cells concomitant with reduced activity of inhibitory neurons. Alterations in physiological properties of PrS neurons and their ensuing hyperexcitability could entrain parahippocampal structures downstream of PrS, including the MEA, contributing to temporal lobe epileptogenesis. EXCITABILITY OF PRESUBICULAR NEURONS IN TLE LII-III of PrS are affected, thereby providing specific insights into the excitability of the PrS and its potential to drive aberrant activity in target structures. MATERIALS AND METHODS Animals Slice Preparation and Electrophysiology Rats were deeply anesthetized with urethane (1.5 g/kg ip) before being decapitated. Horizontal slices (350 m), at the level of the hippocampus between ⫺6.6 and ⫺5.3 mm from Bregma (Paxinos and Watson 2007), were prepared using a microslicer (VT1000S; Leica) in a chilled (4°C) low-Ca2⫹, low-Na⫹ “cutting solution” containing the following (in mM): 230 sucrose, 10 D-glucose, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, and 0.5 CaCl2 equilibrated with a 95%-5% mixture of O2 and CO2. Slices were allowed to equilibrate in oxygenated artificial cerebrospinal fluid (aCSF) containing the following (in mM): 126 NaCl, 26 NaHCO3, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 0.25 L-glutamine, and 10 D-glucose (pH 7.4), first at 32°C for 1 h and subsequently at room temperature before being transferred to the recording chamber. Recordings were obtained at 32 ⫾ 1°C from neurons in the PrS (average of 5.4 ⫾ 0.2 and 6.5 ⫾ 0.3 neurons per control and epileptic rats, respectively) under Nomarski optics (Zeiss) using a visualized infrared setup (Hamamatsu) that enabled the identification of their location within the various lamina. Patch electrodes were pulled from borosilicate glass (1.5-mm outer diameter, 0.75-mm inner diameter, 3– 6 M⍀) and contained the following (in mM): 105 potassium gluconate, 30 KCl, 10 HEPES, 10 phosphocreatine, 4 MgATP, 0.3 GTP, and 20 biocytin. Internal solution pH was adjusted to 7.3 with KOH and had an osmolarity of 300 mosM (uncorrected liquid junction potential, 12.6 mV at 32°C). The conditions applicable to recording from control animals were the same as those in epileptic animals. Slices were maintained in oxygenated (95% O2-5% CO2) aCSF, and drugs and chemicals were applied via the perfusate (2 ml/min). Tetrodotoxin (TTX; Sigma, St. Louis, MO) was bath applied as required for specific protocols. Bipolar electrodes (CE-2C75; Fredrick Haer, Brunswick, ME) with 25-m tip diameters were positioned in LII or LIII of PrS in the vicinity of the recorded neurons, and depolarizing constant current pulses, 20- to 200-A in amplitude, were applied at low frequencies (0.1– 0.3 Hz) to stimulate local afferent fibers that synapse with the recorded neurons to evoke action potentials. The latency to response at threshold in a subset of control and epileptic neurons was 1.9 ⫾ 0.1 (n ⫽ 14) and 2 ⫾ 0.1 ms (n ⫽ 21), respectively, typical of synaptic delays observed during intracortical stimulation. Postsynaptic currents and potentials were recorded using a MultiClamp 700B amplifier and pCLAMP software (Molecular Devices, Union City, CA), filtered at 1–2 kHz (10 kHz for current clamp), digitized at 10 –20 kHz, and stored electronically. Series resistance was monitored continuously, and those cells in which this parameter exceeded 20 M⍀ or changed by ⬎20% were rejected. Series resistance compensation was not used. Whole cell current-clamp recordings were obtained in response to injection of 1) 100 pA of depolarizing current (1–10 s in duration), and 2) hyper- and depolarizing current steps, 600 ms in duration and 50 pA in amplitude. Spontaneous (s) and miniature (m) excitatory postsynaptic currents (EPSCs) were obtained by holding the cell at ⫺70 mV, while inhibitory postsynaptic currents (IPSCs) were recorded at a holding potential of 0 mV, close to the reversal potential for glutamate. The postsynaptic current data, obtained from 2-minlong continuous recordings, were analyzed with Mini Analysis (Synaptosoft, Decatur, GA). The threshold for event detection was set at 3⫻ root mean square noise level, and software-detected events were verified visually before measuring their frequency and amplitude. Miniature postsynaptic currents (mPSCs) were isolated with TTX (1 M). Electrophysiological and Cluster Analysis We previously classified neurons from layers II and III of PrS using unsupervised hierarchical cluster analysis based on electrophysiological parameters gathered from neurons in slices from control animals. Clustering enabled establishment of a schema for neuronal classification, ultimately resulting in characterization of seven electrophysiologically distinct cell types in LII-III of PrS in control animals (Abbasi and Kumar 2013). A portion of the control data reported in this study is from our initial sampling of neurons and is included for comparative purposes. Electrophysiological data for our epileptic group were acquired from 52 neurons in acute brain slices from animals that were confirmed epileptic. Clustering was carried out in Origin (v. 8.6; OriginLab, Northampton, MA) with Ward’s method and squared Euclidean distances as the distance measure (Ward 1963). Briefly, neurons were sorted in a multidimensional space based on similarities of the electrophysiological variables considered. Clustering began with individual neurons in separate clusters, with each subsequent stage combining/reducing cluster number by one, based on the distance measure between neurons, until the final stage when a single cluster contained all of the neurons. Ward’s method uses an ANOVA approach in determining between-cluster distances. All variables were standardized before clustering. Cluster membership was determined by calculating the total sum of squared deviations from the cluster mean, and clusters were combined with the intent of minimizing increases in error sum of squares (Burns 2008). The final number of cluster groups in our analysis was determined with the Thorndike procedure (Thorndike 1953), in which large jumps in within-cluster distances associated with the clustering stages served to indicate salient differences between groups and cluster number. Cells from control and epileptic groups were analyzed identically. Nine distinct electrophysiological parameters gathered from currentclamp recordings of single neurons were considered in the analysis. These included the following: 1) early spike frequency adaptation, 2) late spike frequency adaptation, 3) delay, 4) accommodative hump, 5) sag ratio, 6) maximum firing frequency, 7) instantaneous firing frequency, 8) steady-state firing frequency, and 9) standard deviation of the steady-state firing frequency. The choice of parameters was based on empirical observations of the action potential waveforms of a large population of neurons in the PrS in control animals along with consideration of additional variables deemed most valuable in predicting group membership in alternate studies of the electrophysio- J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 Sprague-Dawley rats (male: n ⫽ 10 pilocarpine treated; n ⫽ 34 controls) from postnatal (P) days 40 – 85 were used in this study. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Florida State University Institutional Animal Care and Use Committee. Rats were made epileptic according to previously described protocols for bringing up the pilocarpine model of TLE (Buckmaster et al. 2004). Briefly, rats were treated with pilocarpine (P38; 153 ⫾ 4 g; 380 mg/kg ip) 20 min after atropine methylbromide (5 mg/kg ip). Diazepam (10 mg/kg ip) was administered 2 h after the onset of status epilepticus and repeated as needed. Following recovery from status epilepticus, rats were video monitored (40 h/wk) for spontaneous seizures. Animals used for electrophysiological experiments were confirmed epileptic (9 out of 10, of which 8 were used for electrophysiological experiments), displaying frank spontaneous recurrent seizures scored 3 or greater on the Racine scale (Racine 1972) on two or more observations during the 40 h/wk video monitoring. Recordings from epileptic animals were made on average 25 days poststatus epilepticus (range: 15–35 days; 298 ⫾ 22 g), with initial seizures observed between 9 and 25 days poststatus. Control groups consisted of age-matched naïve rats. 2889 2890 EXCITABILITY OF PRESUBICULAR NEURONS IN TLE NeuN-biocytin Immunohistochemistry Neurons were filled with biocytin during recording (20 mM, included in the internal solution). To visualize biocytin-labeled neurons after recording, slices