Properties of Miniature Glutamatergic EPSCs in Neurons of the Locomotor Regions of the Developing Zebrafish

Declan W. Ali, Robert R. Buss, and Pierre Drapeau

Centre for Research in Neuroscience, Montreal General Hospital Research Institute, and Departments of Neurology and Neurosurgery and of Biology, McGill University, Montreal, Quebec H3G 1A4 Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ali, Declan W., Robert R. Buss, and Pierre Drapeau. Properties of Miniature Glutamatergic EPSCs in Neurons of the Locomotor Regions of the Developing Zebrafish. J. Neurophysiol. 83: 181-191, 2000. As a first step in understanding the development of synaptic activation in the locomotor network of the zebrafish, we examined the properties of spontaneous, glutamatergic miniature excitatory postsynaptic currents (mEPSCs). Whole cell patch-clamp recordings were obtained from visually identified hindbrain reticulospinal neurons and spinal motoneurons of curarized zebrafish 1-5 days postfertilization (larvae hatch after the 2nd day of embryogenesis). In the presence of tetrodotoxin (TTX) and blockers of inhibitory receptors (strychnine and picrotoxin), we detected fast glutamatergic mEPSCs that were blocked by the AMPA/kainate receptor-selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). At positive voltages or in the absence of Mg2+, a second, slower component of the mEPSCs was revealed that the N-methyl-D-aspartate (NMDA) receptor-selective antagonist DL-2-amino-5-phosphonovalerate (AP-5) abolished. In the presence of both CNQX and AP-5, all mEPSCs were eliminated. The NMDA component of reticulospinal mEPSCs had a large single-channel conductance estimated to be 48 pS. Larval AMPA/kainate and NMDA components of the mEPSCs decayed with biexponential time courses that changed little during development. At all stages examined, approximately one-half of synapses had only NMDA responses (lacking AMPA/kainate receptors), whereas the remainder of the synapses were composed of a mixture of AMPA/kainate and NMDA receptors. There was an overall increase in the frequency and amplitude of mEPSCs with an NMDA component in reticulospinal (but not motoneurons) during development. These results indicate that glutamate is a prominent excitatory transmitter in the locomotor regions of the developing zebrafish and that it activates either NMDA receptors alone at functionally silent synapses or together with AMPA/kainate receptors.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Glutamate is the major excitatory neurotransmitter of the vertebrate CNS, where it mediates fast synaptic transmission through activation of N-methyl-D-aspartate (NMDA) and non-NMDA (AMPA/kainate and kainate) receptors. Glutamatergic ionotropic receptors have been suggested to play important roles in synaptic transmission (Edmonds et al. 1995), plasticity (Nicoll and Malenka 1995), and development (Scheetz and Constantine-Patton 1994), although in vivo studies of development have been limited. One of the first central neural networks to develop is that for locomotion, where glutamatergic inputs are essential components of the motor circuits in adult animals (Grillner et al. 1997). Application of glutamate or NMDA can induce fictive activity patterns in the embryonic spinal cord of Xenopus (Dale and Roberts 1984), chick (Barry and O'Donovan 1987), and rat (Ozaki et al. 1996). However, little is known of the properties of synaptic glutamate receptors on neurons in the developing locomotor network. Pure AMPA/kainate and NMDA-mediated excitatory postsynaptic potentials (EPSPs) as well as mixed responses have been observed in spinal neurons of the Xenopus embryo in the absence of Mg2+ (Dale and Roberts 1985; Sillar and Roberts 1991). Recent studies of spontaneous miniature excitatory postsynaptic currents (mEPSCs) (Gao et al. 1998) and evoked responses (Bardoni et al. 1998; Hori and Kanda 1996) recorded in neonatal rat spinal cord slices have indicated an early role for AMPA/kainate receptors. This contrasts with observations in the developing rat hippocampus (Durand et al. 1996; Isaac et al. 1995; Kullmann 1994) or frog tectum (Wu et al. 1996) where functionally silent synapses with an NMDA component but lacking an AMPA/kainate component have been described. Whether changes in the proportion of AMPA/kainate and NMDA components occur in the intact spinal cord in other preparations and in locomotor regions in general remains unanswered.

