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Distinctive Glycinergic Currents With Fast and Slow Kinetics in Thalamus

¹Department of Anesthesiology, Pharmacology, and Therapeutics and ²Department of Cellular and Physiological Sciences, The University of British Columbia, Vancouver, British Columbia, Canada

Submitted 18 November 2005; accepted in final form 15 March 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We examined functional properties of inhibitory postsynaptic currents (IPSCs) evoked by medial lemniscal stimulation, spontaneous IPSCs (sIPSCs), and single-channel, extrasynaptic currents evoked by glycine receptor agonists or -aminobutyric acid (GABA) in rat ventrobasal thalamus. We identified synaptic currents by reversal at E_Cl and sensitivity to elimination by strychnine, GABA_A antagonists, or combined application. Glycinergic IPSCs featured short (about 12 ms) and long (about 80 ms) decay time constants. These fast and slow IPSCs occurred separately with monoexponential decays, or together with biexponential decay kinetics. Glycinergic sIPSCs decayed monoexponentially with time constants, matching fast and slow IPSCs. These findings were consistent with synaptic responses generated by two populations of glycine receptors, localized under different nerve terminals. Glycine, taurine, or -alanine applied to excised membrane patches evoked short- and long-duration current bursts. Extrasynaptic burst durations resembled fast and slow IPSC time constants. The single, intermediate time constant (about 22 ms) of GABA_Aergic IPSCs cotransmitted with glycinergic IPSCs approximated the burst duration of extrasynaptic GABA_A channels. We noted differences between synaptic and extrasynaptic receptors. Endogenously activated glycine and GABA_A receptor channels had higher Cl^– permeability than that of their extrasynaptic counterparts. The -amino acids activated long-duration bursts at extrasynaptic glycine receptors, consistent with a role in detection of ambient taurine or -alanine. Heterogenous kinetics and permeabilities implicate molecular and functional diversity in thalamic glycine receptors. Fast, intermediate, and slow inhibitory postsynaptic potential decays, mostly attributed to cotransmission by glycinergic and GABAergic pathways, allow for discriminative modulation and integration with voltage-dependent currents in ventrobasal neurons.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Since the discovery of glycinergic transmission in the spinal cord, several studies have demonstrated that glycine mediates neurotransmission above the level of the cord (Chery and De Koninck 1999; Donato and Nistri 2000; Dumoulin et al. 2001; Fatima-Shad and Barry 1998). Recently, glycinergic inhibition has become recognized in the ventrobasal thalamus (Ghavanini et al. 2005; cf. Zeilhofer et al. 2005). Medial lemniscal stimulation produced inhibitory postsynaptic potentials (IPSPs) mediated only in part by the prevalent inhibitory transmitter, -aminobutyric acid (GABA; Steriade et al. 1997). A combination of strychnine with a GABA_A antagonist completely blocked this inhibition (Ghavanini et al. 2005). Unexpectedly, glycinergic or GABAergic IPSPs occurred independently in many neurons, which tended to favor cotransmitting pathways rather than corelease of these amino acids.

Glycine receptors are pentameric complexes containing pore-forming subunits, with or without accessory subunits (Lynch 2004). Heteromeric (/) receptors localize to the synaptic membrane (cf. Lynch 2004), consistent with the punctate subunit staining in the thalamus (Ghavanini et al. 2005). In the caudal CNS, glycinergic inhibitory postsynaptic currents (IPSCs) exhibit diverse decay kinetics that correlate to receptor subunit expression. In brain stem and spinal neurons, glycine receptors with ₁ or ₂ subunits have different kinetic properties (Singer et al. 1998; Takahashi et al. 1992). The ₁ subunit predominance in the adult rat bestows fast decay rate for IPSCs. The ₂ subunit confers slow IPSC decay, often seen in developing neurons (Ali et al. 2000; Takahashi et al. 1992). Thus the first objective of the present study was to examine glycinergic IPSC decay in ventrobasal neurons of the juvenile rat for evidence of kinetic heterogeneity. We also searched for evidence of biphasic decays in spontaneous IPSCs (sIPSCs) sensitive to complete blockade by strychnine with GABA_A antagonists, indicative of corelease from glycinergic and GABAergic pathways (cf. Dumoulin et al. 2001).

Previously, we observed strychnine antagonism of responses to exogenous glycine agonists and diffuse staining for glycine receptor ₁ and ₂ subunits (Ghavanini et al. 2005). These observations were consistent with extrasynaptic receptor populations. However, functional receptors on extrasynaptic membranes of thalamocortical neurons would require direct demonstration. The second objective of the present studies was to determine whether extrasynaptic glycine receptors existed and exhibited the expected kinetic diversity.

For the thalamus as elsewhere, it is not known whether extrasynaptic glycine receptors exhibit differences from synaptic glycine channels. In GABAergic pathways, extrasynaptic GABA_A receptor channels exhibit lower Cl^– conductance than synaptic channels (Yeung et al. 2003). Using fluctuation analysis on evoked and spontaneous IPSCs, our third objective was to compare the conductance properties of synaptic and extrasynaptic receptor channels. These studies delineate some unusual facets of glycine receptors and inhibitory transmission in the thalamus.

