Potassium channel subunit Kv3.2 and the water channel aquaporin-4 are selectively localized to cerebellar pinceau

Brain Research 1026 (2004) 168 – 178 www.elsevier.com/locate/brainres

Research report

Potassium channel subunit Kv3.2 and the water channel aquaporin-4 are selectively localized to cerebellar pinceau Marketta Bobika, Mark H. Ellismana, Bernardo Rudyb, Maryann E. Martonea,* a

Department of Neurosciences, National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, CA 92093-0608, United States b Departments of Physiology and Neurosciences and Biochemistry, New York University Medical Center, NY 10016, United States Accepted 31 July 2004 Available online 22 September 2004

Abstract The pinceau is a cerebellar structure formed by descending GABA-ergic basket cell axonal terminals converging on the initial axonal segment of Purkinje cell. Although basket cells exert a powerful inhibitory influence on the output of the cerebellar cortex, the function and mode of action of the pinceau are not understood because the majority of basket cell axons fail to make identifiable synaptic contacts with the Purkinje cell axon. Several proteins were previously reported to cluster specifically in this area, including a number of voltage-activated potassium channel subunits. In this study, we used immunohistochemistry, electron microscopy, and electron tomography to examine the ultrastructural localization of a novel voltage-gated potassium channel subunit, Kv3.2, in the pinceau. We found strong, selective localization of Kv3.2 to basket cell axons. Additionally, because potassium buffering is often conducted through water channels, we studied the extent of a brain-specific water channel, aquaporin-4 (AQP4), using confocal and electron microscopy. As expected, we found AQP4 was heavily localized to astrocytic processes of the pinceau. The abundance of potassium channels and AQP4 in this area suggests rapid ionic dynamics in the pinceau, and the unusual, highly specialized morphology of this region implies that the structural features may combine with the molecular composition to regulate the microenvironment of the initial segment of the Purkinje cell axon. D 2004 Elsevier B.V. All rights reserved. Theme: Motor systems and sensorimotor integration Topic: Cerebellum Keywords: Basket cell axon; Purkinje cell; Initial segment; Astrocyte

1. Introduction The cerebellar pinceau is a highly complex and anatomically unique structure within the mammalian nervous system. It is formed by basket cell axonal collaterals descending from the molecular layer and converging on the Purkinje cell initial axonal segment to form a dense terminal plexus around the initial segment. Despite the dense innervation of the initial segment region, the majority of basket cell terminals in * Corresponding author. Department of Neurosciences, University of California, San Diego, San Diego, CA 92093-0608, United States. E-mail address: [email protected] (M.E. Martone). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.07.088

the pinceau do not make direct contact with the initial segment, as they do on the soma [31]. On each initial segment, only one to two basket cell terminals actually form axo-axonal synapses; the rest of the terminals end freely [31]. Instead, the basket cell terminals appear to contact velous glial processes, which in turn almost completely envelop the Purkinje cell initial segment. The heavy ensheathment of the initial segment by glial processes makes the astrocytes the only intervening cellular element between the basket cell terminals and the initial segment. The basket cell terminals are sometimes joined together by septate-like junctions [43] which likely add rigidity to this structure, along with an astrocytic sheath surrounding the outer plexus boundaries