We have examined the in vivo development of glutamatergic synapses by recording from neurons in the locomotor regions of the embryonic and larval zebrafish. An advantage of the zebrafish for studies of locomotor network development is that there are relatively few types of neurons within these regions. The two regions that are essential for motor behaviors in the developing zebrafish (in the absence of added neuromodulators) are the hindbrain and spinal cord (Saint-Amant and Drapeau 1998), which are common regions for the locomotor networks of all vertebrates (Grillner et al. 1997). In the developing zebrafish, many neurons have been identified morphologically both in the hindbrain (Kimmel et al. 1981; Mendelson 1985, 1986; Metcalfe et al. 1990) and spinal cord (Bernhardt et al. 1990; Kuwada et al. 1990; Myers et al. 1986). In this study, we have characterized the properties of spontaneously occurring mEPSCs, including their AMPA/kainate and NMDA components, in hindbrain reticulospinal neurons and spinal motoneurons as an index of the features of excitatory synapses in the locomotor network. We found that glutamate is a prominent excitatory transmitter in the locomotor regions of the developing zebrafish and that it activates either NMDA receptors alone at functionally silent synapses or together with AMPA/kainate receptors.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparations

Embryos and larvae were raised at 28.5°C and were obtained from a zebrafish (Danio rerio) colony maintained according to established procedures (Westerfield 1995). All procedures were carried out in compliance with the guidelines stipulated by the Canadian Council for Animal Care and McGill University. Embryos and newly hatched larvae were anesthetized in 0.02% tricaine (MS-222) (Sigma Chemical, St. Louis, MO) and dissected as described by Drapeau et al. (1999). Briefly, the entire hindbrain was exposed after removing rostral structures, but leaving the spinal cord intact, and recordings were obtained from reticulospinal neurons located in the central rhombomeres. For spinal neuron recordings, muscle overlaying the spinal cord was removed from one to three somites in the midtrunk region in otherwise intact larvae. The preparations were moved to the recording set-up and the chamber was continuously perfused with an aerated recording solution that contained 15 µM D-tubocurarine (Sigma Chemical) to paralyze the preparations but lacked tricaine. The recording solution contained (Evans 1979) (in mM) 134 NaCl, 2.9 KCl, 2.1 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, osmolarity adjusted to 280-290 mOsm, pH 7.8. For Mg2+-free solution, MgCl2 was omitted from the recording solution without any further change. Preparations remained healthy for several hours under these recording conditions. We did not need to add glycine to the solutions during the recording of NMDA-mediated mEPSCs as these were always apparent in its absence and their frequency was unchanged throughout the recordings, presumably due to a maintained endogenous level of spontaneously released glycine.

Cell bodies were easily identified under Hoffman modulation optics. In preliminary experiments, cells were filled with Lucifer yellow or sulforhodamine B to confirm their identity (Drapeau et al. 1999), and in this study, six spinal neurons located dorsal to the central canal were filled with sulforhodamine B and their identity confirmed as motoneurons. However, as not all spinal neurons were filled it is possible that a small percentage could be interneurons. As no differences were found in the properties of the glutamatergic mEPSCs between the Mauthner cells and other reticulospinal neurons, these cells are referred to collectively as reticulospinal neurons.

Whole cell recordings

Standard whole cell recordings (Hamill et al. 1981) were performed 20-40 min after dissection (Drapeau et al. 1999). Patch-clamp electrodes were pulled from thin-walled, Kimax-51 borosilicate glass and filled with a Cs-gluconate solution and had resistances of 4-7 MOmega . Cs-gluconate intracellular solution was composed of (in mM) 115 Cs-gluconate, 15 CsCl, 2 MgCl2, 10 HEPES, 10 EGTA, and 4 Na2ATP, osmolarity adjusted to 290 mOsm, pH 7.2.

Whole cell currents were recorded using Axopatch 1A and 1D amplifiers (Axon Instruments) and were low-pass filtered at 5-10 kHz (-3 dB) and digitized at 20-25 kHz. The series resistance was monitored during whole cell recordings by applying 2 mV hyperpolarizing pulses. Series resistances (<= 10 MOmega ) were uncompensated, and neurons that had input resistances <100 MOmega or resting membrane potentials less than -45 mV were discarded. In all cells examined, voltage steps of 20-30 mV elicited fast, transient inward currents that were blocked by TTX. The liquid junction potential for Cs-gluconate recording solution was +5 mV and was corrected. Measurements were performed >= 5 min after obtaining the whole cell configuration to ensure cell dialysis. All drugs were dissolved in the aerated recording solution and applied by bath perfusion. Tetrodotoxin (1 µM), strychnine (5 µM) and picrotoxin (100 µM) were obtained from Sigma Chemical,) whereas CNQX (10 µM), AP-5 (50 µM) and L-glutamic acid were obtained from RBI (Natick, MA).

