Differential distribution of glycine receptor subtypes at the rat calyx of Held synapse

Abstract

The properties of glycine receptors (GlyRs) depend upon their subunit composition. While the prevalent adult forms of GlyRs are heteromers, previous reports suggested functional α homomeric receptors in mature nervous tissues. Here we show two functionally different GlyRs populations in the rat medial nucleus of trapezoid body (MNTB). Postsynaptic receptors formed α1/β-containing clusters on somatodendritic domains of MNTB principal neurons, colocalizing with glycinergic nerve endings to mediate fast, phasic IPSCs. In contrast, presynaptic receptors on glutamatergic calyx of Held terminals were composed of dispersed, homomeric α1 receptors. Interestingly, the parent cell bodies of the calyces of Held, the globular bushy cells of the cochlear nucleus, expressed somatodendritic receptors (α1/β heteromers) and showed similar clustering and pharmacological profile as GlyRs on MNTB principal cells. These results suggest that specific targeting of GlyR β-subunit produces segregation of GlyR subtypes involved in two different mechanisms of modulation of synaptic strength.

Figure 1.

MNTB principal cells express functional α1/β heteromeric GlyRs. A, The configuration for recording of glycine-evoked currents from MNTB principal cells. B, Representative examples of the current traces recorded in the absence (control) or presence of 50 μm PTX. C, Current response, evoked by a low concentration of glycine, was potentiated in the presence of 1 μm ICS 205.930. D, GlyR currents recorded in the absence (control) or presence of 5 μm CTB at 1 min intervals. MNTB slices were incubated in CTB solution for 5 min before the first combined applications of glycine and CTB (i). CTB produced a steady-state inhibition of GlyR responses after 10 consecutive coapplications with glycine (ii). E, Representative examples of the current traces recorded in the absence (control) or presence of 100 μm CTZ. F, Bar graph summarizes the effects of subunit-specific drugs on glycine-evoked responses obtained from 35 principal cells. *p < 0.05 or **p < 0.01 (paired t test).

Figure 2.

Synaptic and extrasynaptic GlyRs in MNTB principal cells have similar subunit compositions. A, Configuration for recording of glycinergic IPSCs from MNTB principal cells. B, Left, The plot shows maximal amplitudes of IPSCs recorded in the absence (○) or in the presence (●) of 50 μm PTX. Right, Superimposed traces obtained by averaging of 20 control IPSCs (black) or 20 IPSCs recorded in PTX-treated cells (gray). C, Averages of 20, low-frequency (0.1 Hz) IPSCs, recorded in the absence (i) or presence of CTB, before (ii) and after (iii) 30 trains of 50 IPSCs (100 Hz) elicited every 20 s. For comparison of time courses, the control IPSC (i) was normalized and superimposed as gray trace at the IPSCs recorded in the presence of CTB (ii and iii, black). Note that gray and black traces overlap completely. The numbers at trains denote their sequence of recording. Stimulus artifacts were removed in the train traces.

Figure 3.