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[31]. Basket cell terminals are densely packed with GABA filled vesicles [7] and they likely exert inhibitory control over the axon initial segment. However, the absence of traditional synapses in this area suggests an unconventional mode of transmission between basket and Purkinje cells. Because the Purkinje cell provides the only neuronal output from the cerebellar cortex, it is especially important to understand how the cellular ensheathments of its spike initiation zone are functionally organized. Although this region has not been well-characterized functionally, anatomical studies have shown several proteins to be selectively concentrated in pinceau fibers, including F-actin [2,31], PSD 95 [16], nitric oxide synthase [37], the GABA transporter GAT1 [34,38], and the neuronal calcium sensor NCS-1 [24]. Particularly interesting is the abundance of voltage-gated potassium channel subunits. Studies have shown that pinceau fibers express extremely high densities of Kv1.1, Kv1.2, Kv3.4 [23,25], and to lesser extent, Kvh2 [33]. In this study, we report that another member of the Shaw-like subfamily of potassium channels, Kv3.2, is expressed exclusively in the cerebellar cortex in pinceau axons and perisomatic baskets. The abundance of potassium channels and the unique distribution of astrocytes around the Purkinje cell initial segment suggest that this region experiences large potassium fluxes. Because astrocytic water channels are known to be involved in potassium buffering [8,9], we also examined the distribution of a brain-specific water channel aquaporin-4 (AQP4). AQP4 was recently discovered and reported in glial processes surrounding blood vessels, ventricles, subarachnoid space and glia around Purkinje somas, and granular cells [46,50]. We are extending these findings here to investigate AQP4 labeling in the pinceau.

2. Materials and methods 2.1. Tissue Adult male Sprague–Dawley rats (2-months old) were deeply anesthetized with ketamine (50 mg/kg), rhompum (1 mg/kg), and acetopromazine (5 mg/kg). The vascular system was perfused intracardially with Ringer’s solution at 35 8C, followed by fixative. For light microscopic analysis, rats were perfused either with 4% paraformaldehyde and 0.1% glutaraladehyde or with 3.75% acrolein and 2% paraformaldehyde in 0.1 M PBS, pH 7.4. For standard electron microscopic studies, animals were perfused with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. For EM immunostudies, animals were perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer. The cerebellum was postfixed upon removal for 2 h in the primary fixative at 4 8C, and was cut into 50-Am-thick sagittal sections with a vibratome (Leica).

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2.2. Light-level immunofluorescence Sections were blocked in 3% normal donkey serum, 0.37% glycine, 1% bovine serum albumin, 1% cold water fish gelatin, and 0.2% Triton X-100 for 30 min. Slices were then incubated in primary antibodies in the blocking buffer on a rotator overnight at 4 8C. A rabbit polyclonal antiserum to Kv3.2 was generated against a synthetic peptide corresponding to a sequence in the intracellular amino-terminal domain as described in Refs. [4,40] and was used at a dilution of (1:100). The following antibodies were also used: a mouse monoclonal anti-PSD95 (1:500, Affinity Bioreagents, Golden, CO), an isoform-specific rabbit polyclonal antiserum generated against a synthetic peptide corresponding to a C terminus of the rat AQP4 (1:100, Alpha Diagnostics, San Antonio, TX), a guinea pig polyclonal anti-GFAP (1:100, Advanced Immunochemical, Long Beach, CA), a mouse monoclonal antineurofilament 68 KDa (1:100, Sigma, Chicago, IL), and a mouse monoclonal antineurofilament 200 KDa (1:100, Sigma, Chicago, IL). As a control, the primary antibodies were omitted from the immunolabeling sequence. The blocking buffer was diluted 1:3 with 0.1 M PBS for subsequent washes and secondary antibody dilution. Secondary antibodies were applied for 2 h on a rotator at room temperature. For immunoperoxidase studies, a goat anti-rabbit antibody tagged with biotin (Vector Laboratories, Burlingame, CA) was employed according to manufacturer’s instructions. For single immunofluorescence studies, a goat anti-rabbit conjugated to FITC was employed as a secondary antibody. For double-labeling studies, affinitypurified donkey anti-rabbit Cy5, donkey anti-rabbit FITC and donkey anti-mouse RedX were employed. All fluorescently tagged antibodies were obtained from Jackson ImmunoResearch (West Grove PA) and used at a dilution of 1:100. Fluorescent and transmitted light images were recorded using a Zeiss Axiovert inverted microscope with a laser scanning confocal attachment (MRC-1024; BioRAD Laboratories, Cambridge, MA) and a krypton/argon mixed gas laser. Images were collected digitally using either a 40 (n.a.=1.3) or 63 (n.a.=1.4) objective lens and transferred to a graphics program (Adobe PhotoShop), where brightness and contrast were adjusted. 2.3. EM immunocytochemistry Animals were perfused with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer. The sections were labeled using anti-Kv3.2 and in separate experiments with AQP-4 using the ABC kit (Vector Laboratories, Burlingame, CA) with diaminobenzidine detection. As a control, the primary antibody was omitted from the immunolabeling sequence. Additional experiments for EM Kv3.2 labeling were performed with quantum dot (Qdots) secondary antibody conjugates (QuantumDot, Hayward, CA). Qdots are nanoparticles which possess both