Analysis of mEPSCs

Synaptic activity was monitored using pClamp 6 software (Axon Instruments). The data were refiltered at 2 kHz using a second order Chebychev digital filter and the synaptic events were detected (using the template function for events >2.5 SD above the basal noise) and analyzed with Axograph 3.56 software (Axon Instruments). The software detected all events that could be recognized visually. All events were inspected visually, and those with uneven baselines or overlaying events (<5%) were discarded. The decay time course was analyzed over the first 40 ms for pure AMPA/kainate events and 400 ms for pure NMDA or mixed AMPA/kainate-NMDA events and was fitted with a sum of exponential curves. The presence of one, two, or three exponential components during the decay of the mEPSCs was tested by comparing the sum of squared errors of the fits over the same decay ranges (Clements and Westbrook 1991). The rise times were defined as the time from 20 to 80% of the mEPSC amplitude, and the half-widths were defined as the time between the rising and decay phases of each mEPSC at 50% of the peak amplitude. The amplitude and decay of the NMDA component was determined by analyzing events with both AMPA/kainate and NMDA components. Averages of these events were best fit with three exponential curves; two exponential curves were sufficient in some motoneurons where the AMPA/kainate component was too small to contribute to the peak amplitude of the mEPSC. The fastest exponential had a time course nearly identical to that of pharmacologically isolated AMPA/kainate events. The amplitude of this exponential was subtracted from the total average amplitude, yielding an estimate of the amplitude of the NMDA component. The two slower exponential curves were attributed mainly to the slower AMPA/kainate component and the NMDA component. As there was essentially no developmental change in the kinetics of pharmacologically isolated AMPA/kainate events, any changes observed in the decay of the mixed events could be attributed to the NMDA component. The frequency of synaptic events was determined manually because the Axograph detection software could not always detect the variably shaped pure NMDA or mixed events. Results are presented as means ± SE throughout the text unless otherwise noted. Correlations were tested using the Pearson product moment correlation, and a significant relationship was noted when P < 0.05. The term significant denotes a relationship with P < 0.05 determined using the paired t-test, Student's t-test, Mann-Whitney test, or Kruskal-Wallis test, as appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we examined mEPSCs in 1- to 5-day-old (postfertilization) animals, from when embryos (which hatch on day 2) first show motor behaviors (day 1) to when larvae actively swim to feed (day 4 or 5).

Properties of reticulospinal neurons

The embryonic zebrafish hindbrain is segmented into eight rhombomeres that each possesses only a few bilateral pairs of descending reticulospinal neurons that innervate the spinal cord (Kimmel et al. 1981; Mendelson 1985, 1986; Metcalfe et al. 1990). These include the largest pair, the Mauthner neurons, whose morphological development has been well characterized in the zebrafish (Kimmel et al. 1981). In addition, their physiological properties have been studied in the adult zebrafish (Hatta and Korn 1998), whereas glycinergic miniature inhibitory postsynaptic currents (mIPSCs) have been characterized in the isolated hindbrain of the zebrafish larva (Legendre and Korn 1994). The Mauthner neurons together with other reticulospinal neurons in the caudal rhombomeres are active during the escape response (O'Malley et al. 1996).

Reticulospinal neurons were recorded from 1.2 day-old embryos to 3.1 day-old larvae. The low input resistance of the larger cells in older larvae precluded high-resolution recordings at later times. The values of the electrophysiological parameters are summarized in Table 1. The resting potential was on average -56 ± 1.6 mV (n = 28) and ranged from -45 to -69 mV. The input resistance (Ri) of reticulospinal neurons decreased significantly (~5-fold) during development, from 1.0 ± 0.18 GOmega in embryos to 0.19 ± 0.02 GOmega in 3.1-day-old larva.


                              
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Table 1. Reticulospinal neurons

Properties of spinal motoneurons

The spinal cord of the early zebrafish is segmented into some 30 somites, each half of which contains two anatomic classes of sensory neurons, seven classes of interneurons (Bernhardt et al. 1990; Kuwada et al. 1990), and two classes of motoneurons (Myers et al. 1986). In the adult zebrafish, the two classes of motoneurons underlie tonic and phasic activation of the trunk musculature during swimming (Liu and Westerfield 1988). Motoneurons (29 cells; see Table 2) from 2.3- to 5.4-day-old larvae had a mean resting potential of -63 ± 1.1 mV (range, -50 to -74 mV) and input resistances of 0.35 ± 0.04 GOmega . Resting potentials in motoneurons were significantly more negative in older animals and in comparison with reticulospinal neurons. There was also a trend to decreasing input resistance in motoneurons of older animals. In preliminary current-clamp experiments (n = 4), application of L-glutamic acid (0.7-2.0 mM) in the presence of 1 µM TTX was observed to depolarize spinal neurons close to 0 mV (data not shown). This indicated the presence of glutamate receptors, and we sought to characterize their synaptic properties at different stages of development by voltage-clamp recording of pharmacologically isolated miniature synaptic currents.


                              
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Table 2. Spinal motoneurons

Basal synaptic activity and the AMPA/kainate component of mEPSCs

In Mg2+ and Mg2+-free saline with a Cs-gluconate intracellular solution, all reticulospinal neurons in newly hatched (2-day-old) larvae exhibited a high level of synaptic activity, including regular bursting episodes as shown in Fig. 1A1. Motoneurons generally received a lower level of basal synaptic activity and bursting episodes that occurred spontaneously or could be evoked by dimming the lights (Fig. 1B1). These bursts of synaptic activity are likely related to "fictive" swimming or escape behaviors. In half (10/20) of the motoneurons, small (~10 pA) long-lasting (~1 s) inward currents were observed in the presence of TTX and did not resemble the synaptic currents described here.



