Subtle differences in synaptic transmission in medial nucleus of trapezoid body neurons between wild-type and Fmr1 knockout mice

Abstract

In animal models for fragile X syndrome where the gene for fragile X mental retardation protein is knocked out (Fmr1 KO), neurotransmission in multiple brain regions shifts excitation/inhibition balance, resulting in hyperexcitability in neural circuits. Here, using whole-cell recordings from brainstem slices, we investigated synaptic transmission at the medial nucleus of trapezoid body (MNTB, a critical nucleus in the brainstem sound localization circuit), in Fmr1 KO and wild-type (WT) mice 2–3 weeks of age in both sexes. Surprisingly, neither synaptic excitation nor inhibition in KO neurons was significantly changed. The synaptic strength, kinetics, and short-term plasticity of synaptic excitation remained largely unaltered. Subtle differences were observed in response patterns, with KO neurons displaying less all-or-none eEPSCs. Similarly, synaptic inhibition mediated by glycine and GABA remains largely unchanged, except for a slower kinetics of mixed sIPSCs. In pharmacologically isolated glycinergic and GABAergic inhibition, no significant differences in synaptic strength and kinetics were detected between the two genotypes. These results demonstrate that at the cellular level synaptic transmission at MNTB is largely unaffected in Fmr1 KO mice by 2–3 weeks after birth, suggesting the existence of compensatory mechanisms that maintain the inhibitory output of MNTB to its targets in the auditory brainstem.

1. Introduction

The mutation in fragile X mental retardation protein (FMRP) underlies fragile X syndrome (FXS), the leading single-gene cause for mental retardation (reviewed by Penagarikano et al., 2007). Knockout of the fragile X mental retardation gene 1 (Fmr1 KO) alters synaptic excitation and inhibition in multiple brain regions, disrupting excitation/inhibition (E/I) balance and resulting in hyperexcitability at the synaptic, cellular, and circuit levels (reviewed by Contractor et al., 2015). Among other deficits, Fmr1 KO animals display compromised sensory processing, especially in the higher cerebral cortical regions. For example, auditory cortical neurons exhibit abnormal auditory responses and impaired neural plasticity (Kim et al., 2013). Cells become hyperexcitable and animals become hypersensitive to sound, expressing audiogenic seizures and even death in response to loud sounds (reviewed by Rotschafer and Razak, 2014). The underlying mechanisms are usually attributed to imbalanced E/I, leaning towards a higher E/I ratio due to enhanced excitation or reduced inhibition or both.

In the brainstem sound localization circuit, such a disturbed E/I balance and consequently impaired sound processing in Fmr1 KO mice occur in the lateral superior olive (LSO) (Garcia-Pino et al., 2017). Of particular interest here is the medial nucleus of trapezoid body (MNTB), the inhibitory output of which plays essential roles in the computation of sound localization (reviewed by Grothe et al., 2010). MNTB neurons receive glutamatergic excitation via the giant synapse, calyx of Held (Guinan and Li, 1990; Kuwabara et al., 1991), and synaptic inhibition mediated by glycine and GABA, via bouton terminals originating from the ventral nucleus of trapezoid body and other unidentified sources (Awatramani et al., 2004, 2005; Albrecht et al., 2014; Mayer et al., 2014). FMRP is strongly expressed in MNTB neurons (Ruby et al, 2015; Zorio et al., 2017). In Fmr1 KO animals, calyxes become larger (Wang et al., 2015) with a reduction in calretinin expression in calyx terminals (Ruby et al., 2015), cell size decreases (Rotschafer et al., 2015; Ruby et al., 2015), and voltage-gated potassium channels change their distribution (Strumbos et al., 2010). These morphological changes suggest physiological abnormalities. It is unknown, however, whether and how synaptic excitation and inhibition are affected in animals with FMRP loss. The glutamatergic excitatory input to MNTB is fast and strong, ensuring high-speed signal transmission and precise temporal coding of acoustic stimuli for computation of sound localization (reviewed by Joris and Trussell, 2018). Being strong and secure, this synaptic excitation is still subject to changes in response to multiple manipulations of the afferent inputs (e.g., Grande et al., 2014; Youssoufian et al., 2005).

