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¹ Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
² Center for Brain Experiment, National Institute for Physiological Science, Okazaki, Japan
³ Department of Cell Physiology, National Institute for Physiological Science, Okazaki, Japan
⁴ Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Nada-ku, Kobe, Japan
⁵ Division of Molecular Psychiatry, Department of Psychiatry and Pharmacology, Yale University School of Medicine and Connecticut Mental Health Center, CT, USA

	Abstract

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To elucidate spatial and temporal profiles of the protein kinase C (PKC) activation in relation to neuronal functions including synaptic plasticity, we tried to detect PKC translocation in living brain slices. We first developed brain region-specific and inducible PKC-GFP transgenic mice using a tetracycline (tet)-regulated system. In the transgenic mice, the expression of PKC-GFP was region-specifically regulated by the promoter and abolished by the administration of doxycycline. Cerebellar slices from the mice were utilized for intracellular recording and fluorescence imaging of PKC-GFP in Purkinje cells. GFP fluorescence was uniformly distributed from soma to dendritic arbor. When mGluR agonists were applied, the intensity was transiently increased at the edge of the dendrite and concomitantly decreased in the cytoplasm, indicating that PKC translocated to the plasma membrane. This transient change in the pattern of GFP fluorescence simultaneously occurred throughout the Purkinje cell dendrites by agonist stimulation. Translocation of PKC-GFP was also induced by electrical stimulation of parallel fibres. However, the event was not restricted at the distal dendrites, propagated forwardly along the dendritic tree and reached to the proximal trunk close to the soma. Time course of the propagation was slower than the electrical signal and Ca²⁺ waves and faster than conveying molecules through microtubules. The present results indicate that PKC signals activated locally by parallel fibre input could propagate to the soma through dendrites in living Purkinje neurones. The findings may provide us with a new insight for understanding molecular mechanisms of the synaptic plasticity including cerebellar long-term depression.

	Introduction

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Protein kinase C (PKC) is a family of phospholipid-dependent serine/threonine kinases that consist of at least 10 subtypes (Nishizuka 1984, 1986, 1992). Based on the structure, the PKC family is divided into three groups, classical PKC (cPKC, including , ßI, ßII and PKC), novel PKC (nPKC, , , and ) and atypical PKC (aPKC, and ) (Nishizuka 1988, 1992). PKC is implicated in a variety of cellular functions, such as cell proliferation, cell growth, cell cycle, cell differentiation, oncogenesis, and neural functions (Nishizuka 1984, 1986, 1988, 1992, 1995). In the central nervous system (CNS), various PKC subtypes exist abundantly (Tanaka & Nishizuka 1994) and their individual regional and subcellular localization differs, indicating that each subtype has its own function in CNS (Tanaka & Nishizuka 1994).

To analyse their subtype-specific functions of PKC, we have developed a method to visualize PKC translocation in cultured cells using GFP-tagged PKC (Sakai et al. 1997), because PKC is known to be translocated from the cytosol to the membrane when activated and thereafter recognizes and phosphorylates its target substrates (PKC targeting) (Kraft et al. 1982; Shirai et al. 1998b; Shirai & Saito 2002). With this method, we have demonstrated that PKC targeting depends on the PKC subtype and the PKC activation mechanism (Shirai et al. 1998a; Shirai et al. 2000; Kajimoto et al. 2001; Kashiwagi et al. 2002). These findings suggested that PKC targeting is the molecular basis of PKC functions exerting cellular responses in subtype- and stimulus-specific manners (Ohmori et al. 1998, 2000). Of particular interest are mechanisms underlying the control of excitability in the dendrites, because dendrites are the primary locus of synaptic integration and plasticity. Protein kinases, including PKC, play important roles in the formation of long-term potentiation and depression (LTP and LTD, respectively) (Hu et al. 1987; Malinow et al. 1989; Linden & Connor 1991; Tanaka & Nishizuka 1994; Linden & Connor 1995; Malenka & Nicoll 1999). Positive contribution of PKC to synaptic plasticity and development is also reported from studies using mutant mice. In mice lacking PKC, a CNS-specific subtype of PKC, hippocampal LTP was attenuated (Abellovich et al. 1993) and elimination of synapses between climbing fibres and Purkinje cells was delayed during the post-natal developmental stage of the cerebellum (Kano et al. 1995). In transgenic mice over-expressing PKC inhibitor peptides specifically in a Purkinje cell, cerebellar LTD and adaptation of the vestibulo-ocular reflex did not occur (De Zeeuw et al. 1998). However, many questions still remain unclear, such as how, when and where PKC is targeted in response to synaptic activation which leads to plasticity formation, and when PKC contributes to CNS development most preferentially.

