Corresponding author J. R. Huguenard: Department of Neurology and Neurological Sciences, Room M016, Stanford University, Stanford University Medical Center, Stanford, CA 94305-5300, USA. Email: firstname.lastname@example.org
Synchrony within the thalamocortical system is regulated in part by intranuclear synaptic inhibition within the reticular nucleus (RTN). Inhibitory postsynaptic currents (IPSCs) in RTN neurons are largely characterized by slow decay kinetics that result in powerful and prolonged suppression of spikes. Here we show that some individual RTN neurons are characterized by highly variable mixtures of fast, slow and mixed IPSCs. Heterogeneity arose largely through differences in the contribution of an initial decay component (τD∼10 ms) which was insensitive to loreclezole, suggesting involvement of the GABAA receptor β1 subunit. Single-cell RT-PCR revealed the presence of β1 subunit mRNA only in those neurons whose IPSCs were dominated by a rapid and prominent initial decay phase. These data show that brief, β1-dependent, loreclezole-insensitive IPSCs are present in a subpopulation of RTN neurons, and suggest that striking differences in IPSC heterogeneity within single neurons can result from of the presence or absence of a single GABAA receptor subunit.
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Heterogeneity of synaptic inhibition in hippocampal or cortical neurons (Banks et al. 1998; Hefti & Smith, 2000; Garden et al. 2002; Xiang et al. 1998; Kobayashi & Buckmaster, 2003) is expected based on the expression of supernumerary receptor subunits in these regions. For example, all of the major subunits (except the α6 subunit, which is restricted to cerebellar neurons) are expressed in hippocampal CA1 and CA3 cells (Wisden et al. 1992). Binding of various allosteric modulators, such as benzodiazepines, barbiturates, anticonvulsants and neurosteroids (all of which mainly prolong IPSC duration), is dependent on the specific subunit composition of the receptor (MacDonald & Olsen, 1994). The different clinical actions of these drugs, ranging from myorelaxant to anticonvulsant, anxiolytic and sedative, illustrate the importance and complexity of IPSC regulation in different brain regions.
The functional diversity of GABAergic synaptic inputs in each region should logically be related to the number of locally expressed receptor subunits. Yet the subunit distribution in the RTN predicts minimal functional diversity. Only four of the 19 existing subunits (α3, β1, β3 and γ2) are present above background levels in RTN (Wisden et al. 1992; Fritschy & Möhler, 1995; Huntsman et al. 1996; Pirker et al. 2000). Therefore, GABAA receptors in RTN neurons are likely to be limited to a few subtypes. Here we show that despite this limited GABAA subunit heterogeneity in RTN, IPSC decay is not uniform. In single RTN cell recordings, we found IPSCs with both slow and fast decay rates, and that the relative numbers of fast and slow IPSCs varied on a cell to cell basis. In an attempt to elucidate the molecular basis of this heterogeneity, we examined the effects of two compounds that target specific GABAA receptor subtypes in the RTN: the benzodiazepine clonazepam (whose activity depends on α1,2,3,5 and γ2 subunits; Pritchett et al. 1989), and loreclezole (which depends on β2 or β3 subunits; Wafford et al. 1994; Wingrove et al. 1994). We then combined the information gained from electrophysiology with single-cell RT-PCR experiments to correlate these unique functional properties with GABAA receptor subunit expression. We found that functional heterogeneity is not only present in the RTN, but that it is likely to be determined by the presence or absence of a single subunit (β1). A preliminary report of this work has been presented (Huntsman & Huguenard, 2001).
Preparation of thalamic slices
All experiments were performed in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Stanford University Institutional Animal Care and Use Committee. Thalamic slices were obtained from Sprague-Dawley rats (of either sex) ranging in age from postnatal day 21 (P21) – P30. Animals were fully anaesthetized (50 mg kg−1 sodium pentobarbital, administered i.p.) before decapitation and brains were blocked and removed from the skull. The blocked brain was then placed in ice-cold, oxygen equilibrated (95% O2–5% CO2) sucrose slicing solution for 2–3 min (mm): 234 sucrose, 11 glucose, 24 NaHCO3, 2.5 KCl, 1.25 NaH2PO4.H2O, 10 MgSO4 and 0.5 CaCl2. The brain was glued to a slicing dish, ventral surface down, and submerged in cold sucrose slicing solution. Horizontal slices that included the dorsal thalamus and adjacent reticular nucleus were cut at a 200-μm thickness with a vibrating blade microtome (Leica). Slices were then incubated in preheated (32°C), oxygen equilibrated standard artificial cerebral spinal fluid (ACSF) (mm): 126 NaCl, 26 NaHCO3, 10 glucose, 2.5 KCl, 1.25 NaH2PO4.H2O, 2 MgCl2.6H2O and 2 CaCl2.2H2O for at least 1 h prior to recording.
