Article |
Address correspondence to Franz Hofmann, Institut für Pharmakologie und Toxikologie, Technische Universität, Biedersteiner Str. 29, D-80802 München, Germany. Tel.: 49-89-4140-3260. Fax: 49-89-4140-3261. email: Hofmann{at}ipt.med.tu-muenchen.de
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Abstract |
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Key Words: cGMP kinase; Cre recombinase; intracellular signaling; synaptic plasticity; learning
Abbreviations used in this paper: cGKI, cGMP-dependent protein kinase type I; DSCT, delayed synaptic Ca2+ transient; EPSC, excitatory postsynaptic current; ESCT, early synaptic Ca2+ transient; LTD, long-term depression; OKR, optokinetic reflex; PC, Purkinje cell; VOR, vestibulo-ocular reflex; VVOR, VOR in the light.
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Introduction |
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The molecular and cellular mechanisms of cerebellar NO signaling are not completely understood. Indirect evidence from experiments with cerebellar slices suggested that NO induces LTD via activation of soluble guanylyl cyclase and subsequent cGMP synthesis in PCs (Daniel et al., 1993; Boxall and Garthwaite, 1996; Hartell, 1996; Lev-Ram et al., 1997a; Hartell et al., 2001). The identification of the signaling components downstream of cGMP is complicated by the existence of multiple receptors for cGMP (Beavo and Brunton, 2002) and by the lack of highly specific activators and inhibitors for a given cGMP receptor protein (Smolenski et al., 1998; Schwede et al., 2000). Cerebellar PCs express high levels of cGMP-dependent protein kinase type I (cGKI) (Hofmann and Sold, 1972; Lohmann et al., 1981), whereas cGK type II was not detected in the cerebellum (unpublished data). Interestingly, agents that inhibit cGKI in vitro, particularly the widely used "cGK inhibitor" KT5823, have been shown to impair LTD in cerebellar slices, indicating a role for cGKI in LTD induction (Hartell, 1994; Lev-Ram et al., 1997a). However, it was recently observed that KT5823 may not inhibit cGKI in certain intact cells (Burkhardt et al., 2000), including cerebellar PCs (Rybalkin, S.D., and J.A. Beavo, personal communication). These findings suggest that the effects of cGKI inhibitors should be interpreted with caution, particularly if inhibition of kinase activity was not demonstrated, for example, by monitoring the phosphorylation of a known cGKI substrate protein (Burkhardt et al., 2000; Shimizu-Albergine et al., 2003). It has been noted that it might be difficult to study LTD with pharmacological tools, as they can exaggerate the importance of certain pathways in LTD induction that might be less important, or not even used, in physiological conditions (Daniel et al., 1998). Furthermore, the specific relevance of cGKI in PCs to cerebellar motor learning has not been investigated yet.
As a first step toward an understanding of the in vivo function of cerebellar cGKI signaling, we have used a genetic, rather than a pharmacological, approach, namely PC-specific disruption of the cGKI gene in mice by using Cre/loxP-assisted conditional somatic mutagenesis (Metzger and Feil, 1999). PC-specific cGKI knockout mice perform normal in several tasks testing general motor performance, but exhibit strongly reduced cerebellar LTD and impaired adaptation of the VOR. Thus, cGKI-dependent signaling in PCs contributes to synaptic plasticity and particular forms of motor learning.
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Results |
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To ablate cGKI specifically in PCs, we generated mice carrying the cGKI L2 allele as well as the L7-Cre transgene (Barski et al., 2000), which expresses the Cre recombinase in almost all cerebellar PCs. The expression of cGKI was first analyzed by Western blot analysis of extracts from various tissues. As compared with control mice, cGKIpko mice showed a strong reduction of cGKI protein in the cerebellum, but normal cGKI levels in other brain regions and peripheral tissues, such as hippocampus, aorta, and heart (Fig. 1 A). Immunohistochemical detection of cGKI at the cellular level indicated that the protein was highly expressed in almost all PCs of control animals, whereas <5% of the PCs in cGKIpko animals expressed cGKI (Fig. 1 B). These results correlate well with the recombination pattern of the L7-Cre mouse line as revealed by expression of ß-galactosidase in "Cre indicator" mice (Barski et al., 2000) and of a loxP-flanked calbindin target gene (Barski et al., 2003), i.e., strong Cre activity in cerebellar PCs and weak to undetectable Cre activity in other brain regions or peripheral tissues. The finding that the cGKI protein was not completely absent in extracts from the cerebellar region of cGKIpko mice (Fig. 1 A) can be attributed to its residual expression in few PCs (Fig. 1 B), and to the presence of cGKI in cerebral vessels (Lohmann et al., 1981). Taken together, these data demonstrated that our knockout strategy resulted in efficient and selective ablation of cGKI in cerebellar PCs.