	METHODS

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Tissue slice preparation

The experimental procedures received approval by the Animal Care Committee of University of British Columbia. Sprague–Dawley rats (13- to 15-day-old) were decapitated while under deep halothane anesthesia. The brain was rapidly removed and submerged in oxygenated solution at 4°C containing (in mM): 26 NaHCO₃; 1.25 NaH₂PO₄; 2.5 KCl; 2 MgCl₂; 2 CaCl₂; 25 dextrose; and 250 sucrose. The solution had an osmolality of 330 mOsmol. The brains were dissected into two blocks. Using a Vibroslicer (Campden Instruments, London, UK), the block was sectioned into 250- to 300-µm-thick sagittal slices, showing landmarks of the scaphoid nucleus and medial lemniscus (Paxinos and Watson 1986). The slices were incubated for >3 h in artificial cerebrospinal fluid (aCSF) at room temperature (23–25°C), saturated with 95% O₂-5% CO₂. The aCSF contained (in mM): 124 NaCl; 26 NaHCO₃; 1.25 NaH₂PO₄; 4 KCl; 2 MgCl₂; 2 CaCl₂; and 10 dextrose. The aCSF had a pH of 7.3–7.4 and an osmolality of 305 mOsmol.

IPSC recording

For recording, the slices were placed in a Perspex recording chamber (approximately 2 ml volume) and were immobilized with a polypropylene mesh. They were perfused with oxygenated aCSF at room temperature, at a rate of 1.5–2 ml/min. Ventrobasal neurons were identified under a differential interference contrast microscope at x400 (Axioskop 2, Carl Zeiss, Oberkochen, Germany). Recording microelectrodes were made using a Narishige puller from thin-wall borosilicate glass tubing (World Precision Instruments, Sarasota, FL), and filled with a solution containing (in mM): 133 K-gluconate; 12 KCl; 4 NaCl; 0.5 CaCl₂; 10 EGTA; 3 Mg-ATP; 0.3 Na₂-GTP; 2.7 Na₂-phosphocreatine, and 10 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES). The pH was adjusted to 7.3–7.4. The calculated Nernst potentials for this normal patch solution were –53 mV for Cl^– (E_Cl) and –84 mV for K⁺ (E_K). Electrode resistances ranged between 4 and 5 M.

Whole cell recording of IPSCs was performed using amplifiers (Axoclamp 2A, Axon Instruments, Foster City, CA; and List EPC-7, HEKA, Lambrecht, Germany) in the current- and voltage-clamp modes. The neurons were voltage clamped at V_h = –80 mV to minimize contributions of infrequently occurring GABA_B currents. As previously (Ghavanini et al. 2005), ionotropic glutamatergic currents were blocked with kynurenate (1 mM). In 14 neurons, Cs⁺ (145 mM) and QX-314 (3 mM) were applied intracellularly to suppress K⁺ and Na⁺ currents, and Ni²⁺ (500 µM) was applied extracellularly to block T-type Ca²⁺ currents. In such experiments, the [Cl^–] was adjusted such that E_Cl was 0 mV. Signals, filtered at 3 kHz and digitized at 10 kHz with a 16-bit data acquisition system, were analyzed using pClamp software (Axon Instruments).

IPSCs were evoked by stimulation at <0.5 Hz with a bipolar electrode placed in the medial lemniscus outside the thalamus, at 1–2 mm from the ventrobasal nuclei. The resistance of the stimulating electrode was 5.5 M. We activated glycine receptors by electrical stimulation of the medial lemniscus, evoking IPSCs during ionotropic glutamate receptor blockade. The stimulus intensity was adjusted to evoke stable amplitude responses, without failures. The stimulus parameters were a rheobasic current of 3.9 ± 0.9 µA and a chronaxie of 245 ± 37 µs (n = 21).

Spontaneous IPSCs were recorded during intracellular application of Cs⁺ and QX-314 in neurons that were voltage clamped at –60 mV. Single sIPSCs were visually selected for averaging and creation of search templates. We used the sliding-template procedure of pClamp software, setting the template match stringency to a medium level. Given the observed variability of sIPSC time courses, multiple template searches were required for precise detection of all sIPSCs. The events were monitored visually during the entire procedure, for rejection of sIPSPs with more than a single peak and noise.

We used pClamp, Prism GraphPad, and CorelDraw software for analysis. Exponential functions were fitted to the decay phase of single sIPSCs, and evoked IPSCs averaged from five to ten individual currents. The double-exponential function was the sum of two terms, A₁ · exp(–t/₁) + A₂ · exp(–t/₂), where A₁ and A₂ were the amplitudes with time constants ₁ and ₂, respectively.

Nonstationary noise analysis

We subjected the IPSCs to nonstationary fluctuation analysis to reveal the properties of synaptic channels activated by endogenous transmitters (De Koninck and Mody 1994; Traynelis et al. 1993). We confirmed the stability of quantal release in the IPSCs before subjecting them to nonstationary noise analysis. The amplitude of evoked IPSCs did not significantly change with the stimulus number, which implied no change in quantal release at stimulation frequencies <0.5 Hz. We grouped three successive responses and calculated the coefficients of variation for each triplet. The coefficients of variation did not change with triplet number, confirming stable quantal release (cf. Scheuss and Neher 2001).

