Primary hippocampal cell cultures are widely used in neurobiological experiments but they are often problematic because of non-physiological conditions of neuronal growth. Although cultured neurons have been shown to maintain some features (e.g. developmental changes in expression of synaptic receptors 1, 2, 3), recent studies demonstrated that alteration of neuronal environment, may cause a strong homeostatic modulation (4, 5). Thus to further investigate the impact of homeostatic plasticity on neuronal activity it seems useful to confront the functional characteristics of cultured neurons with properties of those developing in physiological conditions. In order to compare the basic features of GABAergic (GABA--aminobutyric acid) and glutamatergic currents from hippocampal cultures and acute slices, measurements of current responses to rapid GABA or glutamate applications as well as synaptic current recordings in the two models were performed. We show that amplitude ratio of currents elicited by ultrafast applications of saturating GABA or glutamate are nearly one order of magnitude larger in cultured neurons than that in slices, and differences in synaptic currents were also found. We propose that these variations may reflect the impact of a homeostatic plasticity.


MATERIALS AND METHODS
Neuronal culture was prepared as described preciously (6) from postnatal day 2 to 4 (P2-P4) Wistar rats with accordance to Polish Animal Welfare Act. Culture medium was exchanged (ca. 50% of volume) once every 3 days. Experiments were performed on cells between 9 and 15 days in culture.

Slices were prepared from 19-27 days Wistar rats that were anaesthetized with isoflurane (Baxter, UK) and killed by decapitation. This procedure is approved by the Polish Animal Welfare Act and by a local ethical commission. Brains were sliced using vibratome (Leica VT 1000S, Darmstadt, Germany) in ice-cold solution containing (in mM): 87 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 7 MgCl2, 75 sucrose and 25 glucose, pH 7.4. Transverse hippocampal slices (400 µM thick) were transferred to a chamber containing the same solution and kept at room temperature for 15-20 min, and subsequently stored in the artificial cerebro-spinal fluid (aCSF) containing (in mM): 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 25 glucose, pH 7.4 at room temperature. Slices were transferred to a recording chamber continuously perfused with oxygenated aCSF at room temperature and visualized with an upright microscope (DM LFS, Leica, Darmstadt, Germany).

Currents were recorded either in the whole-cell or in the nucleated/excised patch mode of the patch-clamp technique using the Multiclamp 700B (Molecular Instruments, Sunnyvale, CA, USA) at a holding potential (Vh) of -40 mV for current responses and -70 mV for synaptic current recordings. Signals were acquired using Digidata 1440 and pClamp 10.2 software (Molecular Instruments, Sunnyvale, CA, USA).

Solutions used for recordings of current responses to rapid agonist applications from nucleated or excised patches contained (in mM): intrapipette solution – 137 CsCl, 1 CaCl2, 2 MgCl2, 11 1,2-bis(2-aminophenoxy)ethane-N,N,N’-tetraacetic acid (BAPTA), 2 ATP, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.2 with CsOH), external Ringer solution – 137 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 20 glucose, 10 HEPES (pH 7.2 with NaOH). For synaptic current recordings intrapipette solution contained in mM: 140 KCl, 1 MgCl2, 10 HEPES, 0.5 EGTA, 4 MgATP (pH=7.25 with KOH). In case of culture recordings the same external solution as in the case of current responses was used, whereas for slices oxygenated aCSF was employed. 1 µM tetradotoxin (TTX) was added to extracellular solutions. For recordings of GABAergic or glutamatergic synaptic events, selective blockers of GABAA or glutamate receptors were added to the external solutions: 5-10 µM gabazine for miniature excitatory postsynaptic currents (mEPSCs) and 1 mM kinurenic acid (in cultured neurons) or 20 µM DNQX (6,7-dinitroquinoxaline-2,3-dione) for miniature inhibitory postsynaptic currents (mIPSCs) recordings. Pipette resistance was typically 2-4.5 M. Access resistance was monitored and cells exhibiting access resistance larger than 15 M were rejected from the analysis. Chemicals were from Sigma-Aldrich (Steinheim, Germany), except tetrodotoxin (Latoxan, Valence, France) and HEPES and NaOH (Carl Roth, Karlsruhe, Germany). GABA or Glutamate were applied to excised patches using the ultrafast perfusion system based on a piezoelectric-driven (Physik Instrumente with preloaded HVPZT translator 80 µm; Waldbronn, Germany) theta-glass application pipette (7). The open tip recordings of the liquid junction potentials revealed that 10-90% exchange of solution occurred within 50-80 µs.

