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Immunohistochemical study on the distribution of six members of the Kv1 channel subunits in the rat cerebellum
Brain Research 895 (2001) 173–177 www.elsevier.com / locate / bres
Research report
Immunohistochemical study on the distribution of six members of the Kv1 channel subunits in the rat cerebellum Yoon Hee Chung a , Chung-Min Shin a , Myeung Ju Kim a , Byung Kwon Lee a , Choong Ik Cha a,b,c , * b
a Department of Anatomy, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110 -799, South Korea Neuroscience Research Institute, Medical Research Center, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110 -799, South Korea c Biomedical Research Center, KNIH, Seoul, South Korea
Accepted 26 December 2000
Abstract Voltage-gated K 1 (Kv) channels are critical for a wide variety of processes, and play an essential role in neurons. In the present study, we have demonstrated a unique pattern of expression of the six Kv1 channel subunits in the rat cerebellum, for the first time. The greatest concentration of Kv1.2 was found in the basket cell axon plexus and terminal regions around the Purkinje cells. Relatively weak immunoreactivity for Kv1.1 was also found in this area. The somatodendritic Purkinje cell areas were intensely stained with anti-Kv1.5 antibodies. In the cerebellar nuclei, the cell bodies of cerebellar output neurons showed strong Kv1.5 and Kv1.6 immunoreactivities in the nucleus medialis, interpositus and lateralis. Interestingly, Kv1.2 immunoreactivity was found in some neurons with their processes. Our immunohistochemical results may support the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the cerebellum. 2001 Elsevier Science B.V. All rights reserved. Theme: Motor systems and sensorimotor integration Topic: Cerebellum Keywords: Voltage-gated potassium channels; Rat; Cerebellum; Immunohistochemistry
1. Introduction All excitable cells express voltage-gated K1 (Kv) channels. These channels are critical for a wide variety of processes, including action potential preparation and lymphocyte activation, and play an essential role in neurons where they regulate the resting membrane potential, impact dendritic excitability, control the frequency and duration of action potentials, and modulate neurotransmitter release [5]. Recent work in several laboratories has determined that K1 channels are integral membrane, hetero-oligomeric glycoprotein complexes composed of four pore-forming a subunits and four cytoplasmic b subunit polypeptides [8,11]. The a subunits are sufficient to form Kv channels, while the b subunits have an *Corresponding author. Tel.: 82-2-740-8205; fax: 82-2-745-9528. E-mail address: [email protected] (C.I. Cha).
auxillary function in mediating rapid inactivation of Kv channels [13]. Kv channel a subunits, which have been cloned and expressed in vitro, belong to a superfamily on the basis of structural relatedness [4]. Most likely, different combinations of Kv channel a and b subunit isoforms into heteromultimeric Kv channels may substantially contribute to the generation of Kv channel diversity in the nervous system [3]. The cerebellum is perhaps one of the few sites in the central nervous system where the pattern of intrinsic connections is known in considerable detail. This knowledge has been the catalyst for many of the models and theories of cerebellar function. Given that the cellular localizations of the Kv channels are likely to contribute to the unique firing pattern of a particular neuron, we conducted an immunohistochemical study of six members of Kv1 channel subunits in the rat cerebellum. Although there have been previous reports in the cellular and
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02068-6
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Y.H. Chung et al. / Brain Research 895 (2001) 173 – 177
subcellular localizations of various Kv mRNAs and proteins [6,9,14,15,21], the comprehensive study described here is the first in which the expression of six Kv1 channel subunits have been directly compared in the rat cerebellum. In the present study, we have found that staining patterns of the six Kv1 channel subunits overlap in some areas of the rat cerebellum, but each has a unique pattern of expression.