Limited in vivo juxtacellular recording data show minor changes in excitatory spiking activity (Wang et al., 2015). Anatomical studies examining the changes in inhibitory inputs to MNTB have reported mixed results. Based on the increased staining of vesicular GABA transporters (which also transport glycine into synaptic vesicles) in Fmr1 KO MNTB neurons (Rotschafer et al., 2015), one would predict that synaptic inhibition is strengthened in Fmr1 KO MNTB neurons. However, based on another study which showed decreased GAD67 (a marker for GABAergic input) and decreased glycine transporter 2 in Fmr1 KO neurons (McCullagh et al., 2017), one would expect to observe reduced synaptic inhibition. Anatomical changes in protein levels of transporters, postsynaptic receptors, or ion channels, however, do not always cause significant changes in functional expressions examined by electrophysiological approaches (e.g., Curia et al., 2009; Lu et al., 2004). Interestingly, despite these suggested changes in the inputs to MNTB, the inhibitory output from the MNTB to its primary target, the LSO, remains unchanged in Fmr1 KO mice (Garcia-Pino et al., 2017). Our recent study also reveals non-significant changes in neural modulation by metabotropic glutamate receptors (mGluRs) of synaptic inhibition between WT and Fmr1 KO MNTB neurons (Curry et al., 2018). These observations necessitate electrophysiological investigations at the cellular level for both synaptic excitation and inhibition in MNTB neurons between the two genotypes. Here, we report few subtle differences in the basal level of synaptic transmission between WT and Fmr1 KO MNTB neurons, contrasting the more dramatic changes observed in other brain regions especially the neocortex.

2. Results

2.1. Synaptic excitation between WT and Fmr1 KO MNTB neurons

To examine whether and how the excitatory input to MNTB changes in Fmr1 KO, we recorded and compared evoked EPSCs (eEPSC) in WT and KO animals. Subtle differences in the basal excitatory transmission were found between the two genotypes. More than half of the WT neurons showed typical all-or-none eEPSCs in response to gradually increasing stimulus intensity (Fig. 1A). In contrast, more than half of the Fmr1 KO neurons showed graded eEPSCs (Fig. 1B). At lower stimulus intensities, eEPSCs with small amplitude and short latency was elicited. In response to increasing stimulus intensities, the cell responded with eEPSCs of larger amplitude, without the smaller eEPSCs (Fig. 1B). The disappearance of the smaller component may be due to depolarization block on the presynaptic terminals (Bianchi et al., 2012). Proportionally, more KO cells expressed graded pattern (Fig. 1C; WT: 5 out of 14 cells, 36%; KO: 11 out of 16 cells, 69%; Chi-square p = 0.035). No significant difference was detected in the maximal eEPSC amplitude (Fig. 1D; WT: 2162 ± 612 pA, n = 14; Fmr1 KO: 1850 ± 460 pA, n = 16; p = 0.727). The eEPSC latency (the time between the stimulus onset and the peak of the maximal eEPSC) did not differ between the two genotypes (Fig. 1E; WT: 2.191 ± 0.392 ms, n = 12; Fmr1 KO: 2.361 ± 0.197 ms, n = 14; p = 0.689), nor the eEPSC jitter (the standard deviation of the eEPSC peak latency) (Fig. 1F; WT: 0.117 ± 0.040 ms, n = 12; Fmr1 KO: 0.197 ± 0.054 ms, n = 14; p = 0.164). In a paired-pulse paradigm where two identical stimuli were delivered at a time interval of 10 ms, MNTB neurons expressed different forms of synaptic plasticity. We define synaptic depression (SD) when the pair-pulse ratio (PPR, the ratio of the amplitude of the second eEPSC over the amplitude of the first eEPSC) is less than 1.0, whereas for synaptic facilitation (SF), the PPR is larger than 1.0. Although proportionally more KO cells displayed SF than WT cells (WT: 6 out of 12 cells, 50%; KO: 10 out of 15 cells, 67%) (Fig. 1G), the average PPRs did not differ between the two genotypes (Fig. 1H; WT: 1.30 ± 0.19, n = 12; Fmr1 KO: 1.35 ± 0.14, n = 15; p = 0.862).

Figure 1.

Subtle differences in the basal excitatory transmission between WT and Fmr1 KO MNTB neurons. A-B. One sample WT neuron displayed typical all-or-none eEPSCs in response to electrical stimuli with gradually increasing intensity, whereas one KO neuron showed graded eEPSCs. The eEPSC traces color in red are responses at the threshold (minimum stimulus intensity that elicited detectable eEPSC). C. Proportionally, more KO cells display graded response pattern than WT cells (*, Chi- square p < 0.05). D. No significant difference was detected in the maximal eEPSC amplitude (WT, n = 14; KO, n = 16). E-F. No significant difference was detected in eEPSC peak latency (the time between the stimulus onset and the peak of the maximal eEPSC) and jitter (the standard deviation of the peak latency) (WT, n = 12; KO, n = 14). G-H. In response to a paired-pulse paradigm, neurons show either synaptic depression (SD) or synaptic facilitation (SF). Proportionally, KO cells tended to exhibit more SF than WT neurons. However, no significant difference was detected in pair-pulse ratio (PPR) between the two genotypes. I. Sample sEPSCs from a WT and a KO neuron. Detected individual events are shown on the right, with the traces color in red being the averaged responses. J-M. KO neurons tended to have reduced sEPSC frequency with similar amplitude and kinetics (WT, n = 21; KO, n = 20), with large variation in sEPSC frequency among individual cells. In this and subsequent figures, means ± SEM are shown, with each dot representing the value of one cell, showing the spread of the individual data points.