Whole-cell patch-clamp recordings were obtained by previously published methods (Huntsman & Huguenard, 2000). Briefly, after a 1 h incubation period, sections were transferred to a chamber and bathed in a continuous perfusion of ACSF (2 ml min−1) at room temperature (∼24°C). Whole-cell patch-clamp recordings were made from visually identified neurons within the boundaries of the reticular nucleus. The recordings were made under visual control using a fixed-stage upright microscope (Axioskop, Zeiss, Thornwood, NY, USA) equipped with an insulated 63× objective, Nomarski optics and an infrared-sensitive video camera (Cohu, Inc., San Diego, CA, USA). The internal pipette solution contained (mm): 135 CsCl, 5 lidocaine N-ethyl bromide (QX-314), 2 MgCl2, 10 ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetate acid (EGTA) (Sigma, St Louis, MO, USA), and 10 N-2-hydroxy-ethylpiperazine-N′-2-ethanesulphonic acid (Hepes) (Sigma). The solution was adjusted with CsOH to pH 7.3; final osmolarity was 290 mosmol l−1. Glass electrodes (borosilicate glass, Garner Glass Company, Claremont, CA, USA) were pulled in multiple stages using a Flaming-Brown micropipette puller (Model P-87, Sutter Instrument Co., Novato, CA, USA). Pipettes had resistances of 2.5–3.3 MΩ when filled with intracellular pipette solution. Continuous voltage-clamp records containing spontaneous IPSCs were obtained at a holding potential of −60 mV using a patch clamp amplifier (model L/M-EPC 7, List Medical, Darmstadt, Germany) or a Multiclamp amplifier (700A, Axon Instruments, Union City, CA, USA). Series resistance was always less than 15 MΩ and monitored throughout the experiment for stability (< 25% change), and was not electronically compensated.
GABAA receptor-mediated spontaneous IPSCs were pharmacologically isolated by bath application of the ionotropic excitatory amino acid receptor blockers: 6,7-dinitro-quinoxaline-2,3-dione (DNQX, 20 μm, Sigma-RBI, Natick, MA, USA) and 2-amino-5-phos-phonopentanoic acid (AP-5, 100 μm, Sigma-RBI) in physiological saline. The perfusion rate was 2 ml min−1 and it typically took 2–3 min to fully block all excitatory synaptic responses. GABAB receptors were blocked with 135 mm Cs+ and 5 mm QX-314 in the internal pipette solution. GABA modulators clonazepam (Sigma-RBI) and loreclezole (a gift from Janssen Research, Belgium) were dissolved at 1000× final concentration in DMSO and frozen in aliquots. Vehicle controls contained equivalent final DMSO concentration (0.1%). Fresh dilutions of the concentrated stocks were made daily and applied by bath perfusion. As slow wash-in times (> 10 min, Fig. 2) impeded the quantification of equilibrium drug effects, we mainly examined cells after preincubating (cf. Hollrigel et al. 1996) for 1–2 h in clonazepam (100 nm) or loreclezole (10 μm). In order to examine action potential-independent mini IPSCs (mIPSCs), we recorded sIPSCs in the presence of 1 μm TTX (Sigma). TTX did not affect frequency of events, indicating that the vast majority of sIPSCs in our preparation were mIPSCs.
Analysis of β1 subunit mRNA expression was achieved by single-cell RT-PCR. Methods followed similar single-cell RT-PCR guidelines previously described (D. K. O'Dowd, personal communication; Monyer & Jonas, 1995; Massengill et al. 1997; Berger et al. 1998; van Hooft et al. 2000). Gloves were worn at all times and all solutions were autoclaved prior to use. The silver wire electrode was re-chloridized with bleach prior to each day of recording. The cytoplasm of each RTN neuron was aspirated into the pipette after the whole-cell patch clamp recording was complete (usually 10–15 min total recording time). Electrophysiology was carried out in the same manner as described above with the exception that pipettes were pulled to a resistance in the range of 1.8–2.5 MΩ. RT-PCR was always performed on the same day of whole-cell patch-clamp recording in order to avoid any freeze–thaw irregularities. Cell cytoplasm was harvested by applying negative pressure to the recording pipette under visual control and expelled into an autoclaved tube containing 1 mm deoxynucleoside 5′-triphosphate mixture and 100 ng μl−1 random primers. Each cell's contents were placed on ice until the last cell was harvested and then all were processed together. Controls were nuclease-free water, intracellular pipette solution (a pipette filled with intracellular solution and held above a thalamic slice under flowing ACSF for a few minutes) and 40 pg of total RNA isolated from rodent thalamus were included in every reverse transcription reaction set. The solution was brought to a volume of 10 μl with nuclease-free water, incubated at 65°C for 10 min and put on ice. All reactions were added to new autoclaved tubes containing: 1 × First-strand transcription buffer, 10 mm dithiothreitol (DTT), 20 units of ribonuclease inhibitor (RNaseOUT; Invitrogen, Carlsbad, CA, USA), and 50 units of Moloney murine leukaemia virus reverse transcriptase (Invitrogen Life Technologies). This mixture was incubated for 50 min at 37°C, then heat inactivated at 70°C for 15 min. The entire cDNA template reaction (10 μl) was used in the first round of PCR amplification. The primer pairs were chosen to amplify the heterogeneous cytoplasmic loop of the β1 subunit of the GABAA receptor. The forward primer was 5′-GCA-ATA-TTC-TCC-TCA-GCA-CC-3′ and corresponds to nucleotides 1169–1190 of the rodent sequence (Ymer et al. 1989) and the reverse primer was 5′-GTT-CAC-GTC-AGT-CAA-GTC-A-3′ and corresponds to nucleotides 1400–1418. For the second round of PCR, two nested primers (internal to the first round pair) were used. The forward primer was 5′-GAG-GTG-CTC-ACG-GGT-GTA-AG-3′ and corresponded to