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Keywords:

  • Epilepsy;
  • Pilocarpine;
  • Embryonic stem cells;
  • Patch-clamp;
  • Transplantation

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Summary: Purpose: Embryonic stem (ES) cell–based therapy strategies are thought to bear considerable promise in chronic neurologic disorders. Nonetheless, studies addressing the functional properties of ES cell–derived progeny after transplantation into the adult, pathologically modified CNS are scarce.

Methods: We therefore transplanted ES cell–derived neural precursors expressing enhanced green fluorescent protein only in neuronal progeny bilaterally into the hippocampi of pilocarpine-treated chronically epileptic and sham-control rats. Whole-cell patch-clamp recordings of identified ES cell–derived neurons (ESNs) in hippocampal slices were performed 13 to 34 days after transplantation.

Results: Most ESNs were found in clusters at the transplant site and did not migrate into host tissue. However, they gave rise to a dense network of processes extending over large distances into the host tissue. All ESNs possessed the ability to generate action potentials and expressed voltage-gated Na+ and K+ currents, as well as hyperpolarization-activated currents. Likewise, most ESNs received non–N-methyl-d-aspartate (NMDA) and γ-aminobutyric acid (GABA)A receptor-mediated synaptic input. Both types of synapses displayed intact short-term plasticity. An unusual feature of the majority of ESNs was the occurrence of spontaneous pacemaking activity at frequencies ∼3 Hertz. No obvious differences were found between the functional properties of ESNs in sham-control and in pilocarpine-treated rats.

Conclusions: After transplantation into adult control and epileptic rats, ESNs displayed intrinsic and synaptic properties characteristic of neurons. Even though ESNs remained close to the transplant site, the formation of extensive networks of graft-derived processes may be useful for ES cell–based substance delivery.

Loss of specific subpopulations of neurons is a prominent feature of many common neurologic disorders. In a large fraction of mesial temporal lobe epilepsy cases, pronounced neuron loss occurs within specific subfields of the hippocampus, a condition termed Ammon's horn sclerosis (AHS) (1,2). AHS in patients with epilepsy is characterized by a dramatic loss of neurons in the CA1 and CA3 subfields of the hippocampus, as well as the dentate hilus. Further hallmarks of AHS are a pronounced gliosis and axonal sprouting of surviving neuronal populations (2). These major features of AHS are replicated in a number of animal models of chronic epilepsy. In the pilocarpine or kainate models of epilepsy, a prolonged status epilepticus induced by these agents results in a segmental neuron loss within the Ammon's horn reminiscent of AHS (3,4). In both human temporal lobe epilepsy and animal models of chronic epilepsy, progressive neuron loss within the hippocampus was demonstrated to correlate with impairment of hippocampal function (5–7). Loss of specific cellular subpopulations is also thought to play a major pathogenetic role in a multitude of other CNS disorders. For instance, in Parkinson's disease, loss of dopaminergic neurons from the pars compacta of the substantia nigra is thought to contribute substantially to functional deficits. These observations have stimulated research aimed at selectively replacing neurons lost during the disease process and the mediators they produce.

Hitherto, different donor cell types have been exploited for neuronal repair strategies. In Parkinson's disease, most experimental and clinical approaches have used neurons derived from fetal CNS tissue for transplantation (8). More recently, embryonic stem (ES) cell–based approaches have generated considerable interest for reconstructive purposes. ES cell–derived progeny incorporate readily into the developing CNS after transplantation and give rise to functional neurons (9,10) and glial cells (11) that integrate into the host tissue. An ES cell–based reconstructive approach has been implemented in an animal model of Parkinson's disease, in which dopaminergic neurons had been selectively deleted. In these experiments, ES cell–derived progeny gave rise to dopaminergic neurons in the host CNS, and functional recovery was observed after transplantation (12). These studies illustrate a number of major advantages of ES cell–based reconstructive approaches in the CNS: ES cells can be expanded to virtually unlimited numbers in vitro, they are amenable to genetic modification, and they allow the generation of highly purified populations of neural precursor cells. Surprisingly, the study by Kim et al. (2002) has remained the only one in which the potential of ES cells to replace lost neurons has been tested in a model of a human CNS disease.

