Calcium Dynamics and Electrophysiological Properties of Cerebellar Purkinje Cells in SCA1 Transgenic Mice

Takafumi Inoue,1 Xi Lin,2 Kristi A. Kohlmeier,1 Harry T. Orr,3 Huda Y. Zoghbi,2 and William N. Ross1

 1Department of Physiology, New York Medical College, Valhalla, New York 10595;  2Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030; and  3Department of Pathology, University of Minnesota, Minneapolis, Minnesota 55455


    ABSTRACT
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ABSTRACT
INTRODUCTION
Methods
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DISCUSSION
REFERENCES

Inoue, Takafumi, Xi Lin, Kristi A. Kohlmeier, Harry T. Orr, Huda Y. Zoghbi, and William N. Ross. Calcium Dynamics and Electrophysiological Properties of Cerebellar Purkinje Cells in SCA1 Transgenic Mice. J. Neurophysiol. 85: 1750-1760, 2001. Cerebellar Purkinje cells (PCs) from spinocerebellar ataxia type 1 (SCA1) transgenic mice develop dendritic and somatic atrophy with age. Inositol 1,4,5-trisphosphate receptor type 1 and the sarco/endoplasmic reticulum Ca2+ ATPase pump, which regulate [Ca2+]i, are expressed at lower levels in these cells compared with the levels in cells from wild-type (WT) mice. To examine PCs in SCA1 mice, we used whole-cell patch clamp recording combined with fluorometric [Ca2+]i and [Na+]i measurements in cerebellar slices. PCs in SCA1 mice had Na+ spikes, Ca2+ spikes, climbing fiber (CF) electrical responses, parallel fiber (PF) electrical responses, and metabotropic glutamate receptor (mGluR)-mediated, PF-evoked Ca2+ release from intracellular stores that were qualitatively similar to those recorded from WT mice. Under our experimental conditions, it was easier to evoke the mGluR-mediated secondary [Ca2+]i increase in SCA1 PCs. The membrane resistance of SCA1 PCs was 3.3 times higher than that of WT cells, which correlated with the 1.7 times smaller cell body size. Most SCA1 PCs (but not WT) had a delayed onset (about 50-200 ms) to Na+ spike firing induced by current injection. This delay was increased by hyperpolarizing prepulses and was eliminated by 4-aminopyridine, which suggests that this delay was due to enhancement of the A-like K+ conductance in the SCA1 PCs. In response to CF stimulation, most PCs in mutant and WT mice had rapid, widespread [Ca2+]i changes that recovered in <200 ms. Some SCA1 PCs showed a slow, localized, secondary Ca2+ transient following the initial CF Ca2+ transient, which may reflect release of Ca2+ from intracellular stores. Thus, with these exceptions, the basic physiological properties of mutant PCs are similar to those of WT neurons, even with dramatic alteration of their morphology and downregulation of Ca2+ handling molecules.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
Methods
RESULTS
DISCUSSION
REFERENCES

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurological disorder characterized by ataxia and brain stem dysfunction. Like several other neurodegenerative diseases, SCA1 is caused by the expansion of a CAG trinucleotide repeat, which results in an expanded polyglutamine tract in its gene product, ataxin-1 (Orr et al. 1993). It is well established that polyglutamine expansion confers neurotoxicity to the native proteins, which leads to the degeneration of selective groups of neurons. In SCA1, the consistent neuropathological finding is loss of Purkinje cells (PCs) in the cerebellar cortex and loss of neurons in pontine nuclei (Gilman et al. 1996). As part of an effort to understand the role of the expanded CAG polyglutamine tracts in the pathogenesis of SCA1, lines of transgenic mice that express either a normal human SCA1 allele with 30 glutamine (30Q; e.g., the A02 line) or an expanded allele with 82 glutamine (82Q; e.g., the B05 line) have been established (Burright et al. 1995; Clark et al. 1997). Lines of transgenic animals that expressed a wild type (WT) human SCA1 allele did not develop the typical SCA1 phenotypes, but lines of transgenic mice that expressed the mutant form of the SCA1 gene developed ataxia and PC pathology. Importantly, the eventual development of ataxia is not attributable to cell death per se but to cellular dysfunction and morphological alterations that occur long before neuronal death (Clark et al. 1997). To gain insight into the mechanism underlying cerebellar dysfunction, we previously adopted a subtractive cloning strategy to evaluate the alteration of gene expression in the cerebellums of SCA1 mice and found that several key neuronal genes are specifically altered in the PCs of mutant cerebellums prior to any clinicopathologic manifestations (Lin et al. 2000). It is noteworthy that some of these genes are important for regulating intracellular Ca2+ dynamics. At postnatal day 14 (P14), the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2) are greatly reduced. By P21-P28, type 1 inositol phosphate 5-phosphatase, excitatory amino acid transporter type 4 (EAAT4), and transient receptor potential type 3 (TRP3) are dramatically downregulated (Lin et al. 2000). Therefore it was of interest to study the electrophysiological and synaptic properties of SCA1 PCs, particularly those that affect stimulus-evoked [Ca2+]i changes, since they might be sensitive to alterations in the expression levels of these molecules.