2. Materials and methods Twelve adult (4–6-month-old) Sprague–Dawley rats were examined in this study. The authors conformed to the Seoul National University Ethical Committee Guidelines for Laboratory Animals. The animals were perfused transcardially with cold phosphate buffered saline (PBS, 0.02 M, pH 7.4), and then with ice-cold 4% paraformaldehyde for 10 min at a flow rate of 50–60 ml / min. Brains were removed immediately and sliced into blocks 4–6 mm thick. These were immersed in a cold fixative for 6–12 h and then cryoprotected in a series of cold sucrose solutions of increasing concentration. Frozen sections were cut at 40 mm in the coronal plane, and were incubated using the free-floating method for 48–72 h at 48C in primary antiserum containing Triton X-100 (0.3%), bovine serum albumin (0.5 mg / ml) and normal goat serum (3 drops / 10 ml). Polyclonal anti-Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6 (Product No. APC-009, 010, 002, 007, 004 and 003, Alomone Labs, Jerusalem, Israel) were used as primary antibodies at a dilution of 1:200, 1:200, 1:70, 1:200, 1:70 and 1:70, respectively. After incubation, the sections were immersed in the secondary antiserum containing biotinylated anti-rabbit IgG and visualized according to the avidin–biotin complex (ABC) method, using an ABC kit (VectastainE). The sections were then developed for peroxidase reactivity with 3,39-diaminobenzidine (DAB), and after visualization were mounted on gelatincoated slides. A sample of sections was reacted without primary antiserum, and a different sample was exposed to a primary antiserum that had been preincubated for 24 h with control antigen peptides. Sections from these samples did not exhibit any of the immunoreactivity described in this report. Visual assessment and densitometric measurement using a NIH image program (Scion Image) determined the number and extent of dendritic branches.
3. Results There was a high density of Kv1.1, Kv1.2, Kv1.3, Kv1.5, and Kv 1.6, with a much lower density of Kv1.4 in the rat cerebellum (Fig. 1). Perhaps the greatest concentration of Kv1.2 in the entire cerebellum was found in the basket cell axon plexus and terminal regions around the
Purkinje cells (Fig. 1B). Interestingly, immunoreactivity for Kv1.1 was also concentrated in this area although the staining intensity was relatively lower than that of Kv1.2 (Fig. 1A). The somatodendritic Purkinje cell areas were intensely stained with anti-Kv1.5 antibodies, whereas Kv1.6 immunoreactivity was detected in the Purkinje cell bodies, not in the dendrites (Fig. 1E, F). Kv1.3 and Kv1.5 immunoreactive cells were found in the cerebellar white matter (Fig. 1C, E). Cerebellar granule cells are densely packed in the granular layer. These small cells are in fact the numerous cells in the brain. The cerebellar granule cells send ascending fibers into the cerebellar molecular layer, making en passant synapses with Purkinje cell dendrites. The strong Kv1.1, Kv1.3, Kv1.5 and Kv1.6 immunoreactivities were observed in the granular layer (Fig. 1A, C, E, F). On the other hand, the granule cells were weakly stained with Kv1.2 (Fig. 1B), and there was no immunoreactivity for Kv1.4 in the granular layer (Fig. 1D). The selective localization of Kv1.2 in nerve terminals is perhaps most convincingly demonstrated in the cerebellum. The most intense Kv1.2 immunoreactivity of the whole brain is found here, in the plexuses of nerve terminals that ensheathe the base and initial axon segments of Purkinje cells (Fig. 1B, see inset). These plexuses are physically distinguishable from the Purkinje neuron and are uniquely characteristic of the nerve endings of basket cells. In the present study, the extraordinary intensity of immunostaining was found in basket cell nerve terminals, compared to that found in cerebellar Purkinje cells (Fig. 1B). In contrast to basket cells, Purkinje cells showed no immunoreactivity for Kv1.2 in their cell bodies. In the cerebellar nuclei, the cell bodies of cerebellar output neurons showed strong Kv1.5 and Kv1.6 immunoreactivities in the nucleus medialis, interpositus and lateralis (Fig. 2E, F), with relatively weak staining for Kv1.1 and Kv1.3 in the cell bodies (Fig. 2A, 2C). In addition, immunoreactivities for six Kv1 channels were observed in the surrounding neuropil, the region where the Purkinje cell axons terminate. Interestingly, Kv1.2 immunoreactivity was not found in the cerebellar output neurons but in some neurons with their processes (Fig. 2B)
