Glycinergic mIPSCs in mouse and rat brainstem auditory nuclei: modulation by ruthenium red and the role of calcium stores

Abstract

Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in central neurons are usually highly variable in amplitude due to many factors such as intrinsic postsynaptic channel fluctuations at each release site, site-to-site variability between release sites, electrotonic attenuation due to variable dendritic locations of synapses, and the possibility of synchronous multivesicular release. A detailed knowledge of these factors is essential for the interpretation of mIPSC amplitude distributions and mean quantal size. We have studied glycinergic mIPSCs in two auditory brainstem nuclei, the rat anteroventral cochlear nucleus (AVCN) and the mouse medial nucleus of the trapezoid body (MNTB). Our previous results have demonstrated the location of glycinergic synapses on these neurons to be somatic, thus avoiding electrotonic complications. Spontaneous glycinergic mIPSCs were recorded from AVCN and MNTB neurons in brainstem slices, in the presence of TTX to block action potentials, and 6-cyano-7-nitroquinoxaline-2, 3-dione, (+/-)-2-amino-5-phosphonopentanoic acid and bicuculline to block glutamatergic and GABAergic synaptic currents. Ruthenium red (RuR), which was used to increase the frequency of mIPSCs, significantly changed the shape of most (90 %) mIPSC amplitude distributions by increasing the proportion of large-amplitude mIPSCs. The possibility was investigated (following previous evidence at GABAergic synapses) that large-amplitude glycinergic mIPSCs are due to synchronous multivesicular release initiated by presynaptic calcium sparks from ryanodine-sensitive calcium stores. Interval analysis of mIPSCs indicated that the number of potentially undetected (asynchrony < 0.5 ms) multivesicular mIPSCs was low in comparison with the number of large-amplitude mIPSCs. Ryanodine, thapsigargin and calcium-free perfusate did not reduce the frequency of large-amplitude mIPSCs (> 150 pA), arguing against a significant role for presynaptic calcium stores. Our results support previous evidence suggesting that RuR increases miniature postsynaptic current (mSC) frequency by a mechanism that does not involve presynaptic calcium stores. Our results also indicate that at glycinergic synapses in the AVCN and MNTB, site-to-site variability in mIPSC amplitude, rather than multivesicular release, is a major factor underlying the large range of amplitudes of glycinergic mIPSCs.

Figure 1. Ruthenium red (RuR) increases the frequency and mean peak amplitude of glycinergic miniature inhibitory postsynaptic currents (mIPSCs)

Rat anteroventral cochlear nucleus (AVCN) data. A and B, continuous whole-cell recordings of glycinergic mIPSCs in a rat AVCN neuron before and after the application of RuR (100 µm). There was a significant increase in the frequency of mIPSCs following application of RuR. The increase in frequency was maximal or reached a plateau 10 min after adding RuR to the perfusion solution. C, RuR induced a significant increase in the mean amplitude of glycinergic mIPSCs (bold trace). D, histograms of glycinergic mIPSC peak amplitudes in control (shaded) and in the presence of RuR (open) reveal an increase in the number of large quantal events following application of RuR, which is also illustrated by the shift to the right (RuR; bold line) of the cumulative probability distribution (inset).

Figure 2. Interval analysis of mIPSCs does not indicate multiquantal release

Rat AVCN data. A, two pairs of closely spaced quantal events (P1 and P2) in the presence of RuR. The interevent interval was measured as the time between the peak amplitudes of the two closely spaced events. B, histogram of interevent intervals between all mIPSCs recorded in the presence of RuR. The inset shows the distribution of interevent intervals of mIPSCs that occurred within 0.5 and 10 ms of each other (bin size = 1 ms).

Figure 3. RuR does not act via voltage-activated calcium channels to increase mIPSC frequency

Mouse medial nucleus of the trapezoid body (MNTB) data. A, sample continuous recordings of glycinergic mIPSCs from a mouse MNTB neuron in the presence of TTX, under control conditions, with added cadmium (200 µm) and with added cadmium + RuR. A minimum of 10 min was allowed following application of each solution before measurements were made of mIPSCs. B, summary data for mIPSC amplitude and frequency (n = 4 cells) shows no difference between control and cadmium conditions, but a significant increase in both the frequency and amplitude of mIPSCs following addition of RuR.

Figure 4. BAPTA AM does not affect mIPSC amplitude and frequency

Mouse MNTB data. A, amplitude histogram of mIPSCs (n = 551) in the presence of RuR (100 µm). B, amplitude histogram of mIPSCs (n = 431) for the same cell as A, following the addition of BAPTA AM (100 µm). C, summary data (n = 6 cells) showing a significant increase in mIPSC frequency and amplitude between control and following addition (> 10 min after solution change) of RuR (100 µm), but no significant difference between RuR and RuR + BAPTA AM (100 µm).

Figure 5. Ryanodine and thapsigargin do not affect mIPSC amplitude or frequency

Mouse MNTB data. A, summed mIPSC amplitude histograms (RuR, n = 4870 mIPSCs; RuR + ryanodine (Ryan), n = 4790 mIPSCs; 10 cells), showing a lack of affect of ryanodine (100 µm) on the distribution of mIPCS amplitudes. B, summed mIPSC amplitude histograms (RuR, n = 3654 mIPSCs; RuR + thapsigargin (Thaps, n = 3498 mIPSCs; six cells), showing a lack of effect of thapsigargin (10 µm) on the distribution of mIPSC amplitudes. C, summary data showing a lack of effect of ryanodine (100 µm) and thapsigargin (10 µm) on mIPSC mean amplitude (C, n = 10 cells) and mean frequency (D, n = 6 cells) in the presence of RuR (100 µm).