In this study, we have generated brain region-specific and inducible PKC-GFP transgenic mice using a tet-regulated system. By using transgenic mice expressing PKC-GFP in Purkinje neurones, we have successfully performed live imaging of PKC-GFP in a single Purkinje cell in cerebellar slices, and found that PKC translocation induced by parallel fibre activation propagated from the distal region of the dendritic tree to the proximal trunk close to the soma.

	Results

Top Abstract Introduction Results Discussion Experimental procedures Supplementary material References

 
Development of inducible and brain region-specific PKC-GFP transgenic mice

We generated inducible and brain region-specific PKC-GFP transgenic mice according to the strategies described in our previous reports (Chen et al. 1998; Sakai et al. 2002). In brief, transgenic mice that can drive the expression of the tetracycline transactivator (tTA) under the control of neurone-specific promoters including a neurone-specific enolase (NSE) promoter and a Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) promoter (designated as NSE-tTA and CaMKII-tTA, respectively) were generated. Another transgenic mouse that can drive PKC-GFP under the control of the tetracycline-operated promoter (TetOp) was developed independently (designated as TetOp-PKC-GFP). Two mice lines were then crossed to develop NSE-tTA x TetOp-PKC-GFP (designated as NSE-PKC-GFP) or CaMKII-tTA x TetOp-PKC-GFP (designated as CaMKII-PKC-GFP) bitransgenic mice. In bitransgenic mice carrying two manipulated genes, PKC-GFP was expressed under the control of corresponding neurone-specific promoters.

Expression of PKC-GFP in NSE-tTA x TetOp-PKC-GFP (NSE-PKC-GFP) bitransgenic mice

We developed four lines of TetOp-PKC-GFP mice. Among these lines, one mouse line was able to express PKC-GFP in several brain regions when crossed with NSE-tTA mice. In NSE-tTA x TetOp-PKC-GFP (NSE-PKC-GFP) bitransgenic mice, significant PKC-GFP expression was seen in the caudate–putamen and cerebellum (Fig. 1). In the cerebellum, PKC-GFP was mainly expressed in Purkinje cells (Figs 2A, 2B and 3A). The expression of PKC-GFP was seen throughout the cell body, dendritic shafts and dendrites of Purkinje cells. A low magnification view of immunostaining showed that PKC-GFP was highly expressed in the caudal lobules (especially in lobules 8 and 9) of the cerebellum (Fig. 1). GFP fluorescence was also seen in the axons of Purkinje cells and their terminal area, the dentate nucleus (data not shown). These expression patterns of the target gene products were almost consistent with those as previously reported in bitransgenic mice using the NSE-tTA B-line mice (Chen et al. 1998; Sakai et al. 2002).

View larger version (57K): [in this window] [in a new window]   Figure 1  (A) Patterns of PKC-GFP expression in bitransgenic mice. We crossed TetOp-PKC-GFP mice with NSE-tTA or CaMKII-tTA mice to obtain bitransgenic mice with expression of PKC-GFP in a brain region-specific manner. PKC-GFP expression was determined by immunohistochemistry using anti-GFP antibody. In NSE-PKC-GFP bitransgenic mice, the intense staining of GFP was observed in the striatum and cerebellum. The staining was seen throughout the striatum, while it was mainly seen in the caudal lobules (lobules 8 and 9) of the cerebellum. In contrast, CaMKII-PKC-GFP bitransgenic mice showed the GFP staining throughout the forebrain, including the striatum, cerebral cortex and hippocampus. The staining was also seen in the olfactory bulb. No significant GFP staining was found in TetOp-PKC-GFP single transgenic mice. (B) Immunoblot analysis of regional expression of PKC-GFP and endogenous PKC in bitransgenic mice. Immunoblot analysis using anti-PKC antibody revealed the distinct regional distribution of PKC-GFP in NSE-PKC-GFP (NSE) or CaMKII- PKC-GFP (CaMKII) mice. These distributions of the exogenous PKC-GFP were different from those of endogenous PKC (Cont). As shown in Fig. 3(B), exogenous PKC-GFP and endogenous PKC are recognized as bands with molecular mass of 120 and 85 kDa, respectively. Olf, olfactory bulb; Th, thalamus and hypothalamus; Cb, cerebellum; MB, midbrain; Ctx, cerebral cortex; CP, caudate-putamen; Hip, hippocampus; Med, medulla. (C) Effects of doxycycline, a derivative of tetracycline, on the expression of PKC-GFP in NSE-PKC-GFP bitransgenic mice. Immunohistochemistry of GFP was performed to verify the PKC-GFP expression. As mentioned above and in the text, PKC-GFP was seen in the caudate-putamen and cerebellum (left). After the treatment with doxycycline (50 µg in water) for 4 weeks, the expression was almost completely abolished (middle). The PKC-GFP expression was once again observed 4 weeks after the cessation of doxycycline (right). Data are representative of two separate experiments.