We averaged ten successive IPSCs, after aligning their peaks in time. Starting at the IPSC peak, the decays were binned at 1.5 ms. I_mean(t), or the average current of each bin (t), was calculated from the relationship, I_mean(t) = I_(t)j/n, where I_(t)j was the current amplitude for trial j for bin t, and n was the number of trials.

The variance (²) of each bin was calculated from the difference between the scaled average evoked or spontaneous IPSCs, and the individual currents (cf. Traynelis et al. 1993). Using a least-squares algorithm, the resulting plot was fitted with a quadratic function, _(t)² = i_Cl · I_mean(t) – I_mean(t)²/N + _th(t)², where _th(t)² denoted residual noise and i_Cl was the elementary current through the agonist-gated channel. The parameter N (total number of channels at the synaptic site) was not considered further because scaling the average IPSC to individual IPSC increases the accuracy of i_Cl but decreases the accuracy of N (Traynelis et al. 1993). We also obtained i_Cl as the slope of the initial part of the variance-to-mean current relationship, fitted by linear regression. The results of the two estimates were in good agreement.

The channel Cl^– permeability (P_Cl) was calculated from the Goldman–Hodgkin–Katz (GHK) constant-field relationship, P_Cl = i_Cl · (RT/VF²) · {(1 – e^VF/RT)/([Cl^–]_i – [Cl^–]_o · e^VF/RT)}, where R, T, and F had their usual meanings, V was membrane potential, and [Cl^–]_i and [Cl^–]_o were the intracellular and extracellular Cl^– concentrations, respectively. We applied a similar procedure to calculate the P_Cl from single-channel currents.

Dissociated neuron preparation

For single-channel recording, acutely dissociated neurons were prepared from horizontal slices containing the ventrobasal complex. The slices were initially incubated at room temperature for 10 min in oxygenated, Ca²⁺-free media composed of (in mM): 120 NaCl; 5 KCl; 1 MgCl₂; 5 D-glucose; 20 1,4 piperazine-bis-(2-ethanesulfonic acid) (PIPES); ethylene-glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), and 2 mg/ml bovine serum albumin (BSA) at pH = 7.3. The tissue was then stirred at 32°C for 45 min in a solution of composition (in mM): 120 NaCl; 5 KCl; 1 MgCl₂; 1 CaCl₂; 20 PIPES; 2 mg/ml BSA; and 14 units/ml papain at pH = 7.0. The tissue was rinsed and left for 15 min at room temperature. The cells were mechanically dispersed in 2 ml of Ca²⁺- and BSA-free PIPES solution and plated on uncoated 35-mm tissue-culture dishes. The cells remained in PIPES buffered solution at room temperature until needed for recording.

Single-channel recording and data analysis

We recorded single-channel currents at room temperature (Kim et al. 2004). Dispersed ventrobasal neurons were bathed in a saline containing (in mM): 4 KCl; 135 NaCl; 10 CaCl₂; 1 MgCl₂; 10 HEPES; and, 5 D-glucose (pH 7.3). Patch pipettes (10–15 M) contained a solution (pH 7.3) composed of (in mM): 135 CsCl; 1 MgCl₂; 0.267 CaCl₂; 10 HEPES; 3 EGTA; and 5 D-glucose. E_Cl was 0 mV in these recordings.

Outside-out membrane patches were voltage clamped with a List EPC-7 amplifier at a holding potential, V_h = –60 mV. Amino acids were applied by exchange and perfusion in bath. Responses to agonists reached a steady state within 30 s of switching from control to agonist solutions. The duration of each application was about 5 min. The currents were filtered at DC to 1 kHz, digitized (8 kHz), and analyzed off-line with commercial software (Instrutech, Port Washington, NY). Single-channel openings were detected as transients exceeding 50% of the difference between the averaged baseline and open channel currents, disregarding events briefer than 180 µs. Channel open probability, P_o, was calculated as P_o = (T₁ + 2T₂ +... + NT_N)/T_tot · N, where N was the number of channels in the patch, T_tot the total duration of the record, and T₁ + 2T₂ +... + NT_N were the times when 1, 2, ..., N channels were open.

Distributions of open channel times were fitted by a triexponential function. Closed time distributions were fitted by four exponential terms. Exponential fitting was performed using Simplex maximization of likelihood. We defined groups of openings as bursts, provided that the openings were separated by gaps shorter than t_c, a specified critical time. We calculated t_c by solving 1 – exp(–t_c/_c3) = exp(–t_c/_c2). Here, _c2 and _c3 were the time constants of the second and third fastest components in closed time distributions (Colquhoun and Sakmann 1985; Twyman and Macdonald 1991), as appropriate for a DC to 1 kHz recording bandwidth. Using Simplex methods, we fitted one or two Gaussian terms to the amplitude distributions of single-channel currents. Mean channel conductance was calculated as the weighted sum of the Gaussian-fit components.

Chemicals and drugs

All chemicals, including glycine, -alanine, taurine, strychnine, GABA, bicuculline methiodide, gabazine, kynurenate, and QX-314 were purchased from Sigma Chemical (St. Louis, MO). Drugs were applied either by perfusion of slices or to the external face of outside-out membrane patches.

