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. 2014 Jun 11;34(24):8300-17.
doi: 10.1523/JNEUROSCI.0159-14.2014.

Gephyrin clusters are absent from small diameter primary afferent terminals despite the presence of GABA(A) receptors

Louis-Etienne Lorenzo  1 , Antoine G Godin  2 , Feng Wang  3 , Manon St-Louis  4 , Salvatore Carbonetto  5 , Paul W Wiseman  6 , Alfredo Ribeiro-da-Silva  7 , Yves De Koninck  8
Affiliations

Gephyrin clusters are absent from small diameter primary afferent terminals despite the presence of GABA(A) receptors

Louis-Etienne Lorenzo et al. J Neurosci. .

Abstract

Whereas both GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs) play a role in control of dorsal horn neuron excitability, their relative contribution to inhibition of small diameter primary afferent terminals remains controversial. To address this, we designed an approach for quantitative analyses of the distribution of GABA(A)R-subunits, GlyR α1-subunit and their anchoring protein, gephyrin, on terminals of rat spinal sensory afferents identified by Calcitonin-Gene-Related-Peptide (CGRP) for peptidergic terminals, and by Isolectin-B4 (IB4) for nonpeptidergic terminals. The approach was designed for light microscopy, which is compatible with the mild fixation conditions necessary for immunodetection of several of these antigens. An algorithm was designed to recognize structures with dimensions similar to those of the microscope resolution. To avoid detecting false colocalization, the latter was considered significant only if the degree of pixel overlap exceeded that expected from randomly overlapping pixels given a hypergeometric distribution. We found that both CGRP(+) and IB4(+) terminals were devoid of GlyR α1-subunit and gephyrin. The α1 GABA(A)R was also absent from these terminals. In contrast, the GABA(A)R α2/α3/α5 and β3 subunits were significantly expressed in both terminal types, as were other GABA(A)R-associated-proteins (α-Dystroglycan/Neuroligin-2/Collybistin-2). Ultrastructural immunocytochemistry confirmed the presence of GABA(A)R β3 subunits in small afferent terminals. Real-time quantitative PCR (qRT-PCR) confirmed the results of light microscopy immunochemical analysis. These results indicate that dorsal horn inhibitory synapses follow different rules of organization at presynaptic versus postsynaptic sites (nociceptive afferent terminals vs inhibitory synapses on dorsal horn neurons). The absence of gephyrin clusters from primary afferent terminals suggests a more diffuse mode of GABA(A)-mediated transmission at presynaptic than at postsynaptic sites.

Keywords: calcitonin gene-related peptide; dorsal root ganglion; dystroglycan; isolectin B4; pain; synaptic inhibition.