nucleotides 1221–1240 of the rodent sequence (Ymer et al. 1989) and the reverse primer was 5′-CTG-AGA-GGC-ACG-TCT-GCG-GAT-G-3′ and corresponds to nucleotides 1361–1382. The nested primers yielded a single PCR product of 162 bp. The first round PCR reaction was done in a master mix with the total number of reactions being determined by the number of cells collected for the day of recording along with all the controls. In addition to amplifying the three controls from the RT reaction, a water control was also included in the master mix for each round of PCR. In a 50-μl reaction the ingredients were as follows: 1 × PCR buffer, 0.2 mm dNTP mixture (each), 1.5 mm MgCl2, 20 pmol μl−1 of the outer primer pair, nuclease free water, the contents of the cDNA template reaction from a single cell (or the contents of cDNA reactions done in the presence of water, pipette solution and total RNA controls) and 2.5 units of DNA polymerase (Platinum Taq, Invitrogen Life Technologies). The PCR conditions were as follows: one denaturing step at 94°C for 1 min, 35 cycles of amplification representing 3 steps of denaturing, annealing and extension (94°C for 30 s; 55°C for 30 s; 72°C for 1 min) and a final extension at 72°C for 7 min. After the reaction was complete, 5 μl of the 50 μl total reaction was diluted with 500 μl of RNase free water. Then 5 μl of this dilution was used as the template for the second round of PCR. All reaction and cycling conditions were identical with the exception of the replacement of the nested (inner) primers. Amplification products were analysed on either a 2% agarose gel or an 8% polyacrylamide gel and always yielded a single ethidium bromide stained-band that migrated at the predicted molecular weight of 162 bp. The molecular weight marker was the pBR322 DNA–MspI digest (New England Biolabs, Ipswich, MA, USA). All products were sequenced at the Stanford University Protein and Nucleic Acid Facility and identified as rodent β1 cDNA. With two rounds of PCR amplification, false positives were our greatest concern. When a negative control was positive, then the entire round of PCR reactions (and thus entire day of recordings) were eliminated from the study. In some cases DNase I (Promega, Madison, WI, USA) was added prior to RT or RT reactions were run without the enzyme to check for contamination from the nucleus. However, this was not a primary issue because all cell aspirations were done under visual control and uptake of the nucleus could be avoided.
Data collection and analysis
IPSCs were filtered at 1 kHz, stored in digitized form on a Neurocorder (model no. DR484, Neurodata, NY, USA) and backed up on VHS tape. Continuous records of spontaneous events were collected with Axotape v. 2 and Clampex v. 8.2 (Axon Instruments). Spontaneous events were sorted and categorized using the customized software, Detector v. 4.8 (J. R. Huguenard) and Metatape v15.1 (J. R. Huguenard) and analysed with Scan software (courtesy of J. R. Dempster) and Clampfit 8.2 (Axon Instruments). Double exponential fits of baseline-subtracted averaged unitary inhibitory events were made using Clampfit, with DC (offset) forced to zero. The decay of averaged IPSCs was fit to the following equation: I=A1e−t/τD1+A2e−t/τD2 and weighted time constant τD,W= (τD1A1+τD2A2)/(A1+A2). Data were analysed with Origin v. 6.0 (OriginLab Corp., Northampton, MA, USA) and Microsoft Excel and statistical significance was measured using Student's t test. In order to classify RTN neurons into groups based on kinetic properties of sIPSCs an unsupervised hierarchical cluster analysis was used, based on Ward's method, with z-score normalization and intervals calculated by Euclidian squared distances (SPSS, v. 12). Following this a multiple discriminant analyis (SPSS), was used to determine which sIPSPC kinetic parameters were most predictive for cluster membership, i.e. those with the highest standardized canonical discriminant function coefficients.
Voltage clamp recordings of GABAA receptor-mediated spontaneous IPSCs (sIPSCs) were made from a total of 66 neurons in the reticular nucleus (RTN) of the thalamus. All recordings were performed under visual control to identify neurons by position, between external medullary lamina and the internal capsule, and cellular morphology unique to RTN neurons, for example, the fusiform shape of the soma (Jones, 1985). These sIPSCs are for the most part miniature IPSCs (mIPSCs) reflecting spontaneous fusion of GABA containing vesicles. Action potential-dependent IPSCs do not contribute significantly to spontaneous IPSC events in this preparation, as neither the frequency nor the amplitude of sIPSCs was affected by the sodium channel toxin TTX (1 μm, n= 5).
IPSCs in RTN neurons are heterogeneous
GABAA receptor-mediated IPSCs with slow decay times are characteristic inhibitory events in the RTN (Ulrich & Huguenard, 1997; Zhang et al. 1997). They are a persistent feature throughout postnatal development on to at least early adulthood (Huntsman & Huguenard, 2000). Although ensemble averages of multiple sIPSCs from single neuron yields mean responses with a slow decay component (Huntsman & Huguenard, 2000), it has been our recent observation that the commonly observed slow IPSCs are interspersed to varying degrees, on a cell to cell basis, with much briefer events. Individual sIPSC duration, as measured by the time required for 90% decay from peak (90%-width), can vary between 10 and 200 ms in a single neuron. This readily ascertained measure (90%-width) provided a simple estimate of IPSC duration that could be ascertained from all individual IPSCs, even from overlapped events, which could not be used for the fitting exponential decay curves.