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fluorescence and electron density and can be used for correlated light and electron microscopic imaging [30]. Qdots have size-tunable emission, high luminescence, narrow spectral line widths and stability against photobleaching [3]. Because Qdots are particulate, they possess better spatial resolution than enzymatic detection for electron microscopic studies. We employed 655 nm emitting Qdots, rod-shaped particles measuring approximately 1015 nm. To increase penetration, we increased the Triton X-100 concentration for Qdot experiments from 0.1% to 0.3%. Thin sections (80 nm) were examined on JEOL 100 CX and JEOL 1200 transmission electron microscopes operating at 80 keV. Thicker sections were examined on JEOL 4000 EX intermediate electron microscope operating at 400 KeV.

14-bit cooled CCD camera (Photometrics). The 1024 1024 pixel images were aligned with the program FIDO [44,47]. Reconstruction was performed using either the SUPRIM suite of software (Ref. [41] or IMOD on Ref. [21]). The final volume was rendered and viewed using ANALYZE AVW, a program developed for the measurement of three-dimensional volumes [35]. Volume segmentation was performed manually by drawing contours on 2D slices through the volume using the program Xvoxtrace, developed by Stephan Lamont in our laboratory. These contours were used to generate surfaced reconstructions using SYNU [13].

2.4. Serial section electron microscopy

3.1. Structure of the pinceau

3D reconstructions of the cerebellar pinceau were performed using serial section electron microscopy. Cerebellar blocks were trimmed to a trapezoid containing the Purkinje cell layer and the entire pinceau area. Serial sections were cut on a Reichert Ultracut III ultramicrotome, collected on formvar-coated slot grids, counterstained with lead citrate, and examined on a JEOL 100 CX electron microscope. Electron micrographs of the pinceau area were recorded at a magnification of 3300. Photographic prints were made of each electron micrograph at a final magnification of 10,000. Pairs of prints were overlaid on a light box to obtain a best fit between structures on consecutive planes. On this basis, three to five fiducial marks were assigned to each plane by penetrating the prints with a pin. Structures of interest, including Purkinje cell body, initial segment, pinceau fibers, and astrocytes were included in each picture. Pinceau fibers were identified by their location surrounding the Purkinje cell initial segment and their high content of neurofilaments and vesicles [31]. The contours of each structure were hand traced and digitized using a high-resolution digitizing tablet [51]. Surfaces were fitted to the contour data and viewed using the SYNU suite of programs [13].

In order to examine the relationships between the various cellular components comprising the pinceau, we performed 3D reconstructions of the pinceau area using serial section electron microscopy. The serial 3D reconstructions (Fig. 1A–C) support previous light and electron microscopy findings about the organization of the pinceau [31,42]. The pinceau is composed of the terminal axon ramifications of basket cell axon collaterals. Distinguishing features of these collaterals include their high concentration of neurofilaments (Fig. 1D–F) and the lack of a myelin sheath (Fig. 1D; Ref. [31]). In the reconstruction shown in Fig. 1A, approximately 50 nonmyelinated neurofilamentcontaining axons were identified in the region of the Purkinje cell initial segment. Many of these axons represented short stretches that could not be traced beyond the pinceau region (Fig. 1A and C, shown in multiple colors). We observed a single synapse between the basket cell terminal and the initial segment in the upper one third of the axon, close to the soma (Fig. 1D, arrowhead). In this instance, this same basket cell axon formed an identifiable synapse on both the Purkinje cell soma, as part of the pericellular basket, and the initial segment, as part of the pinceau (data not shown). The basket cell terminals lack neurofilaments, and contain multiple flattened vesicles (Fig. 1B and D). As previously reported, the individual vesicle-filled varicosities were seen to be linked through septate-like junctions (not shown). The reconstruction in Fig. 1C was performed at higher magnification and showed the relationship of astrocytic processes to basket cell terminals and to the initial segment. The same astrocytic process that intertwined among the basket cell fibers also contacted the initial segment (Fig. 1C).