The kinetics of the rising phase of currents was assessed as 10% to 90% rise time (10-90% RT). Deactivation of evoked currents (time course after agonist removal) or the decaying phase of synaptic currents were fitted with a sum exponents:



where Ak are the amplitudes of respective components and tk are the time constants. For GABAergic currents, n=2, and for glutamatergic currents, n=1. For normalized currents, A1+A2=1. The mean deactivation decay time constant was calculated as



In some cases, the quality of recorded current traces was sufficient to determine only the amplitude while the kinetic analysis (rise time or decay kinetics) was not performed (e.g. due to a transient trace disturbance). For this reason, the number of cells (n) included in the analysis of amplitudes was different from that for the kinetic parameters (see Results).

Data are expressed as mean±S.E.M. and Student’s t-test was used for comparison of data. All experiments were performed at room temperature (22-24°C).


RESULTS
An important issue is the appropriate matching of culture period and the age of animals. It has been demonstrated that in cultures after 9 days in vitro (DIV 9), the acceleration of synaptic current kinetics as well as changes in receptor expression pattern is observed that largely mimic developmental mechanisms in vivo (1, 2, 8). Taking this into account we have confronted the properties of currents measured in the neuronal culture (DIV 9-15) to those observed in slices from juvenile/adult rats (P19-23).

First, we have confronted the amplitudes of current responses evoked by ultrafast applications of saturating agonist concentrations (3-5 ms duration) to the excised or nucleated patches pulled from visually identified pyramidal neurons (in culture) or in the CA1 region (in slices). To reduce the impact of cell-to-cell variability, recording of responses to saturating GABA (10 mM) or glutamate (10 mM) was performed on the same patch and current ratios were calculated (in these conditions paired Student’s t-test was used). For cultured neurons, we used nucleated patches because most of glutamate-evoked currents from excised patches were too small to be reliably analyzed. Importantly, it has been previously shown that responses obtained from nucleated patches show indistinguishable time course in comparison to that observed for excised patches, except for rise time kinetics (6). The averaged amplitudes of currents evoked by saturating GABA or glutamate in the nucleated patches were -866±172 pA (n=12) and -577±164 pA (n=12), for GABA- and glutamate evoked currents, respectively. The amplitude ratio of GABA- to glutamate-evoked currents (Fig. 1A, B) showed considerable variability ranging between 0.70 and 5.51 and the averaged ratio was 2.34±0.45 (n=12), indicating a significantly larger current intensity (p<0.05 paired t-test) mediated by GABAergic currents (Fig. 1A, B, E). In slice recordings, nucleated patches were not feasible and recordings were made from excised patches. In slices, the amplitudes of GABAergic and glutamatergic currents evoked by saturating agonists showed reversed proportions with respect to those in cultures: -174±76 pA (n=5) and -711±293 pA (n=5, Fig. 1A-D), for GABA- and glutamate- evoked currents, respectively while the current ratio was 0.24±0.02 (n=5, p<0.05, paired test, Fig. 1E).

Fig. 1 . Ratios of current response amplitudes evoked by saturating concentrations of GABA or glutamate are substantially different. A and B show typical current responses elicited by saturating (10 mM) GABA (A) or glutamate (10 mM) concentration (B) recorded from the same nucleated patch in neuronal cell culture. In C and D, examples of responses evoked by saturating GABA (C) or glutamate concentration (D) and recorded from the same excised patch in acute brain slice are shown. E, statistics of amplitude ratios of currents evoked by saturating GABA and glutamate concentrations. Insets above current traces depict time of agonist application. Asterisk indicates the statistically significant difference (p<0.05).

Fig. 2. Current responses to glutamate (but not to GABA) in neuronal culture and acute brain slices show different deactivation kinetics. In A, typical normalized current responses to saturating GABA (10 mM) measured in neuronal culture (thick line) and acute slice (thin line) are shown. To make the two currents better discernible in this graph, the traces were slightly shifted with respect to each other. B, Typical deactivation phases of current responses evoked by saturating glutamate (10 mM) recorded from patch excised from a cultured neuron (thick line) or a neuron in slice (thin line). C, Statistics of weighted deactivation time constants (mean) for GABAergic (white bins) and glutamatergic currents (dashed bins) recorded in neuronal culture (cult.) and acute slices (slice). Insets above current traces depict time of agonist application. Asterisk indicates the statistically significant difference (p<0.05).

