Association of gephyrin and glycine receptors in the human brainstem and spinal cord: an immunohistochemical analysis

Neuroscience 122 (2003) 773–784

ASSOCIATION OF GEPHYRIN AND GLYCINE RECEPTORS IN THE HUMAN BRAINSTEM AND SPINAL CORD: AN IMMUNOHISTOCHEMICAL ANALYSIS K. BAER,a H. J. WALDVOGEL,b M. J. DURING,a R. G. SNELL,a R. L. M. FAULLb AND M. I. REESa*

molecules and on an array of subsynaptic cytoplasmic linker proteins (Kim and Huganir, 1999; Moss and Smart, 2001). At glycinergic inhibitory synapses, a 93 kDa postsynaptic anchoring protein, gephyrin, is physically bound to the ␤ subunit of the heteropentameric glycine receptor (GlyR; Pfeiffer et al., 1982) and is the key mediator of glycinergic receptor anchoring and clustering. The GlyR forms a chloride-selective transmembrane channel composed of ␣1 and ␤ subunits contributing to a formation of pentameric ion channels in the ratio of 3␣:2␤ (Langosch et al., 1990). Gephyrin binds simultaneously to GlyR ␤ subunit (Meyer et al., 1995; Kneussel et al., 1999b) and to tubulin (Kirsch et al., 1991) and forms a submembranous lattice structure to dynamically trap recycled GlyRs (reviewed in Kneussel and Betz, 2000). In mutant mice lacking gephyrin, GlyR aggregation is lost (Feng et al., 1998), and their phenotype mimics the human neurological disorder hyperekplexia (Shiang et al., 1993; Rees et al., 1994, 2002). In addition to GlyR clustering, there is evidence to suggest gephyrin-dependent GABAA receptor clustering as demonstrated by transgenic mouse analysis (Essrich et al., 1998; Feng et al., 1998; Baer et al., 1999; Kneussel et al., 1999a; Fischer et al., 2000), in rat brain (SassoePognetto et al., 1999) and in cultured spinal cord neurons (Dumoulin et al., 2000). Whereas the majority of GABAA receptors are stabilized at synaptic sites via a gephyrindependent mechanism, a direct biochemical interaction between gephyrin and GABAA receptor subunits has not been established (Meyer et al., 1995; Kannenberg et al., 1997). The description of gephyrin distribution and function has been documented in brain tissue of mice, rats, cats, primate retina or cell lines (Grunert and Wassle, 1993; Kirsch et al., 1995; Alvarez et al., 1997; Feng et al., 1998; Lin et al., 2000; Geiman et al., 2002) and gephyrin was originally localized postsynaptically at glycinergic synapses in rat spinal cord (Triller et al., 1985). The distribution of gephyrin in rodent and cat brain regions is associated with a high number of glycinergic synapses (Wenthold et al., 1988; Cabot et al., 1995; Todd et al., 1995; Alvarez et al., 1997) and colocalization of GlyR a1 subunit and gephyrin was observed in vivo in rat and cat spinal neurons (Triller et al., 1985, 1987; Kirsch and Betz, 1993; Alvarez et al., 1997; Geiman et al., 2000, 2002), and in culture (Kirsch et al., 1993). Virtually complete colocalization of GlyRimmunoreactivity (IR) and postsynaptic gephyrin-IR has been established in the ventral horn of adult rat spinal cord (Triller et al., 1985, 1987; Todd et al., 1995, 1996; Colin et al., 1998).

a Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, Private Bag 92019, University of Auckland, Auckland, New Zealand b Department of Anatomy with Radiology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand

Abstract—Gephyrin is a postsynaptic clustering molecule that forms a protein scaffold to anchor inhibitory neurotransmitter receptors at the postsynaptic membrane of neurons. Gephyrin was first identified as a protein component of the glycine receptor complex and is also colocalized with several GABAA receptor subunits in rodent brain. We have studied the distribution of gephyrin and glycine receptor subunits in the human brainstem and spinal cord using immunohistochemistry at light and confocal laser scanning microscopy levels. This study demonstrates the novel localization of gephyrin with glycine receptors in the human brainstem and spinal cord. Colocalization of immunoreactivities for gephyrin and glycine receptor subunits was detected in the dorsal and ventral horns of the spinal cord, the hypoglossal nucleus and the medial vestibular nucleus of the medulla. The results clearly establish that gephyrin is ubiquitously distributed and is colocalized, with a large proportion of glycine receptor subunits in the human brainstem and spinal cord. We therefore suggest that gephyrin functions as a clustering molecule for major subtypes of glycine receptors in the human CNS. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: CNS, inhibitory synapse, receptor clustering, postsynaptic membrane.