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Transgenic mice

Adult (3-7 mo old) heterozygous SCA1 transgenic mice (B05 line) and WT littermates were used. Transgene configuration and establishment of the B05 line carrying a mutant SCA1 allele with 82 CAG repeats have been described in Burright et al. (1995).

Slice preparation and electrophysiology

Sagittal cerebellar slices 300 µm thick were prepared according to standard procedures (Inoue et al. 1998; Tsubokawa and Ross 1997). PCs were visually identified under differential interference contrast (DIC) optics using a 40× water immersion objective (numeric aperture 0.80) attached to an upright microscope (BX50WI, Olympus). A model P-97 puller (Sutter Instrument, Novato, CA) was used to pull patch electrodes from fiber-filled capillary tubing with an outside diameter of 1.5 mm (no. 1511-M, Friedrich and Dimmock, Millville, NJ). The resistance of the patch electrodes was 3-6 MOmega when they were filled with an intracellular solution composed of (in mM) 130 K-gluconate, 10 Na-gluconate, 4 NaCl, 2 Mg-ATP, 0.3 Na-GTP, 0.2 bis-fura-2 (Molecular Probes, Eugene, OR), and 10 HEPES (pH 7.2). Bis-fura-2 was replaced with 0.3 mM of the lower affinity indicator fura-6F (Molecular Probes) in experiments measuring parallel fiber (PF) responses. The composition of the artificial cerebrospinal fluid (ACSF) bathing solution was (in mM) 124 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 20 glucose. The bathing solution was kept at about 32°C and bubbled continuously with a mixture of 95% O2 and 5% CO2. Recordings were made with an AxoClamp 2A amplifier (Axon Instruments, Foster, CA) in the current-clamp mode, digitized at 20 KHz, and stored in a computer. For stimulation of climbing fiber (CF) and PF responses, a glass pipette (tip diameter of 5-10 µm) filled with standard saline was used. Square pulses (0.4 ms duration) were applied for focal stimulation.

Soma size measurements

DIC images of PC somata were taken with a video camera (VE1000, DAGE MTI, Michigan, IN) and stored in a computer with a frame grabber (Axon Image Lightning 2000, Axon Instruments). The projected areas of the PC somata were measured as numbers of pixels and converted to area.

Calcium concentration measurements

High-speed fluorescence measurements using a cooled charge coupled device (CCD) camera were made as described in Lasser-Ross et al. (1991). Fluorescence was excited and detected using the 40× objective. The use of an Opti-Quip Model 1600 (Highland Mills, NY) power supply (ripple <0.02%) and a Hamamatsu L2481-01 75-W Mercury-Xenon arc lamp improved the excitation intensity stability. Typical intensity fluctuations were <0.3%. Excitation of bis-fura-2 or fura-6F was at 380 nm selected with a 15-nm-wide interference filter (Omega Optical, Brattleboro, VT). Fluorescence changes due to bleaching and baseline drift were corrected by subtracting an identical measurement without stimulation. Optical changes are expressed as Delta F/F (change in fluorescence from resting levels divided by the resting fluorescence corrected for autofluorescence). The contribution of background (autofluorescence) was corrected by making comparable measurements at locations in the slice away from the filled cell and then subtracting this measurement from the experimental ones.

Sodium concentration measurements

Dynamic changes in [Na+]i were measured using the Na+-sensitive indicator sodium binding benzofuran isophthalate (SBFI) (Callaway and Ross 1997; Lasser-Ross and Ross 1992; Minta and Tsien 1989). These experiments were made using the same techniques and apparatus used for measuring [Ca2+]i changes, with the exception that the Ca2+ indicator was replaced by 2 mM SBFI (Molecular Probes) and 0.5 mM EGTA in the pipette solution. Excitation and emission wavelengths for detecting SBFI fluorescence were the same as for bis-fura-2 and fura-6F.


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Shape of PCs, size of somata, and membrane resistance

Most mutant PCs had reduced dendritic arbors that were clearly revealed in fluorescence images of cells filled with bis-fura-2 (Fig. 1). Their somata differed from the somata of WT cells in shape, size, and location, as has been reported (Clark et al. 1997). In WT mice, the cell bodies of PCs are found in a clear layer between the granule cells and the molecular layer (PC layer). In SCA1 mice, the somata were more widely distributed. Many were found in the middle of the molecular layer (Fig. 2A) while others were located in the PC layer. The cell bodies of the mutant PCs appeared to be smaller than those of control PCs when observed with DIC optics (Fig. 2A). The projected areas of the somata of mutant and WT PCs were 170 ± 46 µm2 (n = 50) and 286 ± 58 µm2 (n = 33), respectively (mean ± SD, P < 0.01, t-test). The smaller size of the cell bodies, together with the reduced dendritic arborization, suggested that the input impedance (Rin) of SCA1 PCs would be higher than the impedance of WT cells, even if the specific membrane resistance (Rm) of the two cell types was the same. To test this hypothesis, we measured Rin with small hyperpolarizing current pulses in the soma. Rin was estimated as the peak voltage response divided by the current. A correction for the sag current (Ih) (Crepel and Penit-Soria 1986) was not applied since this response was the same in SCA1 and WT PCs (Fig. 2B). Rin of the mutant PCs was much higher than that of control PCs (147 ± 75 MOmega , n = 57, and 46 ± 13 MOmega , n = 43; P < 0.01). The soma sizes and membrane conductances (1/Rin) of both types of PCs were positively correlated (Fig. 2C), suggesting that the high Rin of the mutant PCs is primarily due to their smaller somata.



