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The Journal of Neuroscience, November 15, 2002, 22(22):9668-9678
Properties of Unitary Granule Cell
Purkinje Cell Synapses in Adult Rat Cerebellar Slices
Philippe Isope and Boris Barbour Neurobiology Laboratory, Ecole Normale Supérieure, 75230 Paris Cedex 05, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
The cerebellar cortex contains huge numbers of synapses between granule cells and Purkinje cells. These synapses are thought to be a major storage site for information required to execute coordinated movements. To obtain a quantitative description of this connection, we recorded unitary synaptic responses between granule cell and Purkinje cell pairs in adult rat cerebellar slices. Our results are consistent with parallel fiber
Purkinje cell synapses having high release probabilities and modest paired pulse facilitation. However, a wide range of response amplitudes was observed. Indeed, we detected many fewer parallel fiber connections (7% of the granule cells that were screened) than expected (54%), leading us to suggest that up to 85% of parallel fiber
Key words: cerebellum; Purkinje cell; granule cell; synapse; glutamate; motor control
INTRODUCTION
Mossy fibers, the most abundant input to the cerebellar cortex, carry multiple modalities of sensori-motor and contextual information in a complex somatotopic arrangement (Shambes et al., 1978
; Oscarsson, 1979
The divergence of parallel fibers onto Purkinje cells is thought to underlie the coordination of multiple body parts by the cerebellum (Thach et al., 1992
This work has been presented in preliminary form (Isope and Barbour, 2000
MATERIALS AND METHODS
Slice preparation. The following procedures were adopted to minimize hypoxic, ischemic, mechanical, and excitotoxic damage to the fragile adult cerebellar tissue. They conform to national and National Institutes of Health guidelines on animal experimentation. Adult male Wistar rats (2-3 months old, 250-450 gm) were anesthetized with ketamine/xylazine (75 and 10 mg/kg, respectively) intraperitoneally. Additional (half) doses were administered if required to ensure attainment of the surgical plane of anesthesia (total abolition of plantar withdrawal reflex to nocive stimuli). Adjuvant atropine sulfate (0.2 mg/kg) usually was given intraperitoneally also. A thermocouple was positioned to monitor tympanal temperature. A tracheal catheter was placed quickly, and the rat was ventilated (14 ml ventilator stroke × 72 breaths/min) without recourse to myorelaxants. Positive end-expiratory pressure (3 cm H2O) was used when the thorax was opened in preparation for cardiac perfusion. Some vigilance was required to ensure that the airways remained patent during the thorectomy. Transcardiac perfusion of the rat with two cold, bubbled (95%O2/5%CO2) solutions was established. The first solution (150 ml) contained (in mM): 115 NaCl, 26 NaHCO3, 3 KCl, 0.8 CaCl2, 8 MgCl2, 1.25 NaH2PO4, 10 D-glucose, 1 lidocaine-HCl, 1 ketamine-HCl. The second solution (100 ml) was identical except that sucrose (230 mM) replaced the NaCl. As soon as perfusion was under way, the abdominal aorta and/or the inferior vena cava were/was clamped, and the head of the rat was packed with ice. The tympanal temperature of the rat would fall to 5-10°C during perfusion. After perfusion the rat was decapitated, and the head was chilled over ice while the entire cerebellum was dissected out. Care was taken to avoid cutting or deforming the cerebellum. The cerebellum was cooled and sliced in a solution containing (in mM): 30 kynurenic acid, 230 sucrose, 26 choline-HCO3, 0.8 CaCl2, 8 MgCl2, 10 D-glucose, 30 NaOH, 1.25 NaH2PO4, 1 lidocaine, 1 ketamine, 0.05 D-APV, 0.01 gabazine, 0.1 picrotoxin, 0.005 strychnine hemisulphate.