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Figures

Figure 1.
Figure 1.
Comparison of labeling obtained with two fixation protocols. Top, Micrographs of the GABAAR α2 and α3 subunits, gephyrin, of the GlyR α1 subunit and of the GABAAR β3 subunit using a mild fixation protocol with immersion of frozen sections of fresh tissue in 4% PFA for 10 min. Bottom, Same labeling with a standard 4% PFA perfusion followed by 2 h postfixation. Identical antibody concentrations and confocal laser settings were used for images obtained with both fixation protocols. For SN, the number of clusters per 100 μm2 gives an index of the cluster detection for each marker in both fixation protocols. Values are expressed as mean ± SD. Scale bar, 40 μm.
Figure 2.
Figure 2.
Laminar distribution of glycine and GABAA receptors in the rat superficial dorsal horn. Shown are images of spinal dorsal horn with immunostainings for the GlyR α1 subunit (A1, A2), the GABAA receptor α1 (B1, B2), α2 (C1, C2), and β3 (D1, D2) subunits. Limits of the superficial laminae were obtained based on their distance from the white matter and by comparison with the localization of the band of IB4 staining in the same optical section (data not shown), as described previously (Lorenzo et al., 2008). E, Quantification of the stainings in LI and LII. LIIi, Inner LII. Results are expressed in percentage difference (1-LII/LI) between LI and LII in the intensity and mask area for each staining. Positive values correspond to higher intensities or higher mask areas in LI, and negative values to higher intensities or higher mask areas in LII. *p < 0.05; **p < 0.01; ***p < 0.001. Data are expressed as mean ± SEM (as in all figures). For each subunit, the number of rats varied from n = 5 to n = 14. Scale bars, 175 μm (low magnification) and 40 μm (high magnification).
Figure 3.
Figure 3.
Examples of the multiple labelings used in the study. Stainings for CGRP or IB4 (afferent terminals) combined with the GlyR α1 subunit (A1, A2), gephyrin (B1, B2), KCC2 (C1, C2), GABAAR α1 (D1, D2), or GABAAR α2 (E1, E2) subunits. Scale bar, 40 μm.
Figure 4.
Figure 4.
Illustration of the method of colocalization analysis used in this study. A1, A2, Confocal images of stainings for GAD65 and IB4, known not to be colocalized. A3, Two binary masks derived from the two stainings. On the graph in A4, each point indicates the ratio value of observed versus expected overlap between the two masks for each rat (n = 8 rats). The dotted line represents the relationship for a purely random distribution of the two staining. The values obtained were not significantly higher than that expected from random distributions. B1, B4, Same type of analysis for CGRP and IB4 labelings, which are known to be colocalize in a small number of primary afferents. Both labels showed observed respective overlaps that were significantly higher than expected from random distributions (n = 12 rats). ***p < 0.001. Scale bars: A1, B1, 175 μm; A2–B3, 5 μm.
Figure 5.
Figure 5.
Confirmation of the reliability of the colocalization analysis. Colocalization analysis as in Figure 4 (see also Materials and Methods) applied to labeling for KCC2 (A), known not to be expressed in primary afferents (Coull et al., 2003) and to labeling for GlyR α1 (B), known to highly associate with gephyrin clusters. ****p < 0.0001. For each marker, the numbers of rats were as follows: n = 6 for KCC2/CGRP, n = 12 for KCC2/IB4, and n = 10 for GlyR α1/gephyrin in LI and LII. Scale bar, 5 μm.
Figure 6.
Figure 6.
The glycine receptor α1 subunit and gephyrin clusters were not found in nociceptive primary afferents terminals. A, B, Colocalization analysis for either the GlyR α1 subunit (A) or its anchoring protein gephyrin with either CGRP or IB4 labeling (B). For each marker, the numbers of rats were as follows: n = 5 for GlyR α1/CGRP, n = 7 rats for GlyR α1/IB4, n = 5 for gephyrin/CGRP, and n = 12 rats for gephyrin/IB4. Scale bar, 5 μm.
Figure 7.
Figure 7.
The α1 subunit of the GABAA receptor is absent from small diameter nociceptive primary afferent terminals. A, B, Top, Sets of micrographs representing triple staining for CGRP/gephyrin/GABAAR α1 subunit. Bottom, Sets of micrographs representing examples of binarized masked extracted from the confocal images above. The bottom graphs represent the colocalization analysis of observed values versus those expected from random distributions of each label. The numbers of rats for each colocalization were as follows: n = 6 for GABAAR α1/CGRP, n = 6 for α1/IB4, and n = 12 for GABAAR α1/gephyrin in LI and LII. ****p < 0.0001. Scale bar, 5 μm.