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Fig. 1. Dendritic arbors in spinocerebellar ataxia type 1 (SCA1) and wild-type (WT) Purkinje cells (PCs). Fluorescence images of SCA1 and WT PCs filled with bis-fura-2 from a patch pipette. Left: typical PC from a WT mouse (4 mo old). Middle and right: two typical mutant PCs from 7-mo-old animals. Note the atrophied dendrites of the SCA1 PCs compared with the typical elaborate arborization of the WT PC.



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Fig. 2. Soma size and input impedance (Rin) in SCA1 and WT cells. A: differential interference contrast images of PC somata in cerebellar slices. Left: several WT PCs with cell bodies in the PC layer (PL) of a 6-mo-old animal. Right: smaller mutant cell in the middle of the molecular layer (ML) of a 2-mo-old animal. GL, granule cell layer. Scale: 20 µm. B: response to hyperpolarizing current pulses in WT (left) and SCA1 (right). Top traces indicate potential changes and bottom traces indicate current command patterns. C: soma size (projected area) plotted against membrane conductance (1/Rin). The high Rin of the SCA1 PCs is correlated with the small size of these cells.

Active membrane potentials

We next observed the pattern of action potentials evoked by depolarizing current pulses applied with a patch pipette on the soma. In normal PCs (n = 36), fast action potentials (Na+ spikes) were generated when the depolarization reached a threshold (Fig. 3A, top left). The frequency of the Na+ spikes increased as depolarizing current increased. With larger currents, spikes stopped firing and a plateau potential appeared (Fig. 3A, bottom left). This pattern of Na+ spikes and plateau potentials accords well with that described for guinea-pig PCs (Llinàs and Sugimori 1980). We also observed slower Ca2+ spikes (Llinàs and Sugimori 1980) in some SCA1 Purkinje neurons (n = 5/41 cells) and WT Purkinje neurons (n = 9/35 cells) (data not shown).



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Fig. 3. Active membrane properties of WT and SCA1 PCs. A: action potential firing patterns induced by depolarizing current pulses. Both WT and mutant cells increased their Na+ spike firing rate with increasing current. However, mutant PCs (right) had a delayed onset (arrows) at lower current amplitudes, which diminished with higher amplitude current pulses. This delayed onset was rarely seen in WT PCs (left). Numbers to the right of each plot indicate injected current. B: expanded plots show slower kinetics of Na+ spikes in the SCA1 PC (gray traces) compared with the WT cell (black traces). Left traces correspond to the first spikes of the top traces in A (black arrowheads). Right traces correspond to the tenth spikes of the second traces in A (white arrowheads).

Mutant PCs (n = 53) also showed Na+ spikes and plateau potentials (Fig. 3A, right), but there were clear differences in the active potentials. First, less current was needed to reach Na+ spike threshold in the SCA1 neurons. This is primarily due to the larger Rin of the mutant PCs. Second, the kinetics of the Na+ spikes in SCA1 cells were slower than those in WT cells (Table 1 and Fig. 3B). All the kinetic parameters (width at half height, 10-90% rise time, maximum slope of rising phase, 90-10% fall time, and maximum slope of falling phase) were notably different (Table 1). These differences were significant both for the first Na+ spikes evoked just above threshold (Fig. 3B, top traces) and for Na+ spikes in the middle of the train (Fig. 3B, bottom traces).


                              
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Table 1. Slower Na+ spike kinetics in SCA1 Purkinje cells

Delay in Na+ spike firing

By using moderate amplitude depolarizing pulses, we observed a delayed onset before Na+ spike initiation in many mutant PCs (43/53) (Fig. 3A, right, arrows). This delay ranged from 20 to 300 ms and was characterized as a plateau-like potential with small humps. Although, when threshold depolarization was used, there was a delay before the start of a train of Na+ spikes in WT PCs (Fig. 3A, left, top two traces), in most cases it was a slow, gradual depolarization without a plateau. We occasionally observed that the delay had the plateau and humps characteristic of SCA1 PCs in WT PCs, but the incidence was very low (2/36 cells).

