GABA Uptake and Heterotransport Are Impaired in the Dentate Gyrus of Epileptic Rats and Humans With Temporal Lobe Sclerosis

Peter R. Patrylo, Dennis D. Spencer, and Anne Williamson

Department of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut 06520


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Patrylo, Peter R., Dennis D. Spencer, and Anne Williamson. GABA Uptake and Heterotransport Are Impaired in the Dentate Gyrus of Epileptic Rats and Humans With Temporal Lobe Sclerosis. J. Neurophysiol. 85: 1533-1542, 2001. In vivo dialysis and in vitro electrophysiological studies suggest that GABA uptake is altered in the dentate gyrus of human temporal lobe epileptics characterized with mesial temporal sclerosis (MTLE). Concordantly, anatomical studies have shown that the pattern of GABA-transporter immunoreactivity is also altered in this region. This decrease in GABA uptake, presumably due to a change in the GABA transporter system, may help preserve inhibitory tone interictally. However, transporter reversal can also occur under several conditions, including elevations in [K+]o, which occurs during seizures. Thus GABA transporters could contribute to seizure termination and propagation through heterotransport. To test whether GABA transport is compromised in both the forward (uptake) and reverse (heterotransport) direction in the sclerotic epileptic dentate gyrus, the physiological effects of microapplied GABA and nipecotic acid (NPA; a compound that induces heterotransport) were examined in granule cells in hippocampal slices from kainate (KA)-induced epileptic rats and patients with temporal lobe epilepsy (TLE). GABA- and NPA-induced responses were prolonged in granule cells from epileptic rats versus controls (51.3 and 31.3% increase, respectively) while the conductance change evoked with NPA microapplication was reduced by 40%. Furthermore the ratio of GABA/NPA conductance, but not duration, was significantly >1 in epileptic rats but not controls, suggesting a compromise in transporter function in both directions. Similar changes were observed in tissue resected from epileptic patients with medial temporal sclerosis but not in those without the anatomical changes associated with MTLE. These data suggest that the GABA transporter system is functionally compromised in both the forward and reverse directions in the dentate gyrus of chronically epileptic tissue characterized by mesial temporal sclerosis. This alteration may enhance inhibitory tone interically yet be permissive for seizure propagation due to a decreased probability for GABA heterotransport during seizures.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GABA is the primary inhibitory neurotransmitter in the adult brain, and its effects at receptors are primarily terminated by diffusion and transport (Borden 1996). The major cortical GABA transporters, GAT1 and GAT3, are specific, high-affinity, electrogenic sodium- and chloride-dependent uptake systems that are primarily localized to presynaptic terminals and to glial processes adjacent to the synaptic cleft, including in the hippocampus (Cammack et al. 1994; Mager et al. 1996; Ribak et al. 1996). Several studies suggest that the clearance of extracellular GABA is compromised in the dentate of epileptic humans in particular those characterized with medial temporal sclerosis (During et al. 1995; Williamson et al. 1995). Furthermore anatomical studies have shown that there appear to be changes in the distribution of GABA transporter-immunoreactivity in the hippocampus from epileptic patients with medial temporal lobe sclerosis (Mathern et al. 1999).

Electrophysiological data from in vitro studies have shown that these transporters can also work in reverse, causing GABA efflux under certain conditions in which the ionic gradients are disrupted (Borden 1996; Cammack et al. 1994). Among these conditions is an elevation in [K+]o (Gaspary et al. 1998) that has been observed to occur during seizures (Heinemann et al. 1986; Krnjevic et al. 1982; Somjen and Giacchino 1985). Thus GABA transporters may release GABA into the extracellular environment during seizures and thereby contribute to the termination and containment of hypersynchronized activity. If this heterotransport is also impaired in chronically epileptic tissue because of alterations in GABA transporter function, it could therefore contribute to the spread and severity of seizure activity. This aspect of transporter function has not been well studied in epileptic tissue.

In this investigation, we tested the hypothesis that functional GABA transport is compromised in both the forward and reverse direction in the dentate gyrus of chronically epileptic tissue characterized by mesial temporal sclerosis. The characteristics of GABA- and nipecotic acid (NPA)-evoked responses were examined in dentate granule cells of hippocampal slices from kainate (KA)-induced epileptic rats (an animal model of temporal lobe epilepsy) (Hellier et al. 1998; Patrylo and Dudek 1998), age-matched saline-treated controls, and epileptic humans with medial temporal lobe epilepsy using intracellular and whole cell recordings and drug microapplication techniques. NPA is an uptake inhibitor that can, when applied as a microdrop, induce facilitated transport of GABA from the intracellular to the extracellular environment without a significant concomitant block of uptake (Honmou et al. 1995; Solis and Nicoll 1992). NPA-evoked responses are mediated by GABAA receptors and are blocked by picrotoxin. Therefore both the forward and reverse modes of GABA transport could be examined in this investigation.