Statistics

Using bootstrap methods, we estimated the 95% confidence interval for parabolic fits to variance-to-mean current relationships. Data are expressed as means ± SE and n denotes number of neurons or patches. The Kolmogorov–Smirnov (KS) test was used to assess goodness-of-fit to normal and Gaussian distributions. For normally distributed data, we used ANOVA for multiple comparisons, and a Tukey post hoc test for comparing group pairs. Student's t-test was used for comparing two groups. For nonnormally distributed data, Wilcoxon and Kruskal–Wallis tests were used for comparing two groups or for multiple comparisons, respectively. Significance was defined as P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Characteristics of IPSCs

We studied IPSCs evoked by medial lemniscal stimulation in 21 neurons, recorded with the normal solution in the patch electrode. Complete blockade of IPSCs in 14 neurons required coapplication of strychnine (1 µM) with a GABA_A antagonist, either bicuculline (25 µM) or gabazine (10 µM; Fig. 1A). The IPSCs in seven remaining neurons showed exclusive sensitivity to either strychnine or GABA_A antagonist. Strychnine eliminated the IPSCs in three neurons, unaffected by prior bicuculline application. Bicuculline or gabazine eliminated IPSCs in four neurons, unaffected by strychnine. We refer to currents requiring both strychnine and GABA_A antagonists for elimination, as "mixed IPSCs."

View larger version (21K): [in this window] [in a new window]   FIG. 1. Medial lemniscal mixed inhibitory postsynaptic currents (IPSCs) resolved into glycinergic and GABAAergic currents reversed at ECl. A, top: 10 µM gabazine slightly reduced control IPSC, yielding a large glycinergic current that was eliminated by coapplied gabazine and 1 µM strychnine (Gbz and Str). Subtraction of current during gabazine from control IPSC yielded a small GABAAergic current. A, bottom: 10 µM gabazine greatly suppressed control IPSC in a second neuron, yielding a small glycinergic current eliminated by coapplied gabazine and 1 µM strychnine. Subtraction of current during gabazine from control IPSC yielded a large GABAAergic current. Currents were averaged from 10 IPSCs. B: reversal potentials for glycinergic (11 neurons) and GABAAergic (14 neurons) components of mixed IPSCs were identical to ECl (arrows). Mean peak values of glycinergic and GABAAergic components did not differ at Vh = –80 mV (ANOVA, P > 0.05).

Decay kinetics of IPSCs

Glycinergic currents often displayed more complex decay kinetics than the GABA_Aergic currents, all of which decayed monoexponentially with a normal distribution of constants (Fig. 2, A and C). Eleven of 17 glycinergic IPSCs exhibited a monoexponential decay (cf. Fig. 2A). The decay time constants for these IPSCs were not well fitted by a normal distribution (P < 0.05, KS test), and likely represented two populations. One population had a short decay time constant, _str(short) = 10 ± 1.4 ms (n = 8), whereas the other had a long decay time constant, _str(long) = 70 ± 4.0 ms in three neurons (ANOVA, P < 0.01). These values remained stable over a period of 1.5 h, indicating stationarity of decay kinetics.

View larger version (29K): [in this window] [in a new window]   FIG. 2. Resolved currents had different decays. A: glycinergic and GABAAergic currents were peak aligned and scaled to the same amplitude for comparison of time courses in different neurons. Glycinergic current decayed faster in the neuron of Aa, and slower in the neuron of Ab, when compared with the GABAAergic currents. B: decay phase of the glycinergic current in another neuron was well fitted by the sum of 2 exponential terms (top trace, smooth curve). Bottom trace shows these terms and their time constants. C: frequency distribution histograms of decay time constants for glycinergic and GABAAergic currents. Arrowheads indicate mean values. Vh = –80 mV, ECl = –53 mV. A and B show averages of 10 IPSCs.

Decay kinetics of spontaneous IPSCs

We studied spontaneous IPSCs in seven neurons, recorded with Cs⁺ and QX-314 in the patch electrode. We observed spontaneous inward currents of small amplitude in all neurons. These events occurred at an average frequency of 3.8 ± 0.9 Hz (cf. Fig. 3A). These events reversed at a V_h near E_Cl. There was no correlation between the amplitude, rise time, and decay time constant of the events (R² < 0.01). Thus the events likely represented genuine sIPSCs rather than random noise.

View larger version (30K): [in this window] [in a new window]   FIG. 3. Spontaneous IPSCs (sIPSCs) recorded with Cs+ and QX-314 in patch electrode. A: sample records of about 1-min duration show currents from neuron in control, strychnine (Str, 1 µM), as well as strychnine and gabazine (Gbz, 10 µM) conditions. Strychnine decreased frequency of sIPSCs. Coapplication of Str and Gbz abolished the sIPSCs. B: frequency distribution of decay time constants from 7 neurons. Before strychnine application, the distribution was well described with sum of 3 Gaussian functions with mean time constants (arrowheads) of 11 ± 0.1, 22 ± 0.1, and 74 ± 2.4 ms. Strychnine abolished fast and slow sIPSCs, but not the sIPSCs, fitted with a single intermediate mean time constant (arrowhead) of 22 ± 0.2 ms. C: examples of unaveraged sIPSCs, scaled to the same amplitude, show fast, intermediate, and slow decay time courses. Smooth curves represent single exponential fits to the peak-aligned sIPSCs (indicated decay time constant, ). Slow sIPSC that had a longer decay time constant than the average time constant of the slow sIPSC (cf. D) was selected for illustrative clarity. D: mean decay time constants for glycinergic sIPSCs did not differ from evoked IPSCs (P > 0.05, ANOVA). Numbers of neurons are indicated in parentheses. For sIPSCs, Vh = –60 mV and ECl = 0 mV.