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Fig. 1. Spontaneous synaptic activity. Whole cell recordings of spontaneously occurring synaptic activity in a reticulospinal neuron (A) and a motoneuron (B). Activity before (A1 and B1) and after (A2 and B2) bath application of 1 µM TTX, 1 µM strychnine, and 100 µM picrotoxin. Holding potential for for both neurons was -65 mV. A3 and B3: no miniature excitatory postsynaptic currents (mEPSCs) were recorded after bath application of 10 µM 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX). A4 and B4: individual (overlapped) miniature EPSCs (mEPSCs) recorded in the presence of TTX, strychnine, and picrotoxin. Right traces in A4 and B4 show averaged, normalized mEPSCs (---) overlaid with a similar trace of mEPSCs recorded in the presence of 50 µM DL-2-amino-5-phosphonovalerate (AP-5, - - -).

The chloride reversal potential was approximately -50 mV such that inhibitory currents would be inward at a holding potential of -65 mV. Therefore to distinguish inward, excitatory currents from inhibitory currents we recorded in the presence of 5 µM strychnine and 100 µM picrotoxin to block glycine- and GABA- mediated currents (Triller et al. 1997). Spontaneous miniature events were recorded in the presence of 1 µM TTX to block Na+-dependent action potentials. The preparation was perfused continuously with 15 µM D-tubocurarine to prevent muscle contractions during the recordings. The high level of spontaneous synaptic activity was reduced greatly in the presence of TTX, strychnine, and picrotoxin (Fig. 1, A2 and B2). The remaining spontaneous miniature synaptic events (mEPSCs) recorded in the presence of Mg2+ were abolished completely by 10 µM 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX; Fig. 1, A3 and B3), a potent AMPA/kainate receptor antagonist, suggesting that they were due to the activation of glutamatergic AMPA/kainate receptors.

Application of AP-5 had no effect on the decay of the mEPSCs (Fig. 1, A4 and B4) or on their amplitude or frequency (data not shown). Thus near the normal resting conditions (-65 mV holding potential) NMDA receptors were unlikely to contribute to the synaptic response. AMPA/kainate-dependent mEPSCs (recorded in the presence of AP-5 or Mg2+) had fast rise times (20-80%) of ~0.2 ms, and averaged traces were well fit with two exponential decays in both reticulospinal and motoneurons (P < 0.05). The kinetic parameters of AMPA/kainate events were similar in reticulospinal neurons and spinal motoneurons and were largely unchanged throughout the developmental stages examined (Tables 1 and 2). The mean event amplitudes and frequencies (the latter of which were highly variable) did not differ significantly but tended to increase in older animals.

An amplitude histogram from a representative cell (reticulospinal, Fig. 2A) showed that the large majority of AMPA/kainate events were clustered between 10 and 30 pA, although the distribution was skewed toward larger amplitudes. The 20-80% rise times in both types of neurons were tightly clustered between 0.1 and 0.3 ms (Fig. 2B), although there were some rare events (<1%) with larger rise times. A concern when recording in the whole cell configuration is the effectiveness of the space clamp. Miniature synaptic events that are not space-clamped will be filtered and will appear to have longer rise times, smaller amplitudes, and longer decays (Rall 1969). We therefore examined the correlation between the rise time and half-width (Fig. 2C) or amplitude (Fig. 2D) of events. The lack of correlation (r < 0.4) for the reticulospinal neuron of Fig. 2, C and D, and in the recordings in both reticulospinal neurons and motoneurons suggests that cells at various developmental stages were properly space-clamped. This is consistent with our ability to resolve such fast events; except for a minute fraction of the events that were either less well clamped or perhaps were less-mature synapses.



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Fig. 2. Isolated AMPA/kainate mEPSCs. A: amplitude histogram of mEPSCs from a reticulospinal neuron recorded in the presence of 1 µM TTX, 1 µM strychnine, and 100 µM picrotoxin (average: 24.0 ± 0.5 pA; n = 710 events; binwidth =1 pA). B: 20-80% rise time distribution of mEPSCs (average: 0.172 ± 0.001 ms; binwidth =10 µs). C: scatter plot of the rise time vs. half-width of the events. D: scatter plot of rise time vs. mEPSC amplitude. Note the lack of correlation in C (r = 0.31) and in D (r = 0.16). Holding potential was -65 mV.