In chronic epilepsy, ES cell–based approaches might be promising to replace neuronal populations lost during the course of the disease. Alternatively, ES cell–derived progeny producing inhibitory mediators could be used to inhibit seizure activity. Reduction of seizure susceptibility has already been achieved by intrahippocampal transplantation of fetal noradrenergic or cholinergic grafts (13,14). Transplantation of immortalized cell lines genetically engineered to produce γ-aminobutyric acid (GABA) appears to reduce excitability in chronic epilepsy models (15,16). Similar results were also seen after transplantation of encapsulated fibroblasts genetically engineered to produce adenosine (17).

So far, the potential of transplanting ES cell–derived neural precursors into the chronically altered CNS, either for neuronal reconstruction or for the release of inhibitory mediators, has been poorly investigated. In the present study, we therefore assessed the potential of ES cell–derived neural precursors for functional integration after transplantation into the hippocampus of adult control and chronically epileptic rats.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Pilocarpine model

Pilocarpine treatment of rats was carried out largely as described previously (18). In brief, male Wistar rats (170–180 g) were injected subcutaneously (s.c.) with 370 mg/kg pilocarpine 30 min after pretreatment with 1.1 mg/kg scopolamine s.c. to reduce peripheral cholinergic side effects. After pilocarpine injection, a majority of the animals developed a limbic status epilepticus that was terminated by injection of diazepam (DZP) 40 min after onset (0.5–2.5 mg). In the minority of animals that did not develop status epilepticus after the first injection of pilocarpine, a second, identical s.c. injection of pilocarpine was administered. The rats were tended and fed with glucose solution for 1 day and kept in separate cages. Sham-control animals were treated in an identical manner, but were injected with saline instead of pilocarpine. Characteristic seizure-related changes in the pilocarpine-treated animals were assessed by the macroscopic hippocampal atrophy as well as by Timm stains that visualize the zinc-rich mossy fibers, and thus the characteristic mossy fiber sprouting.

ES cells

In this study, ES cell–derived neural precursors used for transplantation were genetically engineered to express enhanced green fluorescent protein (EGFP) under control of the tau promoter (19). These cells are derived from the J1 ES cell line (20) and carry the complementary DNA (cDNA) for EGFP targeted in-frame into exon 1 of the tau gene. This results in a fusion protein consisting of the first 31 amino acids of tau and EGFP. The generation of ES cell–derived neural precursors from tau EGFP knockin ES cells was performed as described previously (21,22). In brief, ES cells were aggregated to embryoid bodies, which were subsequently plated and propagated in ITSFn medium (DMEM-F12 supplemented with 5 μg/ml insulin, 50 μg/ml transferrin, 30 nM sodium selenite, and 5 μg/ml fibronectin) for 5 to 7 days. Cells were then trypsinized, triturated to a single-cell suspension, and replated in polyornithine-coated dishes. They were then propagated for an additional 4 to 5 days in DMEM-F12 supplemented with 25 μg/ml insulin, 50 μg/ml transferrin, 30 nM sodium selenite, 20 nM progesterone, 100 nM putrescine, 1 μg/ml laminin, and 10 ng/ml fibroblast growth factor 2. Media, supplements, and growth factors were obtained from Invitrogen (Karlsruhe, Germany), R & D Systems (Wiesbaden, Germany), and Sigma (Taufkirchen, Germany). For transplantation, donor cells were trypsinized and triturated through flame-polished Pasteur pipettes. They were then washed in calcium- and magnesium-free HBSS and concentrated to 5–8 x 104 cells/μl. After in vitro differentiation of this cell line, EGFP fluorescence has been found to be restricted to neuronal progeny (22).

Transplantation

One month after pilocarpine treatment, the rats were anesthetized with intraperitoneal injection of ketamine hydrochloride (8 mg/100 g) and xylazine hydrochloride (1 mg/100 g), and placed in a stereotactic frame (Stoelting, Wood Dale, IL, U.S.A.) for intrahippocampal transplantation of ESNs. Two microliters of a cell suspension containing ∼150,000 ES cell–derived neural precursors derived as described earlier, with 0.1% DNAse, were injected bilaterally into the hippocampi of seven control and 11 pilocarpine-treated rats via a thin glass capillary. The animals were immunosuppressed with daily intraperitoneal injections of cyclosporine (1 mg/100 g body weight; Novartis, Basel, Switzerland). Macroscopically, teratoma formation was never observed. Care and use of the animals conformed to institutional policies and guidelines.