Besides eEPSCs, we also compared sEPSCs between WT and Fmr1 KO MNTB neurons. Blocking voltage-gated sodium channels with tetrodotoxin did not change sEPSCs (data not shown), indicative of the miniature release feature of these events. We found no significant changes in sEPSC frequency in KO neurons, even though a trend for decreased release frequency is apparent, with large variations among individual cells (Fig. 1 I–J; Table 1; WT: n = 21; Fmr1 KO: n = 20). Other parameters of the sEPSCs including amplitude, rise time, and decay time constant (tau) were very similar between the two genotypes (Fig. 1 K–M; Table 1).

Table 1.

Comparisons of sEPSCs between WT and Fmr1 KO MNTB neurons.

sEPSC	WT (21)	KO (20)	p
Frequency (Hz)	15.93 ± 6.98	5.92 ± 1.70	0.684
Amplitude (pA)	40 ± 4	47 ± 5	0.162
Rise time (ms)	0.17 ± 0.01	0.18 ± 0.01	0.682
Decay tau (ms)	0.56 ± 0.06	0.48 ± 0.04	0.230

Number in parenthesis indicates the number of cells. sEPSC: spontaneous excitatory postsynaptic current. In this and the subsequent Table, Mean ± SEM are shown. The p values for Mann-Whitney test or un-paired t test are reported.

2.2. Synaptic inhibition between WT and Fmr1 KO MNTB neurons

Most of the basic properties of synapitc inhibition between WT and Fmr1 KO MNTB neurons did not differ significantly. We firstly compared the total synaptic inhibition in the two different genotypes without pharmacological isolation of the two components mediated by glycine and GABA. The mixed eIPSCs in both WT and KO neurons increased in amplitude in response to increasing stimulus intensities (Fig. 2A, B). The maximal amplitude of the mixed eIPSCs did not differ significantly between WT (1318 ± 247 pA, n = 10) and Fmr1 KO neurons (2129 ± 672 pA, n = 14, p > 0.999, Fig. 2C), with a few cells with large amplitudes in the KO. The eIPSC reversed at a membrane potential of −28.3 ± 3.6 mV (n = 4 cells), close to the expected equilibrium potential for chloride (calculated value of −34.9 mV, using Nernst equation, based on chloride concentrations in the ACSF and the internal solution we used) (Fig. 2D), reflecting the nature of Cl⁻ permeability of these receptor channels. For sIPSCs, events with both large and small amplitudes, and both fast and slow kinetics were observed (Fig. 2E). The frequency and amplitude of the mixed sIPSCs did not differ significantly between WT (n = 14) and KO neurons (n = 15). However, the kinetics of sIPSCs were prolonged significantly in KO than WT neurons, as seen in the significantly increased rise time as well as the decay tau (Fig. 2E–I; Table 2). Because GABAergic sIPSCs have much slower kinetics compared to glycinergic sIPSCs in MNTB neurons (Awatramani et al., 2005), the prolonged kinetics in KO neurons may be caused by a proportionally more frequent presence of sIPSCs containing the GABAergic component. To test this prediction, we visually inspected and counted the number of sIPSCs mediated presumably by GABA, glycine, or both, based on their distinct kinetics. Glycinergic sIPSCs were fast, GABAergic sIPSCs slow, whereas the mixed sIPSCs had both components (Fig. 2J). To control for the developmental regulation of the inhibitory transmitter types (Awatramani et al., 2005), we analyzed the presence of these sIPSCs at the same age of the animals between the two genotypes. At P13, the percentage of sIPSCs that contained a GABAergic component in KO neurons had a trend for increase (80 ± 5%, n = 9) compared to WT (70 ± 5%, n = 7), however, no significant difference was detected (Fig. 2K–L; df = 14, p = 0.184). These results suggest that multiple mechanisms may underlie the slowed kinetics of the mixed sIPSCs, with each contributing only partially to the phenomenon.

Figure 2.

Total synaptic inhibition mediated by both glycine and GABA remains largely unaltered in Fmr1 KO MNTB neurons, except for slowed sIPSC kinetics. A-B. Sample mixed eIPSCs without separating the glycinergic and GABAergic components from a WT (left, black) and a Fmr1 KO neuron (right, red), in response to increasing stimulus intensities. C. The maximal amplitude of eIPSC of KO neurons did not significantly differ from that of WT neurons (WT, n = 10; KO, n = 14). D. Sample traces showing that the mixed sIPSCs reverse at a membrane potential that is close to the calculated equilibrium potential for Cl⁻. E. Sample sIPSCs from a WT and a KO neuron. The detected individual traces are shown in the middle, with the traces color in red being the averaged responses. Shown on the most right are two sIPSCs normalized to their peak, with one being fast in kinetics (black trace, presumably a glycinergic event) and the other slow (green trace, presumably a GABAergic event). F-I. KO neurons did not differ from WT neurons in their sIPSC frequency and amplitude. However, the kinetics was significantly prolonged (WT, n = 14; KO, n = 15; * indicates p < 0.05). J-L. At the same age (P13), although KO neurons (n = 9) displayed proportionally more sIPSCs containing the GABAergic component than WT neurons (n = 7), no significant differences were detected in population data. Visual inspection can readily detect GABAergic (indicated by the symbol “+”), glycinergic (symbol “#”), and mixed (symbol “$”) sIPSC events, based on their distinct kinetics.