After we aligned the peaks and scaled the sIPSCs to the same amplitude, three distinct time courses were evident in all neurons (cf. Fig. 3C). Fast sIPSCs completely decayed within 100 ms, whereas intermediate sIPSCs required 100 to 200 ms and slow sIPSCs required 500 to 1,000 ms for complete decay. The majority of sIPSCs had a decay phase that was well fitted with a single exponential function (Fig. 3C). A biexponential function was required for an appropriate fit in <6% of sIPSCs. The mean amplitude of biexponential sIPSCs (–40 ± 1.9 pA) was slightly higher than the mean for monoexponential IPSCs (–32 ± 0.5 pA) IPSCs (P < 0.05, unpaired t-test). We observed a higher percentage of biexponential decays in IPSCs evoked by medial lemniscal stimulation (nearly 24%) of neurons displaying mixed IPSCs than in sIPSCs (<6%). The reasons for the higher percentage are unclear. One possibility is that electrical stimulation coactivated different nerve fibers, producing more biphasic responses.

Figure 3C illustrates the frequency distribution of decay time constants for the sIPSCs. Before strychnine application, the distribution was well described with the sum of three Gaussian functions (P < 0.05, KS test). This fit implied three populations of sIPSCs. Strychnine abolished the fastest and the slowest sIPSCs with average time constants of 11 ± 0.1 and 74 ± 2.4 ms, but not the GABA_Aergic sIPSCs with an average time constant of 22 ± 0.2 ms (Fig. 3B). Thus glycinergic sIPSCs had distinct fast and slow decay kinetics. The decay time constants of glycinergic and GABA_Aergic sIPSCs matched the respective time constants of evoked IPSCs (Fig. 3D, P > 0.05, ANOVA). Thus the three decay time constants of sIPSCs likely represent genuine findings and decay kinetics of synaptic receptors.

Kinetic properties of extrasynaptic receptors

Application of glycine, taurine, and -alanine induced inward, single-channel currents in membrane patches (Fig. 4A). Before agonist application, the outside-out patches infrequently displayed spontaneous currents at –60 mV. Glycine (20 µM) activated currents in 11 of 27 patches. In separate experiments, taurine (20 µM) activated currents in 12 of 28 patches, and -alanine (20 µM) activated currents in eight of 30 patches. The means of P_o for activations by glycine (0.029 ± 0.018, n = 11), taurine (0.029 ± 0.031, n = 10), and -alanine (0.042 ± 0.013, n = 7) were not different (P > 0.05, ANOVA). P_o had a tendency to decline during agonist application and thus we did not test more than one agonist on individual patches. When strychnine (1 µM) was coapplied with glycine, taurine, or -alanine, single-channel currents occurred very infrequently (overall P_o < 0.001). An observed reversal potential near E_Cl and sensitivity to strychnine implicated glycine receptors in the agonist-evoked currents.

View larger version (36K): [in this window] [in a new window]   FIG. 4. Glycine receptor agonists activate short- or long-duration bursts of channel openings. A: representative currents obtained from separate membrane patches. Channel openings appear as downward deflections from baseline (inward currents). Arrows indicate currents associated with a substate conductance. Inset under the long-duration bursts activated by -alanine shows a segment of the main trace with higher amplification and time base. Current levels indicative of closed channel (c), substate conductance (s, arrow), and full open conductance (o) states are indicated by dashed lines. B: distribution of closed times (n = 2,742 events) for glycine-activated currents from an additional patch showing fit by the sum of 4 exponential terms, with areas and time constants (arrowheads): A1 = 0.45, 1 = 0.2 ms; A2 = 0.28, 2 = 8.1 ms; A3 = 0.19, 3 = 19.8 ms; A4 = 0.08, 4 = 173 ms. C: distributions of burst durations for taurine-activated currents in 2 additional patches showing short- and long-duration bursts. Each distribution was well fitted by the sum of 3 exponentials with A1 = 0.03, 1 = 3.0 ms; A2 = 0.88, 2 = 20.1 ms; A3 = 0.09, 3 = 103 ms, c = 9.0 ms and mean duration = 26.7 ms for short-duration bursts (n = 552 bursts), and A1 = 0.42, 1 = 15.7 ms; A2 = 0.40, 2 = 84.7 ms; A3 = 0.18, 3 = 730 ms; c = 5.7 ms and mean duration = 172 ms for long-duration bursts (n = 506 bursts). Arrowheads indicate time constants. D: mean durations of short- and long-duration bursts activated by glycine receptor agonists. Numbers in parentheses indicate patches. Mean durations of short-duration bursts activated by taurine and -alanine were significantly different from long-duration bursts activated by the same agonist (*P < 0.05, ANOVA). Agonists were applied at 20 µM to outside-out patches. Vh = –60 mV, ECl = 0 mV.