Given the difficulties associated with human-tissue studies, including the lack of true controls, prolonged use of medication, etc., and the uncertainties associated with knowing how closely animal models reflect the human condition, parallel studies using both human material and animal models are needed. These studies may provide insight into the role of GABA transporters in the epileptic and control hippocampus and will allow for further well-controlled studies on the molecular mechanism for the loss of GABA uptake in the sclerotic dentate gyrus.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kainic acid treatment

Kainate-induced epileptic rats were generated using a protocol previously described (Hellier et al. 1998; Patrylo and Dudek 1998; Wuarin and Dudek 1996). Briefly, male Sprague-Dawley rats (200-250 g) were given repeated injections of kainic acid (KA; 5 mg · kg-1 · h-1 ip) until class IV/V seizures (Racine 1972) were elicited for >= 3.5 h. The animals were then given subcutaneous injections of lactated Ringer solution and fed moistened rat chow for several days following the KA injections. Age-matched control animals were prepared by giving comparable injections of saline. All of the KA-treated animals used in this study were observed to have spontaneous recurrent motor seizures months following the initial treatment, while none were observed in the saline-treated controls. This protocol was approved by the Yale Animal Care and Use Committee.

Patient classification

Patients were divided into two groups based on data from magnetic resonance imaging (MRI) and positron emission tomography (PET) studies, quantitative cell counts (Dr. J. Kim), and immunocytochemistry for dynorphin, neuropeptide Y, somatostatin, and substance P (Dr. N. de Lanerolle). One group consisted of patients with medial temporal lobe epilepsy showing the typical pattern of cell loss and synaptic reorganization characteristic of mesial temporal sclerosis (MTLE) (de Lanerolle et al. 1994). The second group of patients consisted of temporal lobe epileptics that did not display these anatomical changes and included tissue from patients with mass-associated temporal lobe epilepsy (MaTLE, n = 4) and those with the "paradoxical" type of temporal lobe epilepsy (PTLE, n = 3) (Luby et al. 1995; O'Connor et al. 1998). While there are subtle differences in the synaptically evoked responses between the MaTLE and PTLE groups, the GABA and NPA responses studied here were comparable and distinct from the MTLE group. Therefore we have chosen to group the data and refer to these patients as the comparison group. Patient information, including the drug regimen at the time of surgery, is shown in Table 1. The MaTLE group was distinct in that the duration of intractable seizures for these patients was lower than for patients in the other two groups; this difference did not reach significance, however. All patients involved in this study had given their informed consent, and these experiments were approved by the Yale Human Investigation Committee.


                              
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Table 1. Patient data

Tissue preparation

Rats were anesthetized with a pentobarbital sodium solution (50 mg/kg) and then decapitated. None of the KA-treated rats used in these experiments were observed to have spontaneous motor seizures >= 2 h prior to sacrifice. Their brains were rapidly removed and placed in an ice-cold solution containing (in mM) 135 choline chloride, 20 MgCl2, 0.5 CaCl2, 20 NaH2CO3, 1.3 NaH2PO4, and 10 dextrose for 1-2 min. (modified from Honmou et al. 1995). The brains were then blocked into a section including the hippocampus and mounted on the stage of a vibratome (Ted Pella, Redding, CA), and transverse hippocampal slices were cut at 400 µm. The hippocampus was dissected free from the surrounding cortex using a razor blade and placed in a recording chamber.

The human hippocampi used in these studies were removed surgically as previously described by Spencer (1991). We received a 5- to 10-mm slab of the anterior body of the hippocampus that was immediately placed in cold (4°C) oxygenated artificial cerebrospinal fluid (ACSF) and transported to the laboratory. Slices (400 µm) were then prepared and placed in a recording chamber (~15 min after surgical dissection).

