Perforant path activation of ectopic granule cells that are born after pilocarpine-induced seizures

Neuroscience 121 (2003) 1017–1029

PERFORANT PATH ACTIVATION OF ECTOPIC GRANULE THAT ARE BORN AFTER PILOCARPINE-INDUCED SEIZURES H. E. SCHARFMAN,a,b* A. E. SOLLAS,a R. E. BERGER,a J. H. GOODMANa AND J. P. PIERCEc

CELLS

Key words: dentate gyrus, perforant path, neurogenesis, epilepsy, hippocampal slices.

a Center for Neural Recovery and Rehabilitation Research, Helen Hayes Hospital, New York State Department of Health, Route 9W, West Haverstraw, NY 10993–1195, USA

Granule cells of the dentate gyrus are born throughout life (Altman and Das, 1965; Kaplan and Hinds, 1977; Gould and Cameron, 1996; Gage, 2002). Although some aspects of neurogenesis in the adult brain are not well understood, it is clear that many factors, including neural activity, can modulate neurogenesis. Seizures, for example, increase neurogenesis in all animal models tested thus far. Enhanced neurogenesis has been reported after brief periods of increased neuronal discharge, such as afterdischarges (Bengzon et al., 1997), after kindling (Parent et al., 1998; Scott et al., 1998; Liptakova et al., 1999), and continuous seizures or status epilepticus (Parent et al., 1997; Gray and Sundstrom, 1998; Covolan et al., 2000; Nakagawa et al., 2000). In addition, after pilocarpine-induced status epilepticus, newly generated granule cells do not necessarily migrate into the granule cell layer. Some appear to follow an abnormal course, moving into the hilus (Parent et al., 1997; Scharfman et al., 2000) presumably due to the disruption of normal migratory cues. A small number of ectopic granule cells (EGCs) are also present after ischemia (Hsu and Buzsaki, 1993). Surprisingly, electrophysiological studies of EGCs in the hilus of pilocarpine-treated rats have shown that their intrinsic electrical properties are comparable to granule cells that are positioned normally, in the granule cell layer (GCL GCs; Scharfman et al., 2000). However, they often display spontaneous epileptiform activity, which is never recorded from normal granule cells (Scharfman et al., 2000). The burst discharges appeared to be generated in area CA3, suggesting that EGCs were integrated into host circuitry (Scharfman et al., 2000). Other studies support this possibility, because EGCs were c-fos immunoreactive 3 h after a spontaneous seizure (Scharfman et al., 2002). Additionally, synapses can be observed on the surface of EGCs ultrastructurally (Dashtipour et al., 2001). This study was therefore initiated to further understand how newborn granule cells, after migrating to abnormal hilar locations, integrate into hippocampal circuitry. Specifically, stimulation of the perforant pathway, the major afferent projection to the dentate gyrus, was used to determine whether EGCs and GCL GCs displayed similar patterns of activation. Two stimulus sites in hippocampal slices were used to activate perforant path axons: the outer molecular layer, and a location in the subiculum, where perforant path axons travel as they reach the hippocampal fissure and descend into the dentate gyrus. Intracellular

b

Departments of Pharmacology and Neurology, Columbia University, New York, NY 10032, USA c

Division of Neurobiology, Weill Medical College of Cornell, New York, NY 10021, USA

Abstract—Granule cells in the dentate gyrus are born throughout life, and various stimuli can affect their development in the adult brain. Following seizures, for instance, neurogenesis increases greatly, and some new cells migrate to abnormal (ectopic) locations, such as the hilus. Previous electrophysiological studies of this population have shown that they have intrinsic properties that are similar to normal granule cells, but differ in other characteristics, consistent with abnormal integration into host circuitry. To characterize the response of ectopic hilar granule cells to perforant path stimulation, intracellular recordings were made in hippocampal slices from rats that had pilocarpineinduced status epilepticus and subsequent spontaneous recurrent seizures. Comparisons were made with granule cells located in the granule cell layer of both pilocarpine- and saline-treated animals. In addition, a few ectopic hilar granule cells were sampled from saline-treated rats. Remarkably, hilar granule cells displayed robust responses, even when their dendrites were not present within the molecular layer, where perforant path axons normally terminate. The evoked responses of hilar granule cells were similar in several ways to those of normally positioned granule cells, but there were some differences. For example, there was an unusually long latency to onset of responses evoked in many hilar granule cells, especially those without molecular layer dendrites. Presumably this is due to polysynaptic activation by the perforant path. These results indicate that synaptic reorganization after seizures can lead to robust activation of newly born hilar granule cells by the perforant path, even when their dendrites are not in the terminal field of the perforant path. Additionally, the fact that these cells can be found in normal tissue and develop similar synaptic responses, suggests that seizures, while not necessary for their formation, strongly promote their generation and the development of associated circuits, potentially contributing to a lowered seizure threshold. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. *Correspondence to: H. E. Scharfman, Center for Neural Recovery and Rehabilitation Research, Helen Hayes Hospital, New York State Department of Health, Route 9W, West Haverstraw, NY 10993–1195, USA. Tel: ⫹1-845-786-4859; fax ⫹1-845-786-4875. E-mail address: [email protected] (H. E. Scharfman). Abbreviations: AP, action potential; EGC, ectopic granule cell; EPSP, excitatory postsynaptic potential; GCL GC, granule cell in the granule cell layer; IPSP, inhibitory postsynaptic potential; PB, phosphate buffer.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00481-0

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recordings were obtained from four groups of granule cells. First, in hippocampal slices from pilocarpine-treated animals, EGCs were compared with GCL GCs. These responses were further compared with EGCs and GCL GCs in age-matched, saline-treated control animals. Although rare, EGCs are present in the normal hilus (Gaarskjaer and Laurberg, 1983; Marti-Subirana et al., 1986).

EXPERIMENTAL PROCEDURES Animal care and use followed the guidelines set by the N.I.H. Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and any discomfort. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless stated otherwise. Animals were provided food and water ad libitum and maintained on a 12-h light/dark cycle.

Pilocarpine treatment Adult male Sprague–Dawley rats (180 –240 g) were injected with atropine methylbromide (1 mg/kg s.c.) and 30 min later with pilocarpine hydrochloride (380 mg/kg i.p.). Diazepam (5 mg/kg i.p., Wyeth-Ayerst) was injected 1 h after the onset of status epilepticus. The low dose of diazepam was used because if given after 1 h of status epilepticus, a more reproducible pattern of damage occurs relative to rats without diazepam treatment, and mortality is reduced (Scharfman et al., 2000, 2001). Diazepam does not stop status epilepticus, but decreases its severity. The onset of status was defined as the first stage 5 seizure (Racine, 1972) that did not abate after several minutes. After approximately 5 h, animals were injected with 2.5 ml 5% dextrose in lactate-Ringer’s s.c. An apple was cut in half and laid at the bottom of the cage each day for approximately 7 days. Saline controls received identical treatment (atropine, diazepam, dextrose-saline, apple) as animals that were injected with pilocarpine, but were injected with saline instead. Injection of pilocarpine or saline was made at the same age (approximately 42 days old). All animals in this study that had status epilepticus had multiple spontaneous stage 5 seizures in subsequent months.