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Adaptation of the VOR but not general motor performance is impaired in cGKIpko mice
Visual inspection of cGKIpko mice did not reveal any gross abnormalities or overt behavioral phenotypes. No differences in weight, growth, life expectancy, and activity in the open field test were observed between control mice and cGKI mutants (unpublished data). Despite their defect in cerebellar LTD, cGKIpko mice showed normal motor coordination as analyzed by the footprint, runway, and rotarod test (Fig. 5), suggesting that cerebellar cGKI is dispensable for general motor control.
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Discussion |
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The main finding of the present study is that cGKIpko mice showed nearly complete absence of cerebellar LTD, as measured by whole-cell patch clamping in acute slices, and impaired adaptation of the VOR, while their general eye movement performance was normal. This phenotype demonstrates a specific role for PC cGKI signaling in cerebellar LTD and motor learning. Despite impaired LTD, cGKIpko mice showed no overt behavioral phenotype and performed normal in several tests of general motor coordination, i.e., the footprint, runway, and rotarod test, suggesting that cGKI in PCs is dispensable for general motor coordination. A highly similar phenotype was observed in transgenic mice expressing a PKC inhibitor peptide selectively in PCs (De Zeeuw et al., 1998; Goossens et al., 2001; Van Alphen and De Zeeuw, 2002). Together, these previous and our present results strongly support the concept that cerebellar LTD is involved in specific forms of motor learning, such as adaptation of the VOR, but not in general motor performance. Impaired motor coordination in various knockout mouse models correlates with aberrant multiple innervation of PCs by climbing fibers, which, even though LTD is retained, is expected to impair the function of the cerebellar neuronal circuit (Ito, 2001). Furthermore, these mouse mutants lacked the gene of interest in all cells of the body, questioning the specific relationship of motor discoordination to the cerebellum. In contrast, cGKIpko mice lacked cGKI selectively in cerebellar PCs and showed normal climbing fiber innervation. Thus, the phenotype of the cGKIpko mouse model may be more informative with respect to the specific role of cerebellar LTD in motor learning compared with other mouse mutants in which multiple climbing fiber innervation and noncerebellar defects might also contribute to motor phenotypes.
How could activation of cGKI in PCs contribute to LTD and cerebellum-dependent learning? LTD induction requires an appropriate balance between protein kinases and phosphatases (Ito, 2002) and can be facilitated by inhibition of protein phosphatase 1/2A (Ajima and Ito, 1995). Indeed, cGKI may phosphorylate G-substrate, a well-characterized cGKI target in PCs (Schlichter et al., 1978; Aswad and Greengard, 1981), which would in turn inhibit protein phosphatase 1/2A (Endo et al., 1999; Hall et al., 1999). Inhibition of protein dephosphorylation would increase the levels of phosphoproteins generated by the action of various protein kinases, including PKC and cGKI itself. It is assumed that phosphorylation of the AMPA receptor complex, presumably by PKC, allows the removal of AMPA receptor subunits from the synaptic membrane via clathrin-mediated endocytosis (Wang and Linden, 2000; Chung et al., 2003). Thus, we propose the following molecular model for cerebellar LTD and motor learning: NO/cGMP-dependent activation of cGKI results in phosphorylation of G-substrate, inhibition of protein phosphatases, extended endocytosis of phosphorylated AMPA receptor subunits, LTD, and motor learning. Future studies, for example, the analysis of the effects of phosphatase inhibitors on LTD in cGKIpko mice, should help to validate this model.