4. Discussion The processing of information, which is based on and organized by electrical signals in the brain, requires that neurons are functionally and anatomically polarized. Within a neuron, electrical signals and information flow from dendritic to axon terminal compartments. In spite of the general plan of cellular organization that most neurons share, they occur in a large variety of sizes and shapes. This reflects the wide range of electrical signaling capabilities observed in various neuronal cell types. Since Kv channels are key determinants of membrane excitability,
Y.H. Chung et al. / Brain Research 895 (2001) 173 – 177
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Fig. 1. Cellular localizations of Kv1.1 (A), Kv1.2 (B), Kv1.3 (C), Kv1.4 (D), Kv1.5 (E), and Kv1.6 (F) subunits in the cerebellar cortex. There was a high density of Kv1.1, Kv1.2, Kv1.3, Kv1.5, and Kv1.6, with a much lower density of Kv1.4 in the cerebellar cortex. Note the greatest concentration of Kv1.2 in the basket cell axon plexus and terminal regions around the Purkinje cells (B, arrows). See inset for the relevant features at higher magnification (B). Box within B contained the basket cell axon plexus and was expanded as inset. Interestingly, immunoreactivity for Kv1.1 was also concentrated in this area (A, arrows). M: molecular layer, P: Purkinje cell layer, G: granular layer, WM: white matter. Scale bar550 mm (A–F); 15 mm (inset within B).
different neurons are endowed with a distinct set of diverse Kv channels. The expression of functionally diverse Kv channels implies that Kv channels have specialized functions in the regulation of presynaptic and postsynaptic membrane excitability. Our observation that a neuron may express a distinct combination Kv1 channel subunits is consistent with this hypothesis. The localization of different Kv channel subunits to dendritic and to axonal domains may be recognized in
immunohistochemical experiments in many neuronal cell types at the light microscope level. Therefore, others [6,10,12,17–20] have employed antibodies directed against Kv channel subunits for mapping Kv channels in the rat brain. In the present study, however, we described the regional localization of six members of Kv1 channel subunits in the rat cerebellum, for the first time. In general, there was very good agreement between immunohistochemistry and previous in situ hybridization results except
176
Y.H. Chung et al. / Brain Research 895 (2001) 173 – 177
Fig. 2. Cellular localizations of Kv1.1 (A), Kv1.2 (B), Kv1.3 (C), Kv1.4 (D), Kv1.5 (E), and Kv1.6 (F) subunits in the cerebellar nuclei. The cell bodies of cerebellar output neurons showed strong Kv1.5 and Kv1.6 immunoreactivities in the nucleus medialis, interpositus and lateralis, with relatively weak staining for Kv1.1 and Kv1.3 in the cell bodies. In addition, immunoreactivities for six Kv1 channels were observed in the surrounding neuropil. Interestingly, Kv1.2 immunoreactivity was not found in the cerebellar output neurons but in some neurons with their processes. Scale bar550 mm (A–F).
for Kv1.2. In many cases, e.g. Purkinje cells, Kv1.2 immunoreactivity did not match Kv1.2 mRNA expression. This discrepancy may be due to the transport of Kv1.2 protein to axon fibers. As shown before [10], Kv1.2 protein was localized in cerebellar basket cell terminals (Fig. 1B). This observation may imply that Kv1.2 protein may also be localized in Purkinje cell axons that were not visible in our sections. Combined with previous reports, the major conclusion may be extracted from our results: different neuronal cell types express different combinations of Kv1 channel subunits.