View larger version (96K): [in this window] [in a new window]   Figure 2  Expression of PKC-GFP in bitransgenic mice. Figures show the GFP fluorescence in the cerebellum (A and B) and hippocampus (C and D) in NSE-PKC-GFP and CaMKII-PKC-GFP bitransgenic mice, respectively. In the cerebellum of NSE-PKC-GFP mice, PKC-GFP expression was mainly seen in Purkinje cells. GFP fluorescence and staining were observed throughout the cell, including the cell body, dendritic shafts and dendrites (A). High magnification of GFP fluorescence imaging shows the existence of PKC-GFP in the dendritic spines (B). PKC-GFP was expressed in the cell bodies and their dendrites of hippocampal CA1 region (C). The expression was predominantly observed in the cytosol of CA1 pyramidal cells (D).

Expression of PKC-GFP in CaMKII-tTA x TetOp-PKC-GFP (CaMKII-PKC-GFP) bitransgenic mice

We also crossed this TetOp-PKC-GFP line with CaMKII-tTA mice. In the obtained bitransgenic mice, PKC-GFP was mainly expressed in the forebrain, including the olfactory bulb, hippocampus and cortex, in contrast to NSE-PKC-GFP bitransgenic mice (Fig. 1). In the hippocampus, PKC-GFP fluorescence was observed in the bodies of CA1 pyramidal cells and their dendrites (Fig. 2C,D). The expression of PKC-GFP in the cell bodies was more prominent in CA3 pyramidal cells or granule cells of the dentate gyrus than in the CA1 region. The highest fluorescence was seen in the terminal area of the mossy fibres from the dentate gyrus to the CA3 field (data not shown). These expression patterns of PKC-GFP were almost consistent with those of the target gene products in transgenic mice crossed with CaMKII-tTA mice as previously described (Mayford et al. 1996). Difference in the regional expression of exogenous PKC-GFP between NSE-PKC-GFP and CaMKII-PKC-GFP mice was evidently shown by immunoblot analysis (Fig. 1B). The expression patterns of PKC-GFP was also different from that of endogenous PKC.

Regulation of PKC-GFP expression by doxycycline

The effect of doxycycline, a derivative of tetracycline, on the PKC-GFP expression was examined in order to confirm whether the tet-regulated system was functioning in the developed mice. As shown in Fig. 1(C), an application of doxycycline (50 µg/mL in drinking water) for 4 weeks almost completely blocked the PKC-GFP expression in NSE-PKC-GFP bitransgenic mice. Removal of doxycycline for 4 weeks enabled the PKC-GFP expression to recover, suggesting that application and cessation of doxycycline can regulate the PKC-GFP expression in our bitransgenic mice.

Effects of PKC-GFP expression on endogenous PKC

PKC-GFP was not expressed in all Purkinje cells throughout the cerebellum of NSE-PKC-GFP mice (Fig. 3A, left), while endogenous PKC was abundant in all Purkinje cells (Fig. 3B). The amount of PKC-GFP in the striatum and cerebellum was studied by Western blotting (Fig. 3B) and the amount was compared with that of endogenous PKC. An anti-PKC antibody (Kitano et al. 1987) recognized PKC-GFP with a molecular mass of approximately 120 kDa in transgenic mice as well as endogenous PKC of 85 kDa. The expression of exogenous PKC-GFP did not alter the endogenous PKC expression levels in both the striatum and cerebellum (Fig. 3B,C) and the amount of exogenous PKC-GFP was significantly lower than that of endogenous PKC, suggesting that GFP fluorescence is a good probe to monitor the PKC movement.

View larger version (86K): [in this window] [in a new window]   Figure 3  There was no influence of PKC-GFP expression on the endogenous PKC level. Immunostaining of PKC was performed using cerebellar sections prepared from NSE-PKC-GFP bitransgenic mice. Immunoreactivity of PKC was visualized with Alexa 546 secondary antibody (red) and co-localization of PKC-GFP fluorescence (green) was observed by confocal laser scanning microscope (A and C). PKC-GFP was not expressed in all Purkinje cells of the layer (A). Low and high magnification of PKC-GFP fluorescence and PKC immunostaining show that the staining level of PKC was not distinguishable between GFP-positive and non-positive cells (A and C). Western blotting analysis using anti-PKC antibody demonstrated that the expression of PKC-GFP with a molecular mass of 120 kDa did not alter the level of endogenous PKC of 86 kDa (B).