The delay and plateau before spike firing in the SCA1 PCs resembled that found in other neurons and in PCs of other species (Hounsgaard and Midtgaard 1988; Klee et al. 1995). In these cells, this response has been attributed to the fast activation and inactivation of an A-like K+ conductance. We performed several standard tests to see if a similar mechanism was responsible for the delay in SCA1 PCs. The delayed onset was blocked by 4-aminopyridine (4-AP) (3 cells by 50 µM and 4 cells by 2 mM; Fig. 4A). In contrast, 2 mM 4-AP did not affect the latency of spike firing in WT PCs (Fig. 4A) but slowed spike kinetics (data not shown), as has been reported for hippocampal neurons (Storm 1990). To test whether this alteration of IA-like current in mutant PCs was due to a different level of inactivation of the A-like K+ channels or to a higher expression level of the channels, we added a hyperpolarizing prepulse before the depolarization (Fig. 4B). This protocol should completely activate all A-like channels before stimulation. In mutant PCs, the delay to spike firing was enhanced by the prepulse. In a mutant cell (Fig. 4B, right), the firing delay, which appeared with moderate depolarization (0.1 nA), diminished with higher-amplitude stimulation (0.3 nA). The prepulse restored the delay to firing (n = 3/5). In WT PCs, only 2 of 13 cells showed a delay in spike firing with this prepulse protocol. Thus the delay in Na+ spike firing in mutant PCs was not due to a difference in the inactivation state of the A-like conductance. Most likely, the delay is due to a higher level of the conductance in mutant PCs.



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Fig. 4. Effect of 4-aminopyridine (4-AP) and hyperpolarization on Na+ spike firing in normal and mutant Purkinje cells. A: delay in onset of Na+ spike firing in a mutant PC was blocked by 2 mM 4-AP. There was little effect on WT PCs. B: a hyperpolarizing prepulse did not induce a delay in onset of Na+ spike firing in WT PC (left). In contrast, in a mutant PC, a prepulse suppressed Na+ spike firing. Numbers to the right of each trace indicate prepulse current. The current command pattern is indicated at the bottom of the left column. Gray traces show responses to the same depolarizing pulses after superfusion of 1 µM TTX.

[Na+]i increases evoked by Na+ spikes

The difference in Na+ spike properties in mutant PCs could be a consequence of the different electrotonic properties of these smaller cells and/or a different spatial distribution of Na+ channels in the cells. One way to test these possibilities is to examine the spatial pattern of Na+ entry caused by Na+ spike firing in the PCs. This pattern can be determined by measuring the spike-evoked [Na+]i changes in the cell at a time before Na+ significantly diffuses away from the sites of entry into the neuron. Using the Na+-sensitive dye SBFI, we found that spikes induced [Na+]i increases in the axon and in the soma that peaked at the time of the last action potential (n = 16). In contrast, this stimulation protocol caused little increase in the dendrites of all WT cells tested (8/8) in and half of the mutant PCs tested (8/16) (Fig. 5, A and B). This spatial distribution matches that found in guinea pig (Lasser-Ross and Ross 1992) and rat (Callaway and Ross 1997) PCs. In the rest of the mutant cells (n = 8), an additional spike-associated [Na+]i increase, peaking at the end of the spike train, was clearly observed at the base of the primary dendrites (Fig. 5C). No increase was observed in the more distal arborization. Since the falling phase of the [Na+]i transient in the primary dendrites was faster than that in the soma, the increase in [Na+]i in the primary dendrites was not due to diffusion of Na+ from the soma but resulted from direct Na+ influx into the primary dendrites.



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Fig. 5. Spike-evoked [Na+]i increases in SCA1 and WT PCs. Left: PCs filled with the Na+ indicator sodium binding benzofuran isophthalate (SBFI). Regions of interest are indicated with colored rectangles. Right: electrical responses (bottom traces) and fluorescence changes (Delta F/F) in different regions in response to a depolarizing current pulse. Middle: pseudocolor images of the amplitudes of the fluorescence changes (Delta F) measured at the end of the current pulses (corresponding to vertical dotted lines in the right panels). Color bar indicates the scale for the pseudocolor images (warmer colors for larger changes in fluorescence). A: in a WT PC, a large [Na+]i increase was detected in the axon (orange) and soma (red) with no increase in the dendrites (blue and green). Pseudocolor scale: 1.25-6.0%. B: a similar pattern was found in most mutant PCs. Pseudocolor scale: 0.1-3.9%. C: in some mutant PCs, a larger [Na+]i increase was observed in the proximal dendrite than in the soma, suggesting voltage dependent Na+ entry into this region. Pseudocolor scale: 0.43-2.2%.

Parallel fiber synaptic responses

Normal PCs receive more than 100,000 parallel fiber connections on their elaborate dendritic arbors (Palay and Chan-Palay 1974). The shrunken dendrites of SCA1 PCs could alter the parallel fiber synaptic connections. To test this possibility, we placed a stimulating electrode over the dye-filled dendrites in the molecular layer. Stimulation in this region evoked a smoothly graded excitatory postsynaptic potential (EPSP) as a function of stimulus intensity (Fig. 6A). The response showed paired-pulse facilitation in both mutant (n = 4) and WT (n = 2) PCs (Fig. 6B). These properties are characteristic of this synapse (Konnerth et al. 1990; Perkel et al. 1990). No significant qualitative difference was noted between the properties of mutant and WT PF responses. However, we did not undertake a detailed quantitative analysis of these electrical parameters.