Recently, differential distribution of ion conductances within specific domains of the neuronal membrane has been apparent from electrophysiological and ligand binding studies. A classic example is Na 1 and K 1 channel segregation in myelinated fibers [3]. The cloning of multiple genes encoding ion channels presages a further level of complexity on the neuronal surface. For instance, differential subcellular localization of Na 1 channel isoforms and Ca 21 channel subtypes has been inferred from recent immunohistochemical studies [1,23]. Our results with the six Kv1 channels in the rat cerebellum imply that
Y.H. Chung et al. / Brain Research 895 (2001) 173 – 177
the membrane geography of K 1 channels is also likely to be highly ordered, in agreement with lateral diffusion studies which suggest that Na 1 and K 1 channels are largely immobile in excitable membranes [2,22]. Given that K 1 channels are more diverse than either Na 1 or Ca 21 channels in terms of genes and functional heterogeneity, the surface distribution of K 1 channels may be very complex. In this regard, immunoelectron microscopic studies will be essential not only to confirm the localizations of these Kv1 channels, but also to define potential microheterogeneity of membrane distribution at these sites. Both in vivo and in vitro studies have demonstrated that Kv1 channel subunits form functional heteromultimers [7,16,18]. Co-expression of these channels in the same neurons may lead to the formation of heteromultimeric Kv channels having distinct properties [7,16]. However, it is still conjectural whether the co-localization of different Kv1 channel subunits in distinct neuronal subcellular compartments represents the immunochemical correlate to the biochemically identified assembly of Kv1 subunits in heteromultimeric Kv channels. In this context, it is important to note that certain Kv subunit pairs, e.g. Kv1.1 / Kv1.2 and Kv1.2 / Kv1.4, are co-localized in some areas of the brain although they are expressed at a quite different level. Nevertheless, our immunohistochemical results are consistent with the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the cerebellum. At present, it is obviously difficult to recognize the molecular clues, which control co-localization or Kv channel sorting to dendritic and axonal domains. Additional studies will be required to explore functionally significant distinctions of Kv channels in vivo in relation to their subunit composition and subcellular targeting.
Acknowledgements This study was supported by Seoul National University Hospital Research Fund (1997) and in part by year 2000 BK21 project for Medicine, Dentistry and Pharmacy.
References [1] M.K. Ahlijanian, R.E. Westenbroek, W.A. Catterall, Subunit structure and localization of dihydropyridine-sensitive calcium channels in mammalian brain, spinal cord, and retina, Neuron 4 (1990) 819– 832. [2] K.J. Angelides, L.W. Elmer, D. Loftus, E. Elson, Distribution and lateral mobility of voltage-dependent sodium channels in neurons, J. Cell Biol. 106 (1988) 1911–1925. [3] J.A. Black, J.D. Kocsis, S.G. Waxman, Ion channel organization of the myelinated fiber, Trends Neurosci. 13 (1990) 48–54.
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[4] K.G. Chandy, G.A. Gutman, Nomenclature for mammalian potassium channel genes, Trends Pharmacol. Sci. 14 (1993) 434. [5] B. Hille, Ionic Channels of Excitable Membranes, Sinauer, Sunderland, MA, 1992. [6] P.M. Hwang, M. Fotuhi, D.S. Bredt, A.M. Cunningham, S.H. Snyder, Contrasting immunohistochemical localizations in rat brain of two novel K1 channels of the Shab subfamily, J. Neurosci. 13 (1993) 1569–1576. [7] E.Y. Isacoff, Y.N. Jan, L.Y. Jan, Evidence for the formation of heteromultimeric potassium channels in Xenopus oocytes, Nature 345 (1990) 530–534. [8] L.Y. Jan, Y.N. Jan, Cloned potassium channels from eukaryotes and prokaryotes, Annu. Rev. Neurosci. 20 (1997) 91–123. [9] W.A. Kues, F. Wunder, Heterogenous expression patterns of mammalian potassium channel genes in developing and adult rat brain, Eur. J. Neurosci. 4 (1992) 1296–1308. [10] N.M. McNamara, Z.M. Muniz, G.P. Wilkin, J.O. Dolly, Prominent location of a K1 channel containing the alpha subunit Kv 1.2 in the basket cell nerve terminals of rat cerebellum, Neuroscience 57 (1993) 1039–1045. [11] O. Pongs, Voltage-gated potassium channels: from hyperexcitability to excitement, FEBS Lett. 452 (1999) 31–35. [12] J. Rettig, F. Wunder, M. Stocker, R. Lichtinghagen, F. Mastiaux, S. Beckh, W. Kues, P. Pedarzani, K.H. Schroter, J.P. Ruppersberg et al., Characterization of a Shaw-related potassium channel family in rat brain, EMBO J. 11 (1992) 2473–2486. [13] J. Rettig, S.H. Heinemann, F. Wunder, C. Lorra, D.N. Parcej, J.O. Dolly, O. Pongs, Inactivation properties of voltage-gated K1 channels altered by presence of beta-subunit, Nature 369 (1994) 289–294. [14] K.J. Rhodes, S.A. Keilbaugh, N.X. Barrezueta, K.L. Lopez, J.S. Trimmer, Association and colocalization of K1 channel a- and b-subunit polypeptides in rat brain, J. Neurosci. 