Microelectrode recording and analysis

Fig. 1. Distribution of sampled cells. A. The distribution of EGCs of epileptic rats in this study is shown. In addition, the sites of stimulation (mol. stim⫽outer molecular layer stimulation; sub. stim⫽subiculum stimulation) are indicated by x’s. In addition, the outline of the hilus is shown. GCL⫽granule cell layer; fissure⫽hippocampal fissure. B. The distribution of GCL GCs of epileptic rats is shown. For this purpose, the dentate gyrus was divided into sections as shown. The number of cells impaled in each region is indicated in parentheses. C. The distribution of EGCs in saline control rats is shown. D. The distribution of GCL GCs of saline control rats is shown.

with 4% Neurobiotin (Vector Labs, Burlingame, CA, USA) in 1 M potassium acetate (60 – 80 megaohms). Intracellular data were collected using an intracellular amplifier with a bridge circuit (Axoclamp 2B; Axon Instruments, Union City, CA, USA), and the bridge was balanced whenever current was passed. Extracellular electrodes were made from the same glass, broken to a lower resistance (5–10 megaohms), and filled with NaCl-buffer. Electrical stimulation. The stimulating electrode was monopolar and made from Teflon-coated wire (75 ␮m wide, including the Teflon coating); stimuli were triggered digitally (10 –200 ␮s; Pulsemaster; World Precision Instruments, Sarasota, FL, USA) using a stimulus isolator (100 ␮A; AMPI, Jerusalem, Israel). Stimulus frequency was less than 0.05 Hz. Data were collected using a digital oscilloscope (Pro10; Nicolet Instruments, Madison, WI, USA) and analyzed with accompanying Nicolet software and Origin 6.1 (OriginLab, Northampton, MA, USA). For stimulation of the outer molecular layer, the stimulating electrode was placed in the outer third of the layer, as close as possible to the hippocampal fissure, in the superior (dorsal) blade (Fig. 1A). To stimulate perforant path fibers without directly activating the dentate gyrus, the stimulating electrode was placed in the subiculum near the hippocampal fissure (Fig. 1A). In horizontal sections, perforant path axons can be visualized in the subiculum as white striations. These fibers coalesce at the hippocampal fissure upon entry into the superior blade of the dentate gyrus, and then form a white fiber bundle before diverging in the outer molecular layer. The stimulating electrode was placed on this fiber bundle. For transverse sections from septal hippocampus, the stimulating electrode was placed in the outer molecular layer at the point where the superior and inferior blades of the dentate gyrus merge.

Hippocampal slice preparation and maintenance. Hippocampal slices (400 ␮m thick) were prepared from rats that were deeply anesthetized with ether and decapitated. Slices were cut in ice cold buffer (“sucrose-buffer”) containing (in mM): 126 sucrose, 5 KCl, 2.0 CaCl2, 2.0 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, and 10 D-glucose (pH 7.4), using a Vibroslice (Stoelting Instruments, Wood Dale, IL, USA). Most slices were cut from the ventral half of the hippocampus in the horizontal plane. The others were cut transversely from the septal hippocampus. Slices were placed on a nylon net immediately after slicing, where they were maintained at an interface of sucrose-buffer and warm (31–33 °C), humidified (95% O2, 5% CO2) air using a slice chamber (Fine Science Tools, Foster City, CA, USA) that was modified to increase humidity at the location where slices were placed, and to allow slices to be submerged up to their surface. All slices from a given animal were placed in the recording chamber immediately after the dissection. Thirty minutes after slices were placed in the chamber, buffer was switched to one containing NaCl substituted equimolar for sucrose (“NaCl-buffer”). Recordings began 30 min thereafter until approximately 7 h after the dissection. Flow rate was approximately 1 ml/min.

Data analysis

Microelectrode recording. Intracellular recordings were made with borosilicate glass with a capillary in the lumen (0.75 mm i.d., 1.0 mm o.d.; World Precision Instruments), filled

Granule cells that were impaled were first screened to ensure that they were healthy (stable resting potential ⬎⫺65 mV, overshooting action potential [AP]). Intrinsic (membrane) properties were

H. E. Scharfman et al. / Neuroscience 121 (2003) 1017–1029 characterized using intracellularly injected current steps (0.05–1.5 nA, 200 ms). Resting potential was defined as the difference between the potential while intracellular and the potential reached upon withdrawal from the cell. APs were measured from resting potential to peak, using a directly evoked AP at threshold (Scharfman, 1995, 2000). Excitatory postsynaptic potentials (EPSPs) were measured using the highest stimulus strength that did not evoke APs at approximately ⫺55 to ⫺60 mV. Responses were evaluated at several membrane potentials to evaluate subthreshold EPSPs and, if present, subthreshold inhibitory postsynaptic potentials (IPSPs). For EPSPs without an IPSP component, latency to onset, latency to peak, rise time, peak amplitude and duration were measured from a maximal EPSP evoked at approximately ⫺70 to ⫺80 mV. As shown in Fig. 6A, latency to onset was calculated from the stimulus artifact to the onset of the depolarization. Stimulus artifacts were measured from the midpoint of the artifact. Latency to peak was defined as the time between the stimulus artifact and the peak of the EPSP. Rise time was measured from the onset of the EPSP to the peak of the EPSP. Peak amplitude was measured from baseline to peak. Duration was measured from the onset of the depolarization to the point on the decay phase that was half the peak amplitude, and is referred to as half-duration. If there was more than one peak, the first peak was used. For EPSPs which were followed by IPSPs, measurements of latency to onset and rise time of the EPSP were made from a response at ⫺55 to ⫺60 mV, using the highest stimulus strength that did not evoke an AP. This membrane potential was chosen because the EPSP was easily distinguished from the IPSP at these potentials. Peak amplitude of an EPSP that was followed by an IPSP was based on the synaptic response evoked at resting potential (between ⫺70 and ⫺80 mV). As shown in Fig. 6B, the EPSP peak was defined as the point on the evoked depolarization that corresponded to the EPSP peak at approximately ⫺55 to ⫺60 mV. Measured in this way, a cell with an EPSP followed by an IPSP could be compared with PSPs of other cells, in which peak EPSP amplitude was determined from the response at resting potential. Reversal potentials were based on responses to a fixed stimulus at several membrane potentials, using current injection to change membrane potential. Reversal potential was calculated from the linear regression of the relationship between response amplitude and membrane potential using Origin 6.1 software (OriginLabs). Statistical comparisons of data were made using Microsoft Excel. Significance was set at 0.05 prior to experiments. Average values are expressed as mean⫾S.E.M.