Slices from both the human and rat material were maintained in a gas-liquid interface recording chamber (Fine Science Tools). They were constantly perfused with ACSF (32 ± 1°C; pH 7.4) at 1 ml/min under a stream of humidified 95% O2-5% CO2. The ACSF contained (in mM) 124 NaCl, 3.0 KCl, 2 MgSO4, 1.2 NaH2PO4, 26 NaHCO3, 2 CaCl2, and 10 dextrose. Slices were allowed to recover for >= 2 h prior to recording.

Electrophysiology

Both sharp and whole cell patch recordings were used in this study. Although some differences were noted in the intrinsic membrane properties of granule cells when whole cell versus sharp electrode recordings were used, no significant differences were noted in the GABA- or NPA-evoked responses. Therefore the NPA and GABA response data obtained with sharp and whole cell recording techniques have been combined. Electrodes were formed on a Brown-Flaming type electrode puller (Sutter Instruments, Novato, CA), and sharp electrodes (60-100 MOmega ) were filled with 4 M K-acetate, while patch electrodes (4-10 MOmega ) were filled with a solution containing (in mM) 130 K-methyl sulfate, 10 HEPES, 10 EGTA, 2 MgCl2, 4 MgATP, and 2 CaCl2, buffered to pH 7.25 with KOH. Recordings were made with an Axoclamp II amplifier (Axon Instruments, Foster City, CA), and Axodata and IgorPro software were used for data analysis. Whole cell patch recordings were made using the "blind" patch method, and cells were identified as granule cells based on their location in the granule cell layer and their distinct electrophysiological characteristics (e.g., hyperpolarized membrane potential, spike width >0.7 ms; triphasic spike afterhyperpolarization, etc.) (see Scharfman 1992).

All drugs used in this study were dissolved in ACSF immediately prior to use. GABA (5 mM) and NPA (5 mM) were applied within 50 µm of the recording electrode by applying brief pressure pulses to the back of a micropipette filled with the transmitter using a Picospritzer (General Valve, Fairfield, NJ). This dose was used to ensure a saturating concentration of GABA. In most cases, a double-barreled drug application pipette was used to ensure consistent quantities and location for drug application. In some experiments, a triple-barreled application pipette containing GABA, NPA, and vehicle was used to rule out mechanical artifact. The membrane potential of cells was held between -75 and -80 mV for these experiments so that the amplitude of the GABA and NPA responses would not be significantly affected by the baseline potential. Both GABA- and NPA-evoked voltage responses have previously been demonstrated to be blocked by bicuculline (Honmou et al. 1995; Solis and Nicoll 1992; Williamson et al. 1995). The uptake inhibitor NO-711 (100 µM) was locally bath applied using a Hamilton syringe (Williamson et al. 1995). GABA, NPA, and bicuculline were obtained from Sigma; NO-711 was purchased from RBI.

Because these recordings were done in current-clamp mode the GABA current could not be measured directly. Therefore the percent change in input resistance was determined at the peak of the response relative to rest. The duration of the agonist response was measured from the time the compound was applied to when both the voltage and conductance levels returned to baseline. Averaged data are shown as the means ± SE, and statistical significance (P <=  0.05) was determined using an unpaired, two-tailed t-test (Statview software; Berkeley, CA).

The reversal potential of the GABA and NPA responses was determined by generating three point I-V curves at rest and at the peak of the response. The point where control and agonist I-V plots cross represents the reversal potential for the response (e.g., McCormick 1989). As seen in Figs. 1, 2, and 5, both depolarizing and hyperpolarizing current steps were delivered during the response period; the I-V relationship for these steps as well as at rest were used to construct the curves.

Orthodromic synaptic responses were examined by delivering electrical stimuli to the outer molecular layer using bipolar stimulating electrodes (0.05-ms duration at an intensity equal to 2.5 times the action potential threshold for each cell, usually between 50 and 600 µA). In the majority of dentate granule cells from rats, the excitatory response was followed by a biphasic inhibitory postsynaptic potential (IPSP), and the conductance of these events was determined using techniques previously described (Buckmaster and Schwartzkroin 1995; McCarren and Alger 1985).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

KA-treated rats

Data were collected from 17 granule cells from nine rats with KA-induced epilepsy and 19 cells from six age-matched controls. As described previously by others (Cronin et al. 1992; Wuarin and Dudek 1996), no difference was noted in the membrane properties of granule cells between these two groups when examined within the linear portion of the I-V curve (resting membrane potential, input resistance, time constant and action potential amplitude; Table 2). Therefore any difference in responsiveness to drug application in granule cells from epileptic animals versus controls is unlikely to be due to differences in membrane properties at potentials hyperpolarized to action potential threshold.