Table 2.

Comparisons of sIPSCs between WT and Fmr1 KO MNTB neurons.

	WT	KO	p
Mixed sIPSC (n)	(14)	(15)
Frequency (Hz)	1.06 ± 0.33	1.30 ± 0.52	0.539
Amplitude (pA)	87 ± 15	86 ± 20	0.419
Rise time (ms)	0.35 ± 0.02	0.42 ± 0.02	0.028*
Decay tau (ms)	2.76 ± 0.46	4.14 ± 0.44	0.017*
Glycine sIPSC (n)	(33)	(25)
Frequency (Hz)	0.81 ± 0.31	0.40 ± 0.11	0.094
Amplitude (pA)	76 ± 10	71 ± 7	0.987
Rise time (ms)	0.18 ± 0.01	0.20 ± 0.01	0.898
Decay tau (ms)	1.07 ± 0.05	1.03 ± 0.05	0.552
GABA sIPSC (n)	(10)	(10)
Frequency (Hz)	1.09 ± 0.55	1.26 ± 0.72	0.515
Amplitude (pA)	55 ± 16	54 ± 6	0.280
Rise time (ms)	0.32 ± 0.04	0.29 ± 0.03	0.564
Decay tau (ms)	4.83 ± 0.88	4.05 ± 0.73	0.509

n: number of cells; sIPSC: spontaneous inhibitory postsynaptic current.

^*p < 0.05, significant difference detected by Mann-Whitney or un-paired t test.

Because both the GABAergic and glycinergic components are present in the ages of mice we studied (Awatramani et al., 2005), we pharmacologically isolated the glycinergic and GABAergic inhibition, and compared IPSCs between WT and KO neurons. For glycinergic transmission, eIPSCs displayed a similar response pattern between WT and KO neurons with large variations among individual cells (Fig. 3A). The maximal eEPSC amplitudes did not significantly differ between WT (1168 ± 287 pA, n = 18) and Fmr1 KO neurons (769 ± 152 pA, n = 12) (Fig. 3B; p = 0.812). GABAergic eIPSCs also displayed a similar response pattern (Fig. 3C) and similar maximal amplitudes between WT (306 ± 24 pA, n = 21) and Fmr1 KO neurons (283 ± 46 pA, n = 12) (Fig. 3D; p = 0.620). Similar observations were seen for spontaneous release properties. The frequency, amplitude, rise time, and decay tau of glycinergic sIPSCs did not significantly differ between WT (n = 33) and Fmr1 KO neurons (n = 25) (Fig. 3E–I), nor for GABAergic sIPSCs (WT, n = 10; KO, n = 10) (Fig. 3J–N; Table 2). These results suggest unaltered vesicular release pathway and function between the two inhibitory transmitter systems in MNTB of Fmr1 KO mice.

Figure 3.

Basal synaptic inhibition, mediated by glycine or GABA, is similar between WT and Fmr1 KO MNTB neurons. A. Sample glycinergic eIPSCs from a WT (left, black) and an Fmr1 KO neuron (right, red). B. The maximal amplitudes did not differ for glycinergic eIPSCs between WT (n = 18) and KO neurons (n = 12). C. Sample GABAergic eIPSCs from a WT (left, black) and a KO neuron (right, red). D. The maximal amplitudes of GABAergic eIPSCs did not differ between the two genotypes (WT, n = 21; KO, n = 12). E. Glycinergic sIPSC sample traces from a WT (black) and a KO neuron (red) neuron. Detected events are shown on the right, with the thicker traces being the averaged responses. F-I. sIPSC frequency, amplitude, rise time, and decay tau are similar for glycinergic sIPSCs between WT (n = 33) and KO neurons (n = 25). J- N. GABAergic sIPSCs are also similar between WT (n = 10) and KO neurons (n = 10).

3. Discussion

In the FXS mouse model, loss of E/I balance results in hyperexcitability of neurons in multiple brain regions. Our results, however, revealed only subtle changes in basal synaptic excitation and inhibition in Fmr1 KO MNTB neurons, implying non-disturbed E/I balance and thus constant inhibitory output from MNTB to its targets. Although being somewhat surprising, our observation is consistent with previous studies showing largely unaltered excitatory transmission in vivo at the same MNTB synapses (Wang et al., 2015), and unchanged output from MNTB to the LSO in vitro (Garcia-Pino et al., 2017), in Fmr1 KO mice.