Intracellular labeling and processing Cells were injected with Neurobiotin from the recording electrode as previously described (Scharfman, 1995, 2000). Repetitive depolarizing current pulses (⫹0.3– 0.5 nA, 20 ms, 30 Hz) were delivered for a total of 5–15 min. Current pulses were triggered after recordings were completed. Immediately after each experiment, the slice was removed, placed flat in a Petri dish, covered with filter paper, and immersed in fixative. In preparation for light microscopic analysis, the slice was immersed in 4% paraformaldehyde (pH 7.4) and refrigerated. Slices were then sectioned (50 ␮m) using a vibratome (Ted Pella). Following incubation overnight in 0.5% Triton-X 100, sections were washed in Tris buffer (3⫻5 min), incubated in 0.3% H2O2 in 10% methanol for 30 min, washed, incubated in ABC (ABC standard kit; Vector Laboratories) in 0.1% Triton X-100 in Tris, washed in Tris, incubated in diaminobenzidine (Polysciences; 50 mg/ 100 ml Tris) and 0.1% NiNH3SO4 until the cell could be fully visualized (10 –30 min), washed in Tris, dehydrated in a series of graded alcohols (10 min each: 70%, 90%, 95% then 10 min in

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100% twice), cleared in xylene, and coverslipped in Permount (Fisher Scientific). Select cells and their processes were then reconstructed using a camera lucida attachment. Additionally, some slices were immersed in 2% acrolein and 2% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4), to allow future electron microscopic analysis. After 3 h they were transferred to 0.1 M PB, and then to a storage solution (30% sucrose and 10% ethylene glycol in 0.1 M PB). After refrigeration, each slice was sectioned (40 ␮m), and the cell was visualized as described above, except that Triton X-100 and NiNH3SO4 were not used. Sections were then postfixed in 2% osmium for 1 h, dehydrated in an alcohol series, and embedded in Embed 812 (Electron Microscopy Sciences, Fort Washington, PA, USA) between two Aclar plastic sheets.

RESULTS General results Sample size. Data were collected from 26 intracellularly recorded hilar EGCs and 29 GCL GCs from a total of 23 pilocarpine-treated rats. In 14 age-matched, saline control rats, five EGCs and 10 GCL GCs were impaled. All neurons were recorded at least 1 month after pilocarpine or saline was administered (range, 1–11 months). EGCs of epileptic rats were recorded in animals that were killed 5.5⫾0.5 months after pilocarpine (range, 1–11); GCL GCs of epileptic rats were recorded 3.7⫾0.5 months after pilocarpine (range, 1–10). All pilocarpine-treated rats had multiple recurrent spontaneous stage 5 seizures. The distribution of recorded neurons is presented schematically in Fig. 1. EGCs from epileptic rats were located throughout the hilar region (Fig. 1A). GCL GCs were sampled in comparable portions of the dentate gyrus (Fig. 1B, D). EGCs in saline-treated control rats were rare; however, it was possible to sample five (Fig. 1C). There was no evidence of physiological differences in cells with respect to the septotemporal axis or area of the hilus where they were sampled. As a result, the data were pooled in the analysis below into four groups: EGCs or GCL GCs of epileptic rats, and EGCs or GCL GCs of control rats. Distinguishing characteristics of EGCs and GCL GCs. As previously reported, the membrane properties and firing behavior of EGCs that arise after pilocarpine-induced seizures appear to be the same as the properties of normal adult GCL GCs (Scharfman et al., 2000). These characteristics are distinct from other types of neurons in the hilus (mossy cells or GABAergic neurons), and CA3c pyramidal cells (Scharfman, 1992, 1993, 1995, 1999), making it possible to categorize cells based on intrinsic electrophysiological criteria. All GCs sampled in this study displayed the intrinsic electrophysiological properties of granule cells. Representative characteristics of all four granule cell types included: 1) high resting potential (⫺70 to ⫺80 mV), 2) short time constant relative to mossy cells and pyramidal cells (10 – 15 ms vs. 20 –30 ms for non-granule cells, 3) broad duration AP (“regular spiking” according to terminology in McCormick et al., 1985) with a high ratio of maximum rate of rise/decay (⬎1.0), 4) triphasic afterhyperpolarizations after

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Fig. 2. Morphology of ectopic hilar granule cells with dendrites in the hilus. Reconstruction (412⫻) of the soma and dendritic tree of a neurobiotinlabeled EGC whose dendrites appeared to be restricted to the hilus. Dendrites which extended to the cut surface of the slice are marked with a small perpendicular line. Upper inset: position within the dentate gyrus. Lower inset: part of the mossy fiber projection to CA3c. Scale bars⫽50 ␮m. GCL: granule cell layer.

APs elicited by current injection, and 5) strong spike frequency adaptation in response to prolonged (100 –200 ms) injected current (Fig. 10; Staley et al., 1992; Williamson et al., 1993; Scharfman, 1995; Lu¨bke et al., 1998; Scharfman et al., 2000). Only cells displaying all of these characteristics were included in the study. Indeed, whenever one of these cells was identified, the morphology confirmed the characterization (n⫽11/11 EGCs in epileptic rats, n⫽13/13 GCL GCs in epileptic rats; n⫽5/5 EGCs in saline controls; Figs. 2 , 3, 11). In particular, all filled cells had small, oval somata relative to pyramidal cells, spiny dendrites, and, when an axon was visible, there was a mossy fiber projection to CA3c and giant terminals. In epileptic rats, the dendritic arbor of EGCs varied considerably in both extent and distribution (Figs. 2, 3). Most were impaled over 100 ␮m from the granule cell layer, and had dendrites that were almost exclusively confined to the hilar region (n⫽7). A representative cell of this type, with large spiny dendrites extending from the soma in a bipolar arrangement, is shown in Fig. 2. Other EGCs, impaled closer to the granule cell layer, had spiny dendrites as well, which extended into both the hilus and molecular layer (n⫽4; Fig. 3). There was no significant difference between these two groups in survival time after pilocarpine administration (EGCs with molecular layer dendrites, 4.1⫾1.3 months, n⫽4; EGCs without, 3.7⫾0.6, n⫽7; Student’s t-test, P⬎0.05).

to reach threshold was 56.7⫾4.4 ␮s (100 ␮A) for EGCs versus 57.2⫾4.3 ␮s (100 ␮A) for GCL GCs (tests were made at resting potential). Direct comparisons were also made between EGCs and GCL GCs within the same slice. In these cases, stimuli that were the same or less than those used to evoke responses in GCL GCs were able to evoke responses in EGCs of the same slice (n⫽3/3 slices; data not shown). In eight slices, a comparison was made between stimuli required to evoke a population spike while recording extracellularly in the granule cell layer, and stimuli necessary to evoke a response in an intracellularly-recorded EGC of the same slice. In all cases, the stimulus intensity for the population spike was equivalent or greater than the stimulus required to reach threshold in the EGC (n⫽8/8 slices; data not shown). Representative responses to outer molecular layer stimulation from EGC and GCL GCs of epileptic and control rats are presented in Fig. 4. They demonstrate that all four types of GCs displayed robust depolarizations. The response shown in part A was recorded from the EGC shown in Fig. 2, which had no identifiable dendrites in the molecular layer. Fig. 4B shows a recording from the EGC shown in Fig. 3, which had dendrites that branched extensively in the molecular layer. The other two cells were GCL GCs of either an epileptic rat (Fig. 4C) or a saline-treated control rat (Fig. 4D).

