Abstract
Commentary
The reversal potential for a synaptic response ought to be a straightforward measurement: evoke a synaptic response, change the neuron's membrane potential by injecting current, and then evoke another response. Thus, a plot of the size of the synaptic response versus the membrane potential would provide the reversal potential. However, it is surprisingly easy to end up with a spurious value of EGABA. The most common pitfall occurs during the process of recording and changing the membrane potential, which frequently changes the balance of ions that flow through the GABAA receptor. This alteration was well known to early investigators who used sharp electrodes filled with molar concentrations of potassium salts; some of the anions used permeated the GABAA receptor channel and altered the reversal potential (5). The subsequent development of whole-cell recording techniques utilizing pipettes filled with “physiological” concentrations of potassium and chloride salts to measure EGABA initially seemed ideal (6). However, the whole-cell recording technique presented as many problems as had the sharp electrodes, because the low-access resistance of the pipette allowed free dialysis of the neuronal soma and to a lesser extent, the dendrites (7). The dialysis disrupts not only the ionic balance but also every second messenger system as well. What to do?
The exploitation of antibiotics that can form pores in cell membranes to allow passage of only ions overcame the second messenger problem. The antibiotic gramicidin was hailed as the solution to the measurement of EGABA, because the pores that it forms allow passage of only cations, so that anions that permeate the GABA channel are not affected. However, the balance of anions in a cell is determined by the balance of cations, through the action of cation–anion cotransporters. In adult neurons, cation–anion cotransport is generally handled by the K+/Cl-cotransporter, KCC2, which links the export of one potassium and one chloride ion from the cell, resulting in a low chloride concentration and an EGABA that is usually more negative than the resting membrane potential (RMP). Gramicidin recordings are accurate in adult neurons because the intracellular potassium concentration is high and the potassium reversal potential (which determines the reversal potential for Cl-, via KCC2 co-transport) does not change much if intracellular potassium is altered by a few mM; however, this is not the case with Cl-cotransport in immature neurons where chloride transport is handled by NKCC1, a cotransporter that depends on a low intracellular Na+ concentration to maintain a high intracellular Cl- concentration by linking the import of Na+, K+, and Cl- ions. Gramicidin recordings in such neurons cause major artifacts by altering the intracellular Na+ concentration (4).
Pathak et al. used gramicidin perforated patch recordings to measure EGABA from adult animals; thus, as long as only KCC2 is expressed in these cells, the gramicidin whole-cell recording technique would be an appropriate method to measure EGABA. However, there is ample evidence from resected brain tissue that NKCC1 also is expressed in neurons from patients with intractable epilepsy (8). Pathak et al. studied hippocampal dentate granule cells during the first week after brain injury, which might antedate the expression of NKCC1 in epileptic tissue. Furthermore, they looked for biochemical evidence of NKCC1 and did not find it. So, why do concerns persist regarding gramicidin and NKCC1?
Dentate granule cells have a very negative RMP, about −80 mV (9). While most cells have a GABAA reversal potential that is negative to RMP (as a result of KCC2 cotransport), granule cells have a GABAA reversal that is more positive than the RMP; Pathak et al. found an EGABA of about −75 mV. Although they did not report the RMP in these cells, a figure published in the study (Figure 6) demonstrates depolarizing GABAA receptor responses, suggesting that indeed EGABA was positive to the RMP. It is difficult to explain EGABA in terms of KCC2 when EGABA is more positive than the RMP. As mentioned, KCC2 cotransports K+ and Cl- and thus, comes to equilibrium when ECl = EK. EK is at least −100 mV and is substantially more negative than the RMP. If KCC2 activity is completely blocked, Cl- will be passively distributed and ECl will equal the RMP. So how does EGABA become positive relative to RMP? It would require either a substantial conductance of another anion or an active Cl- accumulation mechanism, such as NKCC1.
GABA receptors are slightly permeable to the anion, HCO3-, which has a much less negative reversal potential of approximately −12 mV (10). Thus, studies measuring Cl- transport by KCC2 or NKCC1 are best performed in the absence of CO2 and HCO3-, for example, by buffering the media with HEPES and removing CO2 from the artificial CSF (4,11). Is the HCO3- permeability sufficient to explain why EGABA is positive to the RMP in granule cells? The answer also would tell us whether another cation–anion cotransporter, such as NKCC1, is active in these cells. Although Pathak et al. performed some experiments in the absence of HCO3-, the impact of HCO3-flux was not systematically studied, so it cannot be assumed that only KCC2-mediated Cl- transport is active. In some experiments, furosemide was used to block KCC2; this diuretic also blocks NKCC1 and carbonic anhydrase (an enzyme that facilitates HCO3- regeneration) at the same or lower concentrations used by Pathak and coworkers (12) and also blocks some GABAA receptors (13). Unfortunately, no more specific KCC2 antagonist has been identified, making it difficult to be sure that changes in EGABA are solely responsible for the disinhibition produced by furosemide (which was shown in a figure [Figure 7] published in the Pathak et al.'s study).
