Differential Short-Term Changes in GABAergic or Glycinergic Synaptic Efficacy on Rat Hypoglossal Motoneurons

Roberta Donato and Andrea Nistri

Biophysics Sector and Istituto Nazionale di Fisica della Materia Unit, International School for Advanced Studies (SISSA), 34014 Trieste, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Donato, Roberta and Andrea Nistri. Differential Short-Term Changes in GABAergic or Glycinergic Synaptic Efficacy on Rat Hypoglossal Motoneurons. J. Neurophysiol. 86: 565-574, 2001. Using whole cell patch-clamp recording from hypoglossal motoneurons of a neonatal rat brain slice preparation, we investigated short-term changes in synaptic transmission mediated by GABA or glycine. In 1.5 mM extracellular Ca2+ [Ca2+]o, pharmacologically isolated GABAergic or glycinergic currents were elicited by electrical stimulation of the reticular formation. At low stimulation frequency, glycinergic currents were larger and faster than GABAergic ones. GABAergic currents were strongly facilitated by pulse trains at 5 or 10 Hz without apparent depression. This phenomenon persisted after pharmacological block of GABAB receptors. Glycinergic currents were comparatively much less enhanced than GABAergic currents. One possible mechanism to account for this difference is that GABAergic currents decayed so slowly that consecutive responses summated over an incrementing baseline. However, while synaptic summation appeared at >= 10-Hz stimulation, at 5 Hz strong facilitation developed with minimal summation of GABA-mediated currents. Glycinergic currents decayed so fast that summation was minimal. As [Ca2+]o is known to shape short-term synaptic changes, we examined if varying [Ca2+]o could differentially affect facilitation of GABA- or glycine-operated synapses. With 5 mM [Ca2+]o, the frequency of spontaneous GABAergic or glycinergic currents appeared much higher but GABAergic current facilitation was blocked (and replaced by depression), whereas glycinergic currents remained slightly facilitated. [Ca2+]o manipulation thus brought about distinct processes responsible for facilitation of GABAergic or glycinergic transmission. Our data therefore demonstrate an unexpectedly robust, short-term increase in the efficiency of GABAergic synapses that can become at least as effective as glycinergic synapses.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GABA and glycine are neutral amino acids that in the mammalian CNS, in addition to their metabolic role, operate as neurotransmitters via activation of distinct postsynaptic receptors gating Cl- permeable channels. Glycine is mainly present in the spinal cord and brain stem, while GABA is prevalent in higher regions of the neuraxis. In brain stem cranial nuclei, such as, for instance, the hypoglossus nucleus, both inhibitory neurotransmitters are present. The hypoglossus nucleus is made up by motoneurons that innervate tongue muscles and are thus essential for functions such as swallowing, suckling, and vocalization (Lowe 1980) and are involved also in maintaining the upper airways patent. A large part of GABAergic or glycinergic inputs to hypoglossal motoneurons originates from the reticular formation (Li et al. 1996, 1997), and the respiratory centers (the pre-Bötzinger complex in the ventrolateral medulla) (Paton and Richter 1995).

The co-existence of GABA and glycine in the hypoglossal nucleus raises the question of their relative contribution to synaptic microphysiology, their interplay as transmitters, and their differential role at functional level. Although GABA and glycine have been proposed to be co-released at certain synapses (Jonas et al. 1998; O'Brien and Berger 1999), this phenomenon does not appear frequently in the hypoglossal nucleus (Donato and Nistri 2000). One striking feature of GABAergic currents (miniature, spontaneous, or evoked by electrical stimulation) recorded from hypoglossal motoneurons of the neonatal rat is that they are approximately two times slower than their glycinergic counterparts (Donato and Nistri 2000).

We wondered whether such different kinetics could lead to distinct changes in short-term efficacy of GABAergic over glycinergic transmission. This seems to be an interesting issue because during normal neuronal activity, synapses are presumably subjected to phasic and tonic signaling and might employ discrete variations in synaptic efficacy to integrate information. In principle, this might mean that the balance between GABA or glycine in information transfer at motoneuron level is a nonstationary process subjected to changes in the relative contribution by each transmitter to cell excitability.

The present study tested these possibilities by examining if the efficacy of synaptic responses mediated by GABA or glycine during high-frequency stimulation could differentially and rapidly change.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Slice preparation

Experiments were carried out on brain stem slices obtained from 0- to 5-day-old Wistar rats terminally anesthetized with 0.2 ml urethan (10%; intraperitoneal injection). The entire procedure (including animal handling and care) is in accordance with the Animal Welfare Act and was approved by the Local Authority Veterinary Service. Thin brain stem slices (200 µm thick) were prepared following the procedure described by Viana et al. (1994) and Donato and Nistri (2000).