15 (1995) 5360– 5371. [15] K.J. Rhodes, B.W. Strassle, M.M. Monaghan, Z. Bekele-Arcuri, M.F. Matos, J.S. Trimmer, Association and colocalization of the Kvbeta1 and Kvbeta2 beta-subunits with Kv1 alpha-subunits in mammalian brain K1 channel complexes, J. Neurosci. 17 (1997) 8246–8258. ¨ [16] J.P. Ruppersberg, K.H. Schroter, B. Sakmann, M. Stocker, S. Sewing, O. Pongs, Heteromultimeric channels formed by rat brain potassium-channel proteins, Nature 345 (1990) 535–537. [17] M. Sheng, M.L. Tsaur, Y.N. Jan, L.Y. Jan, Subcellular segregation of two A-type K1 channel proteins in rat central neurons, Neuron 9 (1992) 271–284. [18] M. Sheng, Y.J. Liao, Y.N. Jan, L.Y. Jan, Presynaptic A-current based on heteromultimeric K1 channels detected in vivo, Nature 365 (1993) 72–75. [19] M. Sheng, M.L. Tsaur, Y.N. Jan, L.Y. Jan, Contrasting subcellular localization of the Kv1.2 K1 channel subunit in different neurons of rat brain, J. Neurosci. 14 (1994) 2408–2417. [20] H. Wang, D.D. Kunkel, T.M. Martin, P.A. Schwartzkroin, B.L. Tempel, Heteromultimeric K1 channels in terminal and juxtaparanodal regions of neurons, Nature 365 (1993) 75–79. [21] M. Weiser, E. Vega-Saenz de Miera, C. Kentros, H. Moreno, L. Franzen, D. Hillman, H. Baker, B. Rudy, Differential expression of Shaw-related K1 channels in the rat central nervous system, J. Neurosci. 14 (1994) 949–972. [22] R.E. Weiss, W.M. Roberts, W. Stuhmer, W. Almers, Mobility of voltage-dependent ion channels and lectin receptors in the sarcolemma of frog skeletal muscle, J. Gen. Physiol. 87 (1986) 955–983. [23] R.E. Westenbroek, D.K. Merrick, W.A. Catterall, Differential subcellular localization of the RI and RII Na1 channel subtypes in central neurons, Neuron 3 (1989) 695–704.
Research report
Immunohistochemical study on the distribution of six members of the Kv1 channel subunits in the rat cerebellum Yoon Hee Chung a , Chung-Min Shin a , Myeung Ju Kim a , Byung Kwon Lee a , Choong Ik Cha a,b,c , * b
a Department of Anatomy, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110 -799, South Korea Neuroscience Research Institute, Medical Research Center, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110 -799, South Korea c Biomedical Research Center, KNIH, Seoul, South Korea
Accepted 26 December 2000
Abstract Voltage-gated K 1 (Kv) channels are critical for a wide variety of processes, and play an essential role in neurons. In the present study, we have demonstrated a unique pattern of expression of the six Kv1 channel subunits in the rat cerebellum, for the first time. The greatest concentration of Kv1.2 was found in the basket cell axon plexus and terminal regions around the Purkinje cells. Relatively weak immunoreactivity for Kv1.1 was also found in this area. The somatodendritic Purkinje cell areas were intensely stained with anti-Kv1.5 antibodies. In the cerebellar nuclei, the cell bodies of cerebellar output neurons showed strong Kv1.5 and Kv1.6 immunoreactivities in the nucleus medialis, interpositus and lateralis. Interestingly, Kv1.2 immunoreactivity was found in some neurons with their processes. Our immunohistochemical results may support the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the cerebellum. 2001 Elsevier Science B.V. All rights reserved. Theme: Motor systems and sensorimotor integration Topic: Cerebellum Keywords: Voltage-gated potassium channels; Rat; Cerebellum; Immunohistochemistry
1. Introduction All excitable cells express voltage-gated K1 (Kv) channels. These channels are critical for a wide variety of processes, including action potential preparation and lymphocyte activation, and play an essential role in neurons where they regulate the resting membrane potential, impact dendritic excitability, control the frequency and duration of action potentials, and modulate neurotransmitter release [5]. Recent work in several laboratories has determined that K1 channels are integral membrane, hetero-oligomeric glycoprotein complexes composed of four pore-forming a subunits and four cytoplasmic b subunit polypeptides [8,11]. The a subunits are sufficient to form Kv channels, while the b subunits have an *Corresponding author. Tel.: 82-2-740-8205; fax: 82-2-745-9528. E-mail address: [email protected] (C.I. Cha).