In addition to current amplitude, the time course of current responses was also analyzed. The weighted deactivation time constant (mean) for currents evoked by saturating GABA in cultured neurons was 23.58±2.98 ms (n=11) and was not significantly different from that in slices (23.98±5.88 ms, n=6, p>0.05, Fig. 2A, C). However, in the case of currents elicited by saturating glutamate, responses evoked from patches excised from neurons in culture were characterized by a significantly faster deactivation kinetics than those in slices (mean=3.47±0.20 ms, n=8 and 5.87±0.31 ms, n=6, in culture and slice, respectively, p<0.05, Fig. 2B, C). The averaged amplitude of GABAergic mIPSCs measured from cultured neurons was -50.91±1.28 pA (n=39) and did not differ significantly from that in slices (-50.35±3.04 pA, n=11, p>0.05, Fig. 3A,C). However, mIPSCs frequency was significantly larger in neurons from slices (0.21±0.02 Hz, n=27, 0.56±0.09 Hz, n=11, for culture and slices, respectively, p<0.05, Fig. 3A, D). In cultured neurons, mEPSCs were characterized by averaged amplitude of -33.69±1.94 pA, n=6 and frequency of 1.17±0.28 Hz (n=6). Surprisingly, in contrast to current responses to glutamate (Fig. 1), the averaged mEPSC amplitude, measured in slices, was significantly smaller than that in culture (-18.48±0.77 pA, n=5, p<0.05, Fig. 3B, C) and mEPSC frequency was strikingly low (0.14±0.03 Hz, n=6, Fig. 2; p<0.05 for comparison with mEPSC frequency in cultures, Fig. 3B, D). The weighted time constant of the mIPSC decaying phase (mean) was significantly faster in slices than in cultured neurons (35.66±1.17 ms, n=39, 23.29±1.95 ms, n=11, for culture and slices, respectively, p<0.05, Fig. 4A, D). On the contrary, the decaying phase of mEPSCs measured from neurons in slices was significantly slower than that in culture (2.36±0.20 ms, n=6, 3.87±0.35 ms, n=6 for neurons from culture and slices, respectively, p<0.05, Fig. 4C, D). The onset kinetics of mIPSCs was significantly faster in neurons from slices (10-90% RT-0.92±0.06 ms, n=21, and 0.52±0.03 ms, n=10, in culture and slice, respectively, p<0.05, Fig. 4B, E) but the rise time kinetics of mEPSCs did not differ in culture and slices (10-90% RT: 0.55±0.04 ms, n=5 and 0.57±0.04 ms n=6, in culture and slice for mEPSCs, p>0.05, Fig. 4E).

Fig. 3. Miniature synaptic currents recorded in neuronal cultures and slices show differences in amplitude and frequency. A, Typical mIPSC recordings from neuronal culture (upper trace) and slice (lower trace). B, Typical mEPSC recordings from neuronal culture (upper trace) and slice (lower trace). C, Statistics of mIPSC and mEPSC amplitudes in culture and slices (mIPSCs–white bars, mEPSCs –dashed bars). D, Statistics of mIPSC and mEPSC frequencies in culture and slices (mIPSCs–white bars, mEPSCs–dashed bars). Asterisks indicate the statistically significant difference (p<0.05).

Fig. 4. Miniature synaptic currents recorded in neuronal cultures and slices show differences in their time course. A, Typical averaged, normalized and superimposed mIPSCs recorded in culture (thick line) or in slice (thin line). B, Typical averaged, normalized mIPSC in an expanded time scale to better visualize the differences in the mIPSC onset kinetics. C, Example of averaged, normalized and superimposed mEPSCs recorded in culture (thick line) or in slice (thin line). D, Statistics of the weighted average time constant (mean) for the decaying phases of mIPSCs and mEPSCs in culture and slices (mIPSCs–white bars, mEPSCs–dashed bars). E, Statistics of the 10%-90% rise time for the onset phases of mIPSCs and mEPSCs in culture and slices (mIPSCs–white bars, mEPSCs–dashed bars). Asterisks indicate the statistically significant difference (p<0.05).