were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) at 4°C for at least 24 h. After fixation, slices were stored in 30% ethylene glycol and 25% glycerol in 50 mM PB at ⫺20°C before being processed with a whole-mount protocol with counterstaining by NeuN immunocytochemistry. Slices were rinsed in 0.5% Triton X-100 and 0.1 M glycine in 0.1 M PB and then placed in a blocking solution containing 0.5% Triton X-100, 2% goat serum (Vector Laboratories, Burlingame, CA), and 2% bovine serum albumin in 0.1 M PB for 4 h. Slices were incubated in mouse anti-NeuN serum (1:1,000; MAB377; Chemicon, Temecula, CA) in blocking solution overnight. After a rinsing step, slices were incubated with the fluorophores Alexa 594 streptavidin (5 g/ml) and Alexa 488 goat anti-mouse (10 g/ml; Molecular Probes, Eugene, OR) in blocking solution overnight. Slices were rinsed, mounted on slides, and coverslipped with Vectashield (Vector Laboratories) before being examined with a confocal microscope (TCS SP2 SE; Leica). Brightness, contrast, and sharpness of photomicrographs were adjusted to highlight anatomical features in Photoshop (Adobe). Biocytin-labeled cells were visualized through a light microscope before reconstruction of soma and dendritic arbor in Neuroleucida (Micro-Brightfield, Colchester, VT). All statistical values are presented as means ⫾ SE. Statistical differences were measured with the unpaired Student’s t-test, unless otherwise indicated. RESULTS We recorded from a total of 52 neurons in layers II and III of PrS (Fig. 1, A and B) in brain slices from epileptic rats. These, combined with 185 neurons from control animals, enabled us to compare and assess the state of PrS in TLE. A portion of the control data is from our previous electrophysiological and morphological characterization of cell types in LII-LIII of the PrS (Abbasi and Kumar 2013). Interlaminar classification within PrS (Caballero-Bleda and Witter 1993) was as follows: PrS was identified in slices based on LII, a cytoarchietectonically distinguishing landmark consisting of a densely packed layer of pyramidal cells, juxtaposed with a less densely packed LIII that extended up to a cell-sparse LIV, a continuum of lamina dissecans in the MEA (Fig. 1B, left). Recorded neurons were selected randomly under differential interference contrast (DIC) optics, and laminar location was determined visually during recording and confirmed through biocytin-labeling and counterstaining for NeuN immunoreactivity. These sections were also used to confirm LIII cell loss in the MEA-a histopathological feature of TLE (Fig. 1B, right). Whole cell current-clamp recordings were used to determine resting membrane potential of neurons (Vm), single action potential properties, and attributes of action potential discharge profiles in response to depolarizing and hyperpolarizing current injections (see MATERIALS AND METHODS; Fig. 1C and Table 1). These parameters enabled classification of neurons into distinct groups via unsupervised hierarchical cluster analysis (Fig. 1, D and E) and assessment of alterations in intrinsic properties of neurons between the control and epileptic groups (Table 1). Additionally, we measured excitability in a subset of currentclamped neurons by determining the number of action potentials evoked as a function of stimulus intensity (Fig. 2). Stimulus intensity was measured as multiples of threshold (T), defined as the current required for evoking a single action potential on ⬃50% of the trails (Kumar and Buckmaster 2006). Whole cell voltage-clamp recordings of sEPSCs and sIPSCs from the same neurons allowed us to evaluate changes in the excitatory and inhibitory synaptic drive to individual neurons in control and epileptic animals (Figs. 3, 4, and 5 and Table 2). To determine if changes in the synaptic drive to neurons were related to alterations in number of synapses and/or probability of neurotransmitter release, mEPSCs and mIPSCs were recorded in the presence of TTX (1 M, added to aCSF bath solution) in a subset of voltage-clamped neurons (Fig. 6 and Table 2). Cell-Type Classification: Cluster Analysis We used unsupervised hierarchical cluster analysis to classify neurons based on electrophysiological parameters determined from whole cell current-clamp data. We previously addressed the electrophysiological heterogeneity of LII-III PrS neurons in control animals based predominantly on their action potential discharge profiles (Abbasi and Kumar 2013). We observed that electrophysiological properties varied significantly among the recorded neurons prompting a systematic quantitative approach to their characterization. Cluster analysis allowed us to classify these neurons without a priori knowledge of the number of groups. Our analysis yielded seven distinct clusters into which PrS neurons could be classified, consistent with our empirical observations. This technique further assists in the uncovering of data structures and the formulation of more objective schema for characterization and classification of neuronal subtypes in brain areas of interest (Cauli et al. 2000; Garrido-Sanabria et al. 2007; Halabisky et al. 2006; Nowak et al. 2003; Pennartz et al. 1998). To assess changes in intrinsic properties of these neurons in TLE, we remeasured these parameters in neurons from epileptic animals and repeated the cluster analysis with neurons from both groups. We reasoned that if intrinsic properties of neurons in the epileptic J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 logical classification of neocortical neurons (Abbasi and Kumar 2013; Cauli et al. 2000; Halabisky et al. 2006; Pilli et al. 2012). Parameters addressing the adaptive properties of cells were gathered from a 1-s long window of action potential trains evoked by a 100-pA depolarizing current pulse. The instantaneous firing frequencies between the first two spikes (finitial), 200 ms after the beginning of the discharge (f200), and at the end of the window (ffinal) were measured. Early and late spike frequency adaptation was calculated using (finitial ⫺ f200)/finitial and (f200 ⫺ ffinal)/finitial, respectively. Instantaneous and steady-state firing frequencies represented averages of the instantaneous firing frequencies of 10 action potentials at the beginning of a train and from a section where the discharge had reached steady state. Sag ratio, a measure of sag conductance (Gs), was determined as [(Vpeak ⫺ Vss)/Vpeak] ⫻ 100, where Vpeak and Vss are measurements of voltage immediately after a negative 100-pA, 600-ms hyperpolarizing current pulse. Vpeak is voltage measured at the negative-most point in the 600-ms window, and Vss is the measurement at the steady state after the initial voltage excursion. Accommodative hump was determined by the difference between the peak of the smallest and the peak of the largest action potential following a depolarizing current pulse and was used to quantify the tendency of some cells to display a characteristic initial reduction followed by an increase in action potential amplitude. Delay, a measure of the time it takes for the cell to fire the first action potential from the onset of current injection, was used to characterize a group of cells recorded from in control animals that fired significantly later than other cells. EXCITABILITY OF PRESUBICULAR NEURONS IN TLE group were unaltered, they would aggregate/cluster with neurons with similar attributes from the control group. We found that neurons from epileptic animals generally clustered with only three of seven cell types encountered in the control group, A spontaneous recurrent seizures male SD rat 40 hr/wk video monitoring CONTROL LATENT PERIOD 1 - 3 weeks DOB electrophysiology EPILEPTIC 3 - 8 days status pilocarpine treatment epilepticus A ME LE A Par AB PrS Su CA1 1 mm B L1 V/VI L2 III L3 II L4 control 250 um 2 1 C epileptic L5/6 8 6 5 3 D 4 10 9 7 2000 squared eucliden distance 1750 RS Cells in Epileptic Animals Fire Multiple Action Potentials 2000 1500 1000 500 0 1 750 5 10 RS IB Hyperexcitability of PrS neurons was assessed based on their propensity for firing multiple synaptically evoked action CONTROL IR LS Stu FA SS TLE 500 IR RS FA LS 250 FA multi-spiking cells SS Stu 0 E 600 600 400 squared euclidean distance 200 0 CONTROL 1 5 TLE 10 IR RS IB IR RS 400 IR 200 0 namely the irregular-spiking (IR), fast-adapting (FA), and regular-spiking (RS) cell types. We carried out two rounds of cluster analysis. As with our original characterization of PrS neurons in control animals, the first round of clustering segregated all cells into five groups (Fig. 1D), four of which [FA, late-spiking (LS), stuttering (Stu), and single-spiking (SS)] had distinct firing profiles that fit with our empirical classification, and one group, consisting of multiple-spiking cells, with high intergroup variability that deviated from our empirical classification. In the first round of clustering, FA cells from the epileptic group clustered with cells of the same type from the control group; IR and RS cells from the epileptic group clustered with cells of the same type from the control group in the multispiking cell category. Introducing cells from the epileptic group did not alter clustering of LS, Stu, and SS cell types encountered only in the control group. A second round of clustering (Fig. 1E), limited to just the multispiking cells, resulted in further classification of the remaining three cell types, RS, IR, and initially bursting (IB) cell types, of which IB cells were encountered only in the control group. IR cells from control and epileptic groups clustered together but not with the RS or IB cell groups. Likewise, with the exception of few cells, RS cells from control and epileptic groups clustered into one group that was well segregated from the IB or IR cell groups. These data suggest that the cell-type-specific classification established in control animals is generally applicable to cell types encountered in the epileptic animals. The following sections describe the alterations in physiology observed in the three cell types encountered in the epileptic animals. RS IB Fig. 1. The presubicular slice preparation used in our study. A: schematic outlining the various steps involved in bringing up the pilocarpine model of temporal lobe epilepsy (TLE). Low-powered images of Nissl-stained sections from control (left) and epileptic (right) animals, identifying the major anatomical landmarks: presubiculum (PrS), parasubiculum (Par), subiculum (Sub), medial entorhinal area (MEA), dentate gyrus (DG), hippocampal CA1, and lateral entorhinal area (LEA). Red arrows indicate the mediolateral extent of LII in PrS (region demarcated by red dashed lines). B: high-powered image of PrS (left) highlighting LII (white arrows) and various lamina (indicated by Roman numerals) and MEA (right) from control (Œ) and epileptic () animals with demarcated regions of interest from A. C: electrophysiological parameters used for cell classification: 1) early spike frequency adaption (%), 2) late spike frequency adaptation (%), 3) instantaneous firing frequency (Hz), 4) steadystate firing frequency (Hz), 5) sag ratio (%), 6) measure of input resistance (M⍀), 7) delay to first action potential (ms), 8) action potential half-width (ms), 9) spike-firing threshold (mV), and 10) afterhyperpolarization (mV). D–E: classification of all neurons (D) and multispiking cells (E) in LII and III of PrS using unsupervised hierarchical cluster analysis based on the above parameters (Table 1 and Fig. 1C). Intersection of dendrogram branches with the x-axis represents individual cells, and the y-axis represents the squared Euclidean distances between group centroids at each branch point (longer vertical lines indicate greater dissimilarity). Dashed lines indicate number of groups as determined by the Thorndike method (see MATERIALS AND METHODS). Inset: Thorndike method suggests 5 groups in a scree plot; x-axis: clustering stages, y-axis: squared Euclidean distance between group centroids. Individual cells from control and epileptic animals are color coded according to group membership (control only cell types are not colored). Cell types: irregularspiking (IR), regular-spiking (RS), initially bursting (IB), late-spiking (LS), stuttering (Stu), fast-adapting (FA), and single-spiking (SS). J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 b DG 2891 2892 EXCITABILITY OF PRESUBICULAR NEURONS IN TLE Table 1. Action potential waveform properties of PrS neurons Electrophysiological Parameter IR-Con (n ⫽ 15) IR-Epi (n ⫽ 5) ⫺69 ⫾ 2 ⫺41 ⫾ 1 60 ⫾ 7 Late spike frequency adaptation, % 25 ⫾ 5 18 ⫾ 5 Delay, ms Accommodative hump, mV Half-width, ms 35 ⫾ 3 1.4 ⫾ 0.4 2.8 ⫾ 0.2 32 ⫾ 8 1.0 ⫾ 0.2 2.2 ⫾ 0.1 Input resistance, M⍀ 264 ⫾ 22 196 ⫾ 16 Sag ratio, % 1.8 ⫾ 0.2 3.5 ⫾ 1.8 Maximum frequency, Hz 13 ⫾ 1.8 22 ⫾ 4.1 Instantaneous firing frequency, Hz Steady-state firing frequency, Hz SD of steady-state firing frequency, Hz 9 ⫾ 1.0 4 ⫾ 0.5 1.7 ⫾ 0.2 13 ⫾ 1.5 6 ⫾ 0.7 2.3 ⫾ 0.4 FA-Epi (n ⫽ 4) ⫺62 ⫾ 1 ⫺63 ⫾ 3 ⫺40 ⫾ 1 ⫺42 ⫾ 2 57 ⫾ 4 61 ⫾ 3 RS-Epi ⬎⬎ RS-Con n/d n/d RS-Con ⬎ RS-Epi 13 ⫾ 2 16 ⫾ 7 3.3 ⫾ 0.9 0.8 ⫾ 0.1 2.5 ⫾ 0.3 2.1 ⫾ 0.3 RS-Con ⬎⬎ RS-Epi 341 ⫾ 35 166 ⫾ 17 FA-Con ⬎ FA-Epi 1.9 ⫾ 0.6 0.7 ⫾ 0.4 RS-Epi ⬎⬎ RS-Con 39 ⫾ 4.5 24 ⫾ 4.4 RS-Epi ⬎ RS-Con 24 ⫾ 3.2 15 ⫾ 2.7 n/d n/d n/d n/d RS-Con (n ⫽ 116) RS-Epi (n ⫽ 43) ⫺66 ⫾ 1 ⫺40 ⫾ 1 40 ⫾ 2 ⫺66 ⫾ 1 ⫺39 ⫾ 1 51 ⫾ 2 8⫾1 6⫾1 25 ⫾ 1 2.2 ⫾ 0.2 2.2 ⫾ 0.1 28 ⫾ 7 2.3 ⫾ 0.3 1.8⫾ 0.1 330 ⫾ 8 306 ⫾ 15 2.2 ⫾ 0.2 4.7 ⫾ 0.8 35 ⫾ 1.7 43 ⫾ 3.1 24 ⫾ 0.9 16 ⫾ 0.7 1.1 ⫾ 0.1 26 ⫾ 1.4 17 ⫾ 0.9 1.1 ⫾ 0.1 Values represent means ⫾ SE; n, number of cells. PrS, presubiculum; Con, control group; Epi, epileptic group; RS, regular-spiking cell; IR, irregular-spiking cell; FA, fast-adapting cell. Electrophysiological parameters were measured as described in MATERIALS AND METHODS. Significant differences between Epi and Con groups are italicized. ⬎, significantly greater with P ⬍ 0.05, t-test; ⬎⬎, significantly greater, with P ⬍ 0.01, t-test; n/d, not determined because number of action potentials was insufficient for reliable analysis. potentials in response to increasing stimulus intensity, expressed as multiples of T, the threshold for evoking a single action potential (Fig. 2). IR (n ⫽ 3 control and n ⫽ 3 epileptic), FA (n ⫽ 5 control and n ⫽ 4 epileptic), and RS (n ⫽ 38 control and n ⫽ 15 epileptic) cells from control and epileptic groups were compared following whole cell current-clamp recording of neurons from LII-III of PrS (Fig. 2A). We found that IR and FA cells from epileptic animals were indistinguishable from corresponding cell types in the control group based either on the mean number of action potential evoked and/or the area under the composite excitatory postsynaptic potential (EPSP; P ⬎ 0.2 for both; Fig. 2B). RS cells from epileptic animals, on the other hand, were found to be hyperexcitable, firing multiple action potentials in response to even modest current increments. Both the number of action potentials evoked in response to increasing stimulus intensity and area under the composite EPSP were significantly increased in epileptic animals compared with controls (P ⬍ 0.001 at the indicated ⫻T intensities; Fig. 2B) despite comparable T (control: 50 ⫾ 11, epileptic: 42 ⫾ 5 A; P ⫽ 0.6) values. These data suggest that the propensity of RS cells, but not IR or FA cells, to fire multiple action potentials is significantly enhanced under epileptic conditions. We next measured changes in the excitatory and inhibitory synaptic drive in these cell types under epileptic conditions. IR Cells are Unaffected in Epileptic Animals IR (Fig. 3, A1 and A2) cells display an action potential discharge profile characterized by the firing of irregularly spaced action potentials in response to current injections or depolarization (Fig. 3B). In control animals, IR cells (n ⫽ 15) were found exclusively in LII of PrS and comprised 8% of the total sampled population. In epileptic animals, IR cells (n ⫽ 5) were again found to be restricted to LII (Fig. 3A) and accounted for 10% of the total sampled population. Currentclamp recordings revealed no significant changes in electrophysiological parameters associated with action potential waveform between the control and epileptic populations (Table 1). IR cells from both groups had characteristically larger standard deviation of the steady-state firing frequency, higher late spike frequency adaptation, and lower instantaneous and steady-state firing frequencies relative to other cell types. Consistent with these observations, IR cell excitability, as assessed by the number of action potentials fired as a function of increasing intensity of current injection, was comparable between neurons from the control and epileptic groups (P ⬎ 0.1). Voltage-clamp data revealed no significant alterations in frequency and amplitude of either spontaneous or miniature EPSCs and IPSCs (Fig. 3, C–F, and Table 2). Together, these data indicate that IR cells are less affected by TLE relative to other cell types in our sampling of PrS neurons from control and epileptic animals. Excitatory Synaptic Drive in FA Cells Is Enhanced During TLE FA (Fig. 