Figure 1 illustrates IPSC heterogeneity in voltage-clamp recordings from two neighbouring neurons in the RTN. Both neurons possess the distinctive slowly decaying IPSCs (Fig. 1A and B, open arrowheads). However, the neuron in Fig. 1B has a greater proportion of fast events (Fig. 1B, *) intermixed with the characteristic slowly decaying events. In addition, neurons with fast IPSCs often displayed biphasic events – events with both fast and slow decay phases (e.g. Fig. 1B, **). Neurons could be roughly classified into two groups based on the kinetics of > 100 ensemble averaged sIPSCs: fast neurons (cells with a fast decay time constant of less than ∼18 ms), such as that shown in Fig. 1B, had IPSCs with a prominent early rapid decay and slightly larger peak amplitudes. Slow neurons had slower events (Fig. 1C and D). The degree of heterogeneity varied among RTN neurons and did not appear to reflect any particular location within the RTN. We used unsupervised hierarchical cluster analysis (see Methods) and the kinetic parameters of averaged sIPSCs (A1, A2, τ1, τ2) to classify individual RTN neurons into two groups. Multiple discriminant analysis was then used to determine which of the kinetic parameters were most predictive in determining cluster membership. The two time constants, τ1 and τ2, were most predictive (> fivefold greater predictive value than either A1 or A2) and a discriminant function using only these two parameters (Z=τ1× 0.42 +τ2× 0.021 – 8.94) was 100% effective in predicting group membership. Neurons with discriminant function (Z) values < 0.85 were classified as type I (Fast) and those with values ≥ 0.85 were type II (Slow). When τ1 is plotted versusτ2 two distinct clusters could be observed (Fig. 1E), with τ1 yielding the less overlapping values than τ2. Figure 1E shows two separate groupings of RTN neurons: those with Z-scores < 0.85 were grouped together in the region to the lower left of the grey line in a ‘fast’ cluster (cluster 1, filled squares) and a second ‘slow’ cluster (Z-scores > 0.5, cluster 2, open circles) with slower τ1 and to a lesser extent τ2, values formed another group to the upper right of the grey line. Mean values for IPSC decay time constants and coefficients are shown in Table 1.
Table 1. Biexponential decay time constants for ensemble averaged IPSCs for neurons from cluster 1 and cluster 2
Values are means ±s.d.***Values significantly different between cluster 1 and cluster 2 (P < 0.0001).
Cluster 1 (fast)
20.24 ± 9.87
14.82 ± 8.41
15.13 ± 1.96
76.6 ± 14.4
Cluster 2 (slow)
19.01 ± 12.34
13.52 ± 6.06
20.86 ± 1.71***
104.6 ± 17.1***
In all cases, ensemble-averaged spontaneous RTN IPSCs (obtained with ≥ 100 individual events) were best fitted to two exponentials (Fig. 1F). Overall mean values for decay and amplitude from a population of 50 neurons (33 fast + 17 slow) were: τ1= 17.1 ± 0.5 ms, τ2= 86.1 ± 2.9 ms, A1= 19.8 ± 1.5 pA, A2= 14.3 ± 1.1 pA, and τD,W (weighted time constant, see Methods) = 46.03 ± 1.9 ms. The sIPSCs in the example fast cell in Fig. 1 decayed faster in terms of both the fast and slow components (τ1= 16.4 ms, τ2= 72.9 ms, τD,W= 43.2 ms) than the slow cell (τ1= 23.1 ms, τ2= 133.0, τD,W= 87.9 ms). The biphasic decay observed with ensemble averaged IPSCs (e.g. Fig. 1F, fast cell) could be observed in individual IPSCs in some RTN cells (Fig. 1B, **).
Heterogeneity of single RTN neuron IPSC kinetics (i.e. the presence of fast and slow IPSCs in the same neuron) persisted in the presence of 2 μm strychnine (n= 10, data not shown), indicating that the presence of glycinergic (Ghavanini et al. 2005) versus GABAergic sIPSCs with different kinetics are not responsible for IPSC heterogeneity that we observe. Bath application of 10 μm bicuculline blocks all synaptic events (both fast and slow, n= 5). Taken together, these results show that both fast and slow events are mediated by GABAA receptors, and suggest that the diverse kinetic responses are mediated by differences in the underlying GABAA receptors.
We performed a series of experiments (n= 12 cells) at more physiological temperatures (34–36°C) in order to determine whether the heterogeneous IPSC kinetics that could be observed within single RTN neurons persisted under these conditions. The kinetics of IPSCs were considerably faster than those obtained at room temperature, consistent with an earlier report (Huntsman & Huguenard, 2000), yet as with room temperature recordings, three types of sIPSCs were observed at higher temperatures, fast events, slow events and hybrid fast–slow events, similar to those shown in Fig. 1B.