The currents activated by the -amino acids displayed either short- or long-duration bursts (Fig. 4A). Glycine activated short-duration bursts in 10 of 11 patches (Fig. 4A), and long-duration bursts in only one patch. Taurine-activated currents were characterized by short-duration bursts of openings in six of ten patches and long-duration bursts in the remaining four patches. -Alanine activated short-duration bursts in four of seven patches and long-duration bursts in the remaining three patches.

As exemplified by taurine (Fig. 4C), burst-duration distributions were well described by the sum of three exponentials. The mean burst duration for glycine-activated channels was 19 ± 4 ms, whereas the sole long-duration burst averaged 87 ms. The taurine-activated short-duration bursts had a mean of 26 ± 4 ms, whereas long-duration bursts averaged 88 ± 8 ms. The -alanine–activated short-duration bursts had a mean of 21 ± 4 ms, whereas long-duration bursts averaged 137 ± 5 ms. The average lifetimes of short-duration bursts did not depend on the nature of the agonist (cf. Fig. 4D). The average lifetimes of long-duration bursts activated by taurine or -alanine did not differ (P > 0.05, ANOVA). For the -amino acids, short and long bursts differed significantly from each other in duration, and likely represented two populations (cf. Fig. 4D, P < 0.05, ANOVA).

Conductance of synaptic and extrasynaptic receptors

To estimate the Cl^– permeability of synaptic receptor channels for comparison with the extrasynaptic channels, the first step was to determine the elementary current, i_Cl, during synaptic activation. The variance-to-mean current relationships for both short- and long-duration glycinergic IPSCs were well described by a quadratic function (Fig. 5A). From these fits, the mean i_Cl for short-duration IPSCs (–0.6 ± 0.2 pA; n = 12) did not differ from the mean i_Cl for long-duration IPSCs (–0.8 ± 0.2 pA, n = 8; ANOVA, P > 0.05). These means were not significantly different from the mean i_Cl for GABA_Aergic IPSCs (–0.6 ± 0.1 pA, n = 18; P > 0.05, ANOVA).

View larger version (35K): [in this window] [in a new window]   FIG. 5. Estimation of elementary channel currents from nonstationary noise analysis of glycinergic currents. A: 10 successive, short- and long-duration glycinergic IPSCs in 2 neurons, and 37 short-duration sIPSCs in one neuron. Current variance was plotted as a function of mean current for the neurons. Variance–mean relationships were fitted with (t)2 = iCl · Imean(t) – Imean(t)2/N + th(t)2 (dark curves). iCl was also obtained from the slopes of the initial part in the variance–average current relationships, fitted by linear regression. Two methods yielded similar values of iCl. Estimates were iCl = –0.9 pA for a short-duration–evoked IPSC (left), iCl = –0.5 pA for a long-duration–evoked IPSC (middle), and iCl = –3.3 pA for a short-duration sIPSC (right). When averaged, iCl did not differ for short and long IPSCs (n = 20; P > 0.05, ANOVA). Gray lines show 95% confidence intervals. For evoked IPSCs, Vh = –80 mV, ECl = –53 mV. For sIPSCs, Vh = –60 mV, ECl = 0 mV. B: I–V relationship for glycinergic and GABAAergic sIPSCs (3 neurons). ECl = 0 mV. C: comparison of PCl values estimated for short- and long-duration sIPSCs and evoked IPSCs. Numbers in parentheses indicate numbers of neurons. There were no differences between fast and slow responses, or between spontaneous and evoked responses (P > 0.05, ANOVA).

For comparison of i_Cl values obtained from IPSCs and sIPSCs under differing holding potentials and E_Cl values, we used the GHK equation to convert the values to chloride permeability, P_Cl. Mean P_Cl for short-duration glycinergic IPSCs was 1.6 ± 0.5 x 10^–13 cm³/s for evoked (n = 12) and 1.1 ± 0.2 x 10^–13 cm³/s for spontaneous (n = 6) responses. Mean P_Cl for long-duration glycinergic IPSCs was 1.7 ± 0.4 x 10^–13 cm³/s for evoked (n = 8) and 0.9 ± 0.3 x 10^–13 cm³/s for spontaneous (n = 6) responses. As shown in Fig. 5C, P_Cl values from evoked and spontaneous IPSCs were not different (P > 0.05, unpaired t-test). The glycinergic P_Cl values did not differ from evoked (1.5 ± 0.3 x 10^–13 cm³/s, n = 18) and spontaneous (0.8 ± 0.2 x 10^–13 cm³/s, n = 7) GABA_Aergic P_Cl values (P > 0.05, ANOVA).