Aggregation of neurotransmitter receptors at the postsynaptic membrane is necessary for efficient signal transduction between neurons. However, the mechanism by which receptors are anchored at their postsynaptic sites is not fully established and is the subject of intense investigation. At present it is known that postsynaptic receptor clustering depends on protein interactions with key organizational *Corresponding author. Tel: ⫹64-9-373-7599x84486; fax: ⫹64-9-3737492. E-mail address: [email protected] (M. I. Rees). Abbreviations: AON, accessory olivary nuclei; CGA, central gray area; CN, cuneate nucleus; DAB, 3,3-diaminobenzidine tetrahydrochloride; DH, dorsal horn of the spinal cord; DMNX, dorsal motor nucleus of the vagus; DR, dorsal raphe; GlyR, glycine receptor; GN, gracile nucleus; HN, hypoglossal nucleus; IO, inferior olive; IR, immunoreactivity; lam II, lamina II in the dorsal horn of the spinal cord; LC, locus coeruleus; LRN, lateral reticular nucleus; PBS, phosphate-buffered saline; PN, pontine nuclei; PRN, medial portion of pontine reticular nucleus; RN, raphe nucleus of the pons; SN, solitary nucleus; STN, spinal trigeminal nucleus; VH, ventral horn of the spinal cord.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00543-8

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Table 1. Human cases used in this study Case

Age, years

Sex

Postmortem interval, hours

Cause of death

H5873 H3070 H330 H6013 H393 H146

52 84 66 69 87 59

Male Female Male Female Female Male

21 7 13 11.5 11 35

Asphyxia Ischaemic heart disease Cor pulmonale Aortic aneurysm Ischaemic heart disease Ischaemic heart disease

Recently, we have described the distribution of gephyrin in the human brain (Waldvogel et al., 2003). In our ongoing investigation, we have utilized immunohistochemical staining methods to analyze the expression and synaptic localization of GlyRs and gephyrin in the human brainstem and spinal cord. The antibodies utilized in this study produce a punctate immunolabelling pattern in the rat, cat or human brain (Triller et al., 1985, 1987; Kirsch and Betz, 1993; Todd et al., 1996; Alvarez et al., 1997; Colin et al., 1998; Geiman et al., 2000, 2002). We have investigated the co-distribution of gephyrin and GlyR immunoreactivities in the human brainstem and spinal cord to establish the role of gephyrin in the formation and stabilization of GlyR clusters.

EXPERIMENTAL PROCEDURES Brain tissue For this study, the human brain tissue was obtained from the Neurological Foundation of New Zealand Human Brain Bank (Department of Anatomy with Radiology, University of Auckland, Auckland, New Zealand). The University of Auckland Human Subject Ethics Committee approved the protocols used in these studies. Brain tissue was obtained from six subjects (average age, 69 years; range 52– 87 years) with no history of neurological or psychiatric disorder which had a postmortem interval between 7 and 35 h after death (mean postmortem interval 16 h; see Table 1 for details). For the immunohistochemical studies, the human brains were fixed by perfusion through the basilar and internal carotid arteries, first with phosphate-buffered saline (PBS) with 1% sodium nitrite, followed by 15% formalin in 0.1 M phosphate buffer, pH 7.4. After the perfusion, blocks were dissected out and kept in the same fixative for 24 h. These blocks were cryoprotected in 20% sucrose in 0.1 M phosphate buffer with 0.1% Na-azide for 2–3 days, and then in 30% sucrose in 0.1 M phosphate buffer with 0.1% Naazide for a further 2–3 days. Sectioning of the blocks was performed on a freezing microtome at a thickness of 50 –70 ␮m. The sections were collected in PBS with 0.1% sodium azide (PBSazide) and stored for immunohistochemical processing.

Immunohistochemical procedures Microtome-cut brain sections of adult human brain were processed for immunohistochemical staining as previously described (Waldvogel et al., 1999, 2003). Primary antibodies. Three different antibodies were screened to detect gephyrin in human brain. These were: (a), the monoclonal antibody Mab7a (Alexis Biochemicals, Switzerland) which recognizes the N-terminal of the gephyrin molecule; (b), a mouse monoclonal antibody gephyrin (TL) raised against amino