3.1. On synaptic excitation

In Fmr1 KO animals, synapitc excitation is usually increased, contributing to hyperexcitability (reviewed by Contractor et al., 2015). However, exceptions exist and the effects of FMRP loss are brain region and cell type specific. For example, loss of presynaptic FMRP did not change the excitatory transmission onto excitatory neurons in the neocortex (Patel et al., 2013). In the auditory system, changes in synaptic transmission also vary dependent on location. Auditory cortical cells in Fmr1 KO animals are hypersensitive to sound stimuli, exhibiting higher spiking rates and broader frequency tuning to sounds (Rotschafer and Razak, 2013). In the auditory brainstem, LSO neurons in Fmr1 KO animals receive increased synaptic excitation and unchanged inhibition, leading to broader tuning of frequencies and shifted interaural level difference function (Garcia-Pino et al., 2017). Our results that synaptic excitation did not change in strength and kinetics are consistent with those reported by Wang et al. (2015), which showed no obvious changes in spiking activity in Fmr1 KO MNTB neurons. This may not be surprising given that fragile X granules are not detected in the calyx in mice (Akins et al., 2012), suggesting the lack of FMRP in the presynaptic terminals and thus subtle changes in synaptic excitation when FMRP was globally manipulated. In addition, it is worth noting that even in congenital deafness mice where the afferent excitatory input is nearly completely absent, the excitatory transmission to MNTB during early development (from P5 to P12) remains largely unchanged (Youssoufian et al., 2005).

Although synaptic strength and kinetics of the excitatory inputs to MNTB remained unchanged, we did observe changes in the eEPSC response patterns. Proportionally more KO neurons showed graded eEPSCs than WT. In young adults (postnatal 3 weeks) and mature animals, each MNTB neuron is reported to have only one calyx innervation (e.g.: Sätzler et al., 2002; Smith et al., 1991; Taschenberger et al., 2002), underlying the all-or-none response pattern of eEPSCs. In younger mice, one MNTB principle cell could receive excitatory inputs from more than one calyx (Bergsman et al., 2004). A delayed development of synaptic innervation may occur under diseased conditions or hearing loss (Grande et al., 2014), resulting in a delayed transition from multiple calyceal inputs to one calyceal innervation. Besides calyceal innervation, MNTB neurons can also receive non-calyceal excitatory inputs, as shown in P13–15 rats (Hamann et al., 2003). When multiple calyxes innervate a single MNTB neuron and/or non-calyceal inputs are prominent, a graded response pattern would be expected because the activation thresholds for different synaptic terminals are likely to differ and they would be recruited under different stimulus intensities. Therefore, it is possible that in the Fmr1 KO animals, proportionally there may be more MNTB cells that receive inputs from multiple calyxes and/or more non-calyceal inputs are present, resulting in the more graded response pattern than the WT. Alternatively, the observation of the difference in the eEPSC response pattern between WT and KO could be due to age-dependent changes, because younger animals may have multiple calyceal inputs and the number of the inputs decreases in older animals. If younger animals were used proportionally more in KO than WT in the experiment, it could result in the difference in the eEPSC response pattern. While there seemed to be a trend for age-dependent changes in the response pattern (data not shown), the average ages for the two genotypes were very similar (WT: 15.8 ± 0.7 postnatal day, n = 14; Fmr1 KO: 15.9 ± 0.5 postnatal day, n = 16; p = 0.798). Therefore, the difference in the eEPSC response pattern between WT and KO was caused by different genotypes.

Additionally, we observed a tendency for changes in the form of short-term plasticity for glutamatergic transmission. Short-term plasticity could display SD or SF in MNTB neurons, with SD being dominant (Hermann et al., 2007; Müller et al., 2010; Taschenberger and von Gersdorff, 2000). There exists a tendency that proportionally more Fmr1 KO neurons displayed SF (therefore proportionally less SD) than WT neurons (Fig. 1G). This may be interpreted by the changes in the morphology of the calyxes. MNTB neurons in P16–19 mice exhibit SD or SF depending on the morphology of calyxes, with SD more prominent in calyxes with simple morphology (no or few bouton-like swellings) and SF more prominent in calyxes with complex morphology (more bouton-like swellings) (Grande and Wang, 2011). In Fmr1 KO neurons, a larger proportion of calyxes display complex morphology (Wang et al., 2015), consistent with our observation that Fmr1 KO neurons tended to more likely display short-term plasticity in the form of SF than WT neurons. These data suggest that loss of FMRP function did not affect the quantal size and kinetics of glutamate release at the calyx during the animal ages studied. The trend for a reduced release probability is consistent with the observation that proportionally more KO neurons showed SF in paired-pulse responses, because lower initial release probability is typically associated with short-term facilitation (reviewed by Jackman and Regehr, 2017). However, because the morphologically distinct calyxes are not tonotopically organized, the neurons we sampled in our recordings were from a heterogeneous group in terms of calyx morphology. Therefore, we cannot exclude the possibility that our observation could be due to an unintentional bias of cell sampling.