Electrophysiology

Electrophysiological recordings were performed 13–34 days after transplantation. Rats were anesthetized with ether and subsequent injection of ketamine hydrochloride and xylazine hydrochloride. After thoracotomy, the descending aorta was clamped, and the animals were perfused via the left ventricle with 40–50 ml of ice-cold solution of the following composition (in mM): 60 NaCl, 100 sucrose, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 1 CaCl2, 5 MgCl2, and 25 glucose. After perfusion, rats were decapitated, the brains rapidly removed, and 300 μm coronal slices were cut with a vibratome. After cutting, slices were stored for 30 min at 36°C in the sucrose-containing solution used for heart perfusion. Subsequently, they were maintained in artificial cerebrospinal fluid (aCSF) of the following composition (in mM): 135 NaCl, 3 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 1 MgCl2, and 25 glucose (pH 7.4, room temperature). For whole-cell patch-clamp recordings, one slice at a time was transferred onto the stage of an upright microscope (Axioskop FS II; Zeiss, Jena, Germany), equipped for infrared differential interference contrast and fluorescence, and perfused with aCSF (0.7 ml/min). Embryonic stem cell-derived neurons (ESNs) were identified by their expression of EGFP, which resulted in a strong fluorescence signal that was readily identified by using a fluorescence camera (Spot Jr.; Diagnostic Instruments/Visitron Systems, Puchheim, Germany). EGFP+ cells were subsequently visualized by using infrared video microscopy and differential interference contrast optics to obtain patch-clamp recordings under visual control. Whole-cell voltage- and current-clamp recordings from ESNs were obtained by using the patch-clamp technique. Borosilicate capillaries (4–5.5 MΩ) were fabricated with a vertical puller (PP-830; Narishige, Tokyo, Japan) and filled with an intracellular solution containing (in mM): 87.5 Cs-methanesulfonate, 10 N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonate) (HEPES), 20 tetraethylammonium chloride, 5 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), 10 glucose, 5 MgCl2, 0.5 CaCl2, 10 Na2-adenosine triphosphate (ATP), 0.5 guanosine triphosphate (GTP), 5 lidocaine N-ethyl bromide, 20 sucrose, 0.1% biocytin (pH 7.2) for recordings of postsynaptic currents; otherwise, they contained (in mM): 20 KCl, 130 K-gluconate, 1 ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, 0.35 CaCl2, 2 MgCl2, 2 Na2-ATP, and 0.1% biocytin (pH 7.2). After obtaining the whole-cell configuration, voltage- and current-clamp recordings were carried out with an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany). Data were sampled at 5–50 kHz (voltage-clamp recordings) and 4–25 kHz (current-clamp recordings), and filtered appropriately. In voltage-clamp recordings, series resistance compensation was used to minimize voltage errors (average series resistance compensation 60%; average series resistance 13 MΩ). For pharmacologic isolation of postsynaptic GABAA receptor–mediated currents, 50 μM 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX) and 50 μMdl-2-amino-5-phosphonopentanoic acid (DL-AP5) were added to the aCSF. Non–NMDA receptor–mediated excitatory postsynaptic currents were isolated by adding 50 μM DL-AP5 and 10 μM bicuculline to the aCSF. All recordings were performed at room temperature. Apart from CNQX and DL-AP5 (Tocris, Ellisville, MO, U.S.A.), all chemicals were obtained from Sigma. Student's t test (significance level 0.05) or nonparametric tests were used for statistical analyses. All data are given as means ± SEM.

Immunohistochemistry

After electrophysiology, slices were fixed in 4% paraformaldehyde phosphate-buffered saline (PBS; Seromed, Berlin, Germany) for 1–2 days. Part of the slices were processed for fluorescent Texas red avidin (Dako, Hamburg, Germany) or permanent diaminobenzidine (DAB) biocytin staining, according to standard techniques. From the remaining slices, 25- to 30-μm cryostat sections were obtained after cryopreservation in 30% sucrose saline. Double immunofluorescence staining for EGFP and M6, a mouse-specific membrane-bound protein, were done according to established protocols. In brief, cryostat sections were pretreated with 5% normal goat serum (NGS; Sigma) in PBS for 30 min. Then slices were incubated overnight with PBS containing 0.1% sodium azide, 3% NGS, and antibodies for M6 (1:10; Developmental studies hybridoma bank, Iowa City, IA, U.S.A.) and EGFP (1:1,000; Dako). The next day, the slices were washed with PBS and secondary antibodies for EGFP (FITC anti-rabbit immunoglobulin G (IgG), 1:200; Jackson, West Grove, PA, U.S.A.) and M6 (biotin goat anti rat, 1:50; Jackson) in PBS with 3% NGS were applied for 2–4 h. Finally, after washing with PBS, Texas Red avidin (1:125) in 10 mM HEPES buffer (pH 7.5) was applied for 1.5 h.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