In the present study, we focused on the metabotropic glutamate receptor (mGluR)-mediated translocation of PKC-GFP as mGluR1-mediated signals are essential to cerebellar LTD formation and developmental elimination of multiple climbing fibre innervation (Aiba et al. 1994; Kano et al. 1995, 1997; Offermanns et al. 1997; Kano et al. 1998; Hashimoto et al. 2001; Miyata et al. 2001). Cerebellar brain slices were prepared from NSE-PKC-GFP bitransgenic mice. We used a two-photon laser-scanning microscope for image distribution of PKC-GFP in a single Purkinje cell, because the system has the advantage of taking images with high resolution in the z-axis and reducing tissue damage by the laser in living brain slices. Superimposed images of consecutive z sections showed that the GFP signal was clearly detected throughout the Purkinje cells, especially in cell bodies and the dendritic shaft, although it was expressed in the peripheral branches of dendrites (Fig. 4A). In the cell body, PKC-GFP was enriched in the cytosol, consistent with our previous immunohistochemical study on the localization of endogenous PKC (Kose et al. 1988). As shown in Fig. 4(B), an application of (1S, 3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (trans-ACPD), an agonist for group I mGluRs, induced reversible and transient translocation of PKC-GFP. The translocation was most prominently observed in the dendritic shaft of Purkinje cells. PKC-GFP started to move and reached the membrane within 15 s after the treatment with trans-ACPD, and then returned to the cytosol. The transient translocation of PKC-GFP simultaneously occurred throughout the dendrites and was completed within approximately 30 s after the translocation started. The time course of translocation was very similar to that of receptor-mediated PKC translocation seen in our previous reports (Sakai et al. 1997; Ohmori et al. 1998; Shirai et al. 1998a). In the branches of dendrites, their outlines became obscure during the evident translocation of PKC-GFP to the plasma membrane in the dendritic shaft (see the figures at 16 and 22 s), indicating that PKC-GFP translocation also occurs at peripheral dendrites. Translocation was not observed in the cell bodies of Purkinje cells. Previous reports demonstrated that PKC-GFP was repetitively translocated between the cytosol and plasma membrane upon mGluR1 stimulation in mGluR1-expressing cultured cells (Dale et al. 2001; Babwah et al. 2003). However, in slices of Purkinje cells, the second application of trans-ACPD never induced PKC-GFP translocation once it was completed by the first application (n = 5). Among other glutamate receptor agonists, quisqualic acid, but not -amino-3-hydroxy-5-methylisoxazole-propionate (AMPA) elicited PKC-GFP translocation (n = 3, data not shown), suggesting that mGluR is mainly responsible for PKC translocation in this area. Video 1 (Video 1 of supplemental videos) shows trans-ACPD (100 µM)-induced translocation of PKC-GFP in Purkinje cells, observed by a high-resolution CCD camera.

View larger version (119K): [in this window] [in a new window]   Figure 4  Live imaging of PKC-GFP translocation in Purkinje cells under a two-photon laser-scanning microscope. Cerebellar slices were prepared from NSE-PKC-GFP bitransgenic mice. Slices were transferred into a chamber and z-stack images of Purkinje cells were made at intervals of 1 µm (A). PKC-GFP was localized throughout the Purkinje cells, especially in the cell bodies and dendritic shaft, although it was clearly expressed in the peripheral branches of dendrites (A). Application of trans-ACPD, an agonist of mGluR1, elicited PKC-GFP translocation (B). PKC-GFP apparently started to translocate to the membrane of the dendritic shaft within 15 s after the drug was applied close to the observed cells. Then, it returned within 30 s after the drug application. While PKC-GFP in the dendritic shaft was translocated to the membrane, outlines of dendritic branches became obscure (see at 11, 16 and 22 s after the treatment). Time-lapse images were acquired every 5.4 s.

Translocation of PKC-GFP in Purkinje cells was further examined using a high resolution CCD imaging system. Figure 5 shows a typical record of translocation induced by electrical stimulation of parallel fibres (PF). Stimulation was delivered through an electrode placed on the pia matter near the imaged cell, and GFP fluorescence around the dendritic shaft was imaged every 1 s (Fig. 5A). At about 3 s after repetitive stimulation (50 Hz, 50 pulses), the GFP signal started to change. The relative intensity of the signal at a trans-sectional area across the dendrite was measured by line scanning at each time period (Fig. 5B). The signal intensity in the midline of the dendrite showed a peak at the start of stimulation. However, 11 s after the stimulation, the peak signal was observed in both edges of the dendritic shaft, indicating that PKC-GFP was translocated to the membrane regions. In this case, the signal pattern returned within 30 s after stimulation. As we have never detected such translocation in the presence of 500 µM MCPG, an antagonist of mGluRs, activation of mGluR must be required for this event.