Effects of GABAA receptor subunit-specific allosteric modulators on IPSCs
In this section we further probe for GABAA receptor heterogeneity using GABAA subunit specific allosteric modulators. For these experiments, we used two different methods of drug application: bath application to individual neurons for a defined wash-in time or preincubation of thalamic slices in drug-containing solutions prior to recording. Bath application of either clonazepam (100 nm) or loreclezole (10–100 μm) produced a progressive IPSC prolongation over an extended wash-in period of more than 10 min, and the prolongation was poorly reversible during wash-out (Fig. 2). The effects of loreclezole took much longer to reach peak values than clonazepam and had minimal reversibility, even during 20 min of wash-out. By contrast, the effects of clonazepam were at least partially reversible during a wash-out period of 10–20 min (Fig. 2).
Pre-incubation of slices in drug-containing solutions (see Methods, also Hollrigel et al. 1996) allows for quantification of the equilibrium effects of GABA modulators on IPSCs in the RTN cell population, at the expense of within cell comparisons. Recordings made after a 1-h preincubation with either clonazepam or loreclezole resulted in IPSC durations that were increased above control levels to values similar to, but slightly above, those obtained after 8–10 min of bath application (Fig. 2, on the right of the graph). This result suggests that single cell pharmacology can be reasonably approximated with prolonged bath application and wash-out, but that equilibrium effects can be better assessed by comparing results between slices preincubated for long periods in drug versus vehicle containing solutions.
The effects of clonazepam on IPSCs in the RTN
To test whether the two types of IPSCs might arise from synapses with different GABAA receptor subunits, we examined the effects of the benzodiazepine clonazepam. GABAA receptors containing the α3 subunit are modulated to a greater degree than those containing other α subunits (Pritchett et al. 1989; Puia et al. 1991), and the α3 subunit is preferentially expressed in the RTN (Huntsman et al. 1996). RTN cells exposed to 100 nm clonazepam by either bath application (n= 6) or preincubation (n= 11) exhibited a significant increase in both the duration and amplitude of IPSCs (Fig. 3). This observation is exemplified in Fig. 3A–C for a cell with a 10 min bath application of 100 nm clonazepam. After application of clonazepam, the entire population of IPSCs (both fast and slow events) shifted toward slower events (Fig. 3B). Both the fast and slow decay components of ensemble averaged IPSCs were prolonged (Fig. 3D; control: n= 50 RTN cells, τ1= 17.1 ± 0.5 ms, τ2= 86.1 ± 2.9 ms; clonazepam: n= 17, τ1= 26.3 ± 1.3 ms, P < 0.0001, τ2= 134.5 ± 7.7 ms, P < 0.0001). The average weighted time constant was also significantly increased (e.g. Fig. 3C) from 46.0 ± 1.9 ms to 94.5 ± 5.7 ms (P < 0.0001). In contrast with the slowing effects of clonazepam on each of the individual time constants of decay, only the slowly decaying component was significantly increased in amplitude (Fig. 3D; control A1: 19.8 ± 1.5 pA, n= 50, versus clonazepam A1: 23.8 ± 3.1 pA; n= 17, P > 0.2; control A2: 14.3 ± 1.1 pA versus clonazepam A2: 39.3 ± 2.8 pA, P < 0.0001).
The prolongation of all (both slow and fast) IPSCs by clonazepam suggests that both types of events are mediated by benzodiazepine-sensitive GABAA receptors, while the selective increase in amplitude of the slowly decaying component suggests that the receptors underlying this part of the IPSC may not be fully saturated by synaptically released GABA (Otis & Mody, 1992; Hájos et al. 2000).
The effects of loreclezole on IPSCs in the RTN
In contrast to clonazepam, loreclezole targets regulatory sites on GABAA receptors composed of β2 or β3 subunits (Wafford et al. 1994; Wingrove et al. 1994). RTN neurons express only β1 and β3 subunits but lack β2 subunits (Huntsman et al. 1996; Pirker et al. 2000). As with the clonazepam experiments, we examined the effects of loreclezole on IPSCs in RTN by either bath application or preincubation. By contrast with clonazepam, the effects of loreclezole were more selective, producing mainly an increase in IPSC duration, as can be seen from the inset histograms of Fig. 4A. Cumulative probability histograms of 90%-width suggested that the slowing of individual IPSCs was largely restricted to events that were initially of long duration – fast events were unchanged (Fig. 4B). In ensemble averaged IPSCs, loreclezole selectively prolonged the slow phase of decay while leaving the fast component completely unaffected (Fig. 4C and D).