NMDA component of the mEPSCs

To overcome the voltage-dependent Mg2+ block of NMDA receptors (Mayer and Westbrook 1987) and detect their possible contribution to mEPSCs, we voltage-clamped cells at +45 mV (Fig. 3A). Under these conditions, we were able to detect mEPSCs that were blocked only by adding both CNQX and AP-5 and that reversed near 0 mV. A recording performed on a day 3 motoneuron (Fig. 3) shows that at +45 mV, averaged mEPSCs had rapid rise times and decayed with a fast component and a much slower, second component. The slow component disappeared in the presence of 50 µM AP-5, and 10 µM CNQX abolished the remaining fast component (not shown), confirming that they were due to the activation of NMDA and AMPA/kainate receptors, respectively. The NMDA component was larger as the holding potential was made more positive. This is shown in Fig. 3B, where averaged traces of mEPSCs at various holding potentials (-75, -55, -35, -15, and +45 mV) are normalized, inverted where appropriate, and overlaid. The inset shows similarly normalized AMPA/kainate mEPSCs at three different potentials (-75, -35, and +45 mV) isolated in the presence of 50 µM AP-5. The later events decayed with similar time courses, indicating that the AMPA/kainate component of the mEPSCs was voltage independent. The preceding observations were confirmed in four other spinal motoneurons and five larval reticulospinal neurons.



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Fig. 3. N-methyl-D-aspartate (NMDA) component of the mEPSCs at positive holding potentials. A: recording from a day 3 motoneuron at 2 different holding potentials (-75 and +45 mV). Note the appearance of a slowly decaying component at +45 mV that is AP-5 sensitive. B: normalized traces at several different holding potentials (-75, -55, -35, -15, and +45 mV). NMDA component is more apparent at positive holding potentials. Inset: normalized, overlaid AMPA/kainate mEPSCs in the presence of 50 µM AP-5 at -75, -35, and +45 mV. Note that the traces all have similar decay time courses. All traces are averages of >= 25 individual events.

Recordings made at positive potentials, where the NMDA component was revealed fully, were noisy due to the opening and closing of background voltage-dependent channels. Therefore to resolve better the NMDA events we recorded mEPSCs in Mg2+-free extracellular solution as shown in Fig. 4. Averages of events in the presence of either 10 µM CNQX or 50 µM AP-5 are shown, respectively, in Fig. 4A, whereas the inset reveals on an expanded time scale the slower rise time of the CNQX-insensitive events. A rise time-amplitude scatter plot (Fig. 4B) suggests that there are two separate classes of events: slower events (>0.5-ms rise times) with smaller amplitudes (<30 pA), and larger events (>30 pA) with faster rise times (<0.5 ms). Application of CNQX (Fig. 4C) followed by washout and then application AP-5 (Fig. 4D) to the same cell revealed separate populations of slow and fast events. The AP-5-resistant events (Fig. 4, A and D) resembled the AMPA/kainate component of the mEPSCs described earlier in the presence of Mg2+(Figs. 1, A4 and B4, and 2D). A clear separation of events based on amplitude and rise time as shown in Fig. 4C was not always observed, thus it was not possible to distinguish AMPA/kainate events from NMDA events based on rise time alone.



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Fig. 4. Isolation and detection of individual AMPA/kainate and NMDA events in Mg2+-free recording solution. A: averaged mEPSCs recorded in a 3-day-old motoneuron in the presence of either 10 µM CNQX or 50 µM AP-5. Inset: peak currents and rising phases on an expanded time scale. B: scatter plots of 20-80% rise time vs. amplitude and in the presence of 10 µM CNQX (C) or 50 µM AP-5 (D). Note the different populations of slower events in C and faster events in D. Holding potential was -65 mV.

To estimate the proportion of the NMDA events without a distinguishable AMPA/kainate component, the frequency of events that were detected after application of AP-5 (AMPA/kainate only) was compared with the frequency of all events (a mixture of AMPA/kainate and NMDA) detected before addition of AP-5. In AP-5 the frequency of events in reticulospinal neurons and motoneurons was reduced significantly by 52 ± 4.4% (P < 0.05, paired t-test for all ages pooled: reticulospinal n = 17, motoneuron, n = 16). This indicated that approximately half of the mEPSCs were due to activation of NMDA receptors at synapses with an undetectable AMPA/kainate component, and thus these synapses would be functionally silent at resting membrane potentials. The proportion of these functionally silent synapses did not change significantly during development in motoneurons where the reduction of mEPSC in AP-5 was 43 ± 9.2%, 43 ± 10%, and 57 ± 15% in 2-3-, 3-4-, and 4-5-day larvae, respectively. In the reticulospinal cells, the event frequency was reduced by 64 ± 5.6%, 59 ± 10%, and 38 ± 16% in 1.2-1.5-, 2.2-2.3-, and 3.0-3.1-day-old animals, respectively.