Electrophysiological recording

Single hypoglossal motoneurons within the XII nucleus were viewed with an infrared video camera and voltage clamped with whole cell patch-clamp electrodes (3-5 MOmega DC resistance) at -70-mV holding potential (Vh). Series resistance (5-25 MOmega ) was routinely monitored and compensated (usually by 30%, range 20-60%). Voltage and current pulse generation and data acquisition were performed with a PC running pClamp 6.1 software (Axon Instruments, Foster City, CA). Currents elicited by voltage steps were low-pass filtered at 3-10 kHz and sampled at 5-10 kHz.

Electrical stimulation and data analysis

For extracellular stimulation of GABAergic or glycinergic cells, a single bipolar tungsten electrode was placed in the lateral reticular formation. After stabilization of the synaptic response, stimulus intensity for low-frequency pulses (0.5 Hz; 0.2 ms) was adjusted to obtain 25-50% failures for 100 stimuli. After discarding no-response trials, synaptic currents evoked by such low-frequency pulses were stored as individual files and averaged (see also Donato and Nistri 2000) to be used as reference for subsequent tests at high-frequency stimulation. The high-frequency stimulation protocol employed either short (4 pulses) or long (100 pulses) trains using the same stimulus intensity that at 0.5-Hz frequency gave 25-50% failures. Long trains consisted of 100 stimuli (range of 2- to 20-Hz frequency) repeated at least five times (depending on the stability of recording) at >= 5-min intervals (to allow for synaptic recovery). The high-frequency (20 Hz), four-pulse protocol was repeated at >= 3 min intervals for >= 10 times. All responses (including failures) to either long or short trains were stored as individual files and averaged over different trials.

Quantitative analysis of changes in synaptic currents evoked by pulse trains was carried out with standard methods (see for example, Seamans et al. 2001). In detail, with the long (100 pulse) train protocol, the peak amplitude of each response was calculated from baseline current just before each stimulus artifact (see Figs. 1A, right, and 2A, right). With the short (4 pulse) train protocol, the peak amplitudes of the four consecutive responses were calculated with respect to the baseline current before the start of the train. Exponential fitting of current decay (excluding any holding current shift during repetitive stimulation) was carried out with the Chebychev algorhythm provided by AxoGraph software (version 3.5; Axon Instruments) as reported earlier (Donato and Nistri 2000).

Using nonlinear regression analysis with minimization of sample errors, an exponential function of the form: a + (1 - a) * exp(-t/b) was fitted to the time course of a series of evoked currents normalized versus the first one in the train (using SigmaPlot software; Jandel Scientific, San Rafael, CA). In this expression, a is the maximal amplitude, b is the time constant, and t is time.

With the short-term protocol, we also measured the synaptic charge induced by GABA or glycine, namely the time integral of the synaptic current. For this purpose, the area of each response was calculated from the stimulus artifact to the next one taking as baseline an arbitrary line originating from the start of the first synaptic current.

Detection of spontaneous postsynaptic currents (sPSCs) was done with AxoGraph software, which uses the method of minimizing the sum of squared errors between data and a template function approximating the width and time course of a typical synaptic event as described by Clements and Bekkers (1997) and further detailed by Donato and Nistri (2000). Unpaired t-test or Mann-Whitney rank-sum test (SigmaStat software) was used to assess differences in mean values; P < 0.05 was considered as the acceptable level of statistical significance.

Solutions and drugs

For slice preparation, the solution (in mM) contained 130 NaCl, 3 KCl, 26 NaHCO3, 1.5 Na2HPO4, 1 CaCl2, 5 MgCl2, and 10 glucose (290-310 mOsm) gassed with O2-CO2 (95/5%). For electrophysiological recording, the extracellular control solution (in mM) containing 140 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4, 290-310 mOsm) was continuously oxygenated and applied by superfusion (2-5 ml/min) to the slice in a recording chamber. Drugs were applied via the extracellular solution for a minimum of 5-10 min to reach equilibrium conditions. Kynurenic acid (2 mM) was routinely added to block glutamatergic ionotropic currents. In accordance with our previous work (Donato and Nistri 2000), strychnine (0.4 µM) was applied to isolate GABAergic synaptic currents, whereas bicuculline (10 µM) was used to isolate glycinergic currents. This concentration of bicuculline is much lower than the one inducing 50% block of the spike late afterhyperpolarization of brain slice neurons (Debarbieux et al. 1998; Seutin et al. 1997). For multiple tests on the same cells, glycinergic transmission was studied before the GABAergic one to exploit faster washout of bicuculline than strychnine (Donato and Nistri 2000). However, in view of the extensive number of tests performed on each cell, the slow washout of receptor antagonists, and the need to obtain closely reproducible responses, it was not always possible to explore, on the same cell, the differential sensitivity of glycinergic and GABAergic transmission to various stimulus protocols. When this was precluded, data were compared across cell groups. The patch pipette solution (in mM) was 120 CsCl, 9 NaCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 10 EGTA, and 2 Mg-ATP at pH 7.2 (270-290 mOsm).