Responses to electrical stimulation of the perforant path

Variation in EPSP latency with morphology. As illustrated by Fig. 4, the latencies to onset of evoked responses appeared to vary in relation to the pattern of dendritic arborization (see also Table 4). The EPSPs of GCL GCs, regardless of the type of animal (control or epileptic), had a 2.51⫾0.33 ms mean latency to onset (n⫽39). This is consistent with the activation of a monosynaptic pathway,

General characteristics of synaptic responses. Remarkably, all EGCs (n⫽26; Table 1) had robust responses to stimulation of the outer molecular layer. Furthermore, high stimulus strengths were not required, relative to GCL GCs of epileptic rats. The mean stimulus strength needed

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Fig. 4. Responses to molecular layer stimulation of EGCs and GCL GCs of epileptic and control rats. A. The response of an EGC to molecular layer stimulation is shown. The morphology of this cell is shown in Fig. 2. It did not have dendrites in the molecular layer. In this and other figures stimulus artifacts are truncated, and marked by dots. For parts A–D, responses were elicited at resting potential (⫺72 to ⫺75 mV). B. The response of a different EGC (shown in Fig. 3) to molecular layer stimulation. This cell did have dendrites in the molecular layer. C. The response of GCL GC of an epileptic rat to molecular layer stimulation. D. The response of a GC from a saline control rat to molecular layer stimulation.

Fig. 3. Morphology of an ectopic hilar granule cell with dendrites in the molecular layer and hilus. Reconstruction (412⫻) of the soma and dendritic tree of a neurobiotin-labeled EGC with dendrites that branched extensively within the molecular layer. Dendrites which extended to the cut surface of the slice are marked with a small perpendicular line. Inset: position within the dentate gyrus. Scale bars⫽50 ␮m. GCL: granule cell layer, ML: molecular layer.

i.e. perforant path fibers innervating the molecular layer dendrites of the recorded granule cell. EGCs from epileptic tissue that had dendrites in the molecular layer had evoked responses with latencies similar to GCL GCs (mean 2.19⫾0.28 ms, n⫽4; Student’s t-test, P⬎0.05). In contrast, EGCs from epileptic rats that had no apparent dendrites in the molecular layer (n⫽7) had

long latencies: six of seven cells had latencies between 5 and 16 ms, and one had a 3 ms latency. The mean latency for all seven cells was 7.29⫾1.61 ms, which was statistically different from the mean latency of EGCs with molecular layer dendrites (Student’s t-test, P⬍0.05, see Table 4). Subiculum stimulation was compared with outer molecular layer stimulation in three EGCs of epileptic rats (Fig. 5). None of the cells had dendrites that penetrated the GCL or molecular layer. The responses to stimulation of the subiculum were similar to those evoked by molecular layer stimulation (Fig. 5). The major distinction was response latency: subiculum stimulation evoked responses with 2–3 ms longer latency to onset than the responses evoked by molecular layer stimulation (Fig. 5). This is similar to results from recordings of GCL GCs in untreated rats (data not shown). The results of these experiments supported the assumption that outer molecular layer stimulation activated a circuit that was initiated by perforant path fibers.

Table 1. Responses to stimulation of the molecular layer recorded in granule cells of pilocarpine and saline-treated rats

EGC epileptic, n⫽26 GCL GC epileptic, n⫽29 EGC control, n⫽5 GCL GC control, n⫽10

Simple EPSP (%)

Complex EPSPs (%)

Epileptiform bursts (%)

EPSPs followed by IPSPs (%)

Late EPSPs (%)

13 (50) 21 (72) 1 (20) 7 (70)

5 (19) 4 (13) 3 (60) 3 (30)

3 (11) 5 (13) 0 0

5 (19) 0 1 (20) 0

5 (18) 6 (20) 0 0

Frequency of responses in ectopic granule cells of epileptic rats (EGC-epileptic), granule cells in the cell layer of epileptic rats (GCL GC-epileptic), ectopic granule cells in control rats (EGC-control) and granule cells in the cell layer of control rats (GCL GC-control). Responses to stimulation had three forms: simple EPSPs refer to depolarizations with a single peak in response to a maximal subthreshold stimulus. Complex EPSPs correspond to depolarizations with multiple peaks. The third type of response was an all-or-none burst. Of these three types of responses, some had additional characteristics: 1) simple EPSPs could be followed by IPSPs, if the cell was depolarized by injected current to ⫺70 mV (EPSP-IPSP), and 2) EPSPs were sometimes followed by long latency depolarizations (late EPSPs).

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Fig. 5. Comparison of molecular layer and subiculum stimulation of an EGC from an epileptic rat. A. Responses of the cell shown in Fig. 2 to a molecular layer stimulus. 1. The stimulus was triggered at several membrane potentials by timing the stimulus during current pulses (200 ms duration). Resting potential⫽⫺72 mV. 2. The response at the most depolarized potential is expanded to more easily view the latency to onset relative to B2. B. Responses of the same cell as in A to a subicular stimulus. Responses triggered at several membrane potentials, as in part A. The response evoked at the most depolarized potential is expanded. The latency to onset is longer than the response to molecular layer stimulation (shown in A2).

Types of EPSPs: “simple” and “complex”. EGCs and GCL GCs demonstrated subthreshold synaptic potentials that were composed of depolarizations at resting potential. They are described below as EPSPs because increased stimulus intensity or depolarization led to APs at the peaks of these depolarizations.