Isolated NMDA events

The CNQX-insensitive, isolated NMDA events were usually small in amplitude and had highly variable rise times and prolonged decays (Fig. 4A, inset). Thus it was difficult to detect the NMDA events in most cells by the automated procedure, which did so with confidence in only five preparations: four embryonic reticulospinal neurons and one motoneuron (Fig. 4) at 2-3 days. Figure 5 shows pooled data for the NMDA events from the four reticulospinal neurons. The majority of the detected events had amplitudes clustered around 10-15 pA (Fig. 5A). The 20-80% rise times (Fig. 5B) were 12-fold slower (0.4-18 ms: 2.4 ± 0.13 ms; n = 4 cells) than those of the AMPA/kainate component. Such ranges of rise times were comparable with the rise times of the slower population of mixed glutamatergic mEPSCs observed in both reticulospinal neurons and motoneurons. Figure 5, C and D, reveals the lack of negative correlation (r < 0.4) between the rise times and amplitudes or half-widths, suggesting that the space clamp was effective. The NMDA events in reticulospinal neurons were best fit with two decay time constants of 14.1 ± 2.1 ms and 132 ± 25 ms (14 ± 4%). The two decay time constants for the motoneuron were 7.3 and 51 ms (21%). The difficulty in detecting these pure NMDA synaptic events prevented a more detailed analysis of developmental changes. However, developmental changes of NMDA synapses were examined indirectly by subtracting the AMPA/kainate component from synaptic events, recorded in the absence of Mg2+ (see following text).



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Fig. 5. Parameters of isolated NMDA mEPSCs taken from 4 separate reticulospinal neurons. A: amplitude distribution (average: 15.8 ± 0.5 pA; n = 246; binwidth =1 pA). B: 20-80% rise time distribution of pooled NMDA mEPSCs. Average: 2.5 ± 0.3 ms (binwidth = 0.5 ms). C: scatter plot of rise time versus amplitude for NMDA mEPSCs (r = 0.38). D: plot of rise time vs. half-width of NMDA mEPSCs (r = 0.36). Holding potential was -65 mV.

Single NMDA channel conductances

Some mixed synaptic events in young embryonic reticulospinal neurons (day 1.2-1.5) with high-input resistances showed single-channel openings (Fig. 6), indicating either a low probability of channel opening or a low density of postsynaptic receptors at these early synapses. The events often started (Fig. 6A) with a large and brief AMPA/kainate current transient and ended with repeated single, presumably NMDA channel openings. The AMPA/kainate current presumably was generated by the opening of several channels as we could not distinguish single-channel events; this is not surprising as the AMPA/kainate channel conductances are reported to be small (Edmonds et al. 1995). In other cases (Fig. 6B), the fast AMPA/kainate component could not be distinguished because of the opening of few or possibly no channels. These events had highly variable time courses that represent NMDA channel openings rather than spurious openings of background channels, as the activity was largely abolished by perfusion with AP-5 (not shown). An amplitude histogram of the late currents of a single mixed event recorded at -65 mV (Fig. 6C) indicated a primary peak of -3.2 pA. Because the mEPSCs reversed near 0 mV (data not shown), this corresponds to a single NMDA channel conductance of 49 pS. The average single channel conductance (13 events from 4 cells) was estimated as 48 ± 2 pS in embryonic reticulospinal neurons. More detailed single channel studies over a range of potentials will be needed to confirm this estimate of the conductance. We were unable to detect single-channel NMDA events in motoneurons due to the lower input resistance of the cells.



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Fig. 6. Late currents show NMDA single-channel events. A: examples of 4 glutamatergic mEPSCs detected in a reticulospinal neuron of a 1.5-day-old embryo held at -65 mV in which the signal to noise ratio is high. B: examples of predominantly NMDA events from the same cell. C: point-by-point histogram of the first mixed event from A (shown is the inset) revealing a principal peak at -3.2 pA, which corresponds to a single-channel conductance of 49 pS.

Developmental changes in the properties of mEPSCs

As previously presented, the changes in the kinetics of isolated AMPA/kainate mEPSCs were minimal or absent during the developmental periods studied. Therefore any developmental changes in those events detected in the absence of Mg2+ without any glutamatergic antagonists present could be attributed to changes in the NMDA component of these mixed events. However, it is not definitively known whether an individual event arose from a synapse containing only AMPA/kainate channels, only NMDA channels, or a mixture of both AMPA/kainate and NMDA channels. To explore the composition of these mEPSCs, all events were detected manually and classified as pure AMPA/kainate, pure NMDA, or mixed AMPA/kainate-NMDA events based on our knowledge of the appearance of the pharmacologically isolated events. Less than 10% of the events resembled pure AMPA/kainate events. The analysis of functionally silent synapses (using AP-5) presented in the preceding text revealed that as many as half of the events could have been pure NMDA. However, as the variably shaped NMDA events were not automatically detected, only a portion of these events would be included in the averages, resulting in an underestimate of their contribution.