The following drugs were used: kynurenic acid (Sigma), bicuculline methiodide (Sigma), strychnine hydrochloride (Sigma), GABA (Sigma), glycine (Sigma), and 3-[[(3,4-dichlorophenyl)methyl]amino]propyl]diethoxymethyl)phosphinic acid (CGP 52432, gift from Novartis Pharma).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

High-frequency stimulation of GABAergic or glycinergic synapses

Under the present recording conditions (symmetrical transmembrane Cl- concentration and Vh = -70 mV), synaptic currents induced by GABA or glycine were inward. Data were collected from 27 hypoglossal motoneurons (HMs) with 270 ± 20 MOmega input resistance. On neonatal HMs GABAergic synaptic currents are consistently slower than glycinergic ones (Donato and Nistri 2000; O'Brien and Berger 1999). We explored whether such slow kinetics of GABA-mediated currents could be responsible for distinct changes in GABAergic synaptic efficacy following trains of electrical stimuli. For this purpose, we compared pharmacologically isolated GABAergic or glycinergic responses evoked by repetitive stimulation (ePSCs).

Figure 1, A-G, illustrates how we assessed the extent and time course of changes in GABAergic transmission. In particular, raw traces (Fig. 1A, left) demonstrate the development of facilitation of GABAergic inward currents following repeated 5 Hz stimuli. Figure 1A (right) exemplifies how single responses were aligned to measure their peak amplitudes (for clarity, only the 1st 20 responses to a single train are shown). The full time course of facilitation is quantified in Fig. 1, B and C, which depicts the profile of changes in peak amplitude of GABAergic ePSCs evoked by trains of 100 stimuli at 5 or 10 Hz (responses observed at each frequency are the mean of >= 5 trials on the same neuron). Note that there was a very large and gradual increment in the peak amplitude of the inward currents that remained enhanced up to the end of the stimulus train. We next tested if activation of GABAB receptors by endogenous GABA might have limited the observed increase in inward currents. To this end, tests were repeated on the same cell in the presence of the selective GABAB antagonist CGP-52432 (10 µM). As shown in Fig. 1D, the extent of synaptic facilitation during the train remained essentially unchanged. In the presence of 10 µM bicuculline, all evoked currents were fully blocked (Fig. 1E), thus indicating that these responses were mediated by activation of postsynaptic GABAA receptors.



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 1. High-frequency stimulation of GABAergic responses. A, left: sample trace of the 1st 20 GABAergic responses to a 100-stimulus train at 5 Hz. A, right: the same 1st 20 traces aligned to measure their peak amplitudes. B and C: examples of GABAergic peak responses to 100 stimuli delivered at 5 Hz or 10 Hz; each response in the train is the average over 5 trials in the same cell. D: average peak GABAergic responses to 10-Hz tetanus in the presence of 3-[[(3,4-dichlorophenyl)methyl]amino]propyl]diethoxymethyl)phosphinic acid (CGP 52432, 10 µM). E: bicuculline (10 µM; bic) fully blocked GABAergic responses to a 10 Hz tetanus. F: peak responses to 100 stimuli normalized to the first one. Responses larger than the value 1 are considered to be facilitated. Top: normalized responses to 5 Hz train. Middle: responses to 10 Hz in control solution and after addition of 10 µM CGP 52432; note that the 2 traces are almost superimposed. Bottom: currents elicited by electrical stimulation are fully blocked in bicuculline solution (10 µM). We used the exponential function: a + (1 - a) * exp(-t/b), where a = amplitude and b = time constant for the data points and expressed the abscissa as number of stimuli to allow comparison between tests done at different frequencies (fitted functions are superimposed to datapoints). G: histograms of exponential fitting of GABAergic facilitation {a + exp[-t/b(1 - a)] where a = amplitude is expressed as current increment (left ordinate) and b = time constant is expressed as s (right ordinate) under various experimental conditions (bic = bicuculline)}.

Direct data comparison between different cells under the four experimental conditions discussed in the preceding text required normalization of the current peak amplitude (with respect to the 1st response in the train) and extraction of objective parameters to quantify the degree and time course of GABAergic response facilitation. Figure 1F (same cell shown in Fig. 1, A-E) shows how we used this approach. The normalized amplitude plots indicate a larger fractional increment at 5- than at 10-Hz stimuli and that such facilitation could be adequately fitted by the monoexponential function of the form f = a + (1 - a) * exp(-t/b), where a quantifies the extent of response potentiation (namely, the fractional increase in amplitude) and b represents its time constant. The average values for a and b are then summarized in Fig. 1G for one cell under various experimental conditions. Pooling data (n = 5 cells) calculated with this method showed that facilitation of GABAergic response amplitude at 5 Hz was many times larger than at 10 Hz but that it was attained more slowly [for 5 Hz a = 10 ± 3 (SD) increment, b = 5 ± 2 s; for 10 Hz a = 4 ± 1 increment; b = 2.9 ± 0.7 s].