Synaptic GlyRs mediate phasic IPSCs. A, Low-frequency IPSCs recorded in the absence (Control) or presence of 1 μm ICS 205.930, as indicated. Insets emphasize fits of three-exponential curves (gray) to early and late phases of IPSC decay [fitted time constants (relative amplitudes) are 2.2 ms (94.9%), 23.6 ms (3.3%), and 114.2 ms (1.8%) for control and 3.0 ms (94.9%), 17.8 ms (3.6%), and 109.3 ms (1.5%) for ICS 205.930]. B, Values of the time constants (left bar graph) and relative magnitudes (right bar graph) of each component in the fitted curves in control and ICS 205.930 for three-exponential fits to decay of low-frequency IPSCs. C, Examples of glycinergic mIPSCs recorded from an MNTB principal cell in the absence (Control) or presence of ICS 205.930 (20 mIPSCs are shown for each treatment). D, Superimposed traces represent averages of 594 events (Control, black) or 560 events (ICS 205.930, gray) obtained from the same cell shown in C. Note that the drug increased both the maximal amplitude and the decay time of mIPSCs. E, Normalized cumulative distributions of maximal mIPSC amplitudes obtained from all eight cells analyzed. In each cell, the data were collected from 10 min long recording periods before (black; 3199 events), during (solid gray; 2829 events), or 10 min after (broken gray; 2923 events) bath application of ICS 205.930. The drug caused a significant shift of mIPSC amplitudes toward higher values (p < 0.001, Kolmogorov–Smirnov test). F, The traces show trains of 50 IPSCs stimulated at 100 Hz in the absence (Control, black trace) or presence of 1 μm ICS 205.930 (gray trace). The arrow marks the decay after the train in ICS 205.930 and the dashed lines indicate levels of the tonic IPSC. G, The plot shows the magnitude of the effects of ICS 205.930 on trains of IPSCs. Data points represent average values of peaks of phasic IPSCs (circles, left y-axis) or currents just preceding the phasic IPSCs (squares, right y-axis) obtained from 10 trains recorded every 20 s in the absence (open symbols) or presence of the drug (filled symbols). The data are from the same cell as in F. H, Bar graph summarizes the effects of ICS 205.930 on trains of IPSCs. P denotes the peak of the phasic IPSC measured from the current level just preceding the stimulus artifact. τ_Single and τ_Train represent amplitude-weighted mean time constants of decays of single low-frequency IPSCs or trains of IPSCs. The data are collected from 8 to 14 cells. *p < 0.05 or **p < 0.01 (paired t test).

Figure 4.

Localization of GlyR α1-subunits in adult MNTB. A, A 9-μm-thick confocal projection showing presynaptic neurons in an MNTB slice double labeled with primary antibodies against CR (green) and Rab3a (red). Note that both antibodies gave specific staining of calyces of Held while anti-CR immunoreactivity is also observed in preterminal axons. Postsynaptic principal cells were both Rab3a and CR immunonegative. B, C, Single confocal plane images show Rab3a- or CR-immunoreactive calyces (red) and anti-α1 punctate staining (green). The α1-immunoractive clusters do not colocalize with presynaptic fluorescence signals. Note ring-like anatomical specializations of presynaptic processes typical for a mature calyx of Held (open arrowheads). D, α1-immunopositive clusters in MNTB principal cells frequently organized into rosette-like groups (square) (a single confocal plane). The dashed line indicates the cell surface. E, The stack of two confocal sections showing MNTB neurons double labeled for GlyR α1 (red) and vGAT (green). Note that both somatic and dendritic (arrowhead) α1-immunoreactive puncta are nearly always adjacent to vGAT-positive inhibitory terminals. F, A single confocal plane image of a principal cell double labeled with a postsynaptic marker CaBP (green) and with anti-α1 (red) indicated an accumulation of GlyRs on a dendritic process (arrowhead). G, A Z-series projection of four images through a principal cell, retrogradely labeled with BDA (green) injected into the ipsilateral lateral superior olive, shows α1-immunoreactive dots (red) on both a short dendrite and in postsynaptic axonal segment (arrowhead). Scale bars: A, 20 μm; B, 8 μm; C–G, 4 μm.

Figure 5.

GlyR α1- and β-subunits colocalize in postsynaptic neurons of adult MNTB. A, B, Single confocal plane images of an MNTB slice double labeled with Rab3a (red) and with GlyR α2 or α3 antibodies (green). No specific labeling associated with GlyRs was found. C, MNTB principal cells double labeled for gephyrin (GE; red) and for GlyR α1-subunit (green) showing a high degree of colocalization of both fluorescent signals (right). Inset shows rosettes of GlyR clusters at a higher magnification. The dashed line indicates the cell surface. Scale bars: A, B, 20 μm; C, 8 μm; inset, 4 μm.

Figure 6.