Neither fast decay constant (τ1) nor amplitude (A1) was altered by loreclezole (Fig. 4D, control τ1: 17.1 ± 0.5 ms, n= 50 versus loreclezole τ1: 16.9 ± 1.0 ms, n= 18, P > 0.9; control A1: 19.8 ± 1.5 pA, n= 50 versus loreclezole A1: 18.7 ± 2.0 pA, n= 18, P > 0.7). In contrast, the slow time constant of decay (τ2) was more than doubled by loreclezole (control τ2: = 86.1 ± 2.9 ms versus loreclezole τ2: 189.5 ± 15.4 ms, n= 14, P < 0.0001). The amplitude of the slow decay component was also increased by loreclezole (control A2: 14.4 ± 1.1 pA versus loreclezole A2: 34.1 ± 3.2 pA, P < 0.0001). sIPSC weighted time constant (τD,W) was increased nearly threefold by loreclezole (control τD,W: 46.0 ± 1.9 ms versus loreclezole τD,W: 130.1 ± 10.7 ms, P < 0.0001). In neurons with prominent biphasic individual IPSCs (as in Fig. 1B, **), such events were observed even after loreclezole treatment (and in preincubated slices) suggesting that loreclezole selectively enhances the GABA responsiveness at a subpopulation of GABAA receptors within an individual synapse. To test whether fast decay might be affected by loreclezole in at least a subpopulation of neurons, we made scatter plots of τ1versus A1 in individual cells and found instead that the distributions of these two parameters were essentially the same for control and loreclezole (Fig. 5A and B). This suggests that in no cell was the fast decay process augmented by loreclezole.
Overall, these loreclezole results suggest that fast IPSC decay in RTN cells is mediated by loreclezole-insensitive GABAA receptors, presumably those lacking either β2 or β3 subunits (Wingrove et al. 1994; see Discussion).
RTN neurons with fast decay kinetics express β1 subunit mRNA
In order to determine the molecular basis of IPSC heterogeneity in the RTN, we used single-cell RT-PCR to identify the presence of GABAA receptor subunit mRNAs. We chose a simple, single message, RT-PCR approach for these experiments, and thus we did not require degenerate primers or compatible multiple primers – reagents that have been used in previous studies utilizing multiplex PCR. Both of these approaches, through competition, reduce primer specificity and the ability to successfully harvest and amplify mRNA from a single cell. The combined message for all ion channel genes is considered to comprise between 0.1 and 1% of the total RNA in a single neuron (Sucher et al. 2000). Thus the reduced primer specificity required for multiplex PCR would pose an especially difficult problem for studies of RTN neurons in which GABAA receptor transcripts in general are extremely low in abundance (Wisden et al. 1992; Huntsman et al. 1996). While we could not rule out false negatives, we hoped to increase the probability of amplifying β1 subunit message by using specific primers in two rounds of simplex RT-PCR reactions using nested primers (see Methods).
Examples of two RTN neurons for which we were able to obtain both physiological recordings and single-cell RT-PCR are illustrated in Fig. 6. These two cells displayed strikingly different proportions of fast and large events, as can be seen in scattergrams of IPSC decay (90%-width) versus amplitude for each neuron (Fig. 6A and B). The cell in Fig. 6B is dominated by IPSCs with fast decay times and large amplitudes, while the cell in Fig. 6A has a much higher proportion of small and slow IPSCs. Ensemble averaged IPSCs for each cell were best fitted by the sum of two exponential functions (Fig. 6C). The main difference between these two neurons was in the first of two decay components, with a larger amplitude (A1) and shorter time constant (τ1) for the ensemble averaged IPSC from the fast cell in Fig. 6B.
Single cell amplification of β1 subunit mRNA was carried out after whole-cell patch-clamp recordings. Reverse transcription and two rounds of PCR amplification were always carried out in the presence of negative (water, pipette solution, lanes 1 and 2, Fig. 6D) and positive controls (40 pg of total RNA, lane 8 Fig. 6D). All cells were processed on the same day of recording, and therefore if negative and positive controls failed, then all cells were eliminated for that day of experiments. Figure 6D shows an example of an ethidium bromide stained polyacrylamide gel with 10 μl of PCR product obtained from cells (lanes 3 through 7) recorded on the same day as the two examples in Fig. 6A (lane 3) and 6B (lane 7). Lanes 4, 5 and 6 were from three other RTN neurons. All β1 positive cells were distinguished by a solitary, 162 bp cDNA product, as predicted by the inner primers of the nested pair. The remainder of the reaction product was sequenced and identified as rodent β1 subunit cDNA by a BLAST (basic local alignment search tool) search in the National Center for Biotechnology Information database.
β1 positive neurons were distinguished from β1 negative neurons by faster decay time constants, especially in the fast component of double exponential fitted IPSCs. (Fig. 7). Neurons positive for β1 mRNA had faster first component time constants (Fig. 7B, τ1: 14.8 ± 0.7 ms, n= 7) compared to β1 negative cells (τ1: 17.6 ± 0.8 ms, n= 15, P < 0.05), while amplitudes for the early decay component (A1) were twice as large in β1 positive (A1: 19.9 ± 1.6 pA, n= 7) than in negative cells (A1: 9.8 ± 1.0 pA, n= 15, P < 0.0001). In β1 positive cells the mean amplitude of first decay component (A1) was greater than second component (A2) by almost twofold (A1: 19.9 ± 1.6 pA versus A2: 10.4 ± 3.7 pA, n= 7, P < 0.05), while in β1 negative cells the two components were of approximately equal weight (A1: 9.8 ± 1.0 pA versus A2: 9.4 ± 1.1 pA, n= 17, P= 0.8). Parameters describing the slow decay component were not different between β1 positive and negative cells (Fig. 7A). Overall, the faster and larger fast decay component resulted in a mean weighted time constant (τD,W) significantly smaller in β1 positive cells (τD,W: 35.3 ± 3.2, n= 7) than in negative cells (τD,W: 48.4 ± 2.6 ms, n= 15, P < 0.01). The limited reduction in the slow component of decay indicates that the significant reductions in τD,W were driven by the fast component.