After transplantation, ESNs were readily identified by their expression of EGFP. This allowed detection of intrahippocampal transplants in four sham-control and five pilocarpine-treated rats. The locations of grafts are summarized in Fig. 1A. Double immunofluorescence staining for both EGFP and M6, a mouse-specific membrane-bound protein, confirmed that a large fraction of transplanted ES cell–derived neural precursors give rise to EGFP+ ESNs (Fig. 1B and C depict representative examples from control and pilocarpine-treated animals, respectively). In both control and epileptic animals, graft-derived ESNs generally remained in clusters at the transplant sites or close to injection canals. On closer examination, the borders of the clusters were not sharply delineated, with individual ESNs migrating short distances into the host tissue.

image

Figure 1. Transplant morphology and localization. A: Intrahippocampal transplants were detected in four control and five pilocarpine-treated rats. Due to differences in hippocampal atrophy in the pilocarpine-treated rats, transplantation sites were more variable. Circles: Clusters; rods: injection canals. The numbers correspond to the transplants depicted in B and C. B, C: Visualization of transplants with double immunofluorescence staining for both enhanced green fluorescent protein (EGFP) and M6, a mouse-specific membrane-bound protein (B: control; C: pilocarpine-treated; scale bar corresponds to 100 μm). ESNs largely remained in clusters at the transplant site. However, their processes extended significantly into the host tissue in most transplants. Numbers in B and C correspond to the transplantation sites in A.

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Contrasting with the lack of significant migration into host tissue, grafted ESNs showed a striking propensity to extend processes into host brain tissue. EGFP+/M6 immunoreactive processes were observed tens to hundreds of micrometers distant from the transplant site within host tissue. A representative example of this phenomenon is depicted in Fig. 2. In this case, the transplant was situated immediately adjacent to the CA1 region (transplant shown as EGFP/M6 immunolabeling superimposed on a phase-contrast image of the hippocampal slice). A dense plexus of EGFP/M6 double-labeled fibers was observed at considerable distances from the transplant site, within the corpus callosum and the CA1 and the CA3 regions (Fig. 2A–C).

image

Figure 2. Formation of an extensively ramified network of ES cell–derived neuron (ESN)-derived processes invading the host brain. An experiment in which transplantation was carried out into a pilocarpine-treated rat is shown. The central portion of the figure depicts the location of the transplant. Overlay of phase contrast (black and white), and immunofluorescence with antibodies to M6 (red) and enhanced green fluorescent protein (EGFP; green) (scale bar corresponds to 200 μm). Squares, Location of close-up photomicrographs in the corpus callosum (A), CA1 region (B), and CA3 region (C; scale bars correspond to 25 μm). The pyramidal region of CA1 in pilocarpine-treated animals shows a pronounced autofluorescence unrelated to the EGFP signal (B). pcl, pyramidal cell layer; cc, corpus callosum.

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Intrinsic membrane properties of graft-derived ESNs