auxillary function in mediating rapid inactivation of Kv channels [13]. Kv channel a subunits, which have been cloned and expressed in vitro, belong to a superfamily on the basis of structural relatedness [4]. Most likely, different combinations of Kv channel a and b subunit isoforms into heteromultimeric Kv channels may substantially contribute to the generation of Kv channel diversity in the nervous system [3]. The cerebellum is perhaps one of the few sites in the central nervous system where the pattern of intrinsic connections is known in considerable detail. This knowledge has been the catalyst for many of the models and theories of cerebellar function. Given that the cellular localizations of the Kv channels are likely to contribute to the unique firing pattern of a particular neuron, we conducted an immunohistochemical study of six members of Kv1 channel subunits in the rat cerebellum. Although there have been previous reports in the cellular and
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02068-6
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subcellular localizations of various Kv mRNAs and proteins [6,9,14,15,21], the comprehensive study described here is the first in which the expression of six Kv1 channel subunits have been directly compared in the rat cerebellum. In the present study, we have found that staining patterns of the six Kv1 channel subunits overlap in some areas of the rat cerebellum, but each has a unique pattern of expression.
2. Materials and methods Twelve adult (4–6-month-old) Sprague–Dawley rats were examined in this study. The authors conformed to the Seoul National University Ethical Committee Guidelines for Laboratory Animals. The animals were perfused transcardially with cold phosphate buffered saline (PBS, 0.02 M, pH 7.4), and then with ice-cold 4% paraformaldehyde for 10 min at a flow rate of 50–60 ml / min. Brains were removed immediately and sliced into blocks 4–6 mm thick. These were immersed in a cold fixative for 6–12 h and then cryoprotected in a series of cold sucrose solutions of increasing concentration. Frozen sections were cut at 40 mm in the coronal plane, and were incubated using the free-floating method for 48–72 h at 48C in primary antiserum containing Triton X-100 (0.3%), bovine serum albumin (0.5 mg / ml) and normal goat serum (3 drops / 10 ml). Polyclonal anti-Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6 (Product No. APC-009, 010, 002, 007, 004 and 003, Alomone Labs, Jerusalem, Israel) were used as primary antibodies at a dilution of 1:200, 1:200, 1:70, 1:200, 1:70 and 1:70, respectively. After incubation, the sections were immersed in the secondary antiserum containing biotinylated anti-rabbit IgG and visualized according to the avidin–biotin complex (ABC) method, using an ABC kit (VectastainE). The sections were then developed for peroxidase reactivity with 3,39-diaminobenzidine (DAB), and after visualization were mounted on gelatincoated slides. A sample of sections was reacted without primary antiserum, and a different sample was exposed to a primary antiserum that had been preincubated for 24 h with control antigen peptides. Sections from these samples did not exhibit any of the immunoreactivity described in this report. Visual assessment and densitometric measurement using a NIH image program (Scion Image) determined the number and extent of dendritic branches.