Then 450 µm slices oriented at 20° to the transverse plane were prepared (the angle makes the slices asymmetric, facilitating correct orientation for recording) and were kept in a bubbled bicarbonate-buffered solution (BBS) to which lidocaine-HCl (1 mM), ketamine-HCl (1 mM), and Na-kynurenate (2 mM) had been added. Note that this solution should minimize any activity-dependent synaptic modifications. After slicing, the slices were maintained at 32°C for 0.5-1 hr and then allowed to cool to room temperature. Drugs were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France) or Tocris-Cookson (Bristol, UK).
Recording. For recording, the slices were held 1.5 mm above the chamber bottom between nylon meshes stretched over and glued to two concentric stainless steel rings to allow solution flow below as well as above the slice. The experimental bathing solution contained (in mM): 125 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 2 MgCl2, 10 D-glucose, 0.1 picrotoxin, and (sometimes) 0.002 gabazine. Slices were superfused (8 ml/min) at 32°C; recordings were begun only after at least 30 min, ensuring the washing of the lidocaine and kynurenate of the storage solution (data not shown). Cells were visualized with a Zeiss Axioskop [40×, 0.75 numerical aperture (NA) water immersion objective and 0.63 NA condenser; Oberkochen, Germany] by using red light (670 ± 40 nm) and video contrast enhancement (Hamamatsu C2400-07 camera and control unit; Hamamatsu Photonics France, Massy, France). Whole-cell patch-clamp recordings in both voltage-clamp and current-clamp modes were obtained by using an Optopatch amplifier (Cairn Research, Faversham, UK) and optimal series resistance compensation (50-70% of 4-8 M
, typically) or capacitance neutralization as appropriate. The pipette solution contained (in mM): 150 K-gluconate, 4 NaCl, 10 HEPES, 10 Mg-ATP, and 13 biocytin pH-adjusted to 7.3 with KOH at 300 mOsm. Recordings were corrected for a junction potential of 10 mV.
Loose cell-attached stimulation and recording of granule cells were performed with a purpose-built amplifier according to Barbour and Isope (2000)
70 mV did not exceed 900 pA, because recordings of compound EPSCs during development of the leak showed that their amplitude was unaffected. In five cells in which the leak increased from 300 ± 60 to 880 ± 30 pA, EPSC amplitude increased 1 ± 17%, and the charge transfer was reduced by 12 ± 6%.
Granule cells in two different regions were tested. They were either in the immediate vicinity of the recorded Purkinje cell or some 300-500 µm distant from the Purkinje cell soma (measured in the parallel fiber direction). Local granule cells are those most likely to form connections to the Purkinje cell via their ascending axon; the properties of their connections will be described below. Distant granule cells, which constitute the great majority of granule cells contacting a given Purkinje cell, can make only parallel fiber connections. Inclusion of stimulated granule cells within the beam of granule cells for which the parallel fibers intersect with the Purkinje cell (see Fig. 1) was ensured by previous delineation of the connected beam by extracellular stimulation. Stimulation of granule cells was performed in the middle (±50 µm) of this beam.
Within selected regions the granule cells were tested at random. Only granule cells in which an action potential could be distinguished clearly and elicited reliably were analyzed. Only connections in which postsynaptic responses and presynaptic action potentials had the same threshold were retained. Stimulation was adjusted to be just suprathreshold so as to minimize the probability of stimulating other nearby granule cells or axons. In calculating the mean EPSCs, we averaged together all sweeps in which granule cell excitation was successful.
Histology. Slices were fixed in 3.7% formaldehyde/PBS, pH 6.8. After ~5 min the pH was alkalinized (Berod et al., 1981
For the experiments in which the plane of the Purkinje cell dendritic tree was reconstructed, the swelling (15 ± 23%; n = 30) of the slices during fixation and treatment was corrected for by comparing the distances between landmarks in the living and treated slices. Distances are corrected to unfixed tissue.