View larger version (86K): [in this window] [in a new window]   Figure 5  Real-time imaging of PKC-GFP translocation in Purkinje cell dendrites by CCD camera system. Translocation was evoked by electrical stimulation (50 Hz, 50 pulses) of the parallel fibres. (A) Fluorescence image of the cell. The region of interest in the dendritic shaft is indicated by a black rectangle. (B) Time-dependent changes in fluorescence pattern at the region of interest (upper panels). Time after stimulation was indicated on top of each image. Fluorescence intensities at a trans-sectional line area (broken line on the image at time 0) were measured by line scanning. Length and width of the line were 10 and 2 µm, respectively. Normalized intensities of the line area at time 0 (thin line), 11 (thick line), and 29 s (broken line) are shown on a graph (lower panel). (C) Electrophysiological responses induced by parallel fibre stimulation used for producing propagation of PKC-GFP translocation. Whole cell recording was made from the soma of a GFP-positive Purkinje cell. Repetitive stimulation (during horizontal bar, 50 Hz, 50 pulses) induced burst firing followed by slow depolarization and long-lasting hyperpolarization. This slow depolarization (arrow on Control) was abolished by application of 500 µM MCPG, an antagonist of mGluRs.

Unlike drug-induced translocation, PF-induced translocation seemed to propagate along the dendritic shaft from the distal region to the proximal region. Figure 6 shows the time-dependent changes in the site of translocation in the same neurone dendrite. Intensity measurement by line scanning was performed at two trans-sectional line areas simultaneously (Fig. 6A). After repetitive stimulations of PFs, translocation at the distal region occurred at 8 s, and PKC-GFP returned within 5 s (Region A, Fig. 6B). However, at the proximal region, translocation started 20 s after stimulation, and PKC-GFP stayed on the plasma membrane until 60 s (Region B, Fig. 6B). The spatiotemporal properties of the translocation are summarized in Fig. 6(C). Total event time from the start of translocation to the recovery tended to be long at the proximal dendrite less than 50 µm from the soma. Local propagation rates were also measured at several regions in the dendritic shaft. Interestingly, the propagation rate showed a step-wise increase with the distance from the soma. Video 2 (Video 2 in supplemental videos) shows the propagation of PKC-GFP translocation elicited by PF stimulation. PKC-GFP translocation was propagated from the bottom (peripheral) to the upper (Purkinje cell soma). Video 3 (Video 3 in supplemental videos) shows PF stimulation-induced propagation of PKC-GFP at the branching dendrites. Interestingly, the propagation started from the side of stimulated PF (right) and advanced toward, not only the Purkinje cell soma (left), but also the peripheral direction of unstimulated branch (bottom), suggesting that regionally elicited PKC translocation could propagate to neighbouring synapses. Once propagation of PKC-GFP was elicited by repetitive PF stimulation, sequential stimulation did not translocate PKC-GFP, while it elicited electrophysiological synaptic responses. However, after more than a 30-min interval, the same stimulation induced the propagation of PKC-GFP again (n = 3), indicating that the PKC-GFP translocation was a regenerative response which propagated through the dendrites from the postsynapses toward the soma.

View larger version (61K): [in this window] [in a new window]   Figure 6  Propagation of PKC-GFP translocation. Along the dendritic shaft, translocation at the distal region occurred earlier than the proximal region. (A) Imaging area. Intensity measurement by line scanning (length and width of the line were 10 and 2 µm, respectively) was performed at two regions (white broken lines indicated A and B) simultaneously. (B) Patterns of fluorescence intensities at region A and region B. Each trace was lined up from bottom to top by time order at 1-s intervals. Time 0 (third trace from the bottom) indicates the start of stimulation (50 Hz, 20 pulses). During translocation, traces show a small plateau around the peak of the signal. Fluorescence images of each region at time 0, 8, 20 (and 65 for region B) are also shown with thick traces (arrows). Scale bar indicates arbitrary 200 unit of intensity. (C) Spatiotemporal properties of the PKC-GFP translocation. Total event time from the start of translocation to the recovery (left panel) and local propagation rates (right panel) were plotted against distance from the soma. Data were sampled from 14 dendritic regions in 11 neurones. Plots of event time were well fitted by a single exponential decay. In contrast, the propagation rate showed a step-wise increase along the dendritic shaft.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures Supplementary material References

One of the most attractive advantages of the tet-regulated system is that the gene expression of interest can be regulated by simple treatment with tetracycline or its derivative. In the present study, we addressed whether the tet-regulated system was functioning in our PKC-GFP bitransgenic mice. The 4-week application of a relatively low dose of doxycycline (50 µg/mL in drinking water) effectively turned off the PKC-GFP expression and 4-week cessation of the drug almost fully recovered the expression, consistent with our transgenic mice previously reported (Chen & Huang 1992; Sakai et al. 2002). These findings raise the possibility that our PKC-GFP TG mice can compensate the phenotypes of PKC knockout mice in inducible and brain region-specific manners when both transgenic and knockout mice are crossed with each other. This will allow us to analyse the PKC functions in CNS more precisely and specifically.