acids 569 –726 of the invariant C-terminal gephyrin region (Transduction Laboratories, Lexington, KY, USA) (Hermann et al., 2001); and (c), a goat polyclonal antibody raised against the carboxy terminus of gephyrin of rat origin (R-20: sc-6411; Santa Cruz Biotechnology, Inc.). Three different GlyR antibodies were tested for the detection of GlyRs in the human brain. These were: (a), the mouse monoclonal antibody Mab4a raised against amino acids 96 –105 in the 48 kDa ␣1-subunit of the GlyR, and which also recognizes the 58 kDa ␤-subunit of the GlyR (Alexis Biochemicals; Pfeiffer et al., 1984; Schroder et al., 1991; Kirsch and Betz, 1993); (b) a mouse monoclonal antibody Mab2b raised against amino acids 1–10 of the 48 kDa GlyR ␣1 subunit, (Alexis Biochemicals; Pfeiffer et al., 1984); and (c), a rabbit polyclonal antibody AB 5052 raised against a peptide from the N-terminus of human ␣1 GlyR subunit with cross reactivity to the ␣2 subunit (Chemicon, Temecula, CA, USA; Geiman et al., 2002). The rabbit polyclonal AB5052 was used mainly for double labeling studies (see below). All antibodies used were dissolved in immunobuffer consisting of either 1% goat or 1% donkey serum in PBS with 0.2% Triton X-100 and 0.4% thimerosal (Sigma). Single immunoperoxidase labeling. Using standard immunohistochemical procedures, adjacent series of sections were selected and processed free-floating in tissue culture wells. Sections were washed in PBS and 0.2% Triton X-100 (PBS-Triton) and pretreated for antigen retrieval using a protocol modified from that of Fritschy et al., 1998 before being processed for immunohistochemistry. Sections for antigen retrieval were transferred to six-well tissue culture plates and incubated overnight in 0.1 M sodium citrate buffer, pH 4.5, and transferred to 10 ml of fresh sodium citrate buffer solution. Following this the sections were microwaved in a 650 W microwave oven for 30 s and allowed to cool before washing (3⫻15 min in PBS-Triton). The sections were then washed in PBS-Triton, incubated for 20 min in 50% methanol and 1% H2O2, washed again (3⫻15 min) in PBS-Triton, and incubated in primary antibodies for 2–3 days on a shaker at 4 °C. The mouse monoclonal antibody against gephyrin (TL) and mouse monoclonal antibody Mab2b (Alexis Biochemicals) were used at dilution 1:250; the goat polyclonal antibody against gephyrin (Santa Cruz Biotechnology) and mouse monoclonal antibody Mab4a (Alexis Biochemicals) were used at a dilution of 1:2000; the rabbit polyclonal antibody AB.5052 (Chemicon) was used at a dilution of 1:500. The primary antibodies were washed off (3⫻15 min; PBS-Triton) and the sections incubated overnight in speciesspecific biotinylated secondary antibodies (1:500; Sigma; Jackson Laboratories). The secondary antibodies were washed off (3⫻15 min; PBS-Triton) and the sections incubated for 4 h at room temperature in ExtrAvidin, 1:1000 (Sigma). The sections were reacted in 0.05% 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.01% H2O2 in 0.1 M phosphate buffer, pH 7.4, for 15–30 min to produce a brown reaction product. A nickel-intensified procedure was also used in which 0.4% nickel ammonium sulfate was added to the DAB solution to produce a blue-black reaction product (Adams, 1981). The sections were washed in PBS, mounted on gelatin chrom-alum coated slides, rinsed in distilled water, dehydrated through a graded alcohol series to xylene, and coverslipped with DPX (BDH, Poole, UK). Routinely a few sections were processed as control sections to determine nonspecific staining using the same immunohistochemical procedures detailed above except that the primary antibody was omitted from the procedure. In addition, sections were stained for Nissl substance with Cresyl Violet according to standard techniques.

Immunofluorescent double labeling For immunofluorescent, double-labeling confocal laser scanning microscopy, sections were incubated in a cocktail of monoclonal

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Fig. 1. Regional localization of gephyrin and GlyRs in human pons and medulla. Light microscopic images of gephyrin- (A, C, E) and GlyR-labeling (B, D, F) in adjacent sections at the level of upper pons (A, B), lower pons (C, D), and upper medulla (E, F) visualized by DAB staining. (A) Mouse-gephyrin and (B) GlyR4a displaying intense levels of labeling in the LC and the PRN; (C) mouse-gephyrin and (D) GlyR4a demonstrating intense levels of labeling in the LC and the CGA, moderate to high levels of gephyrin and GlyR in the DR, the RN and weaker gephyrin and GlyR in the reticular formation (RF) and the PN; (E) mouse-gephyrin and (F) GlyR4a showing moderately high levels of IR in the DMNX and high levels in the HN. Note that the levels of GlyR are similar to those of gephyrin in the HN and the DMNX. (E) Gephyrin and (F) GlyR display high levels of gephyrin and GlyR in the dorsal and medial AON, IO and the STN. Scale bars⫽1 cm (A–D); E, F⫽0.5 cm.