Analysis. Acquisition of data was performed with pClamp software (version 6 or 8). Analysis was performed in the IGOR graphing and analysis environment (Wavemetrics, Lake Oswego, OR), using home-developed macros. Means are reported with SD. On several occasions we applied the nonparametric "bootstrap" method described by Efron and Tibshirani (1993)
RESULTS
Parallel fiber connections
We recorded unitary granule cell
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Figure 1. Slice orientation. The diagram shows the typical orientation of the cell pairs recorded in this study. Purkinje cells for which the soma was near the surface and for which the dendrites descended into the pseudo-transverse slice were whole-cell clamped. Granule cells within the connected beam were recorded in loose cell-attached mode. The thickness of our slices (450 µm) exceeds the combined depths of the molecular and granule cell layers (220 + 180 µm; Harvey and Napper, 1988
A specimen recording of a parallel fiber synaptic connection (Fig. 2A,B) shows the granule cell action potential, recorded as a capacity current, and several EPSCs in the Purkinje cell. In all, 477 on-beam excitable granule cells in this distance range were screened. Of these, 34 generated detectable responses. The mean amplitude for these parallel fiber synapse responses was 8.4 ± 7.1 pA (Fig. 2C). The distribution of mean amplitudes was skewed toward larger values.
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Figure 2. Unitary parallel fiber
To expedite screening of granule cells, the experimenter determined the identification of connected granule cells "by eye," with off-line averaging in cases of doubt. Under these conditions the mean amplitude for the detected connections was usually >2-3 pA. Although the objection can be made that this method is subject to human error, the likely error introduced appears to be small, because off-line averaging of 50-100 sweeps of connections classified as undetectable (n = 13) did not unmask any further connections. For nearly all recordings in which no response was apparent, we elicited a train of action potentials by increasing the stimulation intensity. This eventually induces the granule cell to emit a train of action potentials at frequencies of a few hundred Hertz (possibly by causing partial membrane breakdown). In a few cases (n = 5 of 400+ apparently nonconnected granule cells) this train induced a postsynaptic current. Unfortunately, probably because of damage to the granule cell, it was difficult to apply such stimulation repetitively. We are uncertain as to the mechanisms generating these responses.
To estimate the size of the EPSP that would be generated by EPSCs of given sizes, we performed successive current-clamp and voltage-clamp recordings of compound responses to extracellular stimulation in the granule cell layer (Fig. 3A). The average compound EPSC kinetics values when fit by bi-exponential waveforms were
rise = 1.2 ± 0.7 msec and
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Figure 3. Estimating synaptic weights. A, Subthreshold compound EPSCs and EPSPs evoked by the same extracellular stimulation in the granule cell layer. Both synaptic responses are averages of 20 sweeps. Both are fit with bi-exponential functions (superimposed). For the EPSP that is shown,
We quantified unitary EPSC kinetics by using bi-exponential fits, one for the rising phase and one for the decay phase. These fits proved to be quite affected by the noise, and bootstrap estimation of the SEs of the derived time constants showed them to be rather unreliable. For this reason we excluded from the kinetics analysis all events for which the mean amplitude was below 5 pA. For these larger parallel fiber EPSCs the average time constants were
Some unitary parallel fiber EPSCs appeared to display bi-exponential decays (data not shown). Because each connection normally involves just a single synaptic contact (or two in the same place on the dendritic tree), a mix of differentially filtered EPSCs is probably not the explanation for this time course. One possible explanation is that such synapses are quite proximal, and the first of the decay exponentials represents the redistribution of charge into the dendrites and pipette, with the second decay exponential reflecting recovery of the charge that had redistributed into more distal dendrites.
Generally, although identification of granule cell excitation was secure, the subtraction of the stimulus artifact was not always sufficiently good to permit precise alignment on the peak of the action potential signal. The averages therefore include the effects of jitter in action potential timing. This will lead to some lengthening of the EPSC rising phase. The EPSC decay phase was in most cases too slow for it (or the amplitude measurement) to be affected significantly by action potential timing jitter.