In contrast with the observations concerning GABAergic currents, glycinergic currents underwent less short-term facilitation changes even if we used tests with a broader range of stimulus frequency (2-20 Hz). Figure 2A shows raw traces of the increment in glycinergic inward currents (5-Hz stimuli). The time course of changes in peak amplitude of glycinergic ePSCs evoked by trains of 100 stimuli at 2, 5, 10, or 20 Hz on the same cell is plotted in Fig. 2, B-E. At 2 Hz (Fig. 2B), there was minimal alteration in inward current amplitude, whereas at 5 Hz (Fig. 2C), the inward current increased in a multiphasic fashion. At 10 Hz (Fig. 2D), the current amplitude increase was quite small, whereas at 20 Hz (Fig. 2E), the initial increment was not sustained. Figure 2F shows, for the same cell, the extent and time course of glycinergic response facilitation to 5 or 10 Hz fitted in the same manner as for GABAergic responses (compare with Fig. 1F). Both train stimuli induced a similar degree of glycinergic synaptic facilitation that was quantified in Fig. 2G. When we used the same analysis for four cells, the average parameters a and b were essentially the same for the 5- or 10-Hz stimulation (for 5 Hz, a = 3.4 ± 0.5 increment, b = 2.4 ± 0.7 s; for 10 Hz, a = 3.5 ± 0.6 increment, b = 1.6.4 ± 0.4 s).



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 2. High-frequency stimulation of glycinergic responses. A, left: sample trace of the 1st 20 glycinergic responses to a 100-stimulus train at 5 Hz (same intensity as in Fig. 1A). A, right: the same 20 traces aligned to measure peak amplitudes. B-E: example of glycinergic peak responses during 100 stimuli delivered at 2 Hz (B), 5 Hz (C), 10 Hz (D), and 20 Hz (E), respectively. F: peak responses to 100 stimuli normalized to the first one (for 5- and 10-Hz stimulation) to represent the time course of facilitation; ---, the fitted exponential functions. G: histograms of exponential fitting of glycinergic facilitation: a = amplitude, b = time constant [fitted function: a + (1 - a) * exp(-t/b), see text].

In summary then, GABAergic currents dramatically increased in amplitude with repeated stimuli while glycinergic currents were comparatively less affected.

Slow kinetics of GABAergic currents determine synaptic summation

One possible mechanism for facilitation of GABAergic responses to high-frequency stimulation would be gradual response summation due to the long-lasting tail of these synaptic currents (Donato and Nistri 2000). To check this possibility, we measured the time constant (tau ) of the decay of each GABAergic current during the course of 5- or 10-Hz train stimulation. Figure 3A shows the time profile of tau  changes during the 100-pulse train at 5 Hz. While there was scattering of these data points especially for the early part of the train, there was no significant change in tau  value throughout the train because these results could be fitted by linear regression with r = -0.09, which was not significantly different from 0. The mean value of tau  (27.2 ± 0.8 ms, n = 5 cells; measured across the entire 5-Hz train) was slightly, though significantly (P < 0.001), larger than the one (24 ± 2 ms) of control responses evoked by a low-frequency (0.5 Hz) train. Figure 3C shows raw traces of individual ePSCs (see expanded record taken at the start of the train) indicating that baseline was often re-attained before the arrival of the next stimulus (see also Fig. 1A). Summation of synaptic currents was therefore an infrequent phenomenon at 5 Hz.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 3. Dependence of decay time of GABAergic responses on frequency of stimulation. A and B: scatter plot of the decay time of GABAergic responses to 5-Hz (A) or 10-Hz (B) tetanic stimulation as function of time. Linear regression (fitted function: y = A + Bx) whose parameters are: A = 25 ± 2, B = -0.1 ± 0.1 and A = 26 ± 3, B = 1.6 ± 0.6 for the 5- and 10-Hz stimulation, respectively. For r and P values, see text. C: record of a single trial at 5 Hz (slow time scale, top; fast time scale, bottom). D: as in C for 10-Hz stimulation.