2.5. Electron tomography from single-axis tilt series For antibody labeled tissues, the three-dimensional (3D) volumes of the pinceau area were obtained by tomographic reconstruction derived from single-axis tilt series [6,32]. Thick cerebellar sections (0.5-1 Am) were mounted on blocks, cut and collected onto 50/50 mesh clamshell grids, and 10-nm colloidal gold was applied to the section surface to serve as fiducial cues for subsequent alignment of images. Labeled pinceau fibers were identified at 400 keV on a JEOL 4000EX intermediate voltage electron microscope. Images were typically obtained over a range of F608 using film at a magnification 10,000–15,000. Each photographic negative of the tilt series was digitized with a

3. Results

3.2. Kv3.2 Immunofluorescence microscopy revealed that the pinceau and the Purkinje pericellular basket were the only

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Fig. 1. 3D structure of the cerebellar pinceau. (A) Three-dimensional reconstruction from unstained serial electron micrographs. Basket cell axons and terminals (in various colors) are surrounding the Purkinje cell axon initial segment (yellow). (B) A higher magnification reconstruction of a basket cell terminal showing the complex morphology of individual terminals. (C) Three-dimensional reconstruction of an astrocyte (in gray color) from the pinceau region. In this reconstruction, a single astrocytic process (gray) is observed surrounding pinceau axons (multiple colors) and contacting the Purkinje cell initial segment (blue). (D) Electron micrograph of the pinceau area from which the reconstruction in panels A–C was made. The Purkinje cell body is apparent at the top (PC) with the initial segment (IS) descending from the soma. A single synapse on the initial segment is clearly identifiable (arrowhead). (E and F) Immunolabeled confocal images showing the structure and some molecular constituents of the pinceau. Pinceau fibers are rich in neurofilaments (blue label and arrows in panels E and F). (F) The pinceau (arrowheads) is also enriched in F-actin (red), and in aquaporin-4 (green), as discussed in the results section. PC=Purkinje cell body; IS=initial segment; NF=processes filled with neurofilaments. V=terminals of basket cells filled with vesicles; Scale bars: A and D=1.5 Am; B=~0.5 Am; C=~1 Am; E and F=20 Am.

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Fig. 2. Localization of Kv3.2 and AQP4 in the pinceau using immunofluorescence. (A) Kv3.2 immunofluorescence labeling is punctate and associated with the pinceau (arrows). (B) Double labeling of Kv3.2 (green) and PSD95 (red). PSD95 is highly concentrated in the pinceau but is not coextensive with Kv3.2 labeling. (C and D) AQP4 labeling in the pinceau area of the cerebellar cortex. AQP4 was found throughout the granule cell layer and Purkinje cell layer. Intense labeling was observed surrounding the Purkinje cell soma and axon (large arrows) and in the pinceau (arrowheads; PC=Purkinje cell somas). Scale bars in A, C and D=20 Am; Scale bar in B=5 Am.