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Fig. 6. Parallel fiber (PF) excitatory postsynaptic potentials (EPSPs) in SCA1 and WT Purkinje cells. PF EPSPs were graded according to stimulus intensity (A) and showed paired pulse facilitation (B). Greater facilitation was detected for more closely spaced stimuli. Similar responses were measured in both SCA1 and WT PCs.

Parallel fiber-evoked Ca2+ release in dendrites

Calcium release from intracellular stores mediated by the activation of IP3 receptors is well known in many cell types (Berridge 1993). This process has been observed in PC dendrites following bath application of metabotropic glutamate receptor agonists (Netzeband et al. 1997; Vranesic et al. 1991), flash photolysis of caged-IP3 (Khodakhah and Ogden 1993), and by repetitive parallel fiber stimulation (Finch and Augustine 1998; Takechi et al. 1998). Since molecules important for regulating Ca2+ release, such as IP3R1 and SERCA2, were drastically reduced in SCA1 PCs (Lin et al. 2000), we tested whether Ca2+ release by parallel fiber stimulation could be observed in SCA1 PCs. Seven of fifteen SCA1 PCs showed slow [Ca2+]i increases in addition to a rapid Ca2+ transient following a short train of parallel fiber stimuli (3-8 pulses at 50 Hz). The latency of the slow [Ca2+]i increase was 100-300 ms and the increase was confined to the stimulated dendritic area (Fig. 7). The time course and location of this slow [Ca2+]i increase coincides well with the characteristics of IP3-mediated Ca2+ release reported in normal rats (Finch and Augustine 1998) and mice (Takechi et al. 1998). Consistent with this interpretation, we found that the secondary [Ca2+]i increase was blocked by 1 mM (R, S)-alpha -methyl-4-carboxyphenylglycine (MCPG) (Fig. 7, n = 3). MCPG blocks group I metabotropic glutamate receptors and associated IP3 mobilization (Conn and Pin 1997). The secondary increase is unlikely to be due to Ca2+ entry through voltage-sensitive Ca2+ channels in the dendrites since there was no observed voltage change at the recording electrode at the time of the secondary [Ca2+]i increase. In addition, the secondary increase persisted when the cell was hyperpolarized with current from the patch electrode. However, it is possible that a small part of the increase was due to a localized potential change in the dendrites that was invisible at the soma. No Ca2+ release was observed in control WT PCs from older animals (0 of 7 cells). However, in later experiments, where we preloaded the Ca2+ stores with long depolarizing pulses, we observed Ca2+ release in a younger WT PC (1 of 1 cell) and also in PCs from another WT strain of mice (C57B6, 4 mo old; 3 of 4 cells, data not shown).



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Fig. 7. (R, S)-alpha -methyl-4-carboxyphenylglycine (MCPG) reversibly blocks PF-evoked Ca2+ release in dendrites of SCA1 PCs. Short trains (5 pulses at 50 Hz) of PF stimuli evoked two phases of [Ca2+]i increases in a confined dendritic region near the stimulating electrode in SCA1 PCs in control artificial cerebrospinal fluid and after washing out 1 mM MCPG. Top: indicator-filled neuron with the position of the stimulating electrode indicated by dotted lines. Pseudocolor images: spatial distribution of Delta F/F measured at the time of the vertical dotted lines in the right panels (scale: 0.85-19.3%). Right: time course of fluorescence changes in the selected regions and the electrical response to PF stimulation. There was no significant voltage change at the time of the secondary increases. For these experiments, 300 µM fura-6F was used as the Ca2+ indicator. PC from an 8-mo-old SCA1 mouse.

CF synaptic responses

Stimulation of the CF PC synapse evoked a large and complex spike in an all-or-none fashion, as has been shown previously (Llinàs and Sugimori 1980). This CF-evoked EPSP elicits a [Na+]i increase in the dendritic shafts mainly due to Na+ influx through alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors located at the CF-PC synapse (Callaway and Ross 1997; Lasser-Ross and Ross 1992). Thus the topological distribution of the [Na+]i transient by CF stimulation is a good indication of the distribution of CF-PC synapses in the PC dendrite. We measured the [Na+]i changes by CF stimulation (Fig. 8). In WT PCs, [Na+]i increased over thick dendrites (n = 5) as has been observed in other species (Callaway and Ross 1997; Lasser-Ross and Ross 1992). In SCA1 PCs, the same pattern of [Na+]i changes was observed by CF stimulation (n = 7). Thus we conclude that CF innervates the thick portion of dendrites in SCA1 PCs just as it does in WT PCs, although the dendritic arbor is deformed.