In conclusion, this study demonstrates that cGKI-dependent signaling in PCs contributes to cerebellar LTD and a particular form of motor learning, adaptation of the VOR. To the best of our knowledge, this is the first cell-specific demonstration that cGKI is involved in cerebellar synaptic plasticity and learning in vivo in a way that cannot be compensated for by PKC. Based on these and previous results, we propose that cGKI in PCs is indispensable for cerebellar learning.
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Materials and methods |
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Western blot analysis of cGKI expression
cGKI was detected using a rabbit polyclonal antibody to cGKI (Pfeifer et al., 1998). Equal loading of gels for immunoblots was confirmed by staining with p44/42 MAP kinase antibodies (New England Biolabs, Inc.).
Morphological and immunohistochemical analysis
Animals were deeply anesthetized and perfused through the ascending aorta with either 10% phosphate-buffered formalin (for detection of cGKI), Bouin's fixative, or, for ultrastructural analysis, a buffered mixture of 2% freshly depolymerized paraformaldehyde and 2% glutaraldehyde. Brains were dissected and postfixed in the same fixatives overnight. For light microscopic studies, brains were embedded in paraffin, cut at 10 µm, and routinely stained with hematoxylin and eosin. Immunohistochemistry was performed as previously described (Mertz et al., 2000) using antisera to cGKI (Pfeifer et al., 1998), calbindin D28k (mouse, clone CL-300, 1:400; Sigm-Aldrich), or synaptophysin (rabbit, G95, 1:2,000; a gift of R. Jahn, Max-Planck Institut für Biophysikalische Chemie, Göttingen, Germany). For detection of primary antibodies, we used either the avidin-biotin method with diaminobenzidine as a chromogen (Vector Laboratories) or species-specific secondary antibodies tagged with Cy-3, Cy-2, or Alexa®488. For ultrastructural analysis, specimens were postfixed in 1% osmium tetroxide in PBS. Specimens were rinsed in water, dehydrated, and embedded in Durcopan resin. Semithin (1 µm) sections were stained with toluidine blue. Ultrathin sections were cut with a diamond knife and contrasted with uranyl acetate and lead citrate.
Electrophysiology and calcium imaging
Slices (300 µm) were prepared from mice that were decapitated after anesthesia with CO2. Whole-cell recordings were obtained from PCs in slices perfused with artificial cerebro-spinal fluid composed of (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 20 glucose, and 0.01 bicuculline (Sigma-Aldrich), bubbled with 95% O2 and 5% CO2. Pipettes (24 M resistance) were pulled from borosilicate glass and coated with silicon. The pipette solution contained (in mM) 148 potassium gluconate, 10 Hepes, 10 NaCl, 0.5 MgCl2, 4 Mg-ATP, and 0.4 Na3-GTP, pH 7.3. Oregon green BAPTA-1 (Molecular Probes) was added to the pipette solution (100 µM for LTD experiments and 200 µM for calcium imaging). Synaptic stimulation was performed by using pipettes filled with 1 mM NaCl (1 M
resistance) placed in the molecular layer. The threshold for climbing fiber activation was identified in the voltage clamp mode by gradually increasing the voltage pulse through a stimulation pipette placed over the PC's dendritic tree. In contrast to the parallel fiber responses, the large-amplitude climbing fiber EPSC is characterized by an all-or-none behavior. The stimulus pulse amplitude (150 µs duration) was 333 V for parallel fiber stimulation and 2090 V for climbing fiber stimulation. Parallel fibers were stimulated at 0.2 Hz, and EPSCs were recorded in the voltage clamp mode until a stable baseline amplitude was obtained for at least 10 min. LTD was induced according to published procedures (Barski et al., 2003). In brief, the stimulus intensity was raised to a value at least 20% over climbing fiber threshold (identified beforehand), and 120 stimuli were repeated at 1 Hz in the current clamp mode. The successful stimulation of the climbing fiber was verified by the recording of complex spikes accompanying each stimulation. After pairing and returning to the voltage clamp mode, the stimulus intensity was set to the initial value and the recording of parallel fiber EPSCs at 0.2 Hz was resumed for 20 min. Passive membrane properties of PCs were monitored by applying 5 mV hyperpolarizing pulses. The series resistance was kept constant throughout the measurement at around 1020 M
. The holding current during LTD measurements ranged from -100 to -500 pA and the input resistance from 29 to 111 M
without differences between both experimental groups. For calcium imaging, a confocal laser-scanning microscope (Odyssey; Noran Instruments) attached to an upright microscope was used. Fluorescence images were acquired at 30 Hz and analyzed off-line with custom-made software. Ca2+ transients were recorded in regions of interest in active dendritic regions. The Ca2+-dependent fluorescence signals were expressed as increases in fluorescence divided by the prestimulus fluorescence values (
F/F) and further analyzed using Igor Pro software (Wavemetrics).