strongly and consistently labeled structures in the cerebellar cortex (Fig. 2A and B). Some cell bodies were labeled in the deep cerebellar nuclei, and occasional cells (presumably basket cells) were labeled in molecular layer. Both of these localizations are consistent with in situ hybridization studies of Weiser at al. [48]. Other regions exhibited little-to-no labeling. Control sections in which the primary antibody was omitted were free of labeling. In double-labeling studies with PSD 95 [16], we confirmed that Kv3.2 was confined to the pinceau and not the surrounding neuropil (Fig. 2B). A previous report indicated that PSD-95 colocalizes with Kv1.4 [17], Kv1.1, and Kv1.2 in the septate junctions, but not with Kv3.4 [23]. We examined the colocalization of Kv3.2 with PSD-95 and found the two proteins partially overlap but do not appear to be coextensive within the resolution limits of the light microscope (Fig. 2B). Electron microscopic analysis using a pre-embedding peroxidase-based method showed that Kv3.2 was present in basket cell axons, identified by neurofilaments in the axonal shaft and vesicles in axonal terminals. This labeling was punctate and was generally associated with the plasmalemma in vesicle-containing regions of the basket cell terminals, but was not associated with the plasmalemma in

the main axonal shaft (Fig. 3A–C). Additionally, presynaptic boutons, synapsing onto the Purkinje cell body, were sometimes labeled. However, the reaction product was not associated with the synaptic active zone but was located at membrane sites not apposed to the Purkinje cell (Fig. 3D and E). Three-dimensional reconstructions of the immunoperoxidase stained tissue using electron tomography revealed that Kv3.2 labeling was often associated with vesicle clusters in the basket cell terminals (Fig. 4). In several cases, focal labeling for Kv3.2 was present in basket cell terminals apposed to thin, glial-like processes (Figs. 3 and 4), although labeling was clearly present in regions of basket cell axons apposed to other basket cell processes as well. Because DAB immunocytochemistry has relatively poor spatial resolution compared to particulate markers, we performed additional immunolabeling using a preembedding procedure with Qdots as the label. As with most preembedding techniques, penetration into the tissue was problematic and the labeling density was decreased relative to peroxidase. To increase penetration, we used higher detergent concentration and longer incubation in detergent-containing buffer which somewhat degraded tissue ultrastructure. Nevertheless, the main neurofilament- and vesicle-containing components of the pinceau

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were clearly resolved. Although the overall density of labeling was low, patchy labeling was present along the intracellular surface of the basket cell axon plasma membrane, both in vesicle containing- and neurofilament-containing regions, consistent with the peroxidase results (Fig. 3F,G).

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3.3. AQP4 The overall pattern of AQP4 labeling was consistent with findings of Nielsen et al. [29]. AQP4 was present in astrocytes and blood vessels, but was absent in neurons. In addition to these findings, we report intense AQP4 labeling

Fig. 3. Electron microscopic localization of Kv3.2 in the pinceau. (A) Thin section (80 nm) of Kv3.2-labeled tissue. The labeling is present in basket cell terminals (arrows) and is most apparent in regions containing vesicles. The antibody targets an intracellular epitope. A higher magnification view of the upper marked terminal is shown in the inset. (B) Electron micrograph of labeled pinceau fibers (arrow) in a 0.5 Am section imaged with IVEM. Part of the Purkinje cell soma is visible on the top left (PC) and the initial segment (IS) is descending in a curve from top left to bottom right. (C) Higher magnification stereo-view of the process indicated in panel B. The punctate labeling in basket cell terminals (arrowhead) is clearly visible. (D and E) Kv3.2 labeling in basket cell terminal synapses on the Purkinje cell soma. The membrane of the basket cell terminal is indicated by arrows. The label was generally not associated with the synaptic active zone but was located at membrane sites not apposed to the Purkinje cell (arrowheads). V=vesicles, nf=region of basket cell containing neurofilaments. (F and G) Kv3.2 labeling using quantum dot conjugates (QD 655, arrows). (F) The signal is punctate and associated with plasma membrane of basket cell axons, characterized by neurofilaments (nf). (G) Consistent with the peroxidase results, labeling is also present in terminal regions of basket cells, characterized by clusters of vesicles (v). Scale bars: A=1 Am; B=0.5 Am; D=0.5 Am; E=1 Am; F and G=200 nm.