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Fig. 2. Regional localization of gephyrin and GlyRs in human medulla and spinal cord. Light microscopic images of gephyrin- (A, C, E) and GlyR- (B, D, F) labeling in adjacent sections at the level of lower medulla (A, B), the pyramidal decussation (C, D), and the cervical level of the spinal cord (E, F) visualized by DAB staining. (A) Mouse-gephyrin IR and (B) GlyR4a-IR demonstrating high levels of labeling in the HN and dorsal motor nucleus (DMNX), and moderate-high mouse-gephyrin- and GlyR4a-IR in the AON (A, C). Mouse-gephyrin-IR (A, B) and GlyR4a (B, D) showing patchy labeling in the GN, and the CN (arrows), with intense mouse-gephyrin- and GlyR4a-IR in the STN. (E, F) mouse-gephyrin IR and GlyR4a-IR in the spinal cord show low-to-moderate levels of labeling in the gray matter with especially intense labeling in lam II of the dorsal horn, and moderate to high levels of labeling in the gray matter of the VH and DH. Scale bars⫽2.5 mm (A–D); E, F⫽2.0 mm.

and polyclonal antibodies (monoclonal antibody against gephyrin (TL) diluted 1:250; and the polyclonal rabbit antibody AB5052

against GlyR subunits diluted 1:200). The sections were processed using the same procedure as the single-labeled sections.

Abbreviations used in the figures Lf N

lipofuscin autofluorescence nucleus

RF reticular formation

K. Baer et al. / Neuroscience 122 (2003) 773–784 They were incubated in primary antibodies for 2–3 days on a shaker at 4 °C, washed, and then incubated in species-specific fluorescent secondary antibody directly linked to FITC, Cy2 or Texas Red (1:100; Sigma, Jackson Laboratories, or Rockland). Control sections where the primary antibody was omitted showed no IR. The sections were washed, mounted on slides with Citifluor (Agar Scientific, Stanstead, England), and viewed using a Leica TCS 4D confocal laser scanning microscope. Confocal laser scanning microscope digital images were collected and saved in Tiff format. These images were contrast optimized using Adobe Photoshop software and were printed on an Epson Stylus ProXL inkjet printer.

Analysis To obtain an indication of the amount of gephyrin and GlyR colocalisation that occurs in the human brainstem, randomly selected high resolution confocal laser scanning microscope images (63⫻ or 100⫻ oil immersion) of double-labeled neurons were examined. Serial digital images of neurons and their dendrites from the hypoglossal nucleus, dorsal motor nucleus of the vagus (DMNX), the ventral horn, vestibular nucleus and reticular formation were collected and assigned a different color for each label (green for gephyrin and red for GlyRs). The individual images of the two labels were then overlain using computer software (Image J or Adobe Photoshop). On the resulting images, the doublelabeled puncta generated a yellow color. The single-labeled and double-labeled images were then printed and the immunoreactive puncta along the cell membranes were counted and the percentages of double-labeling were calculated.

RESULTS Immunohistochemical localization of gephyrin and GlyRs in the human pons, medulla and spinal cord Of the six antibodies assessed for this study (three against gephyrin and three against GlyRs), three were selected to investigate the staining patterns of gephyrin and GlyRs based on their staining properties in human tissue. The monoclonal antibody gephyrin (TL) from Transduction Laboratories produced the most consistent and strongest signal with the least background and this antibody was used most extensively for this study. Similarly the GlyR monoclonal antibody Mab4a produced the strongest signal with the least background and was used for the majority of the GlyR staining in this study. It is predominantly the results of these two antibodies Mab4a and gephyrin (TL) that are presented in this paper, although some illustrations from the goat-anti-gephyrin and GlyR Mab2a antibodies are included where appropriate. Of the antibodies assessed, those which have been traditionally used in rat tissue such as the anti-gephyrin Mab7a did not consistently produce optimal results in human tissue and these were not used. In general terms, all of the antibodies against gephyrin displayed a similar overall pattern of distribution. The GlyR antibodies also demonstrated a similar distribution, although Mab4a consistently produced a stronger signal probably due to recognizing all subunits of the GlyR whereas the others selectively recognized ␣ subunits. The principal aim of this study was to investigate the distribution of gephyrin and its association to GlyRs in the human pons, medulla and spinal cord at the regional and