EPSPs with a single peak are described as “simple” EPSPs below. In contrast, EPSPs with multiple peaks are termed “complex” (see Fig. 6). This terminology, as well as measurements of EPSP characteristics, was based on maximal EPSPs evoked at resting potential (approximately ⫺70 to ⫺80 mV), as mentioned above. This distinction is important because five neurons that demonstrated simple EPSPs (one peak) in response to strong stimulation produced complex EPSPs (multiple peaks) after weak stimuli (Fig. 6). Thus, the definition of “simple” and “complex” used here refers to the characteristics of the EPSP evoked by a maximal subthreshold stimulus. There was no apparent relationship between the length of survival time after pilocarpine treatment and the type of response that was generated upon molecular layer stimulation (simple, 4.4⫾0.7 months, n⫽15; complex, 3.2⫾0.4, n⫽8; Student’s t-test, P⬎0.05). Additionally, when more than one neuron was recorded from an animal, one could produce simple and the other complex EPSPs. Simple EPSPs The majority of EPSPs in this study were simple, i.e. there was one peak when stimulus strengths were near threshold. Tables 1 and 3 show the number of cells that exhibited simple EPSPs versus complex EPSPs, as well as the third type of response, an all-or-none burst of APs. The incidence of simple EPSPs did not appear to vary with cell type. For example, 50% of EGCs in epileptic rats and 72% of GCL GCs of epileptic rats demonstrated simple EPSPs,

Fig. 6. Definition and measurement of EPSPs. A. Measurements of EPSPs are shown. L1⫽latency to onset of the EPSP. L2⫽latency to peak. RT⫽rise time. HD⫽half-duration. X⫽peak amplitude. X/2⫽half of the peak amplitude. B. Measurements of EPSPs at different membrane potentials. The measurement of peak amplitude for EPSPs that were followed by IPSPs was made based on the latency to peak defined by the EPSP elicited at a membrane potential that was depolarized, because at this potential, the EPSP was clearly distinguished from the IPSP. C. Simple EPSPs were distinguished from complex EPSPs by the input– output relation. 1. Simple EPSPs were defined as an EPSP that had one peak in response to weak (left) or stronger (right) currents. Stimulus strength increases progressively from left to right. 2. Simple EPSPs also included cells that exhibited multipeaked EPSPs at low stimulus strengths (left), but single peaked EPSPs at higher stimulus strengths (right). 3. Complex EPSPs were defined as PSPs with multiple peaks regardless of stimulus strength.

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Table 2. Characteristics of evoked responses to molecular layer stimulation in EGCs and GCL GCsa Simple EPSPs

EGC-epileptic Mean S.E.M. n GCL GC-epileptic Mean S.E.M. n EGC-control Mean S.E.M. n GCL GC-control Mean S.E.M. n

Complex EPSPs Peak amp (mV)

Halfdur. (ms)

Lat. to onset (ms)

Lat. to peak (ms)

Rise time (ms)

Peak amp (mV)

Halfdur. (ms)

Lat. to onset (ms)

Lat. to peak (ms)

Rise time (ms)

2.65 0.50 13

8.73 0.91 13

6.08 0.84 13

9.83* 2.07 12

21.46 2.88 12

4.0∧ 0.61 5

15.00 3.11 5

11.00@ 3.05 5

9.40 1.24 5

48.40# 8.34 5

2.56 0.58 21

7.57 0.57 21

4.99 0.46 21

13.67* 1.15 21

18.6 1.54 20

2.06∧ 0.33 4

12.75 2.29 4

10.68@ 2.11 4

10.50 2.10 4

25.00# 4.56 4

6.0

11.5

5.5

8.5

27.5

2

3.17 0.93 3

9.67 2.67 3

6.50@ 3.32 3

7.67 1.45 3

25.67 9.94 3

3.50 0.76 3

8.83 1.59 3

5.33@ 2.20 3

9.67 1.45 3

19.17 5.46 3

2

2

2

2.04 0.25 7

6.50 0.70 7

4.46 0.56 7

2

11.71 2.13 7

17.42 1.69 7

a Lat. to onset, latency to onset; Lat. to peak, latency to peak; half-dur., half-duration. Half-duration was only measured for cells that had no evidence of IPSPs when they were depolarized, because when IPSPs occurred, they appeared to truncate the initial EPSP. * Mean peak amplitudes of simple EPSPs of EGCs and GCL GCs of epileptic rats were significantly different (P⫽0.0489). ∧ Mean latency to onset of complex EPSPs of EGCs and GCL GCs of epileptic rats were significantly different (P⫽0.01584). @ Mean rise time of all epileptic cells (EGCs and GCL GCs) and all control cells was significantly different (P⫽0.0402). # Mean half-duration of complex EPSPs of EGCs and GCL GCs of epileptic rats were significantly different (P⫽0.0235).

and this was not statistically significant (␹2 test, P⬎0.05). In control rats, 40% of EGCs and 70% of GCL GCs had simple EPSPs, a difference that was also not significant (␹2 test, P⬎0.05). In addition, there was no significant difference after pooling EGCs (i.e. epileptic and control, 15/31 cells, 48%) and GCL GCs (28/39, 72%; ␹2, P⬎0.05). Small sample sizes of EGCs in control rats precluded a direct comparison between control EGCs and control GCL GCs. Mean latencies, rise times, peak amplitudes and halfdurations of simple EPSPs are shown in Table 2. There were no significant differences among cell types, except that EGCs had smaller peak amplitudes than GCL GCs, in epileptic tissue (Table 2; Student’s t-test, P⬍0.05). The mean peak amplitude of all EGCs was also smaller than all GCL GCs (mean, EGCs, 9.54⫾1.60, n⫽28; mean, GCL GCs, 13.32⫾1.10, n⫽37, Student’s t-test, P⬍0.05). Complex EPSPs A subset of all types of GCs exhibited complex EPSPs, i.e. there were multiple peaks at all stimulus strengths

tested (Fig. 7; Tables 1, 2). APs could arise on any of the peaks (Fig. 7). For a given cell, these EPSPs varied from stimulus to stimulus in amplitude and in the number of peaks (Fig. 7). Examples shown in Fig. 7 include: an EGC from an epileptic rat (Fig. 7A), a GCL GC from an epileptic rat (Fig. 7B), a GCL GC from a control rat (Fig. 7C), and an EGC from a control rat (Fig. 7D). The incidence of complex EPSPs in EGCs and GCL GCs of epileptic tissue was not statistically different, and there was also no difference between the incidence of complex EPSPs in all EGCs versus all GCL GCs (Table 1, ␹2 tests, P⬎0.05). Two features distinguished complex EPSPs of EGCs and GCL GCs in epileptic tissue. First, the mean latency to onset was greater for EGCs (Table 2; Student’s t-test, P⬍0.05). Second, the mean EGC half-duration was longer (Table 2; Student’s t-test, P⬍0.05). These differences were not evident in control rats (Table 2). One explanation is that polysynaptic circuits that developed in epileptic tissue, presumably due to synaptic reorganization, were most robust in EGCs.