Scatterplots of fast versus slow decay components (Fig. 7B) show two groups of cells separated by both fast decay times and expression for the β1 subunit. We then compared these populations to those predicted from the cluster analysis shown in Fig. 1. We found that allβ1 positive cells belonged to the fast cluster (cluster 1), but that β1 negative cells were distributed into both clusters. This latter result suggests the presence of false negatives in the RT-PCR results. If this is the case, then the population data shown in Fig. 7A may underestimate the kinetic differences between β1 containing and β1 deficient neurons.
In the present study, we used a combination of GABAA receptor subunit-selective pharmacology and single-cell RT-PCR in order to classify RTN neurons with differences in IPSC decay kinetics. We report three principle findings. First, under control conditions, IPSCs recorded from neurons in the RTN have heterogeneous decay properties, with individual neurons exhibiting variable mixtures of rapidly and slowly decaying IPSCs. Cells dominated by faster IPSCs also exhibited large amplitude and/or biphasic events during the recording. Second, we found that two GABA modulating anticonvulsant drugs had differential effects on the degree of IPSC heterogeneity in RTN neurons. The first drug, clonazepam, increased the decay kinetics and overall amplitude, and cumulative probability plots of IPSC durations indicated that all events were prolonged. By contrast, during loreclezole exposure, IPSCs were prolonged but the effect was specific to the second exponential decay component, leaving the first unaffected. Thus brief events with little contribution from the slow component were insensitive (Fig. 4B). Third, by using single-cell RT-PCR we found that RTN neurons with large and rapidly decaying IPSCs were much more likely to express β1 subunit mRNA than those cells with mainly slowly decaying IPSCs.
Heterogeneity of IPSC properties is determined by β1 subunit expression
To narrow down this pool we selected two antiepileptic drugs that target different sites on the GABAA receptor. The benzodiazepine clonazepam was chosen because α subunits determine benzodiazepine responsiveness (Pritchett et al. 1989). In the RTN, the likely target is the α3 subunit. This is based on a combination of studies that examine subunit localization. While it is conceivable that another α subunit (possibly α1) may contribute to the fast component, this can be eliminated if one takes into account a number of factors. First, two studies (one immunocytochemical and one of in situ mRNA localization) report that α3 is the only α subunit identified in the RTN (excluding the cerebellar α6 subunit; Fritschy & Möhler, 1995; Huntsman et al. 1996). Second, two other studies indicate that α3 is the primary α subunit with lower levels of α1 mRNA and protein (Pirker et al. 2000).
Additional data obtained with GABAA receptor mutant mice provide further support for a primary, if not exclusive, role for α3 subunits in RTN synaptic GABAA receptors. Recordings from RTN cells in a mouse strain in which the diazepam binding site on the α3 subunit gene has been inactivated by a point mutation (α3H126R, Low et al. 2000) demonstrated that clonazepam increased the duration of GABAA receptor-mediated IPSCs in wild-type but not α3H126R RTN cells (Porcello et al. 2001). By contrast, the modulatory actions of clonazepam were completely intact in RTN cells of α1H101R mice (Huntsman et al. 2000), in which the α1 subunit was rendered benzodiazepine-insensitive by a single point mutation (Rudolph et al. 1999).
In the present study, application of clonazepam to thalamic slices increased the duration and amplitude of IPSCs; however, the degree of heterogeneity in the overall population of events, as measured by the curvature in the cumulative probability plots, was not greatly altered. This suggests that all IPSCs, both brief and long-lasting events, were modulated by clonazepam, and that the receptors underlying fast and slow decay were similarly sensitive, as might be expected if all such receptors contained two copies of the α3 subunit.