We identified EGFP+ cells by using fluorescence microscopy (Fig. 3A1) and subsequently obtained patch-clamp recordings under visual control by using infrared differential interference contrast optics. We recorded preferentially from ESNs that had migrated away from the transplant sites or were situated at transplant borders. We initially examined the intrinsic membrane properties of ESNs. No significant differences in the passive membrane properties of ESNs were noted in control (capacitance 47.6 ± 3.6 pF, n = 13; membrane resistance 1.46 ± 0.37 GΩ, n = 10) versus pilocarpine-treated rats (capacitance 40.0 ± 4.8 pF, n = 13; membrane resistance 1.51 ± 0.38 GΩ, n = 10; see Table 1). In both groups, 80 to 90% of ESNs displayed spontaneous rhythmic action potential discharges at frequencies of 2.9 ± 0.6 Hz (n = 8) and 3.4 ± 0.7 Hz (n = 9) in control and epileptic animals, respectively (not significant, Fig. 3B1). For further analysis of action potential morphology, ESNs were clamped to hyperpolarized membrane potentials of around –75 mV by current injection to suppress the spontaneous discharges. All ESNs were able to generate single action potentials in response to brief current injections of 3-ms duration. From these recordings, we derived the action-potential threshold, amplitude, and half-width. Plotting these values versus the time after transplantation revealed a tendency for action potentials to become narrower and larger with time after transplantation (Fig. 3B2, see C for summary and Table 1 for average values of all measurements, as well as comparison with host granule neurons). Current injections of 250-ms and 1-s duration resulted in trains of action potentials with varying amounts of spike frequency adaptation. Likewise, ESNs showed variable fast (compare upper and lower traces in Fig. 3B3) and, sometimes, slow afterhyperpolarizations. Few ESNs also showed spike afterdepolarizations. Spike afterpotentials were heterogeneous in both pilocarpine-treated and control rats, with no obvious group differences. Finally, >90% of the ESNs displayed inward rectification in response to hyperpolarizing current injections (Fig. 3B3). After hyperpolarizing current injection, part of the ESNs exhibited rebound action-potential discharges (44 and 17% of ESNs in control and pilocarpine-treated rats, respectively; n.s., Fisher's exact test). Voltage-clamp recordings revealed voltage-dependent membrane currents in all ESNs. Depolarizing voltage steps induced large, unclamped inward currents corresponding to voltage-gated Na+ channels. Furthermore, outward K+ currents were present in all ESNs, similar to voltage-gated ionic currents described in ESNs transplanted into hippocampal slice cultures (10, data not shown).

image

Figure 3. Discharge behavior of ES cell–derived neurons (ESNs). A1: Enhanced green fluorescent protein (EGFP) backdiffusion into the recording pipette after attaining the whole-cell configuration (scale bar, 25 μm). A2–A3: Biocytin staining of recorded neurons with Texas red avidin (A2 scale bar, 50 μm) or permanent diaminobenzidine (DAB) staining (A3 scale bar, 50 μm). B: Current-clamp recordings. The upper panel corresponds to recordings from control rats, and the lower panel to recordings in pilocarpine-treated rats (upper trace, voltage recording; lower trace, current injection step). B1: Spontaneous pacemaking activity of ESNs. B2: Single action potentials in response to a 3-ms depolarization. B3: ESNs exhibit repetitive discharges in response to a 1-s depolarization and inward rectification after hyperpolarizing current injections. C: Action-potential threshold, amplitude and half-width plotted against time after transplantation.

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Table 1. Passive membrane properties and action potential parameters of embryonic stem cell–derived neurons incorporated into the hippocampus of control and pilocarpine-treated rats in comparison with those of endogenous granule cells
 Membrane capacitance (pF)Input resistance (GΩ)Threshold (mV)Amplitude (mV)Half-width (ms)
Control47.6 ± 3.61.46 ± 0.37−49.8±1.652.6 ± 2.42.29 ± 0.19
(n = 13)(n = 10)(n = 9)(n = 9)(n = 9)
Pilocarpine-treated40.0 ± 4.81.51 ± 0.38−51.1 ± 2.548.4 ± 2.92.74 ± 0.19
(n = 13)(n = 10) (n = 10) (n = 10) (n = 10)
Endogenous granule cells control (n = 4)120.7 ± 10.50.248 ± 0.039–57.0 ± 3.9107.0 ± 3.11.20 ± 0.02
Endogenous granule cells pilocarpine-treated (n = 3)130.6 ± 19.00.308 ± 0.064–55.3 ± 1.9104.3 ± 2.41.16 ± 0.03

Synaptic input onto ESNs

More than 80% of ESNs received spontaneous synaptic input (Fig. 4). Excitatory non-NMDA and inhibitory GABAA receptor–mediated components were isolated pharmacologically. Addition of 50 μM DL-AP5 and 10 μM bicuculline to the aCSF yielded non–NMDA receptor–mediated postsynaptic currents with frequencies of 0.7 ± 0.3 Hz (n = 7) in control and 1.2 ± 0.5 Hz (n = 8) in epileptic rats. GABAA receptor–mediated currents were isolated by adding 50 μM CNQX and 50 μM DL-AP5 to the aCSF. They had frequencies of 4.6 ± 0.9 Hz (n = 4) and 3.2 ± 0.5 Hz (n = 5) in control and epileptic rats, respectively.

image

Figure 4. Spontaneous postsynaptic currents (PSCs). Examples of PSCs in control (A) and epileptic (B) animals; 80% of CTRL and 90% of PILO ES cell–derived neurons (ESNs) received spontaneous synaptic input. Currents are filtered offline at 2.5 kHz.