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Fig. 3. (continued).

in the pinceau area and the pericellular basket (Fig. 2C,D). Lighter staining was observed in the molecular layer and in the neuroglial sheath enveloping the glomeruli of the granular cell layer, as previously described [29]. Labeling in the molecular layer was punctuate and associated with blood vessels, most likely in astrocytic end-feet. No immunostaining was detectable in control sections incubated with secondary antibodies only. Electron microscopic analysis of the pinceau revealed that AQP4 labeling was present in astrocytes enveloping the Purkinje cell soma, the initial axonal segment, and between basket cell axons of the pinceau (Fig. 5). The labeling in

pinceau astrocytes was sometimes apposed to vesiclecontaining regions of the neighboring basket cell axonal terminals. No obvious difference was found in either the intensity or pattern of immunolabeling across cerebellar lobules for either Kv3.2 or AQP4.

4. Discussion In our study, we localized another member of the voltage-gated potassium channel family to the pinceau region, the Shaw-related potassium channel subunit Kv3.2.

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Fig. 4. 3D tomographic reconstruction of several labeled basket cell terminals in the pinceau areas showing locations of synaptic vesicle clusters and patches of Kv3.2 labeling. Discrete patches of Kv3.2 labeling (red) are associated with vesicle-containing regions (yellow spheres) of the pinceau fibers. Basket cell processes are shown in different colors while the initial segment (IS) is shown in brown. A small part of an astrocyte (blue) is shown between the initial segment and the green basket cell terminal (arrowheads) to demonstrate the interposition of astrocytes between the basket cell terminals and the initial segment.

We found Kv3.2 localized specifically in pinceau basket cell terminals. Our results are consistent with the in situ hybridization study of Weiser et al. [48,49] who reported that the only Kv3.2 message in the cerebellum was localized to the proximity of the Purkinje cell layer. Anatomical studies have shown that basket cell somata are distributed primarily in the inner third of the molecular layer close to the Purkinje cell layer [31]. Although pinceau fibers express at least four voltageactivated potassium channel subunits, each subunit examined so far appears to have a somewhat different distribution in pinceau fibers (Fig. 6). Kv1.1 and 1.2 form clusters with PSD-95 in the septate junctions [18], while Kv3.4 is more evenly distributed throughout the axon [23]. We found Kv3.2 distributed to focal regions distinct from the septate junctions, corresponding to vesicle clusters at the terminal, suggesting that the pinceau fibers may possess a mosaic of potassium channel subunits. Voltage-gated potassium channels show a similar mosaic distribution at the node of Ranvier as well [26]. The results of this study provide additional evidence that potassium ion fluxes play an important role in the functioning of the pinceau. The central role of potassium channels in the pinceau is further underscored by the results

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of Laube et al. [23] who found no immunolabeling for sodium channel a-subunits in the pinceau. The same report showed that sodium channel immunoreactivity was present in basket cell and Purkinje cell somas, and the Purkinje cell axon, including the initial segment. The role of potassium channel subunits in the functioning of the pinceau is not known. Potassium channels in neurons function as regulators of neuronal excitability and synaptic transmission. Voltage-gated potassium channels open in response to a depolarization of the cell membrane, hence allowing an efflux of potassium ions from the terminal. Basket cells are fast spiking GABAergic interneurons, and the Kv3 subfamily of K channel subunits is characterized by very fast deactivation rates, enabling fast repolarization of action potentials [39]. The high rate of activity [39] and the dense plexus of pinceau fibers in the restricted area of the pinceau suggest that large potassium fluxes occur in this area. Glial cells play an important role in potassium spatial buffering from the interstitial space after neural excitation [15]. High neuronal activity is coupled with potassium buffering and subsequent water flux [14]. Because potassium channels do not admit water, the water flux is mediated through distinct water channels. To this end, astrocytes express the brain-specific water channel AQP4 [27]. As predicted, we observed at the ultrastructural level intense staining for AQP4 in glia wrapping the initial segment and Purkinje soma, as well as in perivascular astrocytic processes, as previously reported [29]. Aquaporin could aid in repolarization-induced water flux in this area following the release of potassium from basket cell terminals. At least one voltage-gated potassium channel subunit, Kv2.1, has been reported to be preferentially located on neuronal membrane apposed to glial processes in the cortex and hippocampus [5]. However, it did not appear from our findings that labeling for Kv3.2 was tightly coupled to glial processes in the pinceau, although we did observe that some patches of Kv3.2 labeling were found in basket cell membrane apposed to glial membrane.