Figure 3B shows, on the same cell, the scatter plot of tau  values for GABAergic current decay at 10 Hz. Their average was significantly larger (34 ± 2 ms, P < 0.001, n = 5 cells) than the tau  value at 0.5 Hz and incremented throughout the train as confirmed by linear regression analysis with r = 0.26, a value significantly different from 0 (P < 0.008). Inspection of the expanded trace (Fig. 3D) indicates that there was gradual summation of GABAergic currents for this frequency of stimulation as the tail of each event did not fully dissipate before the subsequent one. Thus GABAergic currents could be enhanced via two mechanisms: facilitation (typically seen at 5 Hz) whereby individual currents peaks strongly grew from a steady baseline and summation (at 10 Hz) when currents did not fully dissipate before the subsequent response, which just took off from a shifting baseline.

The phenomenon of summation is clearly demonstrated in Fig. 4A. In this example, to ensure that pulses were delivered at a rate higher than the GABAergic current full decay (~75 ms as decay tau  value = 24 ± 2 ms), we applied a short 20-Hz train. In the case of these short, high-frequency trains, we measured peak responses in terms of total inward current generated by GABAergic synaptic activation with respect to the baseline level before the train. On a sample of 11 cells paired-pulse facilitation (ratio between the 2nd and 1st response) was 2.6 ± 0.3 and grew significantly in a stepwise fashion when the ratios between subsequent responses and the first one were considered (Fig. 4C).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4. GABAergic or glycinergic responses at 20-Hz short pulses. A: sample trace of GABAergic responses evoked by 4 pulses at 20 Hz (average over 15 trials). Note clear response summation occurring for subsequent stimuli and apparent lack of desensitization. B: sample trace of glycinergic responses to 4 pulses at 20 Hz (average over 12 trials). Note that facilitation occurs at the 2nd pulse; the amplitude of subsequent responses is only slightly increased. C and D: histograms summarizing the amplitude ratio of consecutive (a1, a2, ··· an) GABAergic (C, , n = 11 cells) or glycinergic (D, , n = 9 cells) responses to the 1st one. In C, **, a significant difference with respect to the 1st response and between consecutive ones. In D, *, a significant difference vs. the 1st response only; no difference was detected between consecutive ones.

Figure 4B shows (on the same cell) that the 20-Hz short train enhanced the second glycinergic current without much further increment for subsequent responses. Glycinergic currents were comparatively short lasting and fell to baseline before the next pulse was delivered. Any increase in glycinergic transmission was thus not due to summation but to facilitation. Figure 4D shows that for an average of nine cells, the same degree of increase in amplitude of glycinergic currents was present throughout the four-pulse train. We also compared the cumulative increment in synaptic charge (pC) for GABA- or glycine-mediated responses. In this way, it was possible to analyze the temporal effectiveness of synaptic signals induced by the two transmitters. The total synaptic charge for the cluster of four synaptic responses was 5 ± 1 and 7 ± 2 pC for glycine and GABA, respectively (n = 9). These results suggest that during a very short period of high-frequency stimuli, the effectiveness of GABA-mediated synaptic activity became very similar to glycine-mediated one despite the fact that spontaneous glycinergic events are faster and occur at higher frequency than their GABAergic counterparts (Donato and Nistri 2000).

Furthermore, while enhancement of glycinergic currents by high-frequency pulses was a stereotypic process, the process for the increase in GABAergic currents relied on a dynamic mechanism, enabling progressively larger synaptic efficacy for the same synaptic input.

Role of extracellular Ca2+

It is known that several types of short-term synaptic changes are largely dependent on extracellular Ca2+ ([Ca2+]o) (for review, see Zucker 1989). The role of [Ca2+]o in GABAergic synaptic facilitation was tested by changing the concentration of this cation within the 1.5- to 5-mM range while stimulating with 5-Hz trains. In the example of the cell shown in Fig. 5A, plots of normalized data for the degree and time course of facilitation with 1.5, 2.5, or 5 mM [Ca2+]o demonstrate that only responses in 1.5 mM [Ca2+]o underwent sustained facilitation. The responses in 2.5 mM [Ca2+]o had transient facilitation, later replaced by depression. The responses in 5 mM [Ca2+]o were unable to show any facilitation. Similar data were collected from five cells.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 5. Effect of increasing [Ca2+]o on GABAergic currents. A: example of GABAergic response facilitation to 5-Hz stimulation while increasing [Ca2+]o from 1.5 mM (left), to 2.5 mM (middle), or to 5 mM (right). Note that increasing [Ca2+]o turns facilitation into depression. Values of a and b used for fitting data with 1.5, 2.5, or 5 mM [Ca2+]o are: a = 6.9 ± 0.8, 1.7 ± 0.9, or -0.6 ± 0.9 increments and b = 11.8 ± 0.9, 0.6 ± 0.4, or 0.9 ± 0.1 s for the 3 [Ca2+]o conditions. B: sample traces of spontaneous events recorded for the same cell at different [Ca2+]o. C: histograms summarizing the effect of increasing [Ca2+]o on the peak amplitude (; top) and inter-event interval (; bottom) of spontaneously occurring GABAergic currents (n = 5, *, P < 0.001 vs. control values).