There are more accurate electrophysiological means to measure EGABA and RMP, but they have limited time resolution and involve painstaking recordings of the reversal potential of channel currents measured from cell-attached electrodes (14). The intracellular Cl- concentration also can be measured using ion-sensitive dyes, including genetically expressed dual-wavelength dyes, such as Clomeleon. To date, these dyes have restricted regions of expression in the CNS (but, fortunately they are expressed in the hippocampus!), limited affinity for Cl-, exquisite pH sensitivity (which can be a problem in light of the HCO3- permeability of the GABA receptor), and must be calibrated to a known Cl- concentration at the end of the experiment (15).
Measuring the transport of Cl- can be useful even if the exact measurement of EGABA is likely to be offset by some of the factors described above (10). For example, if exclusively inward Cl- transport is recorded, it can be concluded that EGABA is positive to RMP, so that GABAA receptor activation will produce membrane depolarization. If the rate of inward transport increases after an experimental intervention, it can be assumed that the intervention caused EGABA to become less negative. Pathak et al. measured Cl- transport by evoking a train of synaptic GABAA receptor responses and then measuring the change in EGABA at the end of the train. If the synaptic influx of Cl- exceeds Cl- efflux via KCC2, Cl- should accumulate and EGABA should become less negative. This shift in EGABA provides a measure of KCC2 activity. Because this measure of KCC2 depends on the difference between synaptic GABA-evoked Cl-influx and KCC2 Cl- efflux, the outcome is affected by a number of factors that impact the synaptic GABA-evoked Cl- influx, including the rate of IPSC decay and receptor desensitization, facilitation and depression of presynaptic GABA release, as well as the rate of GABA reuptake (which affects the spread of GABA to nearby extrasynaptic receptors). Thus, it is important to report the total IPSC current (i.e., the charge transfer during the train of responses that loads the Cl-), which provides the actual amount of Cl- that flowed into the cell and the rate at which it flowed (presuming the experiment is performed in bicarbonate-free solutions). This calculation removes all the synaptic variables from the measurement of KCC2 function and allows quantitation of KCC2 activity (4,10).
Another advantage of calculating the charge transferred during the train of IPSCs is that the value can be combined with the shift in intracellular Cl- concentration, which can be calculated from the change in EGABA using the Nernst equation. Dividing the amount of Cl- influx by the change in Cl- concentration gives the actual volume into which the Cl- current flows (4). The resulting value is important, because the sub-cellular distribution of GABAA receptors that are active during the IPSCs is not known and smaller volumes will accumulate Cl- more rapidly. Stated in another way, cells with a low total GABA activated conductance will need to have more GABAA receptor channels activated over a larger area to generate the same amplitude IPSC as a cell with a higher density of GABA receptors. Thus, in a cell with a lower density of GABAA receptors, the Cl- currents will flow into a larger intracellular volume. The larger volume will effectively dilute the incoming Cl-, decreasing the rate of intracellular Cl- accumulation and decreasing the rate of change of EGABA. The somatic versus dendritic balance of GABAergic innervation of hippocampal neurons changes during epileptogenesis (16), altering the balance of subcellular GABA flux—so calculation of the volume of distribution of the Cl- influx is an important control in studies of Cl- transport during epileptogenesis. The volume of distribution of the Cl- influx also is important because diffusion operates in parallel with KCC2 to reduce the changes in subsynaptic intracellular Cl- concentration caused by GABA-mediated Cl- influx. Widely spread GABAergic synapses will facilitate such diffusion, leading to an overestimate of KCC2 function. Diffusion effects are best controlled by repeating the experiment after transport has been blocked and by subtracting the apparent rates of Cl- transport under these two conditions to derive the transport-specific rate (4). Finally, calculating the volume into which the IPSC currents flow provides an answer to the question raised by Pathak et al. as to whether the nucleus of dentate granule cells should count as part of the volume into which Cl- diffuses (i.e., whether or not Cl- is freely diffusible across the nuclear membrane).
Using Western blots, Pathak et al. found a 25 percent decrease in KCC2 expression in the dentate after status epilepticus. Is this finding sufficient to explain the measured reductions in Cl- transport? The investigators used furosemide, an inhibitor of KCC2, to reduce transport. Only maximal concentrations of furosemide, corresponding to 100 percent block of KCC2 (11), inhibited KCC2 to the same extent as was observed in granule cells after status epilepticus, suggesting that there is essentially no KCC2 function in the granule cells. As the authors conclude, this finding further suggests that the 25 percent decrease in KCC2 expression is not sufficient to cause the observed reduction in KCC2 function and that posttranslational modifications, membrane trafficking, and phosphorylation also are likely to be important determinants of KCC2 function after status epilepticus.
To measure the impact of the change in EGABA on signal processing, Pathak et al. elicited a postsynaptic GABA response (i.e., an IPSP) followed by somatic current injection; they found that more action potentials were triggered from granule cells by this protocol in the first week after status epilepticus. The sub-cellular location of the conductance evoked by the GABAergic IPSP is an important variable in this experiment. Depolarizing GABA-activated currents can both activate voltage-dependent cation currents in the axon hillock and shunt the resulting cation currents (17), so that the net results of GABAA receptor activation depend not only on EGABA but also on the subcellular distribution of the evoked GABA-activated conductances. Because the subcellular distribution of GABA-activated conductances shifts in epileptic animals (15), it will be important to complement these experiments by Pathak and colleagues with studies of the subcellular location of GABA-activated conductances after status epilepticus. In summary, Pathak et al. have identified an important potential mechanism of epileptogenesis. Clearly, the topic is complex and a promising area for many future investigations.