Table 3. Comparison of synaptic responses of EGCs of epileptic rats, with or without dendrites in the molecular layer

EGC with molecular layer dendrites, n⫽4 EGC without molecular layer dendrites, n⫽7

Simple EPSP (%)

Complex EPSPs (%)

All-or-none bursts (%)

EPSPs followed by IPSPs (%)

Late EPSP (%)

4 (75)

0

0

1 (25)

1 (25)

3 (14)

1 (14)

2 (28)

2 (28)

1 (14)

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Table 4. Simple EPSPs of EGCs: variation with dendritic arborization

EGC with molecular dendrites Mean S.E.M. n EGC without molecular dendrites Mean S.E.M. n

Lat. to onset (ms)

Lat. to peak (ms)

Rise time (ms)

Peak amp (mV)

Half- dur. (ms)

2.19* 0.28 4

10.25 1.84 4

8.06 1.93 4

10.02 5.43 4

18.33∧ 3.76 3

5.20* 0.66 6

13.60 1.69 6

8.40 2.11 6

8.20 1.98 6

34.40∧ 5.08 6

* Mean latencies to onset were significantly different (P⫽0.00428). ∧ Mean half-durations were significantly different (P⫽0.0220). For abbreviations, see legend to Table 2.

IPSPs. Subthreshold IPSPs were rarely observed (Table 2). In this study, “subthreshold IPSP” refers to an IPSP elicited without an AP. This distinction was used to avoid including IPSPs that were confounded by spike afterhyperpolarizations. Indeed, when suprathreshold stimuli were tested, they evoked hyperpolarizations that were far greater in amplitude than subthreshold stimuli. Subthreshold IPSPs only occurred in cells that exhibited simple EPSPs at resting potential. Thus, when cells with complex EPSPs were depolarized, no hyperpolarizations were present. Subthreshold IPSPs occurred in EGCs preferentially (Table 2; ␹2 test, P⬍0.05), indicating that the factors governing inhibition (i.e. GABAergic innervation, GABA receptor expression, etc.) may be different between EGCs and GCL GCs. The characteristics of subthreshold IPSPs were similar to those than have been previously described in hippocam-

pal principal cells. Thus, EPSPs were followed by IPSPs. The mean IPSP reversal potential of EGCs in epileptic tissue was ⫺66.9⫾2.14 mV (n⫽5), similar to previous studies of hippocampal neurons (McCarren and Alger, 1985; Janigro and Schwartzkronin, 1987; Bernardo, 1994).

Fig. 7. Complex EPSPs evoked by molecular layer stimulation of granule cells. A. Responses to two successive stimuli (20 s apart) are shown at three membrane potentials (top, ⫺60 mV; middle, ⫺66 mV; bottom, ⫺73 mV). Recordings were made from an EGC from an epileptic rat. Note that there are multiple components of the synaptic response, they vary from stimulus to stimulus, and increase in amplitude with hyperpolarization. B. A complex EPSP evoked by molecular layer stimulation in a GCL GC of an epileptic rat. The same stimulus is shown at two different membrane potentials (top, ⫺62 mV; bottom, ⫺72 mV). C. A complex EPSP recorded in a GCL GC of a control rat. Membrane potential, ⫺74 mV. D. A complex EPSP evoked in an EGC recorded from a control rat. Membrane potential, ⫺70 mV.

Fig. 8. Late EPSPs and burst discharges in epileptic rats. A. Recordings from an EGC are shown in response to two different stimulus strengths. In response to a 15 ␮s stimulus, a small EPSP followed at a long latency by a large EPSP and two APs occurred. After 5 s, a 40 ms stimulus was triggered, and the large EPSP and APs occurred at a short latency. B. Responses of an EGC to the same stimulus at three different membrane potentials. A complex EPSP was evoked at a short latency, and was followed by a large depolarization at a longer latency. At depolarized potentials, both the early and late EPSP could evoke APs. C. All-or-none burst discharges of a GCL GC. The first stimulus evoked a burst discharge, but the next stimulus, triggered 5 s later, evoked no response.

Evoked burst discharges. Epileptiform burst discharges were evoked in a subset of neurons recorded in epileptic rats (n⫽8/55), but not controls (0/15; Table 2). These bursts had two forms. First, all-or-none bursts were observed. These were composed of large (⬎20 mV, ⬎50 ms) depolarizations with superimposed APs (Fig. 8). They were characterized as all-or-none because a fixed stimulus could evoke no response after an initial test, but after another test, the same stimulus could evoke a burst discharge (Fig. 8). Other bursts were preceded by an initial EPSP that was similar to those described above (n⫽2 EGCs, n⫽2 GCL GCs; Fig. 8). The survival time after pilocarpine treatment did not appear to influence whether burst discharges were observed. Thus, bursts were recorded in cells from animals that were killed 6.0⫾1.1

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Fig. 9. Late EPSPs that were synchronized with area CA3 pyramidal cells. Recordings are shown from an EGC of an epileptic rat. A. The response to three identical stimuli are shown. Each response was elicited 5 s apart. The first two evoked a late depolarization (arrow), but the third did not, indicating its all-or-none nature. B. Top: A lower intensity stimulus evoked a similar late depolarization at a longer latency (arrow). Bottom: Simultaneous extracellular recording in the CA3b pyramidal cell layer shows that the late EPSP (arrow) occurs at a similar time as epileptiform bursts in CA3. C. Top: spontaneous activity in the EGC of parts A, B. Bottom: A simultaneous recording from CA3b shows that the burst discharge in area CA3 precedes the depolarization in the EGC. Calibration: 5 mV, intracellular; 2 mV, extracellular.

months after pilocarpine (n⫽8), whereas other animals were killed 4.5⫾0.4 months after pilocarpine (n⫽46; Student’s t-test, P⬎0.05). Late EPSPs. Depolarizations with a long latency to onset (“late EPSPs”), arising after the decay of a short latency EPSP, occurred in EGCs and GCL GCs from epileptic rats (Table 1). All late EPSPs had multiple peaks (Fig. 9). In some of these recordings, an additional extracellular electrode in the CA3 pyramidal cell layer was used to record the response of area CA3 neurons to the same stimulus. These recordings showed that an epileptiform burst in area CA3 occurred at a similar latency to the all-or-none depolarizations in the GC. Fig. 9 shows an example in which recordings were made simultaneously, illustrating that the epileptiform burst in area CA3 actually preceded the late EPSPs (n⫽5/5 slices tested). Spontaneous epileptiform bursts in area CA3 were also observed in these experiments, and when CA3 burst discharges occurred spontaneously, a GC depolarization also occurred (Fig. 9, n⫽5). This indicates that CA3 activity may have been responsible for late EPSPs, as has been suggested in previous studies of hilar neurons (Scharfman, 1994; Scharfman et al., 2001). The present results extend the previous studies by showing that GCL GCs in the epileptic dentate gyrus also appear to be influenced by epileptiform activity in area CA3. EGCs in control rats. EGCs in control rats, although rare, also responded to stimulation of the outer molecular layer (n⫽5). Recordings from one of these cells are shown in Fig. 10, and its reconstruction is shown in Fig. 11. Morphologically, this cell was indistinguishable from EGCs in epileptic tissue with molecular layer dendrites: spines were observed on dendrites, and a mossy fiber axon formed giant boutons in stratum lucidum of area CA3.