                              
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Table 2. Membrane properties of granule cells from control and epileptic rats

GABA and NPA responses are prolonged in granule cells from KA-induced epileptic rats

If the GABA transport system was functionally altered in the dentate gyrus in an animal model of human MTLE, one would predict that specific differences should exist in GABA- and NPA-evoked responses in control versus epileptic animals. First, the duration of the GABA response should be prolonged in the epileptic tissue relative to control. The other characteristics of the GABA response should be comparable, however, since the amplitude and conductance of the GABA response should not be affected by transport. Second, both the amplitude and peak conductance of the NPA response should be smaller in epileptic tissue because less transmitter would be released into the extracellular space via transporter reversal (heterotransport). Furthermore since NPA microapplication does not significantly block uptake (Honmou et al. 1995: Solis and Nicoll 1992), the duration of the NPA responses should also be prolonged in slices from the KA-treated animals since the re-uptake of released GABA would be impaired because of transporter dysfunction.

Application of a GABA microdrop evoked depolarizing responses in granule cells from both KA-treated and control animals that were associated with a significant decrease in input resistance as shown in Fig. 1A. As predicted, the GABA-evoked responses were 56% longer in granule cells from epileptic animals than those observed in the control tissue (56.7 ± 4.8 vs. 37.4 ± 3.7 s, respectively; P < 0.005). By contrast, the amplitude, conductance change and reversal potential of the peak GABA response did not differ significantly between the two groups of animals (Table 3). Figure 1B shows the distribution and means of the response durations and conductance changes for the control and epileptic groups . 



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Fig. 1. GABA responses in control and epileptic animals. A: examples of the response to a microdrop application of GABA in granule cells from a control and an epileptic animal. Note that the response was greatly prolonged in the cell from an epileptic rat. Membrane potentials (MPs), control, -68 mV; kainate (KA)-treated, -72 mV. B: the data for the duration and conductance change of the GABA response for all the cells studied in both control and epileptic animal populations, but that there was a rightward shift for the duration of the response in cells from epileptic animals (left). In contrast, GABA produced large conductances in cells from both groups (right).


                              
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Table 3. Characteristics of GABA and NPA responses in control and epileptic rats

To assess the ability of the transporter to move GABA from the intra- to the extracellular environment, the response of granule cells to NPA microdrop application was examined in slices from control and epileptic animals. In the majority of experiments, the response to NPA was a marked depolarization associated with an increase in membrane conductance. The NPA-evoked conductance change was ~40% smaller in granule cells from epileptic versus control tissue (57.0 ± 7.3 vs. 79.8 ± 3.4%, respectively; P <=  0.005). Furthermore as predicted, the duration of the NPA response was significantly longer (44.0%) in the epileptic tissue compared with control (47.8 ± 4.6 vs. 33.8 ± 3.4 s; P < 0.04; Fig. 2). While there also appeared to be a trend for these evoked responses to have a smaller amplitude in the epileptic tissue, this difference was not statistically significant. Similar values, which did reach significance, were obtained when the data from each animal were grouped. These values are given in Table 3. Taken together, these data support the hypothesis that GABA transport is impaired in both the forward and reverse directions in the dentate gyrus of rats with KA-induced epilepsy.



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Fig. 2. Nipeciotic acid (NPA) responses in control and epileptic animals. A: examples of the response to an NPA microdrop in granule cell from a control and epileptic animal. Note that there was a robust, brief response in the cell from a control animal but that the response was prolonged and attentuated in the cell from an epileptic rat. MPS: -73 mV control; -75 mV epileptic. B: the NPA data were binned as described in Fig. 1. Note that the distribution of the response duration was similar to that seen for GABA. By contrast, there was a great deal of variability in the conductance change associated with NPA responses in epileptic but not control tissue as shown (right bar graph). This variability was in part due to a leftward shift in the percent conductance within the population.