Among various brain regions expressing PKC-GFP, we focused on the cerebellar Purkinje cells in NSE-PKC-GFP bitransgenic mice. To obtain the most optimized spatial resolution, we used 2-photon laser scanning microscope for imaging PKC-GFP translocation in brain slices. As shown in Fig. 4, we succeeded in obtaining live imaging of mGluR-mediated translocation of PKC-GFP with high spatial resolution, which could reveal the apparent PKC-GFP translocation in both the dendritic shaft and the branch of Purkinje cells. This is the first investigation to visualize the stimulus-dependent movement of physiologically functioning protein expressed in living Purkinje cells, namely the PKC translocation. In this study, we regionally applied trans-ACPD throughout to the observed Purkinje cells. and the PKC-GFP translocation in dendritic shaft as well as peripheral branch started at the same time. Grandes and colleagues reported that mGluR1 immunoreactivity is localized in both proximal and peripheral dendrites (Grandes et al. 1994) in addition to dendritic spines receiving parallel fibre inputs (Baude et al. 1993), suggesting that the functioning mGluR1 receptors expressed, not only at postsynaptic domain, but also at the shaft of Purkinje cell dendrites.

In addition to agonists of mGluR, we applied ionotropic glutamate receptor agonists, including AMPA. Activation of AMPA receptor induces depolarization of Purkinje cells followed by the increase in intracellular Ca²⁺ through voltage-dependent Ca²⁺ channels. In COS7 cells, an increase in intracellular Ca²⁺ by Ca²⁺ ionophore induced a transient translocation of PKC to the plasma membrane. Therefore, it is expected that activation of AMPA receptors also induce the translocation. However, application of AMPA failed to elicit PKC-GFP translocation in Purkinje cells in the brain slice. It has recently been reported that AMPA induced translocation of the GFP-PH-domain, an indicator of intracellular IP3, in cultured Purkinje cells (Okubo et al. 2001). According to their report, the application of AMPA or climbing fibre stimulation increased [Ca²⁺]i via voltage-gated Ca²⁺ channels, which triggered IP3 production. These findings suggest that Ca²⁺ mobilization or Ca²⁺ entry is not sufficient and an additional DAG production is indispensable for the PKC translocation in Purkinje cells in cerebellar slice. Although the discrepancy with the finding in our present study is not fully clarified, the expression level and patterns of AMPA receptors and voltage-gated Ca²⁺ channels in Purkinje cells are different between our TG mice and the cultured Purkinje cells that were mainly used for analysing AMPA-induced IP3 production in their study. It is also possible that AMPA-induced PKC translocation in brain slice is too weak to be detected by our imaging system.

To obtain PKC-GFP translocation with high time resolution, we used a CCD camera to take live images. As shown in Figs 5 and 6, we succeed in observing the sequential PKC-GFP translocation in the dendritic shaft of Purkinje cells by electrical simulation applied to the parallel fibres. This is the first live image to reveal the neuronal activity-dependent translocation of PKC. This novel type of imaging of neuronal function has shown some unique properties which have never been observed by conventional analytical methods. For example, the propagation rate of PKC-GFP was distributed between 4 and 15 µm/s as shown in Fig. 6(C), indicating that activity-dependent propagation of PKC occurs with totally different time scales from electrophysiological phenomena mostly mediated by ion diffusion. Propagation of PKC by trans-synaptic activation of Purkinje cell is slower than Ca²⁺-diffusion (Nakamura et al. 2002) and significantly faster than sorting by cytoskeletal protein (Nakata et al. 1998). In addition, PKC translocation was a regenerative but not repetitive phenomenon in Purkinje cells. Once trans-ACPD-induced or trans-synaptic translocation of PKC-GFP had completely occurred, it took approximately 30 min until the next stimulation generated the second translocation. These findings suggest that activity-dependent PKC propagation has a relatively long refractory period, which was never seen in electrogenic responses found in neurones, although it has not yet elucidated how these properties are implicated in the cerebellar plasticity and development. It is strongly expected that precise studies using these brain region-specific PKC-GFP transgenic mice and focusing on the relationship between neuronal functions and spatiotemporal profiles of PKC activities will provide new insights into the PKC significance in CNS.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures Supplementary material References