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In subsequent experiments, postsynaptic currents were elicited by stimulation with a monopolar stimulation electrode placed in the vicinity of the recorded cell. Stimulation from more distant sites proved to be difficult and was possible only in some cases. The non–NMDA receptor–mediated postsynaptic currents displayed a reversal potential of -2.2 ± 1.4 mV (control rats, n = 4) and 0.5 ± 5.8 mV (pilocarpine-treated rats, n = 6) with decay time constants of 8.9 ± 1.9 ms (n = 4) and 6.8 ± 1.0 ms (n = 6), respectively (Fig. 5A and C). In all tested ESNs (n = 4 and 5 for control and epileptic animals, respectively), these currents were completely suppressed by additional perfusion of 50 μM CNQX (Fig. 5B).

image

Figure 5. Excitatory postsynaptic currents (PSCs). A: Non–N-methyl-d-aspartate (NMDA) receptor-mediated evoked PSCs at different holding potentials. Averages from five to nine traces are shown. B: PSCs were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX). C: The reversal potential was –2.2 ± 1.4 mV (n = 4) in ES cell–derived neurons (ESNs) of control and 0.5 ± 5.8 mV (n = 6) in ESNs of pilocarpine-treated rats.

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GABAA receptor–mediated synaptic currents displayed a slower decay than the excitatory postsynaptic currents (23.4 ± 5.1 ms, n = 4, control rats and 24.5 ± 4.4, n = 5, pilocarpine-treated rats). Their reversal potentials were –47.9 ± 2.2 mV (n = 4) and –43.9 ± 1.4 mV (n = 5), in the control and epileptic groups, respectively (Fig. 6A and C). These values, as well as the reversal potentials obtained for glutamatergic excitatory postsynaptic currents (EPSCs), reflect our artificial ionic conditions. In the case of GABAA receptors, as expected, these values are close to the calculated chloride reversal potential (–39 mV). The currents were completely suppressed by 10 μM bicuculline in two of two ESNs from control and four of four ESNs from pilocarpine-treated rats (Fig. 6B).

image

Figure 6. Inhibitory postsynaptic currents (PSCs). A:γ-Aminobutyric acid (GABA)A receptor–mediated evoked PSCs at different holding potentials. Averages from five to nine traces are shown. B: PSCs were blocked by bicuculline. C: The reversal potential was –47.9 ± 2.2 mV (n = 4) and –43.9 ± 1.4 mV (n = 5) in ES cell–derived neurons of control and pilocarpine-treated rats, respectively.

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Next, short-term plasticity of these synapses was investigated by applying double pulses of different interpulse intervals ranging from 20 to 160 ms. As shown in Fig. 7, non–NMDA receptor–mediated currents displayed significant short-term facilitation at most interpulse intervals, which was generally more pronounced at shorter intervals (Fig. 7A). Short-term plasticity was less pronounced for GABAA receptor–mediated currents (Fig. 7B).

image

Figure 7. Paired-pulse plasticity of non–N-methyl-d-aspartate (A) and γ-aminobutyric acid (GABA)A(B) receptor–mediated postsynaptic currents (PSCs). A1, B1: Superimposed averaged postsynaptic potentials for different interpulse intervals. The leftmost trace is the reference sweep. A2, B2: Ratios of second to first PSCs at different interpulse intervals (20, 40, 80, and 160 ms).

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No differences between ESNs transplanted into sham-control and pilocarpine-treated rats were observed for any parameter tested with regard to both intrinsic and synaptic properties.

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

In the present study, we show that ES cell–derived neural precursors develop characteristic neuronal properties after engraftment into the adult normal and chronically epileptic hippocampus. Numerous functionally active ESNs were observed at prolonged time intervals after transplantation, suggesting that long-term survival of transplants in this model is possible.

Intrinsic properties of ESNs

After engraftment, virtually all ESNs developed the capability to fire repetitive action potentials. The amplitudes of these action potentials were lower than those in host neurons, possibly indicating an ongoing maturational process (10). Voltage-clamp recordings were contaminated by space-clamp artifacts, but did allow us to confirm the presence of different types of voltage-gated ion channel types in ESNs. All cells exhibited fast sodium currents and delayed outwardly rectifying potassium currents that underlie their capability for repetitive action potential firing. In addition, some ESNs exhibited a fast transient potassium channel component with properties of A-type currents. The capability to generate action potentials and the expression of different types of voltage-gated ion channels has been demonstrated in ES cell–derived neuronal progeny under various conditions. This applies to ES cells differentiated in culture by using retinoic acid (23–25), as well as to ES cell–derived neurons incorporated both into hippocampal long-term slice cultures (10) and into the embryonic (9) or the adult brain (26).