NPA-evoked responses were verified to be mediated through a transporter-dependent mechanism by locally bath applying NO-711, a specific GAT-1 inhibitor (Borden et al. 1994). In control animals, both the amplitude and peak conductance of NPA-evoked responses were decreased with NO-711 (amplitude: 12.8 ± 2.4 to 3.6 ± 2.2 mV; peak conductance change: 90.8 ± 7.0 to 53.7 ± 6.1%; n = 4). In contrast, NO-711 had no effect on either the conductance or amplitude of the GABA responses, yet increased their duration by 56.5% (from 41.0 ± 19.8 to 57.5 ± 17.5 s, n = 3). This effect is comparable to that seen with NO-711 in dentate granule cells from MTLE hippocampi (Williamson et al. 1995). These responses suggest that NPA-evoked responses are largely mediated via the GAT-1 transporter and that prolongation of GABA-evoked responses can be produced in controls, similar to that observed in the epileptic rats, by functionally compromising GABA transport.

To control for variability between preparations, GABA- and NPA-evoked responses were additionally examined within the same cell (17 cells from controls and 15 cells from KA-treated rats) by using double-barrel drug application pipettes. Under these conditions, both the amount of compound applied and the location of application can be controlled. Because the reversal potential of GABA- and NPA-evoked responses differed (see following text), we focused on the duration and conductance change and calculated a GABA/NPA ratio to normalize the data further. In controls, one would predict that this ratio should be close to 1.0 for both conductance and duration since a high concentration of GABA would be available for GABAA receptor binding in both cases. However, in epileptic animals, one would anticipate that the conductance ratio may be >1 since less GABA should be transported out into the extracellular space via NPA-evoked heterotransport, while GABA-evoked responses would still be robust. Furthermore the GABA/NPA duration ratio should approach 1 since once GABA binds to the postsynaptic receptors, the responses should have similar kinetics. The data support this hypothesis as shown in Fig. 3. Specifically, in control animals, the conductance ratio was 1.23 ± 0.02 and the ratio of the duration was 1.24 ± 0.02, while in epileptic animals the conductance ratio was 2.36 ± 0.45 (P < 0.02) and the ratio of the duration was 1.28 ± 0.15. Thus GABA transporter function appears to be impaired in both the forward and reverse direction in epileptic rodent tissue.



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Fig. 3. Ratio of GABA/NPA response characteristics for granule cells from control and epileptic rats. The ratio of the response durations was close to 1 for cells from both the control and epileptic tissue. By contrast, the conductance ratio was significantly different between the 2 groups. These data suggest that NPA induces less heterotransport in the epileptic tissue relative to control.

NPA and GABA responses have different reversal potentials in epileptic and control rats

Although one would predict that GABA and NPA should evoke comparable responses in controls, our data indicate that a significant difference existed in their reversal potentials and therefore the amplitude of their responses (Table 3). The calculated reversal potential for NPA-evoked responses was consistently hyperpolarized relative to that for GABA in both groups (by 7.5 ± 5.6 mV in controls; 11.1 ± 8.7 mV in slices from KA-treated rats). No difference was noted, however, in the reversal potentials for GABA or NPA when comparing control versus epileptic rats. Nevertheless there was a trend toward smaller-amplitude NPA responses in the epileptic tissue relative to control that is consistent with a reduced transporter function.

Depolarizing GABA responses have been attributed to a bicarbonate efflux due to differences in buffering in somata versus dendrites (Staley et al. 1995; however, see Grover et al. 1993); a GABA-evoked K+-mediated depolarization (Kaila et al. 1997), or activation of different subtypes of GABAA receptors with different ionic conductances (Perkins 1999). We hypothesized that direct application of GABA may have activated one of these mechanisms while the NPA-evoked response reflected a Cl- conductance primarily. We began to test this hypothesis by examining evoked IPSPs in granule cells from controls and KA-induced epileptic rats to assess the characteristics of synaptically evoked GABA release.

Polysynaptic inhibition was examined in a total of 11 cells from seven KA-treated animals and 11 cells from eight saline-treated controls. IPSPs were seen in all of the granule cells from the saline-treated controls studied and in 10/11 cells from the KA-treated rats. As shown in Table 4, no difference was noted in the reversal potentials for either the fast or slow IPSPs between these two group. Furthermore the reversal potential for the fast IPSP was very close to that seen for NPA and was ~12 mV hyperpolarized to the reversal for the response to applied GABA.


                              
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Table 4. IPSP conductances are reduced in granule cells from epileptic rats

It is interesting to note that while dentate granule cells in KA-treated rats receive inhibitory input, a significant decrease in the conductance of polysynaptic IPSPs was observed in granule cells from the KA-treated rats relative to controls. This change was seen for both the fast and the slow IPSPs (Table 4), suggesting that inhibitory tone may be decreased when examined in the transverse slice preparation. Moreover, these data also suggest that NPA and GABA activate somewhat different ionic mechanisms when applied at these doses in rat tissue.