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Fig. 10. Recordings from an EGC in a slice of a control rat. A. Responses to increasing currents to the outer molecular layer (from top to bottom: 10, 20, 30, 40, 50, 60 70 ␮s) demonstrate an EPSP with multiple peaks at low stimulus strengths and a simple EPSP at higher stimulus strengths. B. Testing the 70 ␮s stimulus at three different membrane potentials showed that it evoked an EPSP followed by an IPSP. C. Responses to current pulses (⫾0.1, 0.2, 0.3 and ⫹0.4 nA) demonstrate membrane properties and AP waveform of a typical GCL GC. D. Responses to increasing current pulses (from left to right) demonstrate strong spike frequency adaptation of a granule cell.

Evoked responses to molecular stimulation were robust, and the stimulus strengths that were required were within the range used for GCL GCs. The cell in Fig. 10 had a complex EPSP at low stimulus strengths, and an EPSP with one peak at higher stimulus strengths (Fig. 10A). The EPSP was followed by an IPSP at depolarized membrane potentials (Fig. 10B). EGCs in control rats were also similar electrophysiologically to EGCs in epileptic rats (Fig. 10C, D). Other EGCs in control tissue had evoked responses that were similar to EGCs in epileptic tissue, except that there were no spontaneous or evoked bursts, consistent with the fact that the tissue was from control rats. Thus, EPSPs were either simple (n⫽3) or complex (n⫽2). Comparisons of EPSP latency, rise time, peak amplitude, and half-duration are shown in Table 2. There were no statistical differences between EGCs of epileptic versus control rats in any of these characteristics, when all cells were considered. When EGCs were subdivided into those that had simple versus complex EPSPs, sufficient sample sizes for statistical comparisons were only available for complex EPSPs. In this comparison, there was only a significant difference in EPSP duration. EGCs of epileptic rats had significantly longer EPSP half-durations (Student’s t-test, P⬍0.05), but latencies and peak amplitudes were not statistically different (Student’s t-test, P⬎0.05).

DISCUSSION This study examined synaptic potentials evoked by perforant path stimulation of granule cells located in either the hilus or granule cell layer, in both epileptic and control rats. Surprisingly, many characteristics of the evoked responses were quite similar across all the groups examined. In particular, comparable levels of stimulation evoked robust synaptic potentials, regardless of whether the cell had dendrites extending into the molecular layer, or the

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Efficacy of stimulation

Fig. 11. Morphology of an EGC from a control rat. Reconstruction (400⫻) of the soma and dendritic tree of a neurobiotin-labeled EGC in a slice from a normal rat, whose responses are shown in Fig. 10. Dendrites which extended to the cut surface of the slice are marked with a small perpendicular line. Upper inset: position within the dentate gyrus. Lower inset: part of the mossy fiber projection to CA3c. Scale bars⫽50 ␮m. GCL: granule cell layer, ML: molecular layer.

slice was from a control versus epileptic rat. EGCs thus appear to develop a pattern of afferent input within the hilus that can support relatively normal levels of activation in response to perforant path stimulation. However, there were also distinct differences in the evoked responses: the latency to onset was usually much longer, suggesting that polysynaptic connections were involved. Epileptic tissue may in general include more complex circuitry, given that perforant path stimuli to both EGCs and GCL GCs evoked longer complex EPSPs in slices from epileptic rats relative to control rats. Furthermore, burst discharges were evoked only in epileptic tissue. Some of the data suggest that the pathways mediating these responses may involve area CA3 pyramidal cells, but additional pathways, such as those produced by mossy fiber sprouting, are also likely to contribute. These results provide another example of the extensive restructuring that can occur in the epileptic dentate gyrus: not only are substantial numbers of new granule cells generated after seizures, but it appears that those which migrate into the hilus can insert themselves into novel, functional circuits.

Although it is common to stimulate the molecular layer in slices to gain insight into the responses of dentate neurons to the perforant path, it also should be acknowledged that activating the perforant path may not evoke the same responses as stimulation of the outer molecular layer. Certainly one concern is whether stimulus current spreads to the inner molecular layer if an electrode is placed in the outer two-thirds of the molecular layer. In our experience, stimulus currents can spread unless the electrode is placed in the outer one-third, which is why that location was used for the entire study. To gain direct insight into potential differences, responses to molecular layer stimulation were compared in a subset of neurons to responses elicited by stimulation in the subiculum. The subiculum stimulus site was close to the fissure, in the area where the perforant path can be visualized as white striations that coalesce just before entering the dentate molecular layer and subsequently diverging. The premise was that responses that were elicited by both stimulus sites would be those that reflected perforant path activation. Responses that were dissimilar would indicate that other neurons in the vicinity of the electrode, and not necessarily the perforant path, were responsible for the evoked responses. For example, fibers might be activated in stratum lacunosum-moleculare by a subicular electrode close to CA1. These could potentially activate interneurons that cross the fissure or CA3 neurons that innervate hilar cells. The results showed that responses were similar regardless of stimulus site, except that an increased latency occurred in response to subicular stimulation relative to outer molecular layer stimulation. Although all granule cells were not examined in this way, so there may be exceptions, this suggests that responses to molecular layer stimulation reflected activation of entorhinal input to the dentate gyrus through the perforant pathway. Granule cells displayed robust responses regardless of position The primary finding of this study is that EGCs exhibited robust synaptic potentials in response to perforant path stimulation. For most of the characteristics examined, evoked responses in EGCs were comparable to responses in GCL GCs from both control and epileptic animals. This held true regardless of the position of the GC within the hilus. Similar synaptic potentials were recorded in GCs with somata relatively close to the granule cell layer that had molecular layer dendrites, and cells situated deeper in the hilus that did not appear to develop molecular layer dendrites. These data strongly suggest that axonal processes within the dentate gyrus reorganize to densely innervate the new EGCs. This is perhaps not that surprising, since GCL GCs in epileptic animals are known to substantially restructure, developing new axon collaterals and novel termination sites. Mossy fibers sprout into the inner molecular layer to form connections with granule cells and also