A clear developmental trend that we observed was an increase in the amplitude of the extrapolated NMDA component of the reticulospinal (but not motoneuron) mixed synapses. Mixed synapse NMDA event amplitudes in reticulospinal neurons (Table 2) more than doubled (4.9-11.6 pA, P < 0.05) in the transition from embryonic to larval stages. No such trend was observed in the motoneurons where the average NMDA component was large (ranging from 9.6 to 12.3 pA, Table 2) at all stages examined. In both reticulospinal cells and motoneurons, the difference was statistically significant when average rise times were compared in the same cells before and after AP-5, and average half-widths were two to four times slower for mixed events (not shown). These rise times were nonetheless faster than observed for the isolated NMDA events due to the mixed contribution of NMDA and AMPA/kainate receptors under these conditions. The average frequency of these mixed events increased significantly in larval (4.2 ± 1.4 Hz) compared with embryonic (1.2 ± 0.4 Hz) reticulospinal neurons (Table 1), whereas in motoneurons (Table 2), the frequency remained at ~1 Hz throughout the stages of larval development examined.

In some motoneurons (see METHODS) the AMPA/kainate component did not contribute to the peak amplitude, but its presence was inferred from the presence of fast rise times in the mixed events. Furthermore two exponentials were used to fit the averaged events, both being attributed to NMDA receptor activation. The decay of the majority of averaged, mixed events was best fit with three exponentials where the fast exponential was attributed to the AMPA/kainate component and the slower two exponentials to the NMDA component. The slower two exponentials attributed to the NMDA component are labeled tau off1 and tau off2 in Tables 1 and 2. The time course of tau off1 and tau off2 was variable at all stages of development in both reticulospinal and motoneurons, making it difficult to distinguish developmental trends. The NMDA component of all the mixed events (calculated from the means presented in Tables 1 and 2) was tau off1 = 11 ms, tau off2 = 60 ms (34%) and tau off1 = 7.4 ms, tau off2 = 37 ms (22%) in reticulospinal neurons and motoneurons, respectively.


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We observed that reticulospinal neurons and motoneurons of the curarized zebrafish embryo and larva had spontaneous mEPSCs that were entirely glutamatergic. This is not surprising as glutamate is the main excitatory transmitter of the vertebrate nervous system. However, because we included D-tubocurarine, this would have blocked cholinergic synapses if these occurred, as reported in spinal interneurons (Perrins and Roberts 1995a) and between motoneurons (Perrins and Roberts 1995b) of the Xenopus embryo. In preliminary (unpublished) experiments (n = 8) using a high magnesium/low calcium solution instead of D-tubocurarine to suppress muscle contractions, we failed to detect spontaneous cholinergic mEPSCs. This indicates that glutamate is indeed the main if not sole excitatory transmitter in the developing zebrafish. In the absence of TTX, both types of neurons we examined showed bursts of PSCs, suggesting that these may be related to fictive swimming or escape behaviors, although we did not examine this in any detail. The amplitude histograms of the mEPSCs were skewed (non-Gaussian) toward larger events, as described for glycinergic mIPSCs in the larval zebrafish hindbrain (Legendre and Korn 1994). Such variability in quantal size may be accounted for by presynaptic factors, such as variation in quantal content or multiquantal release, or by postsynaptic factors, including receptor properties and densities (Bekkers and Stevens 1996; Bekkers et al. 1990; Legendre and Korn 1994; Nusser et al. 1997; Ulrich and Luscher 1993).

AMPA/kainate component of the mEPSC

In the presence of TTX and at -65 mV, near the normal resting potential, the mEPSCs were entirely due to AMPA/kainate receptor activation, and these were well space clamped in the whole cell recordings. The AMPA/kainate component of the mEPSCs showed voltage-independent kinetics that were fast, both in their rise times and biexponential decay time constants. These fast kinetics are consistent with those reported for synaptic potentials in the Mauthner neuron of the goldfish (Wolszon et al. 1997) and at central synapses in the mammalian brain (reviewed by Edmonds et al. 1995) where the decay time course of EPSCs is usually monoexponential. However, a second decay phase has been reported for cerebellar granule cells (Barbour et al. 1994; Silver et al. 1992).

NMDA component of the mEPSC

An NMDA component of the mEPSCs was revealed only in the absence of Mg2+ or at depolarized potentials, as expected if Mg2+ blocks NMDA receptors at the resting membrane potential in zebrafish as in other preparations. Recently the NR1 subunit has been cloned in another teleost and shows a high degree (overall 88%) of homology with the mammalian subunit, including essentially identical (>98%) ligand binding sites and pore region (Bottai et al. 1998), the latter being the presumed Mg2+ binding site. The kinetics of the NMDA component of the mEPSCs were an order of magnitude slower than those of the AMPA/kainate component with respect to both the rise times and biexponential decays. In reticulospinal neurons of the youngest embryos examined, single-channel openings with a conductance of ~48 pS were estimated. This is similar to the conductances described for other native NMDA receptors activated at single synapses in the immature mammalian brain (Gibb and Colquhoun 1991; Lester and Jahr 1992; LoTurco et al. 1991; Silver et al. 1992), in cultured spinal neurons (Robert et al. 1998) and in embryonic Xenopus spinal neurons (Zhang and Auerbach 1995).