Human tissue

Taken together, these data support the hypothesis that functional GABA transport is compromised in the dentate gyrus of KA-induced epileptic rats. However, as with any animal model study, it is important to compare the results with those from the human condition. We have the opportunity to obtain hippocampi from patients with MTLE and comparison tissue from patients without synaptic reorganization and region specific cell loss (see METHODS). Thus a comparable series of experiments was performed in tissue resected from epileptic patients, expanding on our previous work on GABA responses in this material (Williamson et al. 1995).

Data were collected from a total of 17 cells from nine MTLE cases and 13 cells from seven comparison cases. As previously described, no difference was noted in the membrane properties (membrane potential, input resistance, time constant, and action potential amplitude) between these groups (Williamson et al. 1999). These data are from a different group of patients than those included in the previous study (Williamson et al. 1995).

GABA and NPA responses in MTLE and comparison tissue

Our results parallel those obtained in the rat as shown in Fig. 4 and Table 5. In the first series of experiments, the responses to GABA and NPA microapplication were examined in both MTLE and comparison tissue (Fig. 4). As in the rat, the GABA- and NPA-evoked responses were very different between the two groups and confirmed our prior findings with GABA microapplication in the MTLE dentate gyrus (Williamson et al. 1995). Specifically, the duration of the GABA and NPA-evoked responses was 78.9 and 64.5% prolonged, respectively, in MTLE granule cells versus comparison tissue. In addition, the amplitude and percent conductance change of the NPA-, but not GABA-, evoked response were diminished in the MTLE tissue by 81.4 and 38.1%, respectively. These differences were also seen when the data were group by patient.



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Fig. 4. Characteristics of GABA and NPA responses in tissue from epileptic patients. A: examples of the responses to microdrop-applied GABA in a cell from a paradoxical type of temporary lobe epilepsy (PTLE) patient (comparison) and a mesial temporal sclerosis (MTLE) patient. Note that the duration of the response was prolonged in the MTLE tissue but that the other response characteristics were similar. The population data are shown in A2 for both the duration and conductance data. Note that the distributions are similar to those seen in the rat tissue. B1: examples of the response to NPA. The NPA response was greatly attenuated in the cell from a MTLE hippocampus relative to a cell from a mass-associated temporal lobe epilepsy (MaTLE, comparison) hippocampus. The population data are shown in B1 and are similar to those in the rat model of MTLE. The calibrations are the same for A1 and B1.


                              
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Table 5. Characteristics of GABA and NPA responses in comparison and MTLE patients

The GABA/NPA ratio was examined in a total of six cells from MTLE hippocampi and five from the comparison hippocampi, Fig. 5. When the GABA/NPA ratios for the conductance change and duration were examined, a significant difference was noted for the conductance (1.03 comparison, 1.64 MTLE, P < 0.05) but not for the duration in MTLE granule cells. As described in the preceding text, these data support the hypothesis that there is a decrease in the ability of NPA to evoke GABA heterotransport from the intra to the extracellular environment.



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Fig. 5. Ratio of GABA/NPA response characteristics for granule cells from comparison and MTLE hippocampi. The ratio of the duration of the GABA and NPA responses was larger for the MTLE cases than for the controls; however, this was not significant. In contrast, there was a significant difference between the 2 groups for the conductance ratio. These data provide further support for the hypothesis that MTLE is associated with a loss of transporter function in the dentate gyrus.

Comparison between NPA and GABA in human tissue

When we examined the data, no significant difference was noted between the GABA and NPA responses in the comparison tissue, although there was a nonsignificant trend toward smaller amplitudes and percent conductance change and a more hyperpolarized reversal of the NPA-evoked responses, similar to the results observed in the rodent model. A different pattern was seen, however, when the effects of NPA and GABA were compared in tissue from MTLE hippocampi. Specifically, significant differences were noted in all features of GABA- versus NPA-evoked responses save duration, Table 5.