4, A1 and A2) cells respond to current injection or depolarization by firing action potentials at regularly spaced intervals for a brief period of time before becoming quiescent for the remainder of cell depolarization (Fig. 4B). The discharge profile for FA cells was generally unaffected by increased current injection. In both control and epileptic animals, FA cells were restricted to LII of PrS, accounting for 6% (n ⫽ 12) and 8% (n ⫽ 4) of total sampled cell types in PrS, respectively. Current-clamp data revealed that with the exception of a significant reduction in input resistance in epileptic animals (341 ⫾ 35 vs. 166 ⫾ 17 M⍀; P ⬍ 0.05), intrinsic firing properties remain unchanged between the control and epileptic groups (Table 1). FA cell excitability, as assessed by the number of action potentials fired as a function of increasing intensity of current injection, was qualitatively different from IR cells, but comparable between neurons from the control and epileptic groups (P ⬎ 0.3). Voltage-clamp data revealed that the average fre- J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 ⫺69 ⫾ 1 ⫺41 ⫾ 1 47 ⫾ 6 Resting membrane potential, mV Spike firing threshold, mV Early spike frequency adaptation, % FA-Con (n ⫽ 12) EXCITABILITY OF PRESUBICULAR NEURONS IN TLE RS Cells Are Less Inhibited Under Epileptic Conditions control - RS A T 2T 4T R . S -65mV epileptic - RS epileptic - IR 20mV 200ms mean evoked action potentials 3 RS Control RS Epileptic IR Epileptic 3.5 *** 2 area under EPSP (mV * ms, x1000) -69mV 2.5 *** *** *** *** 1.5 1 0.5 T 2T 4T stimulus intensity T 2T 4T stimulus intensity Fig. 2. Hyperexcitability of RS cells in TLE. A: representative examples of action potentials evoked in RS and IR cells under the indicated conditions in response to stimulation at threshold (T) and increasing multiples (⫻2 and ⫻4) of T. Resting membrane potential for cells is indicated along with placement of stimulating (S) and recording (R) electrode in brain slices (inset). B: scatter plots for mean number of action potentials evoked and area under the composite excitatory postsynaptic potential (EPSP) in response to increasing stimulus intensity for the indicated cell types. ***P ⬍ 0.001, t-test. quency of sEPSCs in FA cells from epileptic rats was ⬃215% of control rats (1.3 ⫾ 0.3 vs. 2.8 ⫾ 0.6 Hz; P ⬍ 0.05; Fig. 4C and Table 2). The increase in sEPSCs frequency may have resulted from increased activity of presynaptic excitatory circuits as no significant changes in frequency or amplitude of mEPSCs (P ⫽ 0.1 and 0.8, respectively) were observed. The frequency and amplitude of sIPSCs and mIPSCs were unaffected (frequency: P ⫽ 0.2 and 0.1; amplitude: P ⫽ 0.4 and 0.4; Fig. 4D and Table 2) in FA cells suggesting that synaptic inhibitory drive to these neurons is not compromised under epileptic conditions. Overall, these data suggest that FA cells are not hyperexcitable in the epileptic animals, but an enhanced excitatory synaptic drive is seen under these conditions. RS (Fig. 5, A1 and A2) cells are characterized by sustained firing of regularly spaced action potentials in response to current injections or depolarization (Fig. 5B). RS cells were the predominant cell type in the PrS found in both LII and LIII of control and epileptic animals, comprising 62% (n ⫽ 116) of the sampled population in control animals and 82% (n ⫽ 43) in epileptic animals. Current-clamp data from RS cells in control and epileptic animals revealed similar resting membrane potentials and input resistances (Table 1). RS cells were the only group of PrS neurons in epileptic animals that showed significant changes in action potential discharge profiles resulting from current injection or cell depolarization; half-widths of individual action potentials were reduced (control: 2.2 ⫾ 0.1; epileptic: 1.8 ⫾ 0.1 ms; P ⬍ 0.05), maximum firing frequency of action potentials was increased (control: 35 ⫾ 1.7; epileptic: 43 ⫾ 3.1 Hz; P ⬍ 0.05), early spike-frequency adaptation was increased (control: 40 ⫾ 2; epileptic: 51 ⫾ 2%; P ⬍ 0.001), while late adaptation was decreased (control: 8 ⫾ 1; epileptic: 6 ⫾ 1%; P ⬍ 0.05). These data suggest that RS cells in epileptic animals have an increased initial firing frequency in response to depolarization and a faster firing frequency adaption, which presumably enables them to reach steady-state firing frequency sooner compared with RS cells in control animals. Consistent with these observations, RS cell excitability, as assessed by the number of action potentials fired as a function of increasing intensity of current injection, was enhanced in neurons from the epileptic groups compared with control, although these differences did not reach the level of statistical significance (P ⬎ 0.1, t-test). Additionally, RS cells in epileptic animals had an increased sag ratio (control: 2.2 ⫾ 0.2, epileptic: 4.7 ⫾ 0.8%; P ⬍ 0.001; Fig. 5B), a measure of the hyperpolarization-activated Ih current. We noted that RS cells in control animals showed little to no sag in response to hyperpolarizing current injection compared with RS cells in epileptic animals that displayed a more noticeable sag conductance (Fig. 5B). Although the significance of somatic assessments of sag in relation to hyperexcitability of neurons in TLE is unclear (Notomi and Shigemoto 2004; Shah et al. 2004) and possibly requires a direct assessment of Ih, these differences were clearly manifest in RS cells from the control and epileptic groups. It should be noted that the role of Ih (epileptogenic vs. compensatory) and the underlying hyperpolarization-activated cation nonselective 1 (HCN1) channels in mediating seizure threshold is still under debate (Chen et al. 2001; Huang et al. 2009) given that TLE differentially affects their expression at the soma and dendrite (Shah et al. 2004; Shin et al. 2008) and Ih currents can vary depending on the time of measurement following status (Jung et al. 2007). RS cells in epileptic animals had significantly reduced sEPSCs frequency (control: 2.3 ⫾ 0.2; epileptic: 1.2 ⫾ 0.1 Hz; P ⬍ 0.001) and amplitude (control: 19 ⫾ 0.7; epileptic: 15 ⫾ 1.3 pA; P ⬍ 0.005; Fig. 5, C–F and Table 2). Decreases in sEPSC frequency could potentially arise from loss of excitatory synapses, reductions in probability of neurotransmitter release, and/or reduced activity of presynaptic neurons. However, the frequency and amplitude of mEPSCs in RS cells in epileptic animals were unaltered compared with controls (P ⬎ 0.1 for both; Fig. 6A and Table 2), ruling out the first two alternatives in favor of a decreased excitatory drive to these J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 -68mV B 2893 2894 EXCITABILITY OF PRESUBICULAR NEURONS IN TLE A1 LIII A2 LIII LII LII LI pia LI Irregular Spiking (IR) 100 pA 100 pA -100 pA B 20 mV 20 mV 200 ms 375 ms 100 ms control epileptic 20 # APs 15 10 5 -66 mV -70 mV 0 0 50 100 150 200 Current (pA) C control D sEPSCs (aCSF) sIPSCs 20pA 10s 170ms 100pA E mEPSCs (+TTX) F mIPSCs neurons under epileptic conditions. Interestingly, inhibitory drive to RS cells was also reduced in the epileptic animals (sIPSC frequency: 0.7 ⫾ 0.1 vs. 0.4 ⫾ 0.1 Hz; P ⬍ 0.05; sIPSC amplitude: 45 ⫾ 1.6 to 27 ⫾ 1 pA; P ⬍ 0.001; Figs. 5, D–F, and 6A and Table 2) and mIPSCs frequency and amplitude in RS neurons from epileptic animals were 174% (0.23 ⫾ 0.04 vs. 0.4 ⫾ 0.04; P ⬍ 0.05; Figs. 5F and 6A) and 80% (30 ⫾ 2 vs. 24 ⫾ 1 pA; P ⬍ 0.005) of their values in control, respectively. An increase in mIPSC frequency suggests that the probability of neurotransmitter release from GABAergic inhibitory neurons is increased under epileptic conditions, and reductions in mIPSC amplitude indicate a concomitant decrease in number of GABAergic receptors expressed by these neurons and/or alterations in receptor function. Inclusion of TTX (1 M) in the perfusate enables assessment of the proportion of synaptic events that are action potential dependent. We found that TTX significantly reduced both the frequency and amplitude of EPSCs and IPSCs in RS cells from control animals but only affected the amplitude (not frequency, P ⬎ 0.6 paired t-test; Fig. 6B, Table 2) of the PSCs in epileptic animals. Thus, in epileptic animals, synaptic events were predominantly action potential independent. This suggests that the RS cells are driven less by presynaptic neurons in epileptic 10s 170ms epileptic animals than in the controls, presumable because presynaptic neurons are less active under these conditions. The overall shift in synaptic transmission from action potential dependent to action potential independent