The NMDA component remained essentially biexponential over the stages examined, and the time constants did not vary much with development, remaining relatively fast with the slower decay being between 30 and 60 ms. At several immature (early postnatal) mammalian central synapses, the evoked NMDA responses last several hundred milliseconds, and a decrease in the decay phase has been observed during development (Carmignoto and Vicini 1992; Crair and Malenka 1995; Golshani et al. 1998; Hestrin 1992; Ramoa and McCormick 1994). This also has been observed at the level of single NMDA channels isolated in outside-out patches (Carmignoto and Vicini 1992; Hestrin 1992), indicating a developmental alteration of NMDA channel properties that is likely due to a switch in subunit composition (Flint et al. 1997; Monyer et al. 1994). The mEPSCs in zebrafish embryos and larvae were apparently already fast, suggesting that at these early stages there are no major changes in the kinetic properties of the receptors. Glycinergic synapses in the Mauthner neuron also are thought to be mature by hatching (~52 h postfertilization, hpf) (Triller et al. 1997). It thus appears that the zebrafish has a remarkably rapid maturation of central synapses, occurring during embryonic development. As we recorded from neurons 1.2 days (28 hpf) and older in the hindbrain, where axogenesis starts between 20 and 21 hpf (Mendelson 1985), it may be that the earliest period of functional synaptogenesis is as brief as a few hours.

A developmental trend that was observed in the NMDA component of mixed synapses was a doubling in event amplitudes in reticulospinal neurons but not in motoneurons during the stages examined. A likely explanation is an increase in the number of NMDA receptors at synapses onto reticulospinal neurons. The frequency of mixed glutamatergic mEPSCs also tended to increase in the reticulospinal neurons throughout the stages examined, presumably reflecting synaptogenesis. It thus appears that new synapses are added continually in the hindbrain of the zebrafish embryo and larva and that these vary in their AMPA/kainate and NMDA receptor content with half the synapses lacking the former and thus being functionally silent under resting conditions. Interestingly, the hindbrain appears to mature over a longer period than the spinal cord in the developing zebrafish. Thus swimming, which is mediated by a spinal central pattern generator, begins at 28 hpf and reaches a maximal rate by 36 hpf (Saint-Amant and Drapeau 1998). In contrast, hindbrain Mauthner neurons mediating escape behaviors are responsive to sensory inputs from the auditory and visual systems only a few days after hatching (Eaton and Farley 1973), which is the period when large numbers of afferent synapses are formed onto the Mauthner cell dendrites (Kimmel et al. 1981). Thus the extended development of the NMDA component of glutamatergic synapses may be related to the protracted development of afferent innervation in the hindbrain.

Functionally silent synapses

Approximately half of the mEPSCs had an NMDA component but lacked an AMPA/kainate component both in reticulospinal and motor neurons at all stages examined. A smaller fraction (~20%) of these types of responses also was observed for dorsal horn neurons in the neonatal rat spinal cord (Bardoni et al. 1998). These responses thus resemble "silent" central synapses (Durand et al. 1996; Isaac et al. 1995; Kullmann 1994; Liao et al. 1995; Wu et al. 1996) at which the evoked NMDA component is thought to precede the AMPA/kainate component, thus being functionally silent due to Mg2+ block at the resting potential. The activation of NMDA responses, presumably coincident with a depolarizing event, and consequent calcium influx has been proposed as a mechanism of plasticity for the induction of AMPA/kainate receptor expression at synapses in these brain regions, triggering conversion from functionally silent to mature synapses (Wu et al. 1996). It may be that these functionally silent synapses in zebrafish locomotor regions are activated during swimming once the cells have passed spike threshold and the magnesium block is removed from the NMDA channel. The functionally silent synapses could thus contribute to locomotor drive potentials and be especially important in providing a positive feedback pathway.


    ACKNOWLEDGMENTS

D. W. Ali and R. R. Buss contributed equally to this work.

This work was supported by grants (to P. Drapeau) and awards from the National Science and Engineering Research Council (fellowship to D. W. Ali) and Medical Research Council of Canada (studentship to R. R. Buss).

Present address of D. W. Ali: Programme in Brain and Behavior, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada.


    FOOTNOTES

Address for reprint requests: P. Drapeau, Dept. Neurology, Montreal General Hospital, 1650 Cedar Ave., Montreal, QC H3G 1A4, Canada.

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.

Received 6 July 1999; accepted in final form 9 September 1999.


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