Fig. 5. Electron microscopic localization of brain-specific water channel AQP4. Aquaporin immunoreactivity was associated with astrocyte processes apposed to basket cell axons. BC=basket cells. Scale bar=1 Am.

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4.1. Mechanism of action of the pinceau The observed inhibitory effect of basket cells on Purkinje cell somas can be explained via classical synaptic axosomatic contacts, forming symmetrical junctions similar to basket cells in other parts of the brain [45]. However, considering the density of basket cell axons around the initial segment, basket cell terminals in the pinceau form remarkably few synapses with the Purkinje cell axon. In addition, the majority of basket cell processes in the pinceau are separated from the postsynaptic membrane of the initial segment by intervening astrocytic processes. The function of the terminal regions in the pinceau and their effect on Purkinje cell function are therefore unknown. Based on its geometry and physiological studies, researchers have hypothesized that the pinceau might function by generating an inhibitory electrical field effect by means of imposing a passive hyperpolarizing potential on the Purkinje cell initial segment [19]. This hypothesis is supported by the presence of septate-like junctions between basket cell terminals, which are unique to this part of mammalian brain [10]. Amongst other functions, septate-like junctions are known to play a stabilizing and insulating role between cells [22]. Septate junctions might therefore stabilize the entire pinceau

region, and along with the astrocytic sheath encapsulating the entire pinceau, could form an isolated, largely impermeable environment suitable for inhibitory field effects. The field hypothesis is also supported by the structural similarity of the pinceau to the Mauthner cell in the teleocyst [36] as noted in [19]. There is physiological evidence that neurons exhibiting passive hyperpolarizing potential in Mauthner cell cap mediate both field inhibition and chemical inhibition of the initial segment [20]. In light of increasing evidence that astrocyte mechanics play a role in modulating interneuronal communication [1,11,12], the interesting geometry of the pinceau suggests that glia may be active participants in the function of the pinceau. First, access of the basket cell axons to the Purkinje cell initial segment may be controlled by intervening astrocytic processes, as has been demonstrated in the hypothalamus [11,12]. In addition, the possible rigidity imposed by the septate junctions is interesting in light of our findings that AQP4 is present in the pinceau region concurrently with large numbers of potassium channels. Astrocytes are known to undergo significant volume changes as a result of neuronal activity [28]. If the pinceau is a semirigid structure, any significant swelling in astrocytes in the pinceau area could exert physical

Fig. 6. Schematic diagram of proteins in the pinceau according to published studies, including the present. The proteins associated with basket cell terminals are pictured on the right side of the initial segment, the proteins associated with septate-like junctions, basket cell axonal ramifications and astrocytes are marked on the left side. Key: Purkinje cell and its initial segment (yellow), basket cell axons (green), basket cell terminals (green) with vesicles (yellow), and astrocytes (blue).

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pressure on the initial segment and/or Purkinje cell, and thereby influence spiking activity. Thus, the geometry and molecular specializations suggest that the function of the pinceau may involve mechanical as well as biochemical dynamics.

Acknowledgements This work was supported by NIH grants DA016602 (Martone) supported by the National Institutes on Drug Abuse, Bioimaging and Bioengineering and General Medicine through the Human Brain Project, NIH NS 14718 and NIH NCRR RR04050 (Ellisman), and NIH NS30989 and NIH NS 045217 (Rudy). The authors thank Mr. Howard Lien for his work on the 3D reconstructions.

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