3. Results There was a high density of Kv1.1, Kv1.2, Kv1.3, Kv1.5, and Kv 1.6, with a much lower density of Kv1.4 in the rat cerebellum (Fig. 1). Perhaps the greatest concentration of Kv1.2 in the entire cerebellum was found in the basket cell axon plexus and terminal regions around the
Purkinje cells (Fig. 1B). Interestingly, immunoreactivity for Kv1.1 was also concentrated in this area although the staining intensity was relatively lower than that of Kv1.2 (Fig. 1A). The somatodendritic Purkinje cell areas were intensely stained with anti-Kv1.5 antibodies, whereas Kv1.6 immunoreactivity was detected in the Purkinje cell bodies, not in the dendrites (Fig. 1E, F). Kv1.3 and Kv1.5 immunoreactive cells were found in the cerebellar white matter (Fig. 1C, E). Cerebellar granule cells are densely packed in the granular layer. These small cells are in fact the numerous cells in the brain. The cerebellar granule cells send ascending fibers into the cerebellar molecular layer, making en passant synapses with Purkinje cell dendrites. The strong Kv1.1, Kv1.3, Kv1.5 and Kv1.6 immunoreactivities were observed in the granular layer (Fig. 1A, C, E, F). On the other hand, the granule cells were weakly stained with Kv1.2 (Fig. 1B), and there was no immunoreactivity for Kv1.4 in the granular layer (Fig. 1D). The selective localization of Kv1.2 in nerve terminals is perhaps most convincingly demonstrated in the cerebellum. The most intense Kv1.2 immunoreactivity of the whole brain is found here, in the plexuses of nerve terminals that ensheathe the base and initial axon segments of Purkinje cells (Fig. 1B, see inset). These plexuses are physically distinguishable from the Purkinje neuron and are uniquely characteristic of the nerve endings of basket cells. In the present study, the extraordinary intensity of immunostaining was found in basket cell nerve terminals, compared to that found in cerebellar Purkinje cells (Fig. 1B). In contrast to basket cells, Purkinje cells showed no immunoreactivity for Kv1.2 in their cell bodies. In the cerebellar nuclei, the cell bodies of cerebellar output neurons showed strong Kv1.5 and Kv1.6 immunoreactivities in the nucleus medialis, interpositus and lateralis (Fig. 2E, F), with relatively weak staining for Kv1.1 and Kv1.3 in the cell bodies (Fig. 2A, 2C). In addition, immunoreactivities for six Kv1 channels were observed in the surrounding neuropil, the region where the Purkinje cell axons terminate. Interestingly, Kv1.2 immunoreactivity was not found in the cerebellar output neurons but in some neurons with their processes (Fig. 2B)
4. Discussion The processing of information, which is based on and organized by electrical signals in the brain, requires that neurons are functionally and anatomically polarized. Within a neuron, electrical signals and information flow from dendritic to axon terminal compartments. In spite of the general plan of cellular organization that most neurons share, they occur in a large variety of sizes and shapes. This reflects the wide range of electrical signaling capabilities observed in various neuronal cell types. Since Kv channels are key determinants of membrane excitability,
Y.H. Chung et al. / Brain Research 895 (2001) 173 – 177
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Fig. 1. Cellular localizations of Kv1.1 (A), Kv1.2 (B), Kv1.3 (C), Kv1.4 (D), Kv1.5 (E), and Kv1.6 (F) subunits in the cerebellar cortex. There was a high density of Kv1.1, Kv1.2, Kv1.3, Kv1.5, and Kv1.6, with a much lower density of Kv1.4 in the cerebellar cortex. Note the greatest concentration of Kv1.2 in the basket cell axon plexus and terminal regions around the Purkinje cells (B, arrows). See inset for the relevant features at higher magnification (B). Box within B contained the basket cell axon plexus and was expanded as inset. Interestingly, immunoreactivity for Kv1.1 was also concentrated in this area (A, arrows). M: molecular layer, P: Purkinje cell layer, G: granular layer, WM: white matter. Scale bar550 mm (A–F); 15 mm (inset within B).