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Fig. 8. [Na+]i increases evoked by climbing fiber (CF) EPSPs. Pseudocolor images show the spatial distribution of Delta F/F in WT and SCA1 PCs measured at the time indicated by the vertical dotted lines in the right panels (scale: 0.08-1.5% for WT and 0.5-2.0% for SCA1). Note that the increases are predominantly along the thicker dendrites. Right: time-dependent increases in the indicated regions of interest. The sharp increases are time locked to the CF responses. The SCA1 PC in the bottom panels is the same cell as shown in Fig. 5C.

The CF-evoked EPSP elicited a [Ca2+]i transient mainly in the dendrites of both WT and mutant PCs (Fig. 9, A and B), as has been reported (Callaway et al. 1995; Miyakawa et al. 1992). Since IP3R1 and the SERCA2 pump are severely underexpressed in the SCA1 PCs (Lin et al. 2000), we carefully measured the characteristics of these CF-induced Ca2+ transients. The average time constant of decay of the [Ca2+]i transient in SCA1 PCs was 112 ± 56 ms (mean ± SD, n = 11), which was not significantly different from that in WT PCs (140 ± 71 ms, n = 14). The amplitude of the transient depends on many factors. These include the density of Ca2+ channels, the surface-to-volume ratio in the dendrites, and the buffering power of the cytoplasm. Therefore it is difficult to draw conclusions from a comparison of this parameter in different cells. Nevertheless, we found no qualitative difference in the Ca2+ transient amplitude in SCA1 and WT PCs.



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Fig. 9. CF-evoked [Ca2+]i changes in SCA1 and WT PCs. A, top: single-phase Ca2+ transient seen in WT PCs. The same rapid response was detected in each of the three indicated dendritic regions. The peak fluorescence change occurs within one frame interval of the electrical response (lowest trace). Inset: expanded CF electrical response. Bottom: similar CF-induced electrical and fluorescence responses in a mutant PC. This kind of response was typical of most SCA1 cells. B: two examples of fast and slow Ca2+ transients seen in some mutant PCs. Pseudocolor images show the spatial distribution of the change in fluorescence (Delta F) during the period indicated by the pairs of dotted lines. Right: fluorescence changes (Delta F/F) in the different regions and the corresponding electrical responses to CF stimulation. Note that the secondary responses (arrows) were detected only in restricted regions of the cells, primarily over thick dendrites.

In 4 of 16 SCA1 PCs, a secondary slow Ca2+ transient was observed after the typical initial fast [Ca2+]i change following CF stimulation. This slow transient was found in only part of the dendritic tree, usually on a thick branch (Fig. 9B). The latency of the initial Ca2+ transient was <0.50 ms; the latency of the secondary Ca2+ transient ranged between 180 and 300 ms. The amplitude of the secondary [Ca2+]i increase was larger than that of the initial [Ca2+]i increase at the locations in dendrites where it occurred. There was no detectable change in membrane potential during this secondary Ca2+ transient. This slow transient was never detected in WT PCs (n = 17) or in young SCA1 PCs (5-6 wk old, n = 8). However, we did not follow the protocol of preloading the stores in these experiments. Therefore we cannot rule out the possibility that CF activation in WT PCs can release Ca2+.

Unfortunately, the low incidence of the CF-induced slow Ca2+ transient prevented us from undertaking a detailed examination of the underlying mechanism. However, the close similarity of this [Ca2+]i increase to that observed following PF stimulation suggests that it was due to IP3-mediated release following activation of metabotropic glutamate (mGlu) receptors.


    DISCUSSION
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INTRODUCTION
Methods
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DISCUSSION
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In this study we compared the basic electrophysiological and Ca2+ handling properties of PCs in SCA1 and WT mice that were 3-7 mo old. Perhaps the most remarkable finding was how similar the properties were in the normal and mutant PCs in spite of their morphologic and molecular differences (Burright et al. 1995; Clark et al. 1997; Lin et al. 2000). After three months of age, SCA1 transgenic mice develop severe ataxia and their PCs have reduced soma size, loss of dendritic arborization, and mislocalization in the molecular layer (heterotopia). Moreover, the expression of a number of PC-specific genes is dramatically reduced, including two critical Ca2+-regulating molecules (IP3R1 and SERCA2). The finding that these two genes, which have opposing functions in regulating intracellular [Ca2+]i, are both downregulated was perplexing and raised questions about the physiological consequences of these molecular changes. Na+ and Ca2+ spike firing, CF and PF synaptic inputs, paired-pulse facilitation, and the time course and amplitude of CF-induced [Ca2+]i changes were similar in the two cell types. Ca2+ release following repetitive PF stimulation was also preserved in SCA1 PCs and occasionally was observed following CF stimulation. These findings might provide insight about the molecular changes. It is possible that only one of the two genes (either SERCA2 or IP3R1) is downregulated because of the mutation and that the downregulation of the second is compensatory. The similarity of the PF responses is particularly interesting because the greatly reduced dendritic arbor of the SCA1 PCs probably means that these cells receive many fewer inputs than the 100,000+ inputs that have been reported for normal PCs (Palay and Chan-Palay 1974). It is within the context of this overall similarity that we discuss the differences between the SCA1 and WT neurons.