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interneurons (Sutula et al., 1992; Dudek et al., 1994; Represa et al., 1994; Okazaki et al., 1999). Basal dendrites have also been observed on some granule cells in epileptic tissue, providing another novel hilar target (Spigelman et al., 1998; Ribak et al., 2000; Dashtipour et al., 2001). Sprouting of GABAergic neurons (Davenport et al., 1990; Mathern et al., 1997) and cholinergic fibers (Holtzman and Lowenstein, 1995) have also been previously reported. EGCs had longer latency responses, suggesting polysynaptic activation The greatest distinguishing feature between the groups of granule cells examined was the latency to onset. GCL GCs of both control and epileptic animals displayed the fastest latencies to onset, while EGC latencies were longer. Additionally, there was a major latency difference between EGCs that had dendrites extending into the molecular layer and those that did not. It seems unlikely that these differences are due to variable pathology, since the pattern of pathology in animals treated with pilocarpine using our methods (diazepam at 1 h after onset of status) appear to have been consistent across animals (Scharfman et al., 2000, 2001). However, we cannot exclude the possibility that some of the differences among cells reported in the present study were due to characteristics of the particular epileptic animals used, and may relate to differential pathology. Since the monosynaptic pathway to GCL GCs requires 2–3 ms, the EGCs with longer latencies were presumably activated through polysynaptic connections. One possible pathway for polysynaptic activation is the perforant pathto-GCL GC synapse, followed by a GCL GC-to-EGC connection (Fig. 12A,B). A possible trisynaptic pathway would involve perforant path-to-GCL GC-to-mossy cell-to-EGC connections (Fig. 12C). This possibility is suggested by previous studies showing that excitatory hilar mossy cells may survive status epilepticus after pilocarpine (Scharfman et al., 2001). A third circuit could involve perforant path-to-GCL GC-to-CA3 pyramidal cell-to-EGC circuitry, because CA3 pyramidal cells appear to drive EGC discharges (Fig. 12C and D). Additional recurrent excitatory pathways can be imagined, since EGCs could potentially form interconnections with each other, and with surviving mossy cells, that might promote reverberating excitatory pathways. In addition, some EGCs, like normal GCL GCs following seizures, have axons that sprout into the inner molecular layer (Scharfman et al., 2000), providing other potential excitatory pathways. Elucidation of the specific pathways involved will require further careful anatomical and physiological analysis. The development of complex recurrent excitatory circuits with multiple latencies could explain why the large percentage of neurons in epileptic tissue displayed complex EPSPs. Some of these complex synaptic responses were evident in granule cells from normal tissue, although the durations were not as long. One explanation for the latency difference of complex EPSPs in EGCs and GCs is that they are mediated by distinct circuits. In control tissue, sprouting may be present at a low level (Molnar and Na-

Fig. 12. Potential polysynaptic pathways that could underlie molecular layer-evoked EPSPs of EGCs without dendrites in the molecular layer. A. A schematic diagram shows some of the cell types in parts B, C, and D. PP⫽perforant path. B. Illustration of disynaptic pathways that would lead to an EPSP in an EGC. A molecular layer stimulus that would trigger an AP in a GCL GC could lead to an EPSP in the EGC, if the GCL GC innervates the EGC, or if the PP innervates a mossy cell (MC) that targets an EGC (Scharfman, 1991) PP-perforant path. C. Illustration of trisynaptic pathways that would activate an EGC. Initial activation of a GCL GC could indirectly excite an EGC by exciting a pyramidal cell (PC) or MC that in turn innervates the EGC. A 4 synapse pathway that could lead to EGC activation. Perforant path activation of GCL GCs that innervate CA3 PCs could lead to EGC excitation by axon collaterals of CA3 which innervate MCs or other EGCs.

dler, 1999) and play a role. In EGCs, proximity to hilar neurons and the complex circuitry among hilar cells may predispose EGCs to complex evoked responses. Other circuits involving GABAergic neurons are also likely to be involved, since a large percentage of EGCs demonstrated IPSPs. It would theoretically be possible to activate dentate GABAergic neurons, either by stimulating their dendrites/axons in the molecular layer, or by activating them indirectly by stimulating perforant path or GC axons that innervate GABAergic neurons. It is acknowledged that IPSPs may have truncated EPSPs, making our estimation of the EPSP peak less accurate. Differences in EPSP characteristics between EGCs and GCL GCs Although many EPSP characteristics were similar between EGCs and GCL GCs, there were three that were not. One was the difference in latency to onset, and another was the apparent selectivity of IPSPs for EGCs. There was also a difference in half-duration: EGCs in epileptic rats had longer complex EPSP half-durations than GCL GCs. This might be due to an increased density of recurrent excitatory connections among hilar neurons, related to the close proximity of EGCs to axons of several glutamatergic cells, such as other EGCs, hilar mossy cells, and CA3 pyramidal neurons. EGCs may readily develop afferent input because they are young cells, and in addition, are situated close to axons that have recently lost their target cell. Hilar cell loss may also be a stimulus for GCL GC axons in the

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hilus, mossy cell axons and CA3 pyramidal cell axons. Indeed, it has been proposed that hilar cell loss is a stimulus for mossy fiber sprouting, although some studies have pointed out that hilar loss is probably not the only consideration (Qiao and Noebels, 1993; Stringer et al., 1997; Gorter et al., 2001; Nissinen et al., 2001). EGCs and epilepsy Much has been written about the development of new granule cells in the dentate gyrus after seizures. However, little is known about them after they mature into adult neurons. Van Praag and Gage (2002) showed that GCs that are born in the normal adult dentate gyrus develop dendritic input that appears similar to existing granule cells. Currently, the present data indicate that, in both the normal and epileptic rodent brain, at least a percentage of the new cells appear to develop strong afferent input, even within an abnormal neural environment. Thus, these findings add to the growing evidence that neurons born in the dentate gyrus in the adult animal are functional, and appear to participate in functional neural circuits. What is the evidence that these findings in rodents can be generalized to man, i.e. humans with temporal lobe epilepsy? The results of studies in humans currently make predictions difficult. Thus, some studies demonstrate robust neurogenesis in man (Eriksson et al., 1998), while others suggest neurogenesis after chronic epilepsy is not pronounced (Blumcke et al., 2001). Additionally, a surprising aspect of these results is that EGCs of control rats had some of the characteristics of EGCs in epileptic rats. This suggests that epileptiform activity is not essential to the development of hilar EGCs, or afferent input to them. It may be that some EGCs are present before seizures, and new EGCs are inserted into developing circuitry, some of which may be pre-existing. However, because the number of EGCs is so much greater in epileptic tissue, it seems likely that something about seizures facilitates the processes that are involved in supporting migration to aberrant locations. In turn, the increased density of EGCs could presumably result in greater recurrent excitatory interconnectivity, a factor that could potentially contribute to a lower seizure threshold. The fact that EGCs and other hippocampal neurons display c-fos immunoreactivity concurrently following seizures (Scharfman et al., 2002) suggests that EGCs participate in circuits that are involved in supporting recurrent seizures in epileptic rats. Acknowledgements—We thank Annmarie Curcio for general assistance and Ruth Marshall for administrative support. This study was supported by NS 38285, NS 37562, NS 41490 and the Helen Hayes Hospital Foundation.

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(Accepted 10 June 2003)