All three agonists evoked extrasynaptic currents of small and large amplitude (Figs. 4A and 6, A and B). Smaller currents were seen only in the presence of larger-amplitude currents. Thus the small currents likely reflected openings to a substate conductance, nearly 70% of the full conductance. Amplitude distributions for the currents were well described by the sum of two Gaussian terms (Fig. 6A). The I–V relationships were linear over the range of 0 to –60 mV (Fig. 6B). From these relationships, the mean conductances from short-duration bursts were 15 ± 1 pS (n = 10), 21 ± 2 pS (n = 6), and 22 ± 3 pS (n = 4) for glycine, taurine, and -alanine, respectively. The mean conductances from long-duration bursts were 19 pS (n = 1), 33 ± 1 pS (n = 4), and 30 ± 4 pS (n = 3) for glycine, taurine, and -alanine, respectively. These conductances were not different (P > 0.05, ANOVA).

View larger version (18K): [in this window] [in a new window]   FIG. 6. Conductance of single channels activated by glycine receptor agonists. A: amplitude distributions for agonist-activated currents (Vh = –60 mV) were fitted by the sum of 2 Gaussian terms (smooth curves). Means (arrowheads) were –1.2 and –0.96 pA for glycine (n = 1,473 transitions), –1.6 and –1.2 pA for taurine (n = 2,465 transitions), and –1.7 and –1.3 pA for -alanine (n = 1,144 transitions). B: single-channel I–V relationships for the full and substate conductances of glycine-activated receptor channels. Data were obtained from 12 patches. Linear regression fits indicated mean values of 13 and 17 pS for the substate and full conductances, respectively. In all panels, agonists were applied at 20 µM to outside-out patches with ECl = 0 mV. C: comparison of PCl values for short- and long-duration channel bursts, activated by glycine receptor agonists. Numbers in parentheses indicate number of patches. There were no significant differences in values obtained (P > 0.05, ANOVA).

For comparison, we converted extrasynaptic single-channel currents to P_Cl values. In view of the similar values of P_Cl from evoked and spontaneous IPSCs, as well as the similar extrasynaptic P_Cl values for the agonists, we pooled the data into synaptic and extrasynaptic categories. Table 1 shows the distinct nature of P_Cl values of synaptic and extrasynaptic channels. The short- and long-duration, glycinergic synaptic channels yielded values of P_Cl that were higher than the estimates from short- and long-duration bursts activated by the agonists (P < 0.05, unpaired t-test). The GABA_Aergic synaptic channels yielded P_Cl values that were higher than the estimates from extrasynaptic channels (P < 0.05, unpaired t-test) for currents obtained from Kim et al. (2004).

	DISCUSSION

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These studies have revealed some unusual facets of inhibitory transmission in thalamus. Spontaneous IPSCs mediated by glycine- and GABA_A receptors were monophasic, showing fast, intermediate, and slow decays. IPSCs evoked by medial lemniscal stimulation also showed fast, intermediate, or slow decays, alone or in combination. Biphasic IPSCs with an intermediate decay component likely resulted from cotransmission by a GABA_A-receptor–mediated pathway. Strychnine antagonized the fast and slow synaptic currents, mediated by glycinelike amino acids. Extrasynaptic receptors, activated by glycine agonists in membrane patches, displayed the predicted short- or long-duration burst openings (cf. INTRODUCTION). The dual kinetics of synaptic and single-channel currents implicate functional diversity in glycine receptors at a juvenile stage of rat development.

The chief finding of these studies was glycinergic sIPSCs and components of mixed IPSCs, decaying with fast or slow kinetics. Most sIPSCs (>94%) exhibited monoexponential decays with fast (11 ms) or slow (74 ms) time constants. The fast and slow time constants of sIPSCs, which largely represented monoquantal packets of transmitter, matched the fast (12-ms) and slow (80-ms) time constants of evoked IPSCs. This finding provided assurance that spontaneous and evoked IPSCs were attributable to the same glycine-receptor populations. The observations were consistent with the activation of two kinetically distinct populations of glycine receptors. Another important finding was that fast or slow synaptic currents occurred separately in different neurons. These IPSCs also decayed in a monoexponential manner, suggesting that the slow IPSCs were not likely attributable to a spillover of transmitter to perisynaptic receptors (see Chery and De Koninck 1999). Based on the observations on spontaneous and evoked IPSCs, we suggest that the receptor populations are predominantly localized under separate nerve terminals.

Our observations of fast and slow monoquantal sIPSCs contrast with the literature. In embryonic zebrafish (Ali et al. 2000), sIPSCs have biexponential decay arising from co-localization of receptors with fast and slow kinetics at the same synaptic sites. Spontaneous IPSCs decay monoexponentially with a fast (4 to 8 ms) time constant in rat spinal neurons (Chery and De Koninck 1999; Gonzáles-Forero and Alvarez 2005) and with a slow (about 63 ms) time constant in mouse retinal ganglion cells (Tian et al. 1998). Apparently, thalamic neurons in juvenile rats have a predominant ability to segregate two populations of glycine receptors with fast and slow kinetics.