In contrast to clonazepam, loreclezole sensitivity is determined primarily by the β subunits, with β2- and β3-, but not β1-containing receptors being highly sensitive (Wafford et al. 1994; Wingrove et al. 1994). Because neurons in this nucleus completely lack β2 message (Huntsman et al. 1996), loreclezole effects should depend completely on the β3 subunit. The effect of loreclezole in RTN cells was an overall increase in the duration of IPSCs. Analysis of double exponential fits of ensemble averaged IPSCs revealed that this enhancement was achieved mainly through prolongation of the slowly decaying component leaving the fast component completely unaffected (Fig. 5B). This is perhaps the strongest evidence in this report for differential involvement of β subunits in fast versus slow decay components of RTN IPSCs. Notably, some IPSCs still showed a prominent fast decay component, even after several minutes of loreclezole exposure with bath application or preincubation, thus suggesting that two populations of GABAA receptors, β1 containing and β1 deficient, can coexist at individual synapses in RTN. While α subunits have been shown to regulate the duration of GABA responses (Gingrich et al. 1995; Verdoorn, 1994; Lavoie et al. 1997; Serafini et al. 1998), the role of β subunits in this context has not been fully studied. However, the β subunits have been shown to influence the rate of desensitization (Burgard et al. 1996). In that study, recombinant receptors with α5, β, and γ2L subunits had different desensitization rates (at hyperpolarized potentials) with the following rank order of desensitization rates: β2 > β1 > β3. Both β and α subunits make up the structural domain for the GABA binding site (Amin & Weis, 1993), and thus both may influence binding affinity. It is apparent that changes in GABA affinity coincide with the β subunit (Hadingham et al. 1993; Ebert et al. 1994; Ducic et al. 1995), with β3 containing receptors having a much higher GABA affinity than β1 containing receptors (Burgard et al. 1996). This may be important since it has been noted by Jones et al. (1998) that the binding of GABA is especially critical at low GABA concentrations and that ‘receptors that mediate fast IPSCs may be less efficient at binding GABA and thus have a lower occupancy than those underlying slower IPSCs’ (Jones et al. 1998). Further support for differential roles of β1 and β3 receptor subunits in mediating fast and slow IPSCs, respectively, comes from the finding that residual IPSCs in RTN cells of β3 knockout mice consist of a uniform population of rapidly decaying events (Huntsman et al. 1999), quite different from the heterogeneous distribution of IPSCs observed in wild-type mice. Furthermore, mIPSC durations are shortened in cortical neurons of mice devoid of β3 subunits (Ramadan et al. 2003). Even though these data suggest a direct role for β1 subunits in the regulation of IPSC duration, other factors that could also influence IPSC duration such as phosphorylation, release sites or receptor occupancy have not been explicitly studied here (Poisbeau et al. 1999; Hinkle & Macdonald, 2003).
GABAA receptor mRNA expression is extremely low in the RTN (Huntsman et al. 1996), thus creating a formidable technical challenge when attempting to successfully amplify specific subunit messages. The design of an experiment with single RT-PCR amplification of one product was crucial to the success of the study. In a previous study from this laboratory using multiplex PCR and nested primers (Browne et al. 2001), we were unable to detect message for any β subunits in RTN cells, even though both β1 and β3 have been previously detected in this nucleus (Wisden et al. 1992; Huntsman et al. 1996; Pirker et al. 2000). Our results are consistent with occasional false negatives (Fig. 7B) whose incidence may be higher than that expected from other single cell RT-PCR studies (Brorson et al. 1999; van Hooft et al. 2000). Even with these putative false negatives included in the β1-negative pool, the two cell populations exhibited significantly different IPSC decay kinetics (Fig. 7A), supporting the robustness of the result.
Functional heterogeneity requires minimal variation of GABAA receptor subtypes
Basis and function of heterogeneous IPSCs in the RTN
Variability in RTN neuron IPSCs would have important physiological consequences for both intra-thalamic and thalamocortical interactions. Intra-RTN inhibition mainly relies on slowly decaying GABAA receptor mediated IPSCs. The majority of inhibitory synapses in rodent RTN arise from sparse axon collaterals of other RTN neurons (Scheibel & Scheibel, 1966; Deschênes et al. 1985; Yen et al. 1985; Cox et al. 1996; Pinault et al. 1997; Steriade et al. 1997). There is also evidence for extrinsic GABAergic input to the RTN – mainly arising from the globus pallidus, basal forebrain, and the substantia nigra pars reticulata (Asanuma & Porter, 1990; Paréet al. 1990; Hazrati & Parent, 1991; Asanuma, 1994). However, pathway specific inhibition is not likely to contribute to heterogeneous IPSC decay rates. For example, a higher proportion of rapidly decaying IPSCs might arise from extrinsic versus intrinsic inhibitory connections). Extrinsic GABAergic projections typically make up a minor proportion of synaptic contacts in comparison to the connections arising within the RTN and are often segregated and located in different sectors of the nucleus (Paréet al. 1990; Steriade et al. 1997). We found that neurons with heterogeneous IPSCs occurred throughout the RTN and were not located in a defined location.
The difference in IPSC duration (measured by τD,W at room temperature) between β1 positive and β1 negative neurons is roughly 13 ms (this is all cells examined with single cell PCR, including the presumed false negatives). Why might a 13-ms difference make a difference? In the hippocampus, the precise timing of fast GABAA receptor mediated IPSCs is required for the synchronization of neuronal networks (Buzáki et al. 1983; Soltesz & Deschênes, 1993; Whittington et al. 1995). Dual modelling and physiological results indicate that the generation of oscillatory networks depends on a narrow range of (6–12 ms) IPSC decay (Whittington et al. 1995). The synapses between inhibitory neurons in particular, appear to require a fast conductance change (mediated by fast decay and large amplitude IPSCs) for the generation of synchronized network activity in the gamma frequency range (Bartos et al. 2001). A large initial amplitude and fast decay is also a feature of β1 positive neurons in the RTN and may play a role in the observed voltage-dependent 40-Hz oscillations in this nucleus (Pinault & Deschênes, 1992). Target specific expression of GABAA receptor subunits is an emerging characteristic feature in inhibitory networks (Nyiri et al. 2001; Klausberger et al. 2002; Bacci et al. 2003).
The authors thank Matthew V. Jones for careful reading of a preliminary version of this manuscript. This study was supported by NIH grants NS0647, NS072280, NS34774, NS10768-01, and the Pimley Research Fund.
Author's present address
M. M. Huntsman: Department of Pharmacology, Georgetown University, Washington, DC 20057, USA.