It was next examined how these changes in [Ca2+]o might have affected GABAergic transmission pre- and/or postsynaptically. Figure 5B shows spontaneous GABAergic currents (sPSCs) recorded in varying [Ca2+]o from the same cell. Changing [Ca2+]o had a relatively slight influence on the average peak amplitude of sPSCs because a small increase was observed with 5 mM [Ca2+]o only (see average data in Fig. 5C, top). The decay time of sPSCs was not significantly changed (tau  = 23 ± 1, 23 ± 1 or 26 ± 2 ms with 1.5, 2, or 5 mM [Ca2+]o, respectively; P > 0.05; n = 5). Conversely, the frequency of sPSCs (expressed as inter-event interval) was strongly enhanced with 5 mM [Ca2+]o (Fig. 5C, bottom). These results suggest that changes in [Ca2+]o had a mainly presynaptic effect on GABAergic sPSCs.

Similar experiments were carried out with glycinergic currents as shown in Fig. 6A in which normalized response amplitudes in 1,5, 2.5, or 5 mM [Ca2+]o are presented. In 2.5 mM [Ca2+]o, facilitation (normally observed with 1.5 mM [Ca2+]o) was still present, although reduced. In particular, the parameter a related to synaptic amplitude went from 4.0 ± 0.1 (at 1.5 mM [Ca2+]o) to 2.01 ± 0.02, while the time course b went from 4.5 ± 0.3 to 1.8 ± 0.2 s. The latter value indicated faster attainment of steady-state facilitation level in 2.5 [Ca2+]o. With 5 mM [Ca2+]o, there was no apparent facilitation of glycinergic currents. Changing [Ca2+]o had also clear effects on spontaneous glycinergic currents as shown in Fig. 6B where amplitude and particularly frequency went up with 5 mM [Ca2+]o. Like in the case of GABAergic sPSCs, the decay time was not significantly changed by increasing [Ca2+]o (tau  = 12 ± 2, 10 ± 2, or 13 ± 1 ms, respectively, for [Ca2+]o = 1.5, 2.5, or 5 mM; P > 0.05; n = 3). These observations are summarized in Fig. 6C for peak amplitude (top) and for the spontaneous inter-event frequency (bottom). Analogous data were collected from five cells.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 6. Dependence of glycinergic facilitation on [Ca2+]o. A: normalized responses to 5-Hz tetanus (100 stimuli) at different [Ca2+]o (1.5 mM, left; 2.5 mM, middle; 5 mM, right). B: sample records of spontaneous glycinergic currents at different [Ca2+]o. C: histograms of amplitude (; top) and inter-event interval (; bottom) of spontaneous glycinergic events at different [Ca2+]o (n = 3; *, a significant difference vs. control, P < 0.001).

These data indicate that raising [Ca2+]o altered facilitation of evoked GABAergic transmission more strongly than facilitation of evoked glycinergic transmission. However, the sensitivity of GABAergic or glycinergic sPSCs to [Ca2+]o was similar, a fact suggesting to us that [Ca2+]o operated chiefly via a presynaptic mechanism, as the main effect was a strong change in PSC frequency coupled with slight amplitude increase, and no change in kinetics.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The principal finding of the present study is the demonstration that GABAergic transmission on neonatal HMs could be strongly facilitated during high-frequency stimulation with a repertoire of changes depending on the frequency and number of electrical stimuli. On the same cells glycinergic transmission could also be facilitated but to a lesser degree and with a more stereotypic change.

Short-term changes in the efficacy of GABAergic or glycinergic synapses

During repeated stimuli, glycinergic synapses showed less facilitation than GABAergic ones. In particular, in response to 5-Hz stimulation, average facilitation of GABAergic peak amplitude was 10 ± 3 times, a value consistently higher than the one (3.4 ± 0.5) for glycinergic currents. Facilitation of GABAergic currents did not include activation of postsynaptic receptors other than GABAA ones because all postsynaptic responses were eliminated by bicuculline. In other brain areas, GABA release can be regulated by presynaptic GABAB autoreceptors (for review, see Sivilotti and Nistri 1991). Thus, if this situation also applied to neonatal HMs, one might have expected that GABAB receptor activity limited the extent of GABAergic facilitation. However, addition of CGP 52432, a potent and selective GABAB receptor antagonist, had no significant effect on GABAergic responses (evoked at 10 Hz), indicating that GABAB receptors were either absent or nonfunctional at this early postnatal period. This electrophysiological observation accords with immunocytochemical studies that could not detect the two known isoforms of GABAB receptors in the hypoglossal nucleus, although such receptors were readily demonstrable in adjacent areas like the nucleus of the tractus solitarius, dorsal motor nucleus of vagus, and raphe obscurus and magnus (McDermott et al. 1999). Furthermore, the GABAB agonist baclofen was ineffective on HMs at this early postnatal age (R. Lape and R. Donato, unpublished observations). Because GABAB receptors could not be detected histologically or functionally, it is suggested that the comparatively less intense facilitation of glycinergic synapses was unlikely due their preferential depression by GABAB receptor activity.