in RS cells under epileptic conditions is reflected in the observed reductions in mean amplitude of spontaneous events as action potentialdependent events are typically larger in amplitude than action potential-independent events. DISCUSSION The present study characterizes the alterations in physiological properties of neurons in superficial LII-III of PrS in a rat model of TLE. Our principal findings are the following: 1) only a subset of PrS neurons (1 of 3 major cell types) undergoes alterations in action potential discharge profiles and excitatory and inhibitory synaptic drive, making TLE-related changes in this structure cell type specific; and 2) RS cells are hyperexcitable in TLE. This is the predominant cell type in PrS found in both control and epileptic animals and identified previously as a class of projection neurons with the potential to entrain downstream structures, particularly the MEA, in TLE (Abbasi and Kumar 2013). J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 Fig. 3. IR cells. A1: representative photomicrograph captured on a confocal microscope of a biocytinlabeled IR neuron in LII of PrS from an epileptic rat. Roman numerals (I-III) indicate lamina and scale bar ⫽ 100 m. A2: neurolucida reconstruction of the IR cell in A1 showing laminar location of somata and dendritic morphology. B: representative examples of action potential discharge in IR cells from control and epileptic animals. Action potential (AP) waveforms in response to sustained current injections of 100 pA (1to 10-s duration; left) and hyper- and depolarizing current pulses (⫾100 pA, 600 ms duration; middle) from resting membrane potential (Vm). C–F: excitatory and inhibitory synaptic drive to IR cells from control and epileptic animals, measured using voltage clamp. Graph of the mean number of action potentials as a function of depolarizing current injection from control and epileptic neurons (0 –200 pA in 50-pA increments, 600-ms duration; right). C: trace (1 min long) of spontaneous excitatory postsynaptic currents [sEPSCs, inward events recorded at ⫺70-mV holding potential in artificial cerebrospinal fluid (aCSF)] recorded from IR cells in control (left) and epileptic animals (right). In this and all subsequent figures, embedded insets offer an expanded view of the indicated portions of traces (dotted lines). D: spontaneous inhibitory postsynaptic currents (sIPSCs, outward events recorded at 0 mV in aCSF). E: miniature excitatory postsynaptic currents [mEPSCs, inward events recorded at ⫺70 mV in the presence of 1 M tetrodotoxin (TTX)]. F: miniature inhibitory postsynaptic currents (mIPSCs, outward events recorded at 0 mV in TTX). EXCITABILITY OF PRESUBICULAR NEURONS IN TLE A1 A2 LIII LIII LII LII LI pia LI 100 μm Fast Adapting (FA) 100 pA 100 pA -100 pA B 20 mV 20 mV 200 ms 280 ms 100 ms control epileptic 20 # APs 10 5 -66 mV -63 mV 0 0 50 100 150 Current (pA) D control 20pA 10s 170ms sEPSCs (aCSF) epileptic sIPSCs 200 Fig. 4. FA cells. A1: representative photomicrograph captured on a confocal microscope of a biocytinlabeled FA neuron in LII of PrS from an epileptic rat. Roman numerals (I-III) indicate lamina and scale bar ⫽ 100 m. A2: neurolucida reconstruction of the FA cell in A1 showing laminar location of somata and dendritic morphology. B: representative examples of action potential discharge in FA cells from control and epileptic animals. Action potential waveforms in response to sustained current injections of 100 pA (1- to 10-s duration; left) and hyper- and depolarizing current pulses (⫾100 pA, 600-ms duration; middle) from resting membrane potential (Vm). C–F: excitatory and inhibitory synaptic drive to FA cells from control and epileptic animals, measured using voltage clamp. Graph of the mean number of action potentials as a function of depolarizing current injection from control and epileptic neurons (0 –200 pA in 50-pA increments, 600-ms duration; right). C: trace (1-min long) of sEPSCs (inward events recorded at ⫺70-mV holding potential in aCSF) recorded from FA cells in control (left) and epileptic animals (right). Embedded insets offer an expanded view of the indicated portions of traces (dotted lines). D: sIPSCs (outward events recorded at 0 mV in aCSF) E: mEPSCs (inward events recorded at ⫺70 mV in the presence of 1 M TTX). F: mIPSCs (outward events recorded at 0 mV in TTX). mEPSCs (+TTX) E 100pA F 10s 170ms mIPSCs Compared with RS cells, IR and FA cells in the PrS appeared to be relatively unaffected in epileptic animals based on intrinsic firing properties and synaptic drive with the exceptions of an increase in frequency of sEPSCs noted in FA cells along with a decrease in input resistance, similar to that reported for subicular pyramidal neurons in pilocarpine-treated rats (Knopp et al. 2005; Wellmer et al. 2002). Note that these observations for IR and FA cells are from a small number of neurons (n ⬍ 10), owing to the relatively small proportion of these neuronal types in our sampled population of neurons from epileptic animals. RS cells in epileptic animals, on the other hand, displayed changes in action potential waveform properties supporting higher rates of action potential discharge and enhanced early spike frequency adaptation. Although we did not directly assess underlying mechanisms, previous studies have attributed similar changes in TLE to altered gating, increased levels of Na⫹ channel expression (Hargus et al. 2011), and modifications to Ca2⫹-activated BK class of K⫹ channels (Brenner et al. 2005). RS cells from epileptic animals had significantly reduced excitatory and inhibitory synaptic drive, and data from voltageclamp experiments suggest reduced activity in both excitatory and inhibitory circuits presynaptic to the recorded RS cells. An overall reduction in synaptic drive to RS cells conforms to a model in which reductions in excitatory drive to these neurons may also manifest in presynaptic GABAergic interneurons that are capable of providing feed-forward inhibition keeping their excitability in check (Fig. 7). Future work includes identification of GABAergic neurons that target RS cells and the direct assessment of synaptic drive to these neurons under control and epileptic conditions. Alternatively, GABAergic interneurons providing feedback inhibition might also be affected, although this appears unlikely because RS cells are hyperexcitable in epileptic animals. Although the precise sources of excitation to RS cells remain unknown, previous studies examining TLErelated alterations in neuronal populations suggest neuron loss in a number of structures that project to the PrS, including retrosplenial granular cortex, thalamus, and subiculum. It is likely that alterations in the synaptic drive to RS cells that were observed in the epileptic animals arise as a consequence of neuron loss in these structures (Cardoso et al. 2008; Chen and Buckmaster 2005; Covolan and Mello 2000; Dube et al. 2001; Scholl et al. 2013). The RS cells of PrS are not the only neurons found to be hyperexcitable in epileptic animals; previous studies of TLE using animal models have also reported hyperexcitability of J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 15 C 2895 2896 EXCITABILITY OF PRESUBICULAR NEURONS IN TLE A1 A2 Regular Spiking (RS) LII LII LII LI pia LI pia LI 100 μm 100 pA 100 pA -100 pA B 20 mV 20 mV 200 ms 270 ms 100 ms control epileptic 20 # APs 15 10 5 -67 mV -63 mV 0 0 50 100 150 200 Current (pA) C D E 20pA control sEPSCs (aCSF) epileptic 10s 170ms sIPSCs mEPSCs (+TTX) 100pA F MEA neurons (Bear et al. 1996; Scharfman et al. 1998; Tolner et al. 2007) mediated by modifications to both synaptic and intrinsic properties (Hargus et al. 2011; Kumar et al. 2007; Shah et al. 2004). Alterations in synaptic inhibition, in particular, have been identified as the major underlying cause for pathophysiological alterations associated with TLE (El-Hassar et al. 2007; Kobayashi et al. 2003; Kumar and Buckmaster 2006; Obenaus et al. 1993), and suppression of network inhibition through pharmacological blockade of GABAA synaptic transmission in either the MEA or PrS was found sufficient to promote hyperexcitability (Funahashi et al. 1999; Menendez de la Prida and Pozo 2002), suggesting similar pathophysiological processes underlying hyperexcitability in the two regions. Cell Loss and Classification of Neurons in the Epileptic PrS We previously identified seven electrophysiologically distinct cell types in LII-III of PrS in normal animals (Abbasi and Kumar 2013). However, our assessment of the PrS under mIPSCs 10s 170ms epileptic conditions