An unusual feature of ESNs incorporated into adult brain tissue was the generation of spontaneous pacemaking activity at frequencies ∼3 Hz in the vast majority of these cells. Such an activity was very rarely observed in ES cell–derived progeny after transplantation into immature host tissue (9,10). Interestingly, current-clamp recordings revealed the presence of significant hyperpolarization-activated currents in ESNs transplanted into the adult brain, but not into immature host tissue. Hyperpolarization-activated currents are known to contribute to pacemaking in diverse neuron types, for instance in thalamocortical relay neurons or hippocampal stratum oriens interneurons (27).

These data indicate that ESNs exhibit a broad spectrum of membrane currents after incorporation into adult brain tissue. It is intriguing that only ESNs incorporated into adult, but not into immature brain tissue (9,10), exhibit spontaneous pacemaking activity. This finding may indicate the presence of specific cues in adult brain tissue that are present both in control and in pilocarpine-treated rats. Alternatively, the properties of ESNs in our experiments could be influenced by the fact that these cells were situated in or in close proximity to a dense cluster of other ESNs. Both scenarios may explain why we did not observe any differences between ESNs incorporated in a normal versus an epileptic adult host brain.

Synaptic properties of ESNs

In addition to developing characteristic intrinsic neuronal properties, ESNs received both excitatory and inhibitory synaptic input. Non-NMDA and GABAA receptor–mediated postsynaptic currents were isolated pharmacologically. Both excitatory and inhibitory postsynaptic currents exhibited short-term facilitation to paired-pulse stimulation, thought to indicate a presynaptic form of short-term plasticity. As for the intrinsic membrane properties, the characteristics of inhibitory and excitatory synaptic currents proved to be similar in control compared with pilocarpine-treated rats. In contrast to our previous studies (9,10), these experiments did not allow us to differentiate whether synaptic input onto ESNs was derived from endogenous neurons or from neighboring ESNs. Because most ESNs were located in or close to the transplant site, it was virtually impossible to stimulate synaptically in an area devoid of EGFP+ profiles.

Transplant structure

The lack of significant migration into host tissue contrasted sharply with the behavior of ES cell–derived neural precursors after transplantation onto slice cultures or into the ventricles of embryonic rats, where migration occurred over long distances (9,10). However, ESNs incorporated into the adult brain gave rise to long and ramified processes that extended far (up to hundreds of micrometers) into host tissue. The striking outgrowth of such fibers did not appear to be different in control compared with pilocarpine-treated rats, but the variability in location and size of the transplants precluded a quantitative comparison in this study. Outgrowth of graft-derived fibers was also observed over significant distances after transplantation of ES cell–derived neural precursors into the developing brain (9). Additionally, processes of dopaminergic neurons derived from Nurr1-transfected ES cells appear to invade adult host tissue after transplantation into the striatum (12). In these, as well as other previous studies, it has remained unclear what the properties of the efferent projections of incorporated ESNs might be. Such analyses would require paired recordings from both transplanted ESNs and their host target neurons. Nevertheless, the striking invasion of host tissue by a dense plexus of graft-derived fibers suggests that grafts of ESNs could be used for widespread delivery of inhibitory mediators to the host brain, despite their limited propensity to migrate away from the transplantation site. The potential merit of transplantation-based delivery of inhibitory substances is illustrated by a number of studies in which transplantation of donor cell populations genetically engineered to release adenosine or GABA has anticonvulsant effects in chronic seizure models (15–17). Future experiments may therefore be aimed at improving migration and targeting efferent projections of ES cell–derived progeny to specific sites of the host tissue by appropriate modification of the donor cell population.

Acknowledgments

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Acknowledgment:  We thank Rachel Buschwald, Michaela Segschneider, and Barbara Steinfarz for their technical support, and K.L. Tucker for providing the tau:EGFP ES cells. This work was supported by the Gemeinnützige Hertie Stiftung, the SFB/TR3, and the BONFOR Program.

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES
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