Other receptors known to regulate synaptic transmission at presynaptic level are metabotropic glutamate receptors (GluRs) (see Ozawa et al. 1998 for a recent review). On neonatal HMs, GluR activity enhances rather than depresses Cl--mediated synaptic transmission (Donato and Nistri 2000). We cannot firmly rule out that in the present study repetitive stimulation did not release glutamate to activate such receptors. Nevertheless, if repeated stimuli had indeed released enough glutamate, this effect should have been restricted to glycinergic terminals, where these receptors are functional, as GABAergic terminals do not possess them (Donato and Nistri 2000). Yet it was GABAergic transmission that demonstrated the largest facilitation increase. These considerations led us to think that the difference in facilitation observed between GABAergic and glycinergic synapses was unlikely due to activation of mGluRs.

One further possibility to explain the difference in the extent of facilitation between glycinergic and GABAergic synapses was that the much slower kinetics of GABA mediated events enabled response summation. Since the decay time constant of GABAergic events is ~100 ms (Donato and Nistri 2000), synaptic currents induced at >= 10-Hz frequency of stimulation would have been long enough to start from the tail of the preceding response. This prediction was indeed borne out as the tails of GABAergic currents did not fully return back to baseline at 10-Hz stimulation so as subsequent currents emerged from a rising baseline (see Figs. 3 and 4). Nevertheless, this phenomenon was not consistently observed with lower stimulation frequency, implying the GABAergic facilitation did not merely involve summation.

Glycinergic currents showed less intense facilitation with minimal evidence for response summation. Using a short train of high-frequency (20 Hz) pulses, glycinergic current facilitation appeared fully developed by the second response and did not further increase (longer trains actually led to depression; see Fig. 2E). Conversely, summation of GABAergic currents with analogous short protocols was so strong that, in the case of a short, high-frequency train, the membrane charge generated by GABAergic transmission was as much as the one due to glycinergic transmission. In other words, short-term changes in the efficacy of GABAergic synapses could compensate for the initially slower and smaller amplitude of single GABAergic events (Donato and Nistri 2000). Thus, short trains of GABA or glycine synaptic signals seemed to elicit comparable synaptic charge increases.

Role for GABAA receptor desensitization?

It is somewhat surprising that, in control solution, high-frequency stimulation did not cause depression of GABAergic currents. GABAA receptors are known to be prone to rapid desensitization during sustained exposure to this neurotransmitter (Jones and Westbrook 1995, 1996; Jones et al. 1998). This condition would result in smaller response amplitude and increased decay time of the macroscopic currents. The present data showed that the decay time of responses at 5 Hz did not significantly change during the stimulus train while their amplitude was actually augmented. Lengthening of tau  values appeared with 10-Hz trains, indicating that some desensitization of GABAA receptors might have taken place together with the inevitable prolongation of decay due to tail current summation. Hence, it seems noteworthy that consecutive responses to high-frequency stimulation (>= 10 Hz) did summate (see, in particular, Fig. 4A) in contrast to the rapidly developing depression of GABAergic transmission often observed on other brain neurons (Sivilotti and Nistri 1991).

Effects of [Ca2+]o on synaptic facilitation

It is known that Ca2+ is involved in the induction of short (and long)-term changes in synaptic efficacy (Zucker 1989). One explanation is that during intense neuronal firing, free intracellular Ca2+ is not fully buffered by internal stores ("residual Ca2+ hypothesis") so as it can build up after successive action potentials, leading to enhanced transmitter release (Katz and Miledi 1968; Rahamimoff and Yaari 1973).

In our experiments on glycinergic responses to 5-Hz stimuli, we found that with high [Ca2+]o, facilitation, even though decreased, was still present. This finding was accompanied by an increase in the underlying spontaneous activity recorded at rest. Conversely, raising [Ca2+]o blocked facilitation of GABAergic transmission and actually led to synaptic depression even if the frequency of GABAergic sPSCs was enhanced. On neonatal rat motoneurons, changing [Ca2+]o is known to transform facilitation of excitatory synapses into depression (Lev-Tov and Pinco 1992; Pinco and Lev-Tov 1993): thus GABAergic transmission appeared to respond to changes in [Ca2+]o in the same way as other synapses do.