α1 homomeric GlyRs on calyces of Held mediate slow presynaptic facilitation. A, The scheme shows recording configuration. B, GlyR current responses obtained by recording from a calyx terminal in the absence (Control) or in the presence of 50 μm PTX. C, Calyceal responses evoked by glycine applications repeated at 1 min intervals in the absence (control) or presence of 5 μm CTB. CTB eliminated GlyR responses after two consecutive coapplications with glycine (i, ii). D, Representative traces of calyceal GlyR currents recorded in the absence (Control) or in the presence of 10 μm PXN. E, Summary of effects of subunit-specific drugs on glycine-evoked responses obtained from 18 calyces. F, Configuration for recording of EPSCs from MNTB principal cells. G, Maximal amplitudes of control EPSCs (○) and EPSCs obtained in the presence of 100 μm glycine (●) or in the presence of 100 μm glycine and 50 μm PTX (). Note relatively slow time courses of both the onset and the offset of glycine-induced EPSC potentiation. H, Superimposed traces show averages of five consecutive EPSCs in the absence (Control) or presence of glycine (+Gly) or in the presence of glycine and PTX (gray trace). I, Trains of EPSCs recorded from a cell treated with drugs indicated. Averages of three trains elicited in 1 min intervals are shown. J, PTX-sensitive effects of glycine on ratios between the second (P2) and the first (P1) EPSCs in a train or between the last (P10) EPSC and P1. *p < 0.05 or **p < 0.01 (paired t test).

Figure 7.

GBCs contain functional α1/β heteromeric GlyRs. A, A Z-series projection of five images through the AVCN shows GBCs, retrogradely labeled with BDA. Scale bar, 20 μm. B, Configuration for recording of glycine-evoked currents from the soma of a GBC. C, D, GlyR currents recorded from GBCs in the absence (Control) or presence of 50 μm PTX or 1 μm ICS 205.930, respectively. E, GlyR currents recorded at 1 min intervals in the absence (control) or presence of 5 μm CTB. Responses to the first and the tenth combined applications of glycine and CTB are shown (i, ii). F, Summary of the effects of subunit-specific drugs on glycine-evoked responses obtained from 19 GBCs. *p < 0.05 (paired t test).

Figure 8.

Diffuse versus clustered distribution of presynaptic versus postsynaptic GlyRs in MNTB. A, Double labeling of MNTB slice with antibodies raised against CR (red) and with antibodies recognizing an intracellular part of GlyR α1-subunit (α1; green). Note the similar staining pattern to what has been observed with the antibodies raised against extracellular part of α1 (mAb2b). B, A single ultrathin section through a segment of the calyx (CH) surrounding the soma of the MNTB principal cell (PC). C, C′, Two consecutive ultrathin sections of a CH establishing asymmetric synaptic contacts (asterisks) with the soma of an MNTB PC. Calyceal processes were immunostained for VGluT1 (peroxidases reaction end product) and also showed immunoreactivity for α1 (immunogold particles; arrows) along the extrasynaptic plasma membrane. Moreover, GlyR subunits were occasionally observed on plasma membrane of PCs (arrowheads). D, 3D alignment of 22 serial ultrathin sections (70 nm thick) of the same calyceal segment as in C showing disperse membrane distribution of presynaptic GlyRs (white dots) relative to glutamate release sites (green). E, Immunoreactivity for α1 (the same antibody as in A, C) was detected over the postsynaptic specialization (arrowhead) at symmetrical putative inhibitory synapses between vGluT1-negative terminals (Inh.) and somata of MNTB PCs. F, Electron micrograph of MNTB slice double labeled with anti-gephyrin (gold particles) and anti-vGluT1 (peroxidase). Several neighboring clusters of densely packed gold particles (arrowheads) were typically observed at the membrane of principal cells under single vGluT1-negative bouton (Inh.). Inset shows an example of symmetric synaptic contacts (asterisk) between vGluT1 negative terminal (Inh.) and a PC. Scale bars: A, B, 5 μm; C, E, F, 0.5 μm; inset in F, 0.25 μm.

Figure 9.

α1/β heteromeric GlyRs on somata of mature GBCs. A, A single confocal plane image of ventral cochlear nucleus double labeled with anti-Rab3a and anti-α1 (mAb2b). Note that α1-immunoreactive clusters (green) on GBCs do not colocalize with Rab3a-positive presynaptic nerve terminals (red). B, A Z-series projection of eight images through GBCs, retrogradely labeled with BDA (green), with numerous postsynaptic gephyrin-immunoreactive dots (red). Scale bars: 10 μm.