3.2. On synaptic inhibition

Besides the well-known “mGluR theory” of FXS, which states that exaggerated activation of mGluR5 in face of FMRP loss causes the phenotypes of FXS (reviewed by Bear et al., 2004), more recent studies have clearly demonstrated complex roles and interactions of multiple neurotransmitter systems underlying the disease (reviewed by Sethna et al., 2014). Of particular importance is the GABAergic transmission. Among the neural circuits examined, most of them show reduced GABAergic inhibition, while an enhanced inhibition has also been reported in other circuits (reviewed by Braat and Kooy, 2015), indicating that in Fmr1 KO neurons the inhibitory system is altered in a brain region specific manner.

For most of the parameters examined, we did not detect significant differences in synaptic inhibition between WT and Fmr1 KO MNTB neurons. Such a largely lack of physiological differences in synaptic inhibition between the two genotypes thus did not support anatomical studies implying either reduced inhibitory inputs to MNTB in FXS mice (McCullagh et al., 2017), or strengthened inhibition (Rotschafer et al., 2015). The anatomical changes, regardless of direction (either increase or decrease), may not be sufficient to lead to significant functional changes, as observed in other brain systems. For example, a decreased density of GABA synapses has been reported in striatal projection neurons of Fmr1 KO mice, but the amplitude of GABAergic IPSCs is not changed (Centonze et al., 2008). In the somatosensory cortex, postsynaptic GABA signaling shows no changes in Fmr1 KO neurons even though the expression of various GABAA receptor subunits is down-regulated (Curia et al., 2009; Paluszkiewicz et al., 2011). These studies along with our current one support that the effects of FMRP on neural functions are not universal at the cellular level in different brain regions. The only significant difference we observed is the slowed kinetics of the mixed sIPSCs in Fmr1 KO neurons. The mechanisms underlying this observation are unclear. Our results did not confirm our prediction that this could be explained by more frequent presence of the slower GABAergic component in KO neurons, but a trend was seen (Fig. 2L). Under normal conditions, the two inhibitory transmitter systems stagger in development, with the GABAergic component being dominant in early ages, and the glycinergic component becoming dominant later (Awatramani et al., 2005). A delayed development of the inhibitory circuits in Fmr1 KO animals could thus lead to slower kinetics of the inhibitory responses when compared to age-matched WT animals. The loss of postsynaptic FMRP does not seem to underlie the slowed mixed sIPSCs because when the two inhibitory transmitter systems were examined independently no significant differences were detected in the sIPSC kinetics. Therefore, at the postsynaptic MNTB neurons, the subunit composition of the receptor channels for each of the two inhibitory transmitter systems may remain unchanged in Fmr1 KO mice. The slow kinetics of the mixed sIPSCs is unlikely to be caused by the postsynaptic receptors. The prominent presence of the GABAergic component in the mixed sIPSC recordings in Fmr1 KO may be the most feasible interpretation. These results suggest that multiple mechanisms may be at play, with each contributing only partially to the phenomenon.

3.3. On physiological implication

Because synaptic excitation and inhibition in MNTB neurons of Fmr1 KO animals showed no significant differences from WT, the E/I balance and thus the output of MNTB neurons is expected to remain unchanged. Indeed, the synaptic inhibition from MNTB to LSO, in both eIPSCs (amplitude and short-term plasticity) and sIPSCs (frequency, amplitude, and kinetics), remain unchanged (Garcia-Pino et al., 2017), through the animal ages of P8, P14, to P21, approximately the same age range as used in our study. For MNTB, the importance of maintaining a constant inhibitory output to its target nuclei may reside in its essential functions in binaural neural processing for sound localization (reviewed by Grothe et al., 2010). The mechanism underlying the achievement of such a constant inhibition may be via a cellular process of rapid rebalancing of E/I after disturbance, as shown in the auditory cortex (Moore et al., 2018).

4. Experimental Procedure

4.1. Animals

All animal procedures were approved by the Institutional Animal Care and Use Committee at the Northeast Ohio Medical University (NEOMED) and were performed in accordance with the National Institutes of Health polices on animal use. WT and Fmr1 KO mice (with a background of C57BL/6J) were purchased from the Jackson Laboratory and bred at NEOMED. Genotype was confirmed with standard PCR protocol provided by the Jackson Laboratory. Some genotyping was performed by Transnetyx (Cordova, TN). All mice were housed in a vivarium with a normal light-dark cycle (12 h light/12 h dark).