different neurons are endowed with a distinct set of diverse Kv channels. The expression of functionally diverse Kv channels implies that Kv channels have specialized functions in the regulation of presynaptic and postsynaptic membrane excitability. Our observation that a neuron may express a distinct combination Kv1 channel subunits is consistent with this hypothesis. The localization of different Kv channel subunits to dendritic and to axonal domains may be recognized in
immunohistochemical experiments in many neuronal cell types at the light microscope level. Therefore, others [6,10,12,17–20] have employed antibodies directed against Kv channel subunits for mapping Kv channels in the rat brain. In the present study, however, we described the regional localization of six members of Kv1 channel subunits in the rat cerebellum, for the first time. In general, there was very good agreement between immunohistochemistry and previous in situ hybridization results except
176
Y.H. Chung et al. / Brain Research 895 (2001) 173 – 177
Fig. 2. Cellular localizations of Kv1.1 (A), Kv1.2 (B), Kv1.3 (C), Kv1.4 (D), Kv1.5 (E), and Kv1.6 (F) subunits in the cerebellar nuclei. The cell bodies of cerebellar output neurons showed strong Kv1.5 and Kv1.6 immunoreactivities in the nucleus medialis, interpositus and lateralis, with relatively weak staining for Kv1.1 and Kv1.3 in the cell bodies. In addition, immunoreactivities for six Kv1 channels were observed in the surrounding neuropil. Interestingly, Kv1.2 immunoreactivity was not found in the cerebellar output neurons but in some neurons with their processes. Scale bar550 mm (A–F).
for Kv1.2. In many cases, e.g. Purkinje cells, Kv1.2 immunoreactivity did not match Kv1.2 mRNA expression. This discrepancy may be due to the transport of Kv1.2 protein to axon fibers. As shown before [10], Kv1.2 protein was localized in cerebellar basket cell terminals (Fig. 1B). This observation may imply that Kv1.2 protein may also be localized in Purkinje cell axons that were not visible in our sections. Combined with previous reports, the major conclusion may be extracted from our results: different neuronal cell types express different combinations of Kv1 channel subunits.
Recently, differential distribution of ion conductances within specific domains of the neuronal membrane has been apparent from electrophysiological and ligand binding studies. A classic example is Na 1 and K 1 channel segregation in myelinated fibers [3]. The cloning of multiple genes encoding ion channels presages a further level of complexity on the neuronal surface. For instance, differential subcellular localization of Na 1 channel isoforms and Ca 21 channel subtypes has been inferred from recent immunohistochemical studies [1,23]. Our results with the six Kv1 channels in the rat cerebellum imply that
Y.H. Chung et al. / Brain Research 895 (2001) 173 – 177
the membrane geography of K 1 channels is also likely to be highly ordered, in agreement with lateral diffusion studies which suggest that Na 1 and K 1 channels are largely immobile in excitable membranes [2,22]. Given that K 1 channels are more diverse than either Na 1 or Ca 21 channels in terms of genes and functional heterogeneity, the surface distribution of K 1 channels may be very complex. In this regard, immunoelectron microscopic studies will be essential not only to confirm the localizations of these Kv1 channels, but also to define potential microheterogeneity of membrane distribution at these sites. Both in vivo and in vitro studies have demonstrated that Kv1 channel subunits form functional heteromultimers [7,16,18]. Co-expression of these channels in the same neurons may lead to the formation of heteromultimeric Kv channels having distinct properties [7,16]. However, it is still conjectural whether the co-localization of different Kv1 channel subunits in distinct neuronal subcellular compartments represents the immunochemical correlate to the biochemically identified assembly of Kv1 subunits in heteromultimeric Kv channels. In this context, it is important to note that certain Kv subunit pairs, e.g. Kv1.1 / Kv1.2 and Kv1.2 / Kv1.4, are co-localized in some areas of the brain although they are expressed at a quite different level. Nevertheless, our immunohistochemical results are consistent with the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the cerebellum. At present, it is obviously difficult to recognize the molecular clues, which control co-localization or Kv channel sorting to dendritic and axonal domains. Additional studies will be required to explore functionally significant distinctions of Kv channels in vivo in relation to their subunit composition and subcellular targeting.
Acknowledgements This study was supported by Seoul National University Hospital Research Fund (1997) and in part by year 2000 BK21 project for Medicine, Dentistry and Pharmacy.
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