 
Development of transgenic mice

The methods for generating inducible and brain region-specific transgenic mice using the tet-regulated system are described in our previous reports (Chen et al. 1998; Sakai et al. 2002). For the present studies, we used the NSE-tTA B line generated by Chen et al. (1998). The CaMKII-tTA mice, which were originally generated by Mayford and colleagues (Mayford et al. 1996), were purchased from JAX mice (strain no. 003616). The method for developing TetOp-PKC-GFP mice was as follows. The cDNA fragments encoding PKC-GFP were excised from BS 336 (Sakai et al. 1997; Shirai et al. 1998a) by EcoRI digestion and inserted into the EcoRI site of the TetOp-splice plasmid (Invitrogen, Tokyo, Japan). The DNA fragments containing the TetOp promoter, PKC-GFP and SV40 poly A signals were excised from this plasmid by digestion with XhoI/NotI and microinjected into the pronuclei of oocytes from C57BL/6 J mice. Genotyping was performed by PCR using tail DNA. Out of four transgene-positive lines, one mouse line was confirmed to express PKC-GFP when crossed with NSE-tTA mice.

Immunohistochemistry

Preparation of tissue sections and immunohistochemical procedures were as previously described (Itouji et al. 1996; Obata et al. 1997). Briefly, the brains were fixed by perfusion with 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB, pH 7.4). After post-fixation with the same fixative for 24 h, the brains were immersed in 30% sucrose in 0.1 M PB for 2 days and cut into sections of 20 µm thickness by a cryostat. Sections were pre-incubated with 0.3% H₂O₂ and 5% normal goat serum (NGS) in 0.01 M phosphate-buffered saline (PBS) containing 0.03% Triton-X-100 (PBS-T), then incubated with primary antibodies for 18 h. Primary antibodies used were rabbit polyclonal anti-GFP antibody (Molecular Probes, Eugene, OR, diluted 1 : 2000) and mouse monoclonal anti-PKC antibody (Kitano et al. 1987) (diluted 1 : 5000). For GFP immunostaining, sections were incubated with anti-rabbit IgG (MBL, Nagoya, Japan) for an additional 4 h after washing with PBS-T, then with rabbit peroxidase-anti-peroxidase complex (ICN, Lisle, IL). After three rinses, the sections were developed with 0.02% diaminobenzidine (Sigma, St Louis, MO) and 0.2% nickel ammonium sulphate in 50 mM Tris-HCl (pH 7.4) with 0.0005% H₂O₂. The sections were observed and photographed under a light microscope (Axioplan II, Carl Zeiss, Jena, Germany). For PKC staining, sections were incubated with anti-mouse IgG conjugated with Alexa Fluor 546 (Molecular Probes, diluted 1 : 500). The fluorescence of PKC-GFP and Alexa Fluor 546 was observed with a confocal laser scanning fluorescent microscope (LSM 510 invert, Carl Zeiss) at 488-nm argon excitation using a 510- to 525-nm band pass barrier filter and 588 nm excitation using a 590-nm-long pass barrier filter, respectively.

Immunoblotting

Dissected tissues of each brain regions from NSE-tTA x TetOp-PKC-GFP bitransgenic mice were homogenized and sonicated in RIPA buffer [10 mM Tris-HCl, 1% NP40, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.15 M NaCl, 1 mM EDTA, 20 µg/mL leupeptin and 1 mM PMSF, pH 7.4]. Samples with 50 µg protein were subjected to 7.5% SDS–polyacrylamide gel electrophoresis (PAGE) and the separated proteins were electrophoretically transferred on to polyvinylidene difluoride (PVDF) filters (Millipore, MA, USA). Non-specific binding sites on the PVDF filters were blocked by incubation with 5% non-fat milk for 18 h. The PVDF filters were then incubated with anti-PKC monoclonal antibody (diluted 1 : 5000) for 2 h at room temperature. After washing with PBS-T, the filters were incubated with goat anti-mouse IgG conjugated with peroxidase (Jackson Immuno-Research, West Grove, PA, diluted 1 : 10 000) for 1 h. After three rinses, the immunoreactive bands were visualized with a chemiluminescence detection kit (ECL, Amersham, Buckinghamshire, UK).