yielded only three of these cell types (FA, IR, and RS). PrS cell types that were not encountered in epileptic animals included IB and Stu cells, a class of putative inhibitory interneurons (Abbasi and Kumar 2013). One explanation for this discrepancy is the greater vulnerability of certain neuronal populations to perish during TLE. Indeed, previous studies have identified differences in the susceptibility of PrS neurons including reductions in the number of parvalbumin and calretinin-positive GABAergic interneurons, reductions in total cell counts, and increased labeling of markers of neuronal degeneration (Cardoso et al. 2011; Drexel et al. 2011; Scholl et al. 2013; van Vliet et al. 2004). The absence of IB neurons from our sample of the epileptic PrS is reminiscent of a study in the subiculum of pilocarpine-treated, chronically epileptic rats that reported drastic reductions in the population of bursting neurons compared with controls (Knopp et al. 2005), although other studies have reported an upregulation of bursting neurons in the subiculum following pilocarpine-induced J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 Fig. 5. RS cells. A1: representative photomicrograph captured on a confocal microscope of biocytin-labeled RS neurons in LII and LIII of PrS from an epileptic rat. Roman numerals (I-III) indicate lamina and scale bar ⫽ 100 m. A2: beurolucida reconstruction of RS cells in A1 showing laminar location of somata and dendritic morphology. B: representative examples of action potential discharge in RS cells from control and epileptic animals. Action potential waveforms in response to sustained current injections of 100 pA (1- to 10-s duration; left) and hyper- and depolarizing current pulses (⫾100 pA, 600-ms duration; middle) from resting membrane potential (Vm). C–F: excitatory and inhibitory synaptic drive to RS cells from control and epileptic animals, measured using voltage clamp. Graph of the mean number of action potentials as a function of depolarizing current injection from control and epileptic neurons (0 –200 pA in 50-pA increments, 600-ms duration; right). C: trace (1-min long) of sEPSCs (inward events recorded at ⫺70-mV holding potential in aCSF) recorded from RS cells in control (left) and epileptic animals (right). Embedded insets offer an expanded view of the indicated portions of traces (dotted lines). D: sIPSCs (outward events recorded at 0 mV in aCSF) E: mEPSCs (inward events recorded at ⫺70 mV in the presence of 1 M TTX). F: mIPSCs (outward events recorded at 0 mV in TTX). LIII LIII LIII EXCITABILITY OF PRESUBICULAR NEURONS IN TLE mIPSC RS sEPSC mEPSC sIPSC mIPSC spontaneous (aCSF) miniature (aCSF + TTX) 3 3 2 2 *** ns 1 1 * * 0 0 Control Control Epileptic ns Epileptic 30 Values represent means ⫾ SE. The total number of cells tested (n) is indicated for each group, reported for spontaneous and miniature experiments, respectively. Frequency is reported in hertz (Hz), and amplitude is reported in picoamp (pA). Values are for spontaneous (s) and miniature (m) excitatory postsynaptic current (EPSC) and inhibitory postsynaptic current (IPSC). *P ⬍ 0.05, †P ⬍ 0.01, ‡P ⬍ 0.001, t-test. 50 25 40 20 15 ** *** 30 ** 20 10 Control Control Epileptic Epileptic Frequency mEPSCs mIPSCs B ** 120 80 80 60 60 40 40 20 20 % in TTX (aCSF = 100%) 100 0 * 120 100 control epileptic 0 control epileptic Amplitude 120 120 ns 100 % in TTX (aCSF = 100%) status (Wellmer et al. 2002). It is plausible to attribute these differences to the short- and long-term consequences of an epileptogenic treatment (Kumar and Buckmaster 2006). Additionally, there might be differences in the effects of pilocarpine treatment and TLE along the proximal-distal axis of the subiculum, with Knopp et al. (2005) recording preferentially in the middle portion of the subiculum that projects to the PrS, in contrast with Wellmer et al. (2002) whose recordings are in proximal subiculum that does not to project to the PrS (O’Reilly et al. 2013). It has been suggested that an increased Ca2⫹ conductance, deemed important for bursting activity (Jung et al. 2001), contributes to the vulnerability of these neurons to excitotoxic cell death; a hypothesis also proposed for explaining vulnerability of neuronal populations in the MEA (Gloveli et al. 1999). Alternatively, differences in the proportional distribution of cell types encountered in PrS between control and epileptic groups might be related to alterations in intrinsic properties of neurons that modify their action potential discharge properties resulting in their reclassification. However, we consider this a remote possibility because cells in PrS appear not be interconvertible between cell types (Abbasi and Kumar 2013). The fact that two of the three cell types encountered in epileptic animals (IR and FA cells) were unperturbed from control conditions and that all cell types from both groups clustered according to the classification schema for control animal supports this assertion. Finally, we acknowledge the possibility of not recording from certain cell IPSCs 4 100 80 80 60 60 40 40 20 20 0 control epileptic 0 ns control epileptic Fig. 6. Measurements of synaptic activity in RS cells from control and epileptic animals. A: changes to frequency and amplitude of excitatory and inhibitory postsynaptic currents under the indicated conditions. Statistical significance (voltage-clamp) between control and epileptic groups: *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001, t-test; before and after of TTX. ⫹P ⬍ 0.05, ⫹⫹P ⬍ 0.01, ⫹⫹⫹P ⬍ 0.001, paired t-test. B: bar plots comparing s- and mEPSC frequency and amplitude under control and epileptic conditions (for each plot 100% ⫽ magnitude of the response in aCSF). *P ⬍ 0.05; **P ⬍ 0.01, t-test. J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 sIPSC 4 ++ mEPSC EPSCs A ++ FA sEPSC +++ mIPSC + sIPSC n ⫽ 5; 5 1.8 ⫾ 0.5 Hz 13 ⫾ 1.1 pA 1.2 ⫾ 0.3 Hz 12 ⫾ 1.2 pA 0.3 ⫾ 0.1 Hz 31 ⫾ 3.2 pA 0.3 ⫾ 0.1 Hz 26 ⫾ 1.9 pA n ⫽ 4; 4 2.8 ⫾ 0.6 Hz* 14 ⫾ 2.0 pA 1.2 ⫾ 0.3 Hz 13 ⫾ 2.4 pA 0.2 ⫾ 0.1 Hz 29 ⫾ 3.1 pA 0.2 ⫾ 0.1 Hz 26 ⫾ 2.0 pA n ⫽ 39; 29 1.2 ⫾ 0.1 Hz‡ 15 ⫾ 1.3 pA† 0.9 ⫾ 0.2 Hz 12 ⫾ 0.9 pA 0.4 ⫾ 0.1 Hz* 27 ⫾ 1.0 pA‡ 0.4 ⫾ 0.1 Hz† 24 ⫾ 1.2 pA† +++ mEPSC n ⫽ 15; 5 2.0 ⫾ 0.3 Hz 17 ⫾ 1.3 pA 1.1 ⫾ 0.2 Hz 11 ⫾ 0.6 pA 0.4 ⫾ 0.2 Hz 34 ⫾ 2.0 pA 0.2 ⫾ 0.1 Hz 23 ⫾ 2.6 pA n ⫽ 12; 5 1.3 ⫾ 0.3 Hz 19 ⫾ 1.9 pA 0.7 ⫾ 0.1 Hz 14 ⫾ 2.4 pA 0.4 ⫾ 0.1 Hz 37 ⫾ 4.8 pA 0.4 ⫾ 0.1 Hz 25 ⫾ 1.4 pA n ⫽ 113; 37 2.3 ⫾ 0.2 Hz 19 ⫾ 0.7 pA 1.1 ⫾ 0.1 Hz 14 ⫾ 0.8 pA 0.7 ⫾ 0.1 Hz 45 ⫾ 1.6 pA 0.2 ⫾ 0.1 Hz 31 ⫾ 1.7 pA +++ sEPSC Epileptic Frequency (Hz) IR Control types on account of their limited representation in the control population (SS, 5%; LS, 2%; Stu: 4%) leading to sampling errors in the population of neurons sampled from epileptic animals, although the absence of IB cells, is likely not attributable to errors in sampling because this cell type accounted for as much as 13% of our control population. Note on sufficiency of neurons sampled. The number of cells sampled in our control and epileptic groups was adequate to make inferences and conclusions about changes in their physiological properties. Thus getting more cells to increase ns in any category would not only be cumbersome, but impractical, Amplitude (pA) Table 2. Summary of frequency and amplitude of PSCs in PrS neurons from control and epileptic groups 2897 2898 EXCITABILITY OF PRESUBICULAR NEURONS IN TLE epileptic control direct excitation ? ? feed-forward inhibition RS normal hyperexcitable pyramidal neuron excitatory synaptic input inhibitory synaptic input normal synaptic drive decreased synaptic drive Fig. 7. A summary of possible changes in excitatory and inhibitory synaptic drive rendering RS cells in the PrS hyperexcitable under epileptic conditions. The brake analogy refers to synaptic inhibition of RS cells. Open questions (?) include the identity of GABAergic neurons that target RS cells and the direct assessment of synaptic drive to these neurons under control and epileptic conditions. given the difficulty of “searching” for particular cell types in brain tissue under DIC optics and that doing so might also preclude the obtaining of an unbiased random sampling of neurons from the two populations for comparative purposes. Given that certain cell types account for only a small percentage of all sampled neurons in PrS, boosting ns might only minimally affect statistical inferences and may not resolve the issue of missing cell types in the epileptic sample. For instance, IB cells accounted for 13% of total sampled neurons in control animals, a significantly