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cellular levels. Sections from representative regions of the human brainstem and spinal cord, were immunohistochemically labeled for gephyrin and GlyR subunits, and were microscopically examined to determine the distribution of specific immunoreactivities generated by these antibodies. Light and confocal laser scanning microscope analysis of the distribution of gephyrin and GlyR subunits revealed robust and punctate gephyrin- and GlyR-IR in the human brainstem and spinal cord regions. The GlyR punctate labeling was consistently more distinct than that of the gephyrin-IR. Intracellular labeling for gephyrin was observed to a lesser extent in certain cell populations. Gephyrin-IR and GlyR-IR in the pons Within the pons, the regional gephyrin labeling (Fig. 1B, D) was very similar to that of the GlyR labeling. The highest intensities of GlyR-IR and gephyrin-IR were in the reticular formation and central gray area (CGA). At the regional level, the most intensely labeled nuclei for both gephyrin and GlyR were the locus coeruleus (LC) and the medial portion of the pontine reticular nucleus (PRN; Fig. 1A). At the cellular level, both of these nuclei had high levels of labeling of dendritic membranes. Both pigmented and nonpigmented cell bodies in the locus coeruleus displayed less GlyR labeling on their soma than gephyrin-IR (Figs. 1C, D; 3G, H). Neurons in the CGA were intensely immunoreactive for gephyrin, and punctate GlyR-IR was observed on membranes of cell bodies and processes in the CGA (Fig. 3E, F). The dorsal raphe nucleus (DR) and the raphe nucleus of the pons (RN) demonstrated high levels of gephyrin and GlyR-IR (Fig. 1C, D). Throughout the pontine nuclei (PN) a dense network of cell bodies and dendritic processes were labeled with punctate gephyrinIR, while GlyR-IR was more scattered in this region. Gephyrin- and GlyR-IR in the medulla Gephyrin- and GlyR-IR (Fig. 1E, F) produced a heterogeneous distribution in the gray matter of the upper and lower medulla (Figs. 1E, F; 3A, B). The hypoglossal nucleus (HN) labeled with high intensities of gephyrin-IR (Fig. 1E), and intense levels of GlyR-IR (Fig. 1F). The DMNX consistently showed high intensity of gephyrin- and moderate GlyR-IR (Fig. 1E, F). At the cellular level a dense network of intensely labeled punctate gephyrin- and GlyR-IR outlining small and large sized dendritic processes was observed in both of these nuclei (arrows, Fig. 3A–D). The network of GlyR-IR was especially dense in the hypoglossal nucleus and was observed on the membranes of dendritic processes and on cell bodies (Fig. 3D). The membranes of cell bodies revealed gephyrin-IR along their membranes, although to a lesser degree than dendritic processes (Fig. 3C). In the lower medulla, the spinal trigeminal nucleus (STN) was highly immunoreactive for gephyrin and GlyR (Fig. 2A–D). The gephyrin labeling was mainly restricted to lamina II of this nucleus, with GlyR-IR distributed throughout the remaining regions of the nucleus. The gracile nucleus (GN) and the cuneate nucleus (CN) revealed small patches of high GlyR-IR (arrows in Fig. 2B, D), which, in the dorsal regions of the gracile

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Fig. 3. Gephyrin and glycine-receptor IR in the human pons and medulla at the cellular level. Light microscopic images of mouse-gephyrin- (A, C, E, G) and GlyR4a- (B, D) and GlyR2b- (F, H) labeling visualized by DAB staining. (A) Gephyrin- and (B) GlyR-IR in the DMNX showing a dense network of intensely small and large diameter dendritic processes with punctate-IR outlining the membranes of dendritic processes (arrows) and to a lesser degree the membranes of cell bodies; (C) punctate gephyrin- and (D) GlyR (arrows) along the membrane of dendrites in the hypoglossal nucleus; (E) gephyrin and (F) GlyR labeling (arrows) of neuronal membranes in the CGA; (G) gephyrin-IR and (H) punctate GlyR (arrows) in the LC showing a pigmented neuron with strong punctate gephyrin-IR on its membrane (G) and moderate GlyR on dendritic membranes of this region (H). Scale bars⫽50 ␮m (A, B, E, F); C, D, G, H⫽10 ␮m.

K. Baer et al. / Neuroscience 122 (2003) 773–784 Table 2. Intensity of gephyrin- and GlyR-IR in human pons, medulla and spinal corda Gephyrin

GlyR Mab4a

IR on IR on IR on IR on processes processes cell cell bodies bodies Pons CGA ⫹⫹⫹ ⫹⫹⫹⫹ LC ⫹⫹⫹ ⫹⫹⫹⫹ PRN ⫹⫹⫹ ⫹⫹⫹ PN ⫹⫹ ⫹⫹ DR ⫹⫹⫹ ⫹⫹⫹ RN ⫹⫹ ⫹⫹ Medulla DMNX ⫹⫹⫹ ⫹⫹⫹⫹ HN ⫹⫹⫹ ⫹⫹⫹ SN ⫹⫹ ⫹⫹ GN ⫹⫹ ⫹⫹ CN ⫹⫹ ⫹⫹ STN ⫹⫹⫹ ⫹⫹⫹ AON ⫹⫹⫹ ⫹⫹⫹ IO ⫹⫹⫹ ⫹⫹⫹ LRN ⫹⫹ ⫹ Spinal cord Substantia gelatinosa (lam II) ⫹⫹⫹⫹ ⫹⫹⫹⫹ DH ⫹⫹⫹ ⫹⫹⫹ VH ⫹⫹ ⫹⫹⫹

⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹

⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹

⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹

a

Density of immunoreactivity: ⫺, not detectable; ⫹, weak; ⫹⫹, moderate; ⫹⫹⫹, high; ⫹⫹⫹⫹, most intense.