Observation of PKC-GFP translocation in cerebellar slices

All slice experiments were carried out according to the guidelines laid down by the animal welfare committee in National Institute for Physiological Sciences. Three- to four-week-old TG mice were deeply anaesthetized with ether and decapitated. The brains were quickly removed and hemisected on filter paper moistened with cutting solution of the following composition (mM): 120 choline-Cl, 3 KCl, 8 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, 20 glucose, equilibrated with 95% O₂–5% CO₂. Brain tissues containing the cerebellum were dissected out and put in the cutting chamber filled with ice-cold cutting solution. This block was sliced into 250-µm sagittal sections using a vibrating slicer (Campden Instruments). The slices were immediately placed in a reservoir chamber filled with normal solution, and incubated at room temperature (25 °C) for about 1–2 h and then maintained at room temperature. The normal recording solution was composed of (in mM): 125 NaCl, 2.5 KCl, 2 CaCl₂, 2 MgCl₂, 1.25 NaHPO₄, 26 NaHCO₃, 10 glucose, bubbled with a mixture of 95% O₂, 5% CO₂, making the final pH = 7.4.

Two-photon imaging of Purkinje neurones was performed with an upright microscope (BX50, Olympus, Tokyo, Japan) with a water immersion objective lens (LUMPlanFI 60 water/IR; NA, 0.9). The slice was superfused continuously with the normal solution regulated at room temperature. Mode-locked femtosecond-pulse Ti:sapphire laser (Tsunami, Spectra Physics, Mountain View, CA) with original pulse duration of 70–100 fs was attached to a laser-scanning microscope (FluoView, Olympus). The group velocity dispersion of the microscope was compensated for by chirp compensation optics. The laser power at the specimen was 18–22 mW. Excitation wavelength was 900–905 nm. For CCD imaging, a single slice was transferred to a submerged chamber mounted on the stage of an upright microscope (BX51WI, Olympus). The slice was superfused continuously with the normal solution regulated at room temperature. Time-lapse imaging of the GFP signals (1 frame/s) was made from single Purkinje neurone using a cooled CCD camera system (CoolSnap-HQ, Roper Scientific). The cell was excited every 200 ms at 480 ± 10 nm Trans-ACPD (100 µM) or quisqualate was applied either by changing bath solution or by puffing to their dendrites through a glass pipette. A bipolar stimulation electrode constructed from thin Pt-Ir wire (50-µm OD) was placed on the pia mater to stimulate parallel fibres. All drugs were obtained from Tocris Cookson Inc. (Bristol, UK).

Supplementary material

	Supplementary material

Top Abstract Introduction Results Discussion Experimental procedures Supplementary material References

 
The authors have provided the following supplemental videos, which can be viewed at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC779/GTC779sm.htm. Quicktime video viewer is required. Video S1: PKC-GFP translocation elicited by trans-ACPD treatment. Trans-ACPD at 100 µM induced a reversible and transient translocation of PKC-GFP in the dendritic shaft of Purkinje cells. Sequential 80 images obtained every 1 s using CCD imaging system were compacted to this movie. This movie under higher resolution can be also seen at http://www.pkn.biosig.kobe-u.ac.jp/supplement/login.htm (ID = neuron, password = F94837). Video S2: Propagation of PKC-GFP translocation elicited by PF stimulation. The bottom and upper are the sides of PF and Purkinje cell body, respectively. PKC-GFP translocation was propagated from the bottom to the upper, namely from the PF side to Purkinje cell soma. Sequential 60 images with 1-s intervals were compacted to this movie. This movie under higher resolution can be seen at http://www.pkn.biosig.kobe-u.ac.jp/supplement/login.htm (ID = neuron, password = F94837). Video S3: PF stimulation-induced propagation of PKC-GFP at the region where the dendritic shaft is divided into two branches. The right and left are the sides of stimulated-PF and Purkinje cell body, respectively. Propagation of PKC-GFP translocation started from the right, the stimulated-PF side. It directed not only to Purkinje cell body (the left side), but also to unstimulated peripheral branches (the bottom side). This movie under higher resolution can be seen at http://www.pkn.biosig.kobe-u.ac.jp/supplement/login.htm (ID = neuron, password = F94837).

	Acknowledgements

 
This work was supported by a 21st century COE Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan, a grant from the Ministry of Education, Culture, Sports, Science and Technology in Japan, a Grant-in-Aid for Scientific Research on Priority Areas(C)-Advanced Brain Science Project from Ministry of Education, Culture, Sports, Science and Technology of Japan, the Uehara Memorial Foundation, the Sankyo Foundation of Life Science, Japanese Smoking Research Association and Takeda Science Foundation.

	Footnotes

 
Communicated by: Kozo Kaibuchi

^* Correspondence: E-mail: naosaito{at}kobe-u.ac.jp

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Received: 23 April 2004
Accepted: 6 July 2004

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