These results did not appear to be affected by the antiepileptic drugs that the patients were using at the time of surgery. All of the patients in this study were being treated with either phenytoin or carbemazepine. In addition, three of the seven MTLE patients were on either vigabatrin or gabapentin at the time of surgery; both these compounds have been shown to increase whole-brain GABA levels (Petroff et al. 1996). None of the comparison group were being treated with drugs that interact with the GABAergic system. There were no differences in either the GABA or NPA responses between MTLE patients using either of these drugs and those using just the Na+ channel antagonists (phenytoin, carbemazepine, and valproic acid). Moreover, the significant differences between the MTLE and comparison groups were maintained when the patients were divided into those with and without vigabatrin or gabapentin therapy. Therefore the drug regimen does not appear to be a confounding variable in these data.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular recordings were used to assess the hypothesis that GABA transport is functionally impaired in both the forward and reverse directions (uptake and heterotransport, respectively) in the epileptic dentate gyrus by examining the response to GABA and NPA microapplication in granule cells from KA-induced epileptic rats, an animal model of mesial temporal sclerosis, and humans with TLE (MTLE and the comparison tissue). The primary findings from this investigation were that the responses to GABA and NPA microapplication were prolonged in granule cells from epileptic, KA-treated rats, and MTLE patients compared with comparison tissue; the conductances of NPA-evoked responses were attenuated in granule cells from both epileptic rats and humans with MTLE; and the ratio of GABA/NPA conductance but not duration was significantly >1 in the sclerotic dentate gyri. Taken together, these data corroborate our hypothesis and suggest that the transporter system is functionally compromised in both directions. Furthermore the experiments using NO-711 verified that the altered GABA and NPA responses were transporter dependent. These changes in transport function may be specific to the dentate gyrus since no differences were reported in the duration of GABA-evoked responses in the CA1 region of KA-treated rats (Ashwood and Wheal 1986; Franck et al. 1988), and no significant differences were observed in GABA-evoked responses in CA2 pyramidal cells from MTLE versus MaTLE patients (Williamson and Patrylo 1999) and in neocortex adjacent to epileptogenic lesions (Telfeian et al. 1999).

Consequences of impairment

The overall consequence of this functional alteration in GABA transport in temporal lobe epilepsy is unclear. Our prior data in human and the present data suggest that there is a decrease in uptake that should result in an increase in [GABA]o. Such a change would be expected to help preserve inhibitory tone in the "epileptic" dentate gyrus despite the loss of subpopulation of inhibitory interneurons (Buckmaster and Dudek 1997b; de Lanerolle et al. 1988). Indeed in in vivo experiments on patients with temporal lobe epilepsy (Colder et al. 1996) and in animal models (Buckmaster and Jongen-Relo 1999; Hellier et al. 1999; Sloviter 1991), inhibitory tone appears to be intact or even enhanced interictally. It is important to realize, however, that an increase in [GABA]o may also act on extrasynaptic GABA receptors that may have distinct responses to GABA (Perkins and Wong 1996, 1997; Staley et al. 1995). Additionally, [GABA]o may also act on GABAB receptors on both inhibitory and excitatory terminals synapsing onto interneurons as well as principal cells (Davies et al. 1990; Haas et al. 1996). Therefore the net effect of the decrease in GABA re-uptake is complex and could have both restorative and proconvulsive effects.

The new NPA data presented here suggest that GABA heterotransport is also compromised in the sclerotic dentate gyrus from epileptic humans and rats. Thus any role that the GABA transporters may play in curtailing seizure activity as well as limiting its spread would be compromised in mesial temporal sclerosis. Specifically, Gaspary et al. (1998) demonstrated that increases in [K+]o can induce GABA transporter reversal. Since similar elevations in [K+]o have been reported to occur in the CNS during seizures (Somjen and Giacchino 1985), it is possible that heterotransport will occur and consequently dampen either seizure activity or spread. If reversed transport is compromised in the dentate gyrus in mesial temporal lobe epilepsy, seizures may be prolonged and may propagate more efficiently. This may represent the breakdown of the protective gate normally provided by the dentate gyrus. In conclusion, it appears that a compromise in uptake as well as transporter reversal can act as a double-edged sword that may improve inhibitory tone on the short term (compensatory), but that it may exacerbate epileptiform activity (duration and spread) once seizure activity has begun.