DISCUSSION
In the present report we demonstrate that GABAergic and glutamatergic currents in cultured hippocampal pyramidal neurons show profound differences with respect to their counterparts in Cornu Ammonis 1 (CA1) region in the acute slices. The most apparent difference is that the amplitude ratio of currents evoked by saturating GABA or glutamate differs in the two models by nearly one order of magnitude. Since recordings were performed in the presence of magnesium ions at negative potentials, only non- N-methyl-D-aspartic acid (NMDA) receptors contributed to the glutamatergic currents. The reversal potential was close to 0 mV for both neurotransmitters, indicating that changes in GABA to glutamate current ratios resulted from alterations in respective membrane conductances. However, observed changes might be also related to different subunit composition of receptors in the considered models (9) as suggested by differences in the kinetics of both current responses (to glutamate) and synaptic currents (mIPSCs and mEPSCs). It should be noted, however, that synaptic current time course depends not only on the postsynaptic receptor properties but also on synapse geometry (e.g. 10) and several presynaptic factors. Strikingly, in slices, the current responses to glutamate are particularly large (Fig. 1), but mEPSCs are characterized by a small amplitude and low frequency in comparison to cultured neurons (Fig. 3). Moreover, while in slice, GABA to glutamate amplitude current ratio is much smaller than in culture, mEPSCs show the opposite trend with respect to mIPSCs (Fig. 1, 3). This observation might suggest that pyramidal neurons in the two models are characterized not only by substantially different expression pattern of glutamate and GABAA receptors but also by a different mechanism of segregation between synaptic and extrasynaptic receptors. However, additional studies using morphological and molecular biology methods (e.g. immunohistochemistry, Western blotting) or refined electrophysiology (extracellular single-cell oscillations recordings see 11) will be needed to further characterize the expression pattern and subcellular distribution of these receptors. These approaches would be particularly useful to compare the expression profiles for receptors with different subunit compositions in the two models.

An important message coming from this study is that basic properties of GABAergic and glutamatergic currents (both evoked and synaptic) show profound differences in the two considered models. Assuming that acute brain slices closely mimic the physiological conditions, these results imply that the cell cultures should be used with caution. The major question arising from our study is the mechanism underlying the profound differences in GABAergic and glutamatergic currents in culture and brain slices. Although our data are insufficient to elucidate this problem, several possibilities merit mentioning as likely candidates. Both GABAergic and glutamatergic currents are subject to homeostatic modulation (see e.g. 4, 5, 12). It is known, for instance, that decreased network activity (due to e.g. TTX treatment) gives rise to enhancement of EPSCs and reduction of GABAergic synaptic currents (13-18). Interestingly, the homeostatic modulation of mEPSCs may concern not only postsynaptic but also presynaptic mechanisms. For instance, Murthy et al. (19) provided evidence that the intensity of synaptic activity affected both release probability and the synaptic morphology. However, while in the spinal cord and in the cortex, the up regulation of mEPSCs after network activity blockade is not associated with altered frequency (13-15), in hippocampal neurons, up regulation of mEPSC amplitude is accompanied by an increase in their frequency (20). This difference is probably related to a lower occurrence of silent synapses in the cortex than in hippocampus (21). In the case of GABAergic transmission, similar to excitatory synapses, there is activity-dependent modulation of quantal amplitude and receptor clustering but there is also a clear change in the number of synapses (16). Moreover, expression of GABAA receptors is known to strongly depend on membrane depolarization as well as on the presence of glutamate receptor agonists (22-24). In particular, glutamate receptor agonists were shown to affect both GABA release and size of GABAergic terminals (25). Importantly, GABAARs expression is also regulated by exposure to their agonists or positive modulators (26). In addition to above mentioned stimuli, homeostatic modulation of GABAergic and glutamatergic currents may also depend on activity of trophic factors such as brain-derived neurotrophic factor (BDNF) (4, 27). Although we are unable to indicate the mechanism underlying observed functional differences in pyramidal neurons in culture and in slices, it is interesting to note that the trend observed in our study appears qualitatively opposite to that induced by suppression of network activity (increase in mEPSCs and decrease in mIPSCs). It is thus tempting to speculate that in our cultures, excessive network activity takes place (e.g. due to accumulation of glutamate or increased extracellular potassium) giving rise to an overexpression of GABAARs. Lower synaptic activity in slices might result, however, from severing the synaptic projections upon slice preparation. Another intriguing difference between neurons in culture and slices is that in the latter model there is a much larger disproportion between current responses to exogenous glutamate and the amplitudes of mEPSCs. This observation appears important as extrasynaptic pool of -amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors is widely believed to play a crucial role in synaptic plasticity (13, 14, 28). Thus, as a matter of speculation, we may propose that overexcitability in the network of cultured neurons might shift the balance of extrasynaptic versus synaptic AMPA receptors towards the latter ones.

In conclusion, we show that the hippocampal pyramidal neurons in culture and in slices show profound functional differences that are manifested as reversed proportions of GABAergic and glutamatergic evoked currents and marked differences between respective synaptic currents. We propose that homeostatic plasticity, resulting from profoundly different conditions during neuronal development in the two models, is a likely mechanism that underlies these differences.

Acknowledgements: This work was supported by Wellcome Trust International Senior Research Fellowship in Biomedical Science (grant no. 070231/Z/03/Z).

Conflict of interests: None declared.


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