nucleus were also reactive for gephyrin (arrows, Fig. 2A, C). At the cellular level, these regions in the CN and GN were identified as gephyrin and GlyR immunoreactive cells (Fig. 2B, D). The inferior olive (IO; Fig. 1E) displayed moderate to high levels of gephyrin-IR on neuronal cell bodies and their proximal dendrites. The dorsal and medial accessory olivary nuclei (AON) displayed moderate to weak gephyrin-IR on the membranes of dendritic processes and cell bodies (Fig. 1E, 2A). In contrast, the dorsal and medial AON and the IO had high levels of punctate GlyR labeling on dendritic processes and neuronal cell bodies. The lateral reticular nucleus was moderately labeled with GlyR but showed weaker labeling with gephyrin (Table 2). The solitary nucleus (SN; Fig. 1E) had moderate gephyrin-IR on dendritic processes and cell bodies with high GlyR-IR predominantly on dendritic processes, and moderate labeling on the cell bodies (SN; Fig. 1F). A large part of the remaining gray matter in the medulla including cell bodies and neuropil were labeled with gephyrin-IR; many fine dendritic processes as well as beaded structures could be observed. Similarly, the GlyR labeling in the majority of the remaining gray matter in the medulla was localized as punctate membrane-bound IR in the neuropil as well as on various sized dendritic processes and cell bodies. Gephyrin and GlyR-IR in the spinal cord Gephyrin-IR was observed in sections at the cervical level of the human spinal cord. Gephyrin and GlyR-IR was

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evident throughout the gray matter of the spinal cord (Fig. 2E, F) and was especially intense in the dorsal (DH) and ventral horns (VH; Fig. 2E, F). The labeling of the GlyR was consistently high throughout the spinal cord, and the intensity of gephyrin labeling although lower, was always proportional to the intensity of the GlyR labeling. The neuropil of lamina II of the dorsal horn (lam II) showed the most intense gephyrin and GlyR-IR in the spinal cord (Fig. 2E). At high magnification a dense network of cell bodies and fine dendritic processes was evident with punctate gephyrin and GlyR-IR on their membranes in lam II (Fig. 4A, B) and lamina III (arrows Fig. 4C, D). The gephyrin-IR was moderate in lamina I and III with cell bodies and dendritic processes coated with punctate gephyrin and GlyR-IR. In the ventral horn, moderate to high levels of punctate gephyrin-IR and intense GlyR puncta were observed on cell bodies and dendritic processes (arrows Fig. 4E–H). Association of gephyrin- and GlyR-IR at the cellular level To further investigate the precise distribution of gephyrin and GlyRs, sections which had been double labeled using immunofluorescent markers to detect gephyrin and GlyRs were observed by confocal laser scanning microscopy. At the cellular level, the IR of gephyrin and GlyRs was examined in the hypoglossal nucleus (Fig. 5A–C), the medial vestibular nucleus in the medulla (Fig. 5D–F) and in the ventral horn (Fig. 5G–I). Punctate IR for both markers was observed on the membranes of the soma and neuronal processes. Colocalization of individual immunoreactive puncta for gephyrin and GlyRs was observed on the membranes of cell bodies and dendritic processes (arrows Fig. 5). GlyR-IR was distributed along the entire length of neuronal membranes, whereas gephyrin was localized to ‘hot spots’ on the same membranes. In addition to immunoreactive punctate labeling for gephyrin along the neuronal membranes, intracellular gephyrin-IR was observed scattered throughout the cytoplasm. Semi-quantitative counting of immunoreactive puncta was performed on confocal laser scanning microscope images of randomly selected double-labeled neurons throughout the human brainstem (examples shown on Fig. 5). Images from five serial scans at 1 ␮m intervals from a total of 17 neurons and their dendrites as well as 15 additional dendrites were counted from three human cases. A total of five different nuclei: the hypoglossal nucleus (five neurons and 10 other dendrites); DMNX (three neurons and three dendrites); the ventral horn (five neurons) vestibular nucleus (two neurons), and two neurons and dendrites from the reticular formation. A total of 1399 GlyR-immunoreactive puncta and 1582 gephyrin-immunoreactive puncta were counted on these neurons and dendrites. This revealed that approximately 10% more gephyrin positive puncta were present on these neurons than GlyR-positive puncta. The colocalisation of gephyrin and GlyR puncta was approximately 60%: counts of doublelabeled puncta revealed that on average 57% of gephyrin positive puncta were colocalised with GlyRs and 63% of GlyRs were colocalised with gephyrin. The punctate im-

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Fig. 4. Gephyrin and glycine-receptor IR in the human spinal cord at the cellular level. Light microscopic images of mouse-gephyrin (A, C, E, G), GlyR4a (B, D, F) and GlyR2b (H) labeling are presented from the dorsal (A–D) and ventral (E–H) horns of the human spinal cord visualized by DAB staining. (A) Gephyrin-IR and (B) GlyR-IR demonstrating high levels of labeling on fine dendritic processes. (C) Gephyrin and (D) GlyR on membranes of neuronal processes and cell bodies in the dorsal horn (arrows). (E) Moderate to high levels of punctate gephyrin- (arrows) and (F) GlyR-labeling along the membranes of dendrites and cell bodies in the ventral horn (arrows). (G) High magnification images of neurons in the ventral horn with punctate gephyrin- (arrows) or (H) GlyR-labeling (arrows) on the membranes of cell bodies and processes. Scale bars⫽50 ␮m (A–F); G, H⫽10 ␮m.