4.2. Slice preparation and in vitro whole-cell recordings

Coronal brainstem slices (250 μm in thickness) were prepared from P12-P24 mice of both sexes, using a Linearslicer PRO 7N (Dosaka EM Co., Japan), as described previously with minor modifications (Lu, 2009). Mice were deeply anesthetized with isoflurane and rapidly decapitated. The brainstem was removed and sliced under warm (35 °C) artificial cerebrospinal fluid (ASCF) containing the following (in mM): 250 glycerol, 3 KCl, 1.2 KH2PO4, 20 NaHCO3, 3 HEPES, 1.2 CaCl2, 5 MgCl2, and 10 glucose, pH 7.4 (when gassed with 95% O2 and 5% CO2). Slices were incubated in a custom-made interface chamber at 34–36 °C for > 1 h in normal ACSF containing the following (in mM): 130 NaCl, 20 NaHCO3, 3 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.2 KH2PO4, and 10 glucose, pH 7.4. For recording, slices were transferred to a 0.5 ml chamber mounted on a Zeiss Axioskop 2 FS Plus microscope with a 40× water-immersion objective and infrared differential interference contrast optics. The chamber was continuously superfused with ACSF (2–5 mL/min) by gravity.

Patch pipettes were drawn on a PP-830 Microelectrode Puller (Narishige, Japan) to a 1–2 μm tip diameter using borosilicate glass micropipettes (inner diameter, 0.84 mm; outer diameter, 1.5 mm, World Precision Instruments). For EPSC recordings, the electrodes were filled with a solution containing the following (in mM): 140 K- gluconate, 4.5 MgCl2, 4.4 tris-phosphocreatine, 9 HEPES, 5 EGTA, 4 Na-ATP, 0.3 tris- GTP, pH 7.3 adjusted by KOH (1 M), and osmolarity of about 290 mOsm/L. A voltage- gated sodium channel blocker QX-314 (5 mM) was added to the internal solution to prevent generation of action currents. The liquid junction potential was 9.7 mV, and data were corrected accordingly. Cells were held at −60 mV for EPSC recordings. For IPSC recordings, the electrodes were filled with a solution containing the following (in mM): 105 Cs-methansulfonate, 35 CsCl, 5 EGTA, 10 HEPES, 1 MgCl2, 4 ATP-Mg, 0.46 GTP- Na, 5 QX-314, with pH 7.2, adjusted with CsOH, and osmolarity about 290 mOsm/L. A liquid junction potential of 9.5 mV was corrected accordingly. Cells were held at −70 mV for IPSC recordings. Different internal solutions and holding potentials were used for EPSC versus IPSC recordings in order to maintain consistency with pilot experiments. Electrodes had resistances (Re) between 3 and 6 MΩ, and the series resistance (Rs) was generally two folds of the Re. Only cells with Rs less than 15 MΩ were used. Rs for EPSC recordings was not compensated, whereas Rs for IPSC recordings was compensated at 70–80%. The conclusions are expected not to be affected by whether Rs was compensated or not, because we compared the same parameters between WT and KO animals under the same recording conditions, and thus systematic errors were cancelled out.

Voltage clamp experiments were performed with an AxoPatch 200B (Molecular Devices, San Jose, CA), under near physiological temperatures (34–36 °C) controlled by a single channel temperature controller TC324B (Warner Instruments, Hamden, CT). Data were low-pass filtered at 5 kHz and digitized with a data acquisition interface ITC- 18 (InstruTech, Longmont, CO) at 50 kHz. Recording protocols were written and run using the acquisition and analysis software AxoGraph X (AxoGraph Scientific, Australia). EPSCs were isolated pharmacologically with antagonists for GABAA receptors (10 μM SR95531, or gabazine) and glycinergic receptors (1 μM strychnine). IPSCs were isolated pharmacologically with antagonists for AMPA receptors (50 μM DNQX) and NMDA receptors (50 μM APV). Glycinergic and GABAergic currents were pharmacologically separated by bath application of gabazine (10 μM) and strychnine (1 μM), respectively. All chemicals were purchased from Sigma-Aldrich except for gabazine, which was obtained from Tocris Bioscience, and DNQX, which was obtained from Abcam.

4.3. Recordings of synaptic responses

For recordings of eEPSCs and eIPSCs, extracellular electrical stimulation was performed using concentric bipolar electrodes with a tip core diameter of 127 μm (World Precision Instruments, Sarasota, FL). To record eEPSCs, the stimulating electrode was placed using a NMN-25 Micromanipulator (Narishige, Japan) onto the afferent fibers in the midline between the two MNTBs (von Gersdorff et al., 1997; Grande et al., 2014). To record eIPSCs, the stimulating electrode was positioned lateral and ventral to the MNTB to activate the inhibitory afferent fibers. When a paired-pulse paradigm was used, the stimulus intensity for the two pulses was identical and the stimulus interval was 10 ms, at a stimulus frequency of 0.1 Hz. Spontaneous EPSCs (sEPSCs) and sIPSCs were recorded in the absence of external stimulation.