larger portion of the sample, compared with either IR (8%) or FA (6%) cells. The fact that we were able to record from both IR and FA cells in epileptic tissue (that accounted for 10 and 8% of sampled neurons from epileptic animals, respectively) suggests that recording from IB cells should have been possible. Increasing our sample size to “uncover” IB cells in epileptic tissue could lead to the following scenarios: if we are unsuccessful in recording from these cells, we can more confidently assert the vulnerability of this PrS cell type in TLE. Alternatively, if we are successful in recording from IB cells in any additional recordings, they most likely will end up accounting for a smaller proportion of the sample from epileptic animals than they did for controls, thereby validating the vulnerability of IB cells in TLE. Both these conclusions are not very different from those reached through our experiments. On the other hand, Stu cells accounted for a smaller portion (4%) of our sampled control population, and so it is conceivable that these cells were not recorded from in epileptic animals because of errors in sampling and/or TLE-related changes in their distribution. To the best of our knowledge this is a first attempt at direct assessment of the physiological state of PrS neurons in confirmed epileptic animals, with the potential to shed light on the hyperexcitability of neurons in the neighboring MEA and hippocampus. The PrS itself is capable of triggering hyperexcitability and mediating pathophysiological events in neighboring MEA (Tolner et al. 2005, 2007; Zahn et al. 2008) although details of the underlying mechanisms have remained unknown. Our results pinpoint RS cells as being hyperexcitable under epileptic conditions, a state brought about by and/or exacerbated by reductions in synaptic inhibition. PrS projections to MEA have been worked out anatomically (Caballero-Bleda and Witter 1993; Honda et al. 2011; Honda and Ishizuka 2004; van Haeften et al. 1997) and characterized physiologically (Bartesaghi et al. 2005), and, more recently, shown to form functional connections with cells in all layers of MEA (Canto et al. 2012). Our recent study of the PrS (Abbasi and Kumar 2013) provided detailed electrophysiological characterization for PrS projection neurons, including those targeting MEA, which include RS cells. We have shown that projection neurons in the PrS have bifurcating axons that seem to concomitantly innervate the MEA and more distant target structures (subiculum, retrosplenial granular cortex) via the angular bundle, enabling them to potentially drive multiple structures (Abbasi and Kumar 2013; Honda et al. 2011). Additionally, it has been reported that PrS projections targeting MEA are preserved in epilepsy despite significant loss of neurons in LIII of MEA (Tolner et al. 2005) (see also, Fig. 1B). Our findings therefore implicate the PrS in the entrainment of downstream structures including the MEA under epileptic conditions and identify this parahippocampal structure as a potential locus for implementation of cell-type-specific interventional/therapeutic strategies in combating TLE. ACKNOWLEDGMENTS We are grateful to Richard L. Hyson and Frank Johnson for critically reading the manuscript and for suggestions. We thank Max Richardson, Tiffany Jacobson, John Gray, Mark Basista, and Jyotsna Pilli for contributions to various aspects of this research endeavor. We are grateful to J. Barber, F. Fletcher, and T. Tang of the support groups in the College of Medicine and Department of Psychology at Florida State University for technical assistance. GRANTS This work was supported in part by grants from the Center for Research and Creativity and College of Medicine at Florida State University and the Epilepsy Foundation. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: S.A. and S.S.K. conception and design of research; S.A. and S.S.K. performed experiments; S.A. and S.S.K. analyzed data; S.A. and S.S.K. interpreted results of experiments; S.A. and S.S.K. prepared figures; S.A. and S.S.K. drafted manuscript; S.A. and S.S.K. edited and revised manuscript; S.A. and S.S.K. approved final version of manuscript. J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 GABAergic interneuron Implications for Entrainment of Downstream Structures EXCITABILITY OF PRESUBICULAR NEURONS IN TLE REFERENCES Funahashi M, Harris E, Stewart M. Re-entrant activity in a presubiculumsubiculum circuit generates epileptiform activity in vitro. Brain Res 849: 139 –146, 1999. Funahashi M, Stewart M. Presubicular and parasubicular cortical neurons of the rat: functional separation of deep and superficial neurons in vitro. J Physiol 501: 387– 403, 1997. Garrido-Sanabria ER, Perez MG, Banuelos C, Reyna T, Hernandez S, Castaneda MT, Colom LV. Electrophysiological and morphological heterogeneity of slow firing neurons in medial septal/diagonal band complex as revealed by cluster analysis. Neuroscience 146: 931–945, 2007. Gloveli T, Egorov AV, Schmitz D, Heinemann U, Muller W. Carbacholinduced changes in excitability and [Ca2⫹]i signalling in projection cells of medial entorhinal cortex layers II and III. Eur J Neurosci 11: 3626 –3636, 1999. Halabisky B, Shen F, Huguenard JR, Prince DA. Electrophysiological classification of somatostatin-positive interneurons in mouse sensorimotor cortex. J Neurophysiol 96: 834 – 845, 2006. Hargus NJ, Merrick EC, Nigam A, Kalmar CL, Baheti AR, Bertram EH 3rd, Patel MK. Temporal lobe epilepsy induces intrinsic alterations in Na channel gating in layer II medial entorhinal cortex neurons. Neurobiol Dis 41: 361–376, 2011. Harris E, Witter MP, Weinstein G, Stewart M. Intrinsic connectivity of the rat subiculum: I. Dendritic morphology and patterns of axonal arborization by pyramidal neurons. J Comp Neurol 435: 490 –505, 2001. Honda Y, Furuta T, Kaneko T, Shibata H, Sasaki H. Patterns of axonal collateralization of single layer V cortical projection neurons in the rat presubiculum. J Comp Neurol 519: 1395–1412, 2011. Honda Y, Ishizuka N. Organization of connectivity of the rat presubiculum: I. Efferent projections to the medial entorhinal cortex. J Comp Neurol 473: 463– 484, 2004. Huang Z, Walker MC, Shah MM. Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis. J Neurosci 29: 10979 – 10988, 2009. Jones BF, Witter MP. Cingulate cortex projections to the parahippocampal region and hippocampal formation in the rat. Hippocampus 17: 957–976, 2007. Jung HY, Staff NP, Spruston N. Action potential bursting in subicular pyramidal neurons is driven by a calcium tail current. J Neurosci 21: 3312–3321, 2001. Jung S, Jones TD, Lugo JN, Jr., Sheerin AH, Miller JW, D’Ambrosio R, Anderson AE, Poolos NP. Progressive dendritic HCN channelopathy during epileptogenesis in the rat pilocarpine model of epilepsy. J Neurosci 27: 13012–13021, 2007. Knopp A, Kivi A, Wozny C, Heinemann U, Behr J. Cellular and network properties of the subiculum in the pilocarpine model of temporal lobe epilepsy. J Comp Neurol 483: 476 – 488, 2005. Kobayashi M, Wen X, Buckmaster PS. Reduced inhibition and increased output of layer II neurons in the medial entorhinal cortex in a model of temporal lobe epilepsy. J Neurosci 23: 8471– 8479, 2003. Kohler C. Intrinsic projections of the retrohippocampal region in the rat brain. I. The subicular complex. J Comp Neurol 236: 504 –522, 1985. Kononenko NL, Witter MP. Presubiculum layer III conveys retrosplenial input to the medial entorhinal cortex. Hippocampus 22: 881– 895, 2012. Kumar SS, Buckmaster PS. Hyperexcitability, interneurons, and loss of GABAergic synapses in entorhinal cortex in a model of temporal lobe epilepsy. J Neurosci 26: 4613– 4623, 2006. Kumar SS, Jin X, Buckmaster PS, Huguenard JR. Recurrent circuits in layer II of medial entorhinal cortex in a model of temporal lobe epilepsy. J Neurosci 27: 1239 –1246, 2007. Menendez de la Prida L, Pozo MA. Excitatory and inhibitory control of epileptiform discharges in combined hippocampal/entorhinal cortical slices. Brain Res 940: 27–35, 2002. Notomi T, Shigemoto R. Immunohistochemical localization of Ih channel subunits, HCN1– 4, in the rat brain. J Comp Neurol 471: 241–276, 2004. Nowak LG, Azouz R, Sanchez-Vives MV, Gray CM, McCormick DA. Electrophysiological classes of cat primary visual cortical neurons in vivo as revealed by quantitative analyses. J Neurophysiol 89: 1541–1566, 2003. O’Mara SM, Commins S, Anderson M, Gigg J. The subiculum: a review of form, physiology and function. Progr Neurobiol 64: 129 –155, 2001. O’Reilly KC, Gulden Dahl A, Ulsaker Kruge I, Witter