Small soma size and high Rin

The input resistance (Rin) of SCA1 PCs was significantly higher than the Rin of WT PCs. The most likely reason for this increase is the smaller somata and reduced dendritic arborization of the SCA1 cells. If the specific membrane resistance (Rm) of both cell types were the same, this correlation would result. We tried to quantitate this relationship by measuring the cross-sectional area of the soma. This area should be proportional to the surface area of the cell body if the cell bodies have similar shapes---which they appear to have. We found that the cross-sectional area and the input conductance (1/Rin) were strongly correlated (Fig. 2). However, Rin depends on current flowing through the dendritic membrane as well as the somatic membrane, although the somatic component should dominate (Rapp et al. 1994). Since we could not measure the dendritic contribution to either the surface area or Rin, we could not be more precise about the constancy of Rm. A similar value for Rm in SCA1 and WT PCs suggests that none of the molecular consequences of the polyglutamine expansion have an important impact on the molecules that determine Rm.

One consequence of the higher Rin in SCA1 PCs is that the synaptic currents activated by single PFs will cause a greater potential change in the PC. Therefore activation of fewer PFs will be required to reach the threshold for spike firing. Since the dendrites are much smaller in these cells, they probably receive fewer inputs than do normal PCs. The need for fewer active inputs to reach threshold appears to be a homeostatic mechanism intended to preserve some aspects of PC function in SCA1 mice.

Delay in Na+ spike firing and other parameters affecting spike kinetics

The delay in Na+ spike firing in SCA1 PCs was a surprise since none of the known molecular or morphological changes pointed toward this alteration. The most likely explanation for this delay is an enhancement of an A-like K+ conductance. The rapid activation and inactivation of this conductance (Connor and Stevens 1971) are known to cause similar delays in other cells (Storm 1990). Support for this conclusion comes from experiments in which the delay was eliminated by 4-AP, a known blocker of this conductance (see Hoffman et al. 1997). In SCA1 PCs, the delay in spike firing was restored or enhanced by a hyperpolarizing prepulse (Fig. 4B), which suggests that the A-like conductance is partially inactivated at rest in mutant PCs. In contrast, most WT PCs did not show a delay in spike firing even with a hyperpolarizing prepulse, indicating that a fully active A-like conductance is not enough to generate a delay in spike firing in normal PCs. This shows that the enhanced A-like conductance in SCA1 PCs is not merely due to a difference in the inactivation state of the conductance but that it may result from increased expression of IA-like channels or from altered expression of accessory modulatory proteins for voltage-gated K+ channels (An et al. 2000).

In addition to the delay in spike firing, other aspects of Na+ spike kinetics were altered in SCA1 PCs. These include spike width and rate of rise (Table 1). We have no clear explanation for these significant differences. Some of the variation may be due to the differences in cell morphology since the need to charge the cell membrane will affect the rise time and fall time of the spike independent of the properties of the channels. The spatial distribution of the channels also can affect spike kinetics. In several experiments, we measured the spatial distribution of the spike-evoked [Na+]i changes. This distribution should reflect the distribution of active Na+ channels. We found that the [Na+]i increase in half of the tested SCA1 PCs and in all of the WT PCs was confined to the soma and axon. This result is consistent with previous measurements in rats and guinea pigs (Callaway and Ross 1997; Lasser-Ross and Ross 1992) and is also consistent with experiments that found that Na+ propagation into PC dendrites is completely passive (Stuart and Hausser 1994). The results from the other half of the tested SCA1 PCs are interesting. In these PCs we found clear spike-evoked [Na+]i increases in the proximal dendrites. We did not find dendritic [Na+]i increases in any WT cell. This result suggests that the spatial distribution of Na+ channels in SCA1 PCs may not be as tightly regulated as in WT cells.

It is not clear whether the differences in the kinetics of the Na+ spikes in SCA1 and WT PCs have any significant physiological consequences. Propagation in the axon should be insensitive to small variations in spike parameters. Differences in spike width could affect the release of neurotransmitter. However, we have no information about spike parameters in the terminal arborization.