Possible mechanism of impairment

Several potential mechanisms may be involved with producing both the prolonged GABA- and NPA-evoked responses and the decreased conductance of NPA-evoked events. These mechanisms include changes in the number and distribution of the transporter, alterations in the affinity and/or modulation of the transporter, and changes in GABA receptor-mediated events. Anatomical studies have shown that GAT-1-like immunoreactivity is decreased in the inner molecular layer of the dentate gyrus of MTLE patients, despite an increase in the density of GABA- and GAD-immunoreactive fibers within this region (Mathern et al. 1999). Since this is the region where GABA and NPA were microapplied, it could account for the prolonged responses as well as the reduced conductance of the NPA-evoked responses. While comparable anatomical studies have not been performed in KA-induced epileptic rats; many forms of synaptic reorganization seen within the dentate gyrus of patients with MTLE are also found in this animal model (Buckmaster and Dudek 1997a,b). Binding experiments by During et al. (1995) also suggest that there is a decrease in the number of GABA transporters in the kindling model of TLE since Bmax of NPA binding was reduced in hippocampal tissue. Although the exact mechanism for a decrease in transporter is unknown, it is interesting to note that the expression of this protein is dependent on the [GABA]o (Bernstein and Quick 1999), and several investigators have shown that GABA levels are decreased in patients with temporal lobe epilepsy (Petroff et al. 1996, 1999) and in animal models (Lothman et al. 1991).

A second possibility is that the affinity and/or modulation of the transporter is altered. In control rodents, the GABA transporter can be modulated by a variety of calcium-dependent secondary messengers systems (Beckman et al. 1999; Law et al. 2000). Data from both human temporal lobe epileptics, as well as animal models, suggest that Ca2+ homeostasis is altered (Isokawa 1998; Jeub et al. 1999; Kunz et al. 1999; Nagerl et al. 2000). Thus transporter function may consequently be altered through posttranslational modification. Support for this hypothesis comes from biochemical studies using the GAERS rat (an animal model of absence epilepsy), where an apparent decrement in GAT-1 transporter function was observed with no concurrent change observed in either transporter number or affinity (Sutch et al. 1999). To the best of our knowledge, however, comparable studies have not been performed on tissue from MTLE patients or in animal models.

Changes in the GABA receptors themselves could also contribute to altered GABA and NPA responses. Several studies have reported changes in the GABAA receptor in temporal lobe epilepsy, including alterations in subunit composition (Brooks-Kayal et al. 1998) and receptor density (Shumate et al. 1998). However, electrophysiological data have shown that there is no difference in the potency of GABA at these receptors, although their modulation is altered (Shumate et al. 1998). Therefore it appears that we are dealing with a different and isolated phenomenon. In summary, available data strongly suggest that the prolonged GABA- and NPA-evoked responses and the decreased conductance of NPA-evoked responses were in part due to a change in the number and distribution of GABA transporters, although a potential contribution by an alteration in posttranslational modification cannot be ruled out.

Differences between GABA and NPA responses

The reversal potential and the amplitude of the GABA- versus NPA-evoked responses were significantly different in both control and epileptic rats. The amplitude will be dependent on both the amount of GABA available to bind postsynaptic receptors and on the reversal potential. The data support the hypothesis that the differences in reversal potential between GABA and NPA is the primary reason for the smaller NPA responses. ENPA was noted to be closer to EIPSP than EGABA and therefore probably reflects a preferential activation of synaptic GABA receptors. For instance, exogenously applied GABA may activate both synaptic and extrasynaptic receptors along a significant length of the somata/dendritic axis and would thus have a high probability of eliciting a bicarbonate efflux due to saturation of the Cl gradient in the fine processes (Staley and Proctor 1999; Staley et al. 1995). In contrast, NPA microapplication may result in [GABA]o localized primarily at the inhibitory synapses near the soma/inner molecular layer border and thus activate a smaller bicarbonate flux. This mechanism could also explain why a similar trend was observed for the reversal potentials for GABA- versus NPA-evoked events in slices from humans, although no significant difference was noted. Specifically because the size of the microdrops used in both sets of experiments was similar, these data may simply reflect the differences in the size and dendritic length of dentate granule cells in rats versus humans. While a difference in the degree of bicarbonate flux could account for EGABA being depolarized relative to ENPA and EIPSP, other mechanisms cannot be ruled out.


    ACKNOWLEDGMENTS

We thank the patients without whose informed consent this work could not be performed.

This work was supported by National Institutes of Health Grants NS-139092 and NS-06208 to A. Williamson and AG-00795 to P. R. Patrylo.


    FOOTNOTES

Address for reprint requests: A. Williamson, Dept. of Neurosurgery, Box 208039, Yale University School of Medicine, New Haven, CT 06520 (E-mail: Anne.Williamson{at}yale.edu).

Received 19 September 2000; accepted in final form 22 December 2000.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society