MP. Subicularparahippocampal projections revisited: development of a complex topography in the rat. J Comp Neurol 521: 4284 – 4299, 2013. J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 Abbasi S, Kumar SS. Electrophysiological and morphological characterization of cells in superficial layers of rat presubiculum. J Comp Neurol 521: 3116 –3132, 2013. Bartesaghi R, Di Maio V, Gessi T. Topographic activation of the medial entorhinal cortex by presubicular commissural projections. J Comp Neurol 487: 283–299, 2005. Bear J, Fountain NB, Lothman EW. Responses of the superficial entorhinal cortex in vitro in slices from naive and chronically epileptic rats. J Neurophysiol 76: 2928 –2940, 1996. Boccara CN, Sargolini F, Thoresen VH, Solstad T, Witter MP, Moser EI, Moser MB. Grid cells in pre- and parasubiculum. Nat Neurosci 13: 987–994, 2010. Brenner R, Chen QH, Vilaythong A, Toney GM, Noebels JL, Aldrich RW. BK channel beta4 subunit reduces dentate gyrus excitability and protects against temporal lobe seizures. Nat Neurosci 8: 1752–1759, 2005. Buckmaster PS. Laboratory animal models of temporal lobe epilepsy. Comp Med 54: 473– 485, 2004. Buckmaster PS, Alonso A, Canfield DR, Amaral DG. Dendritic morphology, local circuitry, and intrinsic electrophysiology of principal neurons in the entorhinal cortex of macaque monkeys. J Comp Neurol 470: 317–329, 2004. Burns R. Cluster Analysis. Business. Research Methods and Statistics Using SPSS. London: SAGE, 2008. Caballero-Bleda M, Witter MP. Projections from the presubiculum and the parasubiculum to morphologically characterized entorhinal-hippocampal projection neurons in the rat. Exp Brain Res 101: 93–108, 1994. Caballero-Bleda M, Witter MP. Regional and laminar organization of projections from the presubiculum and parasubiculum to the entorhinal cortex: an anterograde tracing study in the rat. J Comp Neurol 328: 115–129, 1993. Canto CB, Koganezawa N, Beed P, Moser EI, Witter MP. All layers of medial entorhinal cortex receive presubicular and parasubicular inputs. J Neurosci 32: 17620 –17631, 2012. Cardoso A, Lukoyanova EA, Madeira MD, Lukoyanov NV. Seizureinduced structural and functional changes in the rat hippocampal formation: comparison between brief seizures and status epilepticus. Behav Brain Res 225: 538 –546, 2011. Cardoso A, Madeira MD, Paula-Barbosa MM, Lukoyanov NV. Retrosplenial granular b cortex in normal and epileptic rats: a stereological study. Brain Res 1218: 206 –214, 2008. Cauli B, Porter JT, Tsuzuki K, Lambolez B, Rossier J, Quenet B, Audinat E. Classification of fusiform neocortical interneurons based on unsupervised clustering. Proc Natl Acad Sci USA 97: 6144 – 6149, 2000. Chen K, Aradi I, Thon N, Eghbal-Ahmadi M, Baram TZ, Soltesz I. Persistently modified h-channels after complex febrile seizures convert the seizure-induced enhancement of inhibition to hyperexcitability. Nat Med 7: 331–337, 2001. Chen S, Buckmaster PS. Stereological analysis of forebrain regions in kainate-treated epileptic rats. Brain Res 1057: 141–152, 2005. Covolan L, Mello LE. Temporal profile of neuronal injury following pilocarpine or kainic acid-induced status epilepticus. Epilepsy Res 39: 133–152, 2000. Drexel M, Preidt AP, Kirchmair E, Sperk G. Parvalbumin interneurons and calretinin fibers arising from the thalamic nucleus reuniens degenerate in the subiculum after kainic acid-induced seizures. Neuroscience 189: 316 –329, 2011. Du F, Eid T, Lothman EW, Kohler C, Schwarcz R. Preferential neuronal loss in layer III of the medial entorhinal cortex in rat models of temporal lobe epilepsy. J Neurosci 15: 6301– 6313, 1995. Du F, Whetsell WO, Jr., Abou-Khalil B, Blumenkopf B, Lothman EW, Schwarcz R. Preferential neuronal loss in layer III of the entorhinal cortex in patients with temporal lobe epilepsy. Epilepsy Res 16: 223–233, 1993. Dube C, Boyet S, Marescaux C, Nehlig A. Relationship between neuronal loss and interictal glucose metabolism during the chronic phase of the lithium-pilocarpine model of epilepsy in the immature and adult rat. Exp Neurol 167: 227–241, 2001. Eid T, Du F, Schwarcz R. Ibotenate injections into the pre- and parasubiculum provide partial protection against kainate-induced epileptic damage in layer III of rat entorhinal cortex. Epilepsia 42: 817– 824, 2001. El-Hassar L, Milh M, Wendling F, Ferrand N, Esclapez M, Bernard C. Cell domain-dependent changes in the glutamatergic and GABAergic drives during epileptogenesis in the rat CA1 region. J Physiol 578: 193–211, 2007. 2899 2900 EXCITABILITY OF PRESUBICULAR NEURONS IN TLE entorhinal cortex from rats with kainate-induced epilepsy. Neurobiol Dis 26: 419 – 438, 2007. Tolner EA, Kloosterman F, van Vliet EA, Witter MP, Silva FH, Gorter JA. Presubiculum stimulation in vivo evokes distinct oscillations in superficial and deep entorhinal cortex layers in chronic epileptic rats. J Neurosci 25: 8755– 8765, 2005. van Groen T, Wyss JM. The connections of presubiculum and parasubiculum in the rat. Brain Res 518: 227–243, 1990a. van Groen T, Wyss JM. Connections of the retrosplenial granular a cortex in the rat. J Comp Neurol 300: 593– 606, 1990b. Van Groen T, Wyss JM. Connections of the retrosplenial granular b cortex in the rat. J Comp Neurol 463: 249 –263, 2003. van Haeften T, Wouterlood FG, Jorritsma-Byham B, Witter MP. GABAergic presubicular projections to the medial entorhinal cortex of the rat. J Neurosci 17: 862– 874, 1997. van Vliet EA, Aronica E, Tolner EA, Lopes da Silva F.H, and Gorter JA Progression of temporal lobe epilepsy in the rat is associated with immunocytochemical changes in inhibitory interneurons in specific regions of the hippocampal formation. Exp Neurol 187: 367–379, 2004. Vogt BA, Miller MW. Cortical connections between rat cingulate cortex and visual, motor, and postsubicular cortices. J Comp Neurol 216: 192–210, 1983. Ward JH Jr. Hierarchical grouping to optimize an objective function. J Am Stat Assoc 58: 234 –236, 1963. Wellmer J, Su H, Beck H, Yaari Y. Long-lasting modification of intrinsic discharge properties in subicular neurons following status epilepticus. Eur J Neurosci 16: 259 –266, 2002. Yoder RM, Clark BJ, Taube JS. Origins of landmark encoding in the brain. Trends Neurosci 34: 561–571, 2011. Zahn RK, Tolner EA, Derst C, Gruber C, Veh RW, Heinemann U. Reduced ictogenic potential of 4-aminopyridine in the perirhinal and entorhinal cortex of kainate-treated chronic epileptic rats. Neurobiol Dis 29: 186 –200, 2008. J Neurophysiol • doi:10.1152/jn.00406.2014 • www.jn.org Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017 Obenaus A, Esclapez M, Houser CR. Loss of glutamate decarboxylase mRNA-containing neurons in the rat dentate gyrus following pilocarpineinduced seizures. J Neurosci 13: 4470 – 4485, 1993. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates (6th ed.). Burlington, MA: Elsevier/Academic, 2007. Pennartz CM, De Jeu MT, Geurtsen AM, Sluiter AA, Hermes ML. Electrophysiological and morphological heterogeneity of neurons in slices of rat suprachiasmatic nucleus. J Physiol 506: 775–793, 1998. Pilli J, Abbasi S, Richardson M, Kumar SS. Diversity and excitability of deep-layer entorhinal cortical neurons in a model of temporal lobe epilepsy. J Neurophysiol 108: 1724 –1738, 2012. Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32: 281–294, 1972. Scharfman HE, Goodman JH, Du F, Schwarcz R. Chronic changes in synaptic responses of entorhinal and hippocampal neurons after aminooxyacetic acid (AOAA)-induced entorhinal cortical neuron loss. J Neurophysiol 80: 3031–3046, 1998. Scholl EA, Dudek FE, Ekstrand JJ. Neuronal degeneration is observed in multiple regions outside the hippocampus after lithium pilocarpine-induced status epilepticus in the immature rat. Neuroscience 252: 45–59, 2013. Seki M, Zyo K. Anterior thalamic afferents from the mamillary body and the limbic cortex in the rat. J Comp Neurol 229: 242–256, 1984. Shah MM, Anderson AE, Leung V, Lin X, Johnston D. Seizure-induced plasticity of h channels in entorhinal cortical layer III pyramidal neurons. Neuron 44: 495–508, 2004. Shin M, Brager D, Jaramillo TC, Johnston D, Chetkovich DM. Mislocalization of h channel subunits underlies h channelopathy in temporal lobe epilepsy. Neurobiol Dis 32: 26 –36, 2008. Shipley MT, Sorensen KE. On the laminar organization of the anterior thalamus projections to the presubiculum in the guinea pig. Brain Res 86: 473– 477, 1975. Thorndike RL. Who belongs in the family? Psychometrika 18: 267–276, 1953. Tolner EA, Frahm C, Metzger R, Gorter JA, Witte OW, Lopes da Silva FH, Heinemann U. Synaptic responses in superficial layers of medial