The fast and slow kinetics of the synaptic currents are likely explained by structurally distinct receptor populations. The ₁ and ₂ receptor subunits (Ghavanini et al. 2005) determine synaptic decays of fast and slow IPSCs (Singer and Berger 1999; Takahashi et al. 1992). Given their very long burst duration, receptors containing ₂, but not ₁, subunits (Mangin et al. 2003), may account for the long decay tails of two atypical IPSCs in this study. Coassembly of ₁ and ₂ subunits likely occurs in developing neurons, where slow IPSCs are common (Ali et al. 2000; Takahashi et al. 1992). Thus persistence of ₁/₂ receptors in thalamic neurons may have resulted in the slow kinetics. Slow glycinergic inhibition contrasts with metabotropic GABAergic inhibition (Browne et al. 2001), mostly suppressed in our recordings. Another possibility is that posttranslational phosphorylation of glycine receptor channels (Agopyan et al. 1993) produced diverse kinetics.

The kinetics of extrasynaptic glycine receptor channels resembled the decays of glycinergic currents. The average lifetimes of short- and long-duration bursts activated by glycine, taurine, and -alanine were close to decay time constants for fast and slow IPSCs. The multiple congruencies in kinetics seem unlikely to have occurred by chance, although burst duration may depend on high agonist concentrations (cf. Beato et al. 2002) at glycinergic synapses.

There are reasons for postulating differences between synaptic and extrasynaptic glycine receptors. We observed that synaptic channels had higher P_Cl values than those of extrasynaptic channels. The P_Cl estimates were similar when measured with an optimized space clamp and were not likely the result of vagaries in fluctuation analysis (cf. Benke et al. 2001). The unitary conductance obtained from sIPSCs was in the same range as that in other preparations under similar conditions (cf. Poncer et al. 1996; Singer and Berger 1999). The synaptic GABA_Aergic channels had a higher conductance than that of extrasynaptic channels, as found elsewhere (Yeung et al. 2003). The low conductances of extrasynaptic glycine receptors were compatible with embryonic receptor channels (Rajendra et al. 1997) and extrasynaptic receptors on hippocampal neurons (Fatima-Shad and Barry 1995). Given these considerations, we suggest that the conductance differences were genuine. Extrasynaptic receptors, usually considered as high conductance homomers (Lynch 2004), in this case may have reduced conductance, reflecting posttranslational modification (cf. Caraiscos et al. 2002).

The present results are compatible with cotransmission by glycinergic and GABA_Aergic pathways, rather than corelease of glycinelike amino acids and GABA. An appreciable number of neurons showed exclusively glycinergic or GABAergic responses to medial lemniscal stimulation, consistent with cotransmission by independent pathways. If corelease of glycinelike amino acids and GABA were to occur (Jonas et al. 1998), we would expect a prevalence of multiphasic sIPSCs in each neuron, converting on strychnine application to monophasic GABA_Aergic currents (see Dumoulin et al. 2001). In contrast, the majority of sIPSCs in seven tested neurons showed a monophasic decay, with or without strychnine application. We conclude that if present in thalamic inhibition, corelease was a less common occurrence than cotransmission.

Physiological implications

Fast synaptic kinetics allow rapid phasic transfer of information for somatotopic representations of rapidly adapting receptors (cf. Tsumoto and Nakamura 1974). Slow IPSP decays affect hyperpolarization-activated currents, remove Ca²⁺ channel inactivation, and promote low-threshold Ca²⁺ bursting (cf. Steriade et al. 1997). The glycinergic IPSC components were kinetically distinct from GABA_Aergic IPSCs (about 22 ms; cf. Dumoulin et al. 2001). The cooccurrence of fast and slow glycinergic IPSPs with intermediate GABA_Aergic IPSPs would confer fine-tuning of inhibitory transmission by modulation of voltage-dependent currents in somatosensory thalamus. The higher P_Cl of synaptic receptors ensures high transmission efficacy.

Despite the differences in P_Cl, the striking similarities between IPSC decay and extrasynaptic channel burst duration imply that glycine, taurine, and -alanine each could mediate inhibition. When applied at the same concentration, glycine, taurine, and -alanine activated channels with comparable open probabilities. The abilities of -amino acids, relative to glycine, to activate long-duration bursts was greater at extrasynaptic receptors than most receptor variants (cf. Flint et al. 1998; Martin and Siggins 2002). The lower Cl^– permeability may suit extrasynaptic receptors for the detection of ambient -amino acids, tonic inhibition, and receptor modulation (cf. Berger et al. 1998; Flint et al. 1998; Mori et al. 2002).

Thalamocortical neurons segregate ionotropic glycine receptors showing fast and slow decay kinetics. Cotransmission with GABA_A receptors showing intermediate kinetics, and known metabotropic GABA_B receptors, facilitate postsynaptic discrimination of inputs in neurons of somatosensory nuclei.

	GRANTS

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This work was supported by grants from the Canadian Institutes for Health Research to E. Puil and D. A. Mathers and the Jean Templeton Hugill Foundation Chair to E. Puil. A. A. Ghavanini received a University Graduate Fellowship through the University of British Columbia Neurosciences Program.

	ACKNOWLEDGMENTS

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We thank V. Dobrovinska and C. Caritey for technical assistance.

Present address of H.-S. Kim: Department of Anesthesiology, College of Medicine, Seoul National University, Seoul, Korea.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. Mathers, 2146 Health Sciences Mall, Vancouver BC V6T 1Z3, Canada (E-mail: mathers{at}interchange.ubc.ca)

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