Figure 8.
Figure 8.
Expression of the GABAAR α2 subunit in small diameter nociceptive primary afferent terminals. A, B, Triple staining of IB4/gephyrin/GABAAR α2 subunit. The bottom panels of each set or micrographs represent examples of binarized masked extracted from the confocal images above. The bottom graphs represent the colocalization analysis of observed values versus those expected from random distributions of each label. The numbers of rats used for each colocalization were as follows: n = 11 for GABAAR α2/CGRP, n = 14 for α2/IB4, and n = 14 for GABAAR α2/gephyrin in LI and LII. *p < 0.05; ***p < 0.001; ****p < 0.0001. Scale bar, 5 μm.
Figure 9.
Figure 9.
Expression of α3, α5, and β3 subunits of the GABAA receptor in nociceptive afferent terminals and inhibitory postsynaptic sites. Shown is the analysis of colocalization of the α3 subunit with CGRP/IB4 (respectively, n = 6 and 8 rats; A), or gephyrin (n = 6 in LI and 5 rats in LII; B), the α5 subunit with either CGRP/IB4 (n = 5 and 7 rats; C), or gephyrin (n = 5 in LI and 6 rats in LII; D), the β3 subunit with CGRP/IB4 (n = 6 and 9 rats; E), or gephyrin (n = 14 rats in LI and LII; F). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 10.
Figure 10.
Relative expression of α subunits of the GABAA receptors at presynaptic versus postsynaptic sites. A, B, Mean (M) intensity of every GABAAR subunit labeling in gephyrin clusters was subtracted from that of the same subunit in CGRP in LI (A) and in IB4 in LII (B). Positive intensity unit (i.u.) values indicated preferential postsynaptic localization (gephyrin clusters), whereas negative values indicate preferential presynaptic location (CGRP+ or IB4+ afferents). *p < 0.05; **p < 0.01. Numbers of rats were that same as for Figures 7 and 8.
Figure 11.
Figure 11.
Ultrastructural detection of the β3 GABAAR subunit in central boutons of synaptic glomeruli and in dorsal horn neurons. A–C, β3 GABAAR subunit, detected by preembedding immunogold (arrows indicate silver-gold particles), was found in the central boutons of type Ia glomeruli (CIa), which represent the spinal terminations of nonpeptidergic nociceptive afferents (Ribeiro-da-Silva, 2004). D, E, The subunit was also found in the central boutons (CIIa) of type IIa glomeruli (thought to represent the termination of Aδ nonnociceptive afferents; Ribeiro-da-Silva, 2004). Note the presence of GAD+ boutons (labeled GAD+V2 or just V2), revealed using DAB-based immunocytochemistry, in direct apposition to the central element of synaptic glomeruli (based on their typical morphology properties; Ribeiro-da-Silva and De Koninck, 2008). E, Enlargement of the framed area in D. F–H, Representative labeling for the β3 subunit (black arrows) clustered at inhibitory synaptic sites onto dendrites and cell bodies of dorsal horn neurons. C, Cytoplasm; D, dendrite; N, nucleus; V, varicosity.
Figure 12.
Figure 12.
Expression of GABAAR-associated proteins in nociceptive primary afferent terminals and at inhibitory postsynaptic sites. A, Typical staining in the dorsal horn for α-DG with a minor staining of blood vessels, for NL2 (B), and for CB2 (C). D, Analysis of colocalization of α-DG with either CGRP/IB4. E, NL2 in CGRP/IB4. F, CB2 in CGRP/IB4. G, Analysis of colocalization of DG with gephyrin in LI and LII. H, Analysis of colocalization of NL2 with gephyrin. I, Analysis of colocalization of CB2 with gephyrin. J, Percentage of CGRP and DG mask intersection containing the β3 GABAAR subunit (in blue); percentage of IB4 and DG mask intersection containing the β3 GABAAR subunit (in red). K, percentage of IB4 and DG mask intersection containing the α2 GABAAR subunit. Number of rats: n = 6. **p < 0.01; ****p < 0.0001. Scale bar, 40 μm.
Figure 13.
Figure 13.
qRT-PCR of mRNAs encoding α1, α2, and α3 GABAAR subunits and associated proteins. qRT-PCR of Gabra1 (A1), Gabra2 (A2), and Gabra3 (A3) in the liver (control), the DRG, the spinal dorsal horn (DH), and the brain. B, qRT-PCR of GPHN. C–E, qRT-PCR of mRNA encoding for other GABAAR-associated proteins, C, DAG1 (α-DG), D, NLGN2 (NL2), and E, ARHGEF9 (CB2), in the spleen (control), the DRG, the spinal dorsal horn and the brain. Numbers of rats were as follows: for liver, n = 3; for spleen, n = 4; for DRG, n = 4; for spinal DH, n = 4; and for brain, n = 7. For every tissue, we used the same number of animals in all experiments. Øp > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.
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