Motor coordination tests
Footprint patterns of mice were analyzed using a narrow tunnel (10 cm wide, 35 cm long, 10 cm high) with white paper on the bottom. Before traversing the tunnel, the hind- and forepaws of the animals were dipped in nontoxic blue and red ink, respectively. The runway test was performed as follows. The animal was placed at one end of a horizontally fixed beam (1 cm wide, 100 cm long, separated into 11 segments by low obstacles, height from a table 40 cm), facing the opposite end, and allowed to move on the beam to reach an escape platform on its home cage. One investigator counted the number of slips of the forepaw and hindpaw on the right side of the beam, and another investigator counted the slips on the left side. Each day, each mouse underwent one trail (five consecutive runs) on five consecutive days. The ability to maintain balance on a rotating cylinder was assayed by using a computerized rotarod (Technical & Scientific Equipment GmbH) in its accelerating mode. Each mouse was tested once per day on five consecutive days. During each test session, animals were placed on the stationary rod for 30 s, and the rod was started and accelerated continuously from 5 to 58 rpm over 270 s. The rotational speed at which a mouse fell off the rotating cylinder was recorded automatically. Mice that did not fall off during the 270-s trail period were given a score of 58 rpm.
Eye movement recordings
Mice were anesthetized with a mixture of halothane, nitrous oxide, and oxygen. The procedures for implanting a head fixation pedestal and a "mini" search coil were identical to those previously described (van Alphen et al., 2001). Baseline measurements were taken for their OKR, VOR, and VVOR. The OKR and VVOR in response to sinusoidal movement of the drum or table in the light were tested at five different frequencies (0.1, 0.2, 0.4, 0.8, and 1.6 Hz) and two different amplitudes (58 and 108; 0-peak). VOR in response to sinusoidal whole body rotation in the dark was tested at the same set of frequencies and amplitudes except that the stimulus frequency 0.1 Hz was omitted, because at this frequency, the vestibular signals driving the VOR are insufficient to obtain a powerful and reliable response. Subsequently, the animals were subjected to visuo-vestibular training for 5 d, which lasted 1 h per day. Animals were trained to reverse its direction using the "in phase" training protocol, which is the most effective training paradigm (van Alphen et al., 2001). Training began on the first day by rotating the optokinetic drum in phase, i.e., 0° phase difference, with table rotation at 5° amplitude. In the following 4 d, amplitude of the optokinetic drum was increased at 1° per day until it was 9° on day 5. At this point, the optokinetic drum was rotating in phase with the table but at twice the amplitude. For both turntable and drum movement, we chose a stimulus training frequency of 0.6 Hz, which is an optimal compromise to ensure both a reliable vestibular input to the VOR and a visual input with a peak velocity well within the physiological range of the mouse optokinetic system (van Alphen et al., 2001). Gain of the eye movement and phase of eye movement with respect to stimulus movement were calculated by fitting a sine wave to the average response using least-square optimization. When eye movement lagged stimulus movement, phase was expressed with a negative sign. Phase relations of VOR were shifted by 180°, making the phase angle zero for perfectly compensatory responses.
Statistics
Data shown are mean ± SEM, and statistical analysis was performed using ANOVA for repeated measures or the t test for two independent means. Significance was accepted if P < 0.05.
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Acknowledgments |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft, VolkswagenStiftung, Dutch Research Council for Medical Sciences and Life Sciences, Human Frontier Science Program, European Community, and Fonds der Chemischen Industrie.
Submitted: 26 June 2003
Accepted: 8 September 2003
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