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Fig. 5. Gephyrin- and glycine-receptor IR at the cellular level. Confocal laser scanning micrograph single scans from sections of the hypoglossal nucleus (A–C), the medial vestibular nucleus of the medulla (D–F) and the ventral horn (G–I) after double-labeling with mouse-gephyrin (green; A, D, G) and rabbit-anti-GlyR (red; B, D, F). Markers are shown together as merged images in (C, F, I). The labeling for each marker is shown separately and arrows point to colocalizing gephyrin and GlyR in each triplet of images (A–C, D–F, G–I). Note the colocalizing punctate gephyrin and GlyR outlining cell bodies as well as on dendritic processes (yellow; C, F, I), double arrow in A and C indicates intracellular gephyrin labeling. Scale bars⫽10 ␮m (A–F); G–I⫽20 ␮m.

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munoreactive labeling for gephyrin and GlyRs along somatic and dendritic labeling was colocalised in a similar proportion in all regions examined.

DISCUSSION This is the first detailed study comparing the regional and cellular distribution of the anchoring protein gephyrin and GlyRs in the human brain. Using immunohistochemical methods at the light and confocal laser scanning microscopic levels, our results suggest that gephyrin and GlyRs are widely distributed in the human pons, medulla and spinal cord. The methods used were optimized for structural tissue preservation and receptor stability of the postmortem human brain to limit background staining and lipofuscin artifacts. We have tested six antibodies, three raised against gephyrin, and three raised against the GlyR, to demonstrate immunohistochemically the presence of GlyR and gephyrin antigens in human pons, medulla and spinal cord. The staining patterns of the various antibodies were generally similar in the regions tested and the strongest and most specific labeling of the antibodies, gephyrin (TL) and Mab4a (for GlyRs) were adopted for this study. More detailed analysis of the different GlyR subunit antibodies will be the focus of future studies. The goat–anti-gephyrin antibody and the monoclonal antibody are both raised against the conserved sequence of the carboxy terminal and produced similar staining patterns, although the goat– anti-gephyrin antibody generally had a higher background labeling than the monoclonal anti-gephyrin (TL). The GlyR antibodies also produced similar patterns of staining except that the Mab4a which recognizes all subunits was consistently more intense than the other GlyR antibodies which selectively recognize ␣ subunits. GlyR, using Mab4a, has demonstrated localization of GlyR at postsynaptic sites on human cortical neurons (Naas et al., 1991), while the monoclonal antibody against gephyrin (TL), has recently displayed punctate labeling in the human brain (Waldvogel et al., 2003). Gephyrin- and GlyR-IR were observed in most of the human brainstem and spinal cord regions examined. At the regional level, the most intense staining for both markers was observed in the dorsal and ventral horns of the spinal cord, the spinal trigeminal nucleus, and the motor nuclei associated with the autonomic nervous system such as the dorsal motor nucleus of vagus. The patches of high IR in the cuneate and gracile nuclei may be associated with particular sensory inputs as the inputs to these nuclei are all topographically organized. The hypoglossal nucleus revealed intense labeling especially for GlyRs and therefore glycinergic inhibitory control of this nucleus is predominant. The GlyR-IR which we have detected in the human is in agreement with studies in the rat brain showing high levels of IR for the neurotransmitter glycine in the hypoglossal nucleus, gracile nucleus, spinal trigeminal nucleus and raphe nucleus of the rat brain (Rampon et al., 1996). Other brainstem nuclei such as the SN and the IO displayed moderate to high levels of both gephyrin- and GlyR-IR. At