Biphasic [Ca2+]i transients evoked by parallel fiber stimulation

We observed an initial rapid Ca2+ transient and a localized slow [Ca2+]i increase following a brief train of PF stimulation in SCA1 PCs. The slow [Ca2+]i increase was blocked by MCPG, a blocker of Group I metabotropic glutamate receptors. A similar biphasic pattern of [Ca2+]i increase by PF stimulation has been reported in rat PCs (Finch and Augustine 1998) and murine PCs (Takechi et al. 1998). In those reports the secondary Ca2+ transient was attributed to Ca2+ release from IP3 receptor-operated intracellular stores because the [Ca2+]i increase was blocked by intracellular heparin. The unreliability in generating the secondary response prevented us from doing similar experiments. Nevertheless, the similarity in the stimulation pattern (50-60 Hz, 5-15 pulses), the delay following stimulation (0.1-0.3 s), the localized pattern of [Ca2+]i increase, and the sensitivity to MCPG make it likely that the second slow phase in our experiments also was due to Ca2+ release from intracellular Ca2+ stores. We observed this Ca2+ release in half of the SCA1 PCs (7/15 cells). This incidence is lower than the 100% reported by Takechi et al. (1998) . The discrepancy may arise from differences in experimental procedure. In particular, we did not preload the stores with voltage pulses before PF stimulation, a procedure followed by Finch and Augustine (1998) . In support of this possibility we did not observe Ca2+ release by PF stimuli in seven WT PCs in early experiments. But we did observe release in later experiments on WT neurons when we preloaded the PCs (n = 3). However, the main conclusion is that PF-mediated Ca2+ release was observed in SCA1 mice, indicating that mGlu receptors, IP3 receptors, and other molecules responsible for controlling release are present and functional in these mutant animals.

[Ca2+]i transients evoked by CF stimulation

We observed two kinds of Ca2+ transients in response to CF stimuli in SCA1 PCs (Fig. 9). One was a fast transient that peaked at about the time of the CF electrical response and decayed rapidly. This transient was widely distributed in the PC dendrites and closely resembled the CF-evoked Ca2+ transients in other cells (Callaway et al. 1995; Miyakawa et al. 1992). In other cell types, the removal rate of Ca2+ following transient elevation has been attributed to a combination of cytoplasmic buffering and pumps (plasma membrane and endoplasmic reticulum) (Airaksinen et al. 1997; Markram et al. 1995; Neher and Augustine 1992). The similarity in removal rates in SCA1 and WT PCs implies that these mechanisms are equally effective in both types of cells. Since SERCA2 is downregulated in SCA1 PCs, our results imply that uptake by SERCA2 is not the rate-limiting step for restoring the resting [Ca2+]i level after CF excitation. It is noteworthy that the expression of another calcium pump, plasmalemmal Ca2+-ATPase (PMCA2), which is abundantly expressed in cerebellar PCs, is not reduced in SCA1 PCs (Lin et al. 2000).

The second kind of Ca2+ transient was a slower and larger signal that was restricted to parts of the dendritic tree, suggesting Ca2+ release. The localization to regions near the thick dendrites is consistent with the location of CF synapses on the cell (Larramendi and Victor 1967). Because of the low incidence of this response, we were not able to undertake a detailed pharmacological analysis of the underlying mechanism of this secondary [Ca2+]i increase. This phenomenon was not seen in WT PCs or in young (5-6 wk old) mutant PCs in this study and it has never been reported in other species following CF stimulation (Callaway et al. 1995).

One argument in favor of a release mechanism is the localized spatial distribution of the secondary response. The primary response, which has been shown to be caused by the generation of a Ca2+ spike in the dendrites (Callaway et al. 1995; Llinàs and Sugimori 1980), is widely distributed in the dendrites. This distribution is consistent with the generation and propagation of this spike over the dendrites. In contrast, a spatially restricted response is consistent with the localized generation of IP3 following mGluR activation, as also observed following repetitive PF stimulation (see Parallel fiber-evoked Ca2+ release in dendrites). In addition, the secondary [Ca2+]i increase had a slow time course and there was no apparent membrane potential change underlying the Ca2+ transient, as was also observed in the PF response. Because of this similarity, it is likely that the secondary CF-evoked Ca2+ transients observed in this study were also caused by release from calcium stores. However, further experiments are needed to definitively establish this conclusion.

In summary, the electrophysiological responses and synaptically activated [Ca2+]i changes in SCA1 PCs are similar to those in WT PCs even though the expression of some important Ca2+-regulating molecules is different in the two cell types. The most significant exceptions are the secondary [Ca2+]i increase observed following some CF responses, the higher A-like K+ conductance in SCA1 PCs, and the higher Rin of SCA1 PCs. The differences we observed may be adapted to compensate for the atrophied dendrites of the SCA1 PCs. Alternatively, the heterotopic PCs and the atrophic molecular layer suggest that the organization of the PF inputs to the SCA1 PCs are altered. These observations suggest that the mechanisms underlying ataxia in SCA1 transgenic mice may reside in changes to cerebellar neuronal circuitry rather than in alterations of the intrinsic electrophysiological properties of the PCs.


    ACKNOWLEDGMENTS

We thank Dr. Akinori Kuruma for help with some of the calcium release experiments.

This research was supported by National Science Foundation Grant IBN-9514266, National Institute of Neurological Disorders and Stroke Grants NS-27699 and NS-16295, and by the Howard Hughes Medical Institute. T. Inoue was supported by a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science (JSPS). K. A. Kohlmeier was supported by National Institute of Mental Health Grant MH-18825.


    FOOTNOTES

Address for reprint requests: W. N. Ross (E-mail: ross{at}nymc.edu).

Received 14 July 2000; accepted in final form 21 December 2000.


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ABSTRACT
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society