4.4. Data analysis

For eEPSC and eIPSC experiments, the peak amplitude was measured after each stimulus. sEPSCs and sIPSCs were detected by a template function using a function for product of exponentials,

$$f(t)=[1-exp(-t/risetime)]\times exp(-t/decaytau),$$

where t stands for time and tau for time constant. The values of the parameters for the template used to detect sEPSCs are: amplitude of −30 pA, rise time of 0.2 ms, decay tau of 0.4 ms, with a template baseline of 1 ms and a template length of 1 ms. The values of the parameters for the template to detect mixed sIPSCs are: amplitude of −60 pA, rise time of 0.4 ms, decay tau of 3 ms, with a template baseline of 2 ms and a template length of 15 ms. The values of the parameters for the template to detect glycinergic sIPSCs are: amplitude of −50 pA, rise time of 0.2 ms, decay tau of 1 ms, with a template baseline of 2 ms and a template length of 5 ms. For the template to detect GABAergic sIPSCs, the values are: amplitude of −50 pA, rise time of 0.3 ms, decay tau of 4 ms, with a template baseline of 2 ms and a template length of 15 ms. These parameters were determined based on an average of visually detected synaptic events. The detection threshold is threefold the noise standard deviation, which detects most of the events with the least number of false positives. The average of detected events for each cell was obtained using AxoGraph to measure the amplitude, rise time, and decay tau. The decay time of the sIPSCs was fit by single or double exponential functions. For a single component fitting, the tau was reported directly. For a double component fitting, the weighted decay tau was calculated as,

$$\text{τ}=\left({\text{A}}_{\text{fastτfast}}+{\text{A}}_{\text{slowτslow}}\right)/\left({\text{A}}_{\text{fast}}+{\text{A}}_{\text{slow}}\right),$$

where A_fast and A_slow represent the amplitude, and τ_fast and τslow represent the time constants of the fast and slow components of the sIPSC, respectively. The best fit was selected by comparing the sum of squared errors (SSE) between fits with single and double components. If the SSEs differed by > 20%, the fit with double components was used; otherwise, the fit with single component was used (Tang and Lu, 2012).

Statistics were performed using Excel (Microsoft, Redmond, WA) and GraphPad Prism (GraphPad Software, La Jolla, CA), and graphs were made in Igor (Wavemetrics, Lake Oswego, OR). Mean ± SEM (standard error of the mean) are reported. Unless indicated otherwise, data were subjected to either unpaired t test if they passed the D’Agositino-Pearson omnibus normality test, or Mann-Whitney test if they failed the normality test, with p < 0.05 being considered statistically significant.

4.5. Considerations for comparisons between WT and Fmr1 KO MNTB neurons

To achieve valid and meaningful comparisons in neuronal properties of WT and Fmr1 KO MNTB neurons, several considerations were taken into account in experimental design. First, we used WT and KO animals with approximately matched ages in each measured parameter. Both sexes of animals were used, and we did not observe any obvious gender differences, so data were grouped together. Second, we sampled approximately the same number of neurons in each category for WT and KO wherever statistical analyses were performed. Third, we maintained the consistency as much as possible in terms of the MNTB regions where cells were recorded between the WT and KO animals. This is important, because FMRP in normal hearing animals has a tonotopic distribution, with higher expression of FMRP protein level in high-frequency than in low-frequency coding MNTB neurons (Ruby et al., 2015), suggesting that the loss of FMRP function may result in a graded effect in MNTB. Our cells were mostly collected from the lateral half of MNTB in two to three coronal brain slices, the most caudal one of which contained the facial nerve tract visibly running in the dorsomedial to ventrolateral direction.

Research Highlights:

• Neurotransmission in MNTB neurons in Fmr1 knockout mice shows subtle alterations.

• Synaptic excitation by glutamate remains largely unchanged.

• Evoked EPSCs show more graded response pattern than wild-type.

• Synaptic inhibition by GABA and glycine remains largely unchanged.

• Spontaneous IPSCs mediated by mixed transmitters display prolonged kinetics.

ABBREVIATIONS

ACSF

artificial cerebrospinal fluid

EPSC

excitatory postsynaptic current

eEPSC

evoked EPSC

E/I

excitation/inhibition

eIPSC

evoked inhibitory postsynaptic current

Fmr1

fragile X mental retardation gene 1

FMRP

fragile X mental retardation protein

FXS

fragile X syndrome

IPSC

inhibitory postsynaptic current

KO

knockout

LSO

lateral superior olive

mGluR

metabotropic glutamate receptor

MNTB

medial nucleus of trapezoid body

SD

synaptic depression

sEPSC

spontaneous EPSC

SF

synaptic facilitation

sIPSC

spontaneous IPSC

PPR

pair-pulse ratio

WT

wild-type