the cellular level, gephyrin- and GlyR-IR were present on the plasma membranes of the soma and dendrites of neurons throughout the human pons, medulla and spinal cord. Using light microscopy, GlyR- and gephyrin-IR were observed in the same brain regions and on similar neuronal cell types. Punctate labeling for either marker was observed on the membranes of similar types of neuronal somata and processes. This indicates a common cellular distribution of GlyRs and gephyrin in neurons of human pons, medulla and spinal cord. Confocal laser scanning microcopy revealed a colocalization of the gephyrin- and GlyR-IR at punctate sites indicating synaptic positioning, while incomplete colocalization on extrasynaptic regions of the membranes was also observed. Approximately 60% of all gephyrin sites in the human brainstem were colocalised with GlyRs. This would indicate that approximately 40% of all gephyrin puncta are localized with non-glycinergic receptors, presumably GABAA receptors in the human brainstem. This is consistent with previous studies which indicate that glycine is present as a neurotransmitter at those synapses where both gephyrin and glycine are present, but suggests that gephyrin may also be present at non-glycinergic synapses (Todd et al., 1995). GABAA receptors are colocalised with gephyrin on Renshaw cells in addition to other regions of the rat brain (Geiman et al., 2002; Sassoe-Pognetto et al., 1999). Gephyrin is considered a selective marker of postsynaptic sites by virtue of gephyrin and GlyR ‘hot spots’ (brightly stained puncta) reflecting synaptic localization and aggregation (Grunert and Wassle, 1993; Kirsch et al., 1996). The confocal laser scanning microscopy data in the human is consistent with colocalization of gephyrin and GlyRs as observed in rat spinal cord (Geiman et al., 2002). In both cases, in addition to gephyrin and GlyR cluster ‘hot spots,’ GlyR-IR was observed along the entire length of neuronal membranes. A similar observation was reported for cat spinal cord ventral horn neurons where gephyrin and GlyR were clearly colocalized, but the density and topographical organization of gephyrin and GlyR clusters varied in different neurons and in different dendritic regions (Alvarez et al., 1997). The anti-GlyR antibodies used in this study were directed to various different sequences of the GlyR subunits and detected either the a1 subunit only (Mab2b), the a1 and ␣2 subunits (AB 5052), or recognized an epitope conserved in all known ␣ and ␤ subunits (Mab4a; Schroder et al., 1991; Pfeiffer et al., 1984; Kirsch and Betz, 1993). It is established that Mab2b- and 4a-IR represent postsynaptic structures (Triller et al., 1985; Altschuler et al., 1986) and also intracellular sites of GlyR protein synthesis, transport and aggregation (Hoch et al., 1989). Sequence comparison of human and rat GlyR cDNAs reveals a complete conservation of the epitope for the Mab4a antibody (Schroder et al., 1991). In rat brain, Mab4a produces both punctate and diffuse somatic labeling, suggesting that the Mab4a antigens are not restricted to postsynaptic membrane specializations (Kirsch and Betz, 1993). Intracellular labeling was also reported for the anti-GlyR AB 5052 (Geiman et al., 2002). This correlates with findings using cul-

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tured spinal neurons in vitro, where synapse formation corresponds with gephyrin expression (Bechade et al., 1996; Colin et al., 1996). Although the receptor clusters are mainly postsynaptic a proportion of extrasynaptic and cytoplasmic staining is observed (Levi et al., 1999; Dumoulin et al., 2000). The observations made in the present study of some intracellular gephyrin IR suggests that a similar mechanism may exist in the human brain. Using the antibodies Mab2b, Mab4a, and AB 5052, it is not possible to exclusively detect the GlyR ␤ subunit, which contains the gephyrin binding domain (Meyer et al., 1995) or to differentiate between ␣2 and/or ␣3 subunit-containing GlyRs. To establish whether gephyrin is only associated with ␤-subunit containing GlyRs in human brain, differential studies using ␤, ␣2 and ␣3 subunit-specific antibodies are required. The role of gephyrin with regard to the mechanisms of synaptic localization and receptor clustering is not yet established. There is evidence to show that GlyR clustering is regulated by several factors involving interaction with gephyrin for stabilization (Levi et al., 1998) and synaptic accumulation (Kirsch et al., 1993; Kirsch and Betz, 1998), but may also include other gephyrin-independent mechanisms (Meier et al., 2000). It has been suggested that presynaptically released signaling molecules are involved in the formation of postsynaptic receptor clusters on neurons (Seitanidou et al., 1992; Craig et al., 1994; Craig and Boudin, 2001). In vitro studies support the model of random insertion of receptors and accumulation by a diffusion/ trapping mechanism (Craig et al., 1994; Meier et al., 2000). Further studies are necessary in the human brain to determine whether gephyrin is trapped at postsynaptic sites by other anchoring molecules or whether it is recruited by active transport mechanisms. In this context, the existence of a general ‘synaptogenic’ element common to excitatory and inhibitory synapses was recently postulated (Rao et al., 2000), in addition to ‘synapse-specific’ signaling factors. Summary and functional implications This is the first study that establishes the association of gephyrin and GlyRs in human brainstem and spinal cord. The data indicates an association and colocalization of gephyrin and GlyR immunoreactivities, implicating similar functions for gephyrin in human brain as that reported for rodent brain. Thus, gephyrin is likely to play a fundamental role in the organization of major types of inhibitory synapses at postsynaptic membranes in human brain. Acknowledgements—K. Baer and H. J. Waldvogel contributed equally to this study. This work was supported by the Auckland Medical Research Fund (AMRF), the Health Research Council of New Zealand, Freemasons of New Zealand and the Neurological Foundation of New Zealand. We thank the Neurological Foundation of New Zealand Human Brain Bank for providing the human brain tissue and the Biomedical Imaging Research Unit of the University of Auckland for their expert assistance.

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(Accepted 3 July 2003)