The mechanisms through which changes in [Ca2+]o affected synaptic efficacy in the present investigation are complex. On the one hand, the higher rate of spontaneous release in high [Ca2+]o might have reduced the number of vesicles available for stimulus-induced release, a factor potentially leading to synaptic depression (Bailey and Chen 1988). On the other hand, repetitive firing induced by pulse trains might have raised free intracellular Ca2+ to promote facilitation as indeed proposed for hippocampal inhibitory synapses (Jensen et al. 1999).

On HMs the observed discrepancy between the behavior of GABAergic and glycinergic currents in high [Ca2+]o suggests that the pool of GABA available for transmitter release during repeated pulses was probably more limited than the pool of glycine. This observation is consistent with our previous study (Donato and Nistri 2000) demonstrating the much larger sensitivity of GABAergic rather than glycinergic transmission to tetrodotoxin (TTX). Even though the complexity and diversity of synaptic connections to hypoglossal motoneurons make it difficult to calculate release probability (P) with quantal analysis (see Nicholls et al. 2001), our results imply a high release P for glycine and a low one for GABA. It is known that the extent of synaptic facilitation is inversely related to the value of P (McLachlan 1978; Stevens and Wang 1995) as a "low P" synapse is more likely to display facilitation or augmentation following a repetitive stimulation, whereas a "high P" synapse is more likely to exhibit depression for the same stimulation. Such an inverse correlation is possibly due to dependence of the release machinery on presynaptic Ca2+ levels (Thomson 2000). Hence, it seems probable that on neonatal HMs different P values for GABA and glycine were responsible for the dissimilar facilitation of GABAergic and glycinergic currents.

In principle, repeated stimuli in the presence of high [Ca2+]o might have also increased postsynaptic Ca2+ levels to trigger intracellular second messengers to modulate receptor sensitivity to GABA or glycine. Some considerations make this hypothesis unlikely: GABAA or glycinergic receptors are not permeable to Ca2+; even if intracellular Ca2+ was raised via influx through other channels or via internal store release, under our experimental condition (intracellular loading with 10 mM EGTA as buffer), postsynaptic [Ca2+]i is supposed to be maintained at rather constant level (Umemiya et al. 2001); raising [Ca2+]o enhanced glycinergic or GABAergic sPSC frequency with only slight effect on amplitude, and no change in current kinetics.

Collectively, these results indicate that increasing [Ca2+]o mainly affected synaptic transmission at presynaptic level.

Functional significance of facilitation of GABAergic or glycinergic currents

Short-term changes in synaptic efficacy can be caused by presynaptic changes, for example, in Ca2+entry or sequestration, in the number of synaptic vesicles or active release sites, or in the factors that lead to fusion of synaptic vesicles with the plasma membrane, or in the number of functional postsynaptic receptors (Magleby 1987). Further work will be necessary to clarify the mechanisms involved in the facilitation process observed on HMs. Notwithstanding the resolution of this issue, the striking difference found in the temporal information processing of GABAergic and glycinergic synapses opens the possibility for a markedly different functional role for the two neurotransmitters at least as young neonatal rat HMs are concerned. Developmental studies (Paton and Richter 1995) on the neonatal mouse brain stem slice preparation (600-700 µm thick; kept at 29°C) have examined GABAergic and glycinergic inputs from respiratory neurons to HMs: whereas block of neonatal glycinergic receptors does not affect the respiratory rhythm, block of GABA receptors increases burst duration and cycle length of respiratory-driven rhythmic activity, and it induces a sustained depolarization of the motoneuron membrane. Thus in neonatal animals, GABA-mediated activity seems to have the role of pacing the respiratory-driven rhythmic activity, whereas, at the same stage of development, glycine seems not to be involved in regulation of the respiratory rhythm. Since the normal respiratory rhythm of young rodents can be <= 5 Hz (Jacquin et al. 1996), it is plausible that this frequency could physiologically enhance GABAergic transmission to control the respiratory drive to neonatal HMs. Because at warmer ambient temperature the GABAergic current decay kinetics are expected to speed up, synaptic facilitation might predominate over summation as a mechanism to boost GABAergic events.


    ACKNOWLEDGMENTS

This work was supported by grants from Ministero dell'Universita' e della Ricerca Scientifica e Tecnologica and from Istituto Nazionale di Fisica della Materia.

Present address of R. Donato: Div. of Neurosciences, Baylor College of Medicine, Houston, TX 77030.


    FOOTNOTES

Address for reprint requests: A. Nistri, International School for Advanced Studies (SISSA), Via Beirut 4, 34014 Trieste, Italy (E-mail: nistri{at}sissa.it).

Received 13 October 2000; accepted in final form 27 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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