Probability of release
Based on an analysis of paired pulse facilitation at the parallel fiber
Our overall approach was to compare amplitude histograms, which contain a mixture of failures and successes of liberation, with measurements of noise, which have the same distribution as the response failures. To maximize the accuracy of the measurement of response amplitudes, we used a template-based method. The template for each connection was based on the bi-exponential fit of the corresponding average EPSC. So as to minimize disturbance of the measurement by spontaneous EPSCs, we used a relatively short template that covered the rising phase and peak of the EPSC but only a portion of its decay. The measurement stage simply involved adjusting the amplitude of the template to fit each sweep. A specimen trace fit by a template is shown in Figure 4A. Simulations (data not shown) demonstrated that measurements were fairly insensitive to realistic jitter in EPSC onset (SD of <0.5 msec). Noise measurements were obtained by applying exactly the same procedure to portions of traces that contained no evoked responses.
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Figure 4. Method of amplitude measurement for individual EPSCs. A, A truncated version of the bi-exponential fit of the average EPSC was scaled to each individual EPSC by using a least-squares criterion, yielding an automatic amplitude estimate for each sweep. The baseline and scaled template are shown superimposed on an EPSC. B, Amplitude distribution (light shaded histogram) for the same connection, constructed from amplitude measurements as in A. An equal number of identical measurements was performed on trace segments in which the granule cell was not stimulated (or active) to estimate the amplitude distribution expected for failures of transmission. This noise distribution was scaled (heavy open histogram) to fit the response distribution for amplitudes greater than (to right of) zero (shaded range). The scaling provides an estimate of the probability of failure, 0.36 in this case.
Once the EPSC amplitude and noise histograms had been obtained, the probability of failure was estimated by scaling (by the standard least-squares method) part of the noise distribution to fit the corresponding range of the EPSC amplitude distribution. We chose to scale the noise and EPSC amplitude distributions for values greater than zero (EPSCs having negative amplitudes by convention). Figure 4B shows the EPSC amplitudes for the same connection as in Figure 4A and the scaled version of the noise distribution.
Figure 5A shows a scatter plot of the failure probability estimates against mean amplitudes. Although the estimated failure probability is quite low for the largest connections, there is a strong trend for the smaller connections to yield higher estimates of failure probability. We know, however, that the measurement method will always tend to overestimate the failure probability (because noise could cause small successes to have amplitudes greater than zero) and that this problem will be exacerbated for small amplitudes. A simple model (Fig. 5B) can be used to illustrate the expected behavior of our method of estimating release probability. We assumed EPSC amplitudes and noise were Gaussian and arbitrarily set the SD of the amplitudes of successful releases to be 40% of their mean. These assumptions are unlikely to be exact, but it would be difficult to obtain more detailed information. We set the SD of the noise to be 5.8 pA. The noise distributions have a mean SD of 7.3 pA, but they are slightly asymmetric. Because the histogram fitting uses the right-hand side of the noise distribution, we reflected that half of each noise distribution about zero and then recalculated the mean SD (i.e., 5.8 pA). For a given failure probability we then could calculate the proportion of failures that would be estimated by our method. The expected estimates are plotted as a function of mean amplitude in Figure 5A as a family of curves for different underlying failure proportions. Different values of the coefficient of variation of the successes were tested (Fig. 5, see legend), showing the low sensitivity of the model to this parameter.
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Figure 5. Distribution of failure probability estimates for parallel fiber synapses. A, The failure probability estimates are plotted as a function of mean amplitude with their bootstrap SE estimates. The smooth curves (solid lines) are relations obtained by using the model illustrated in B. Each theoretical relation is for a fixed underlying failure probability (labeled). B, Illustration of the model used to calculate the theoretical curves in A. A Gaussian noise distribution (SD of 5.8 pA; noise, shown by a solid line) was convolved with a Gaussian success amplitude distribution [probability of 0.9, amplitude of 11.11 pA, SD of 4.44 pA; therefore, coefficient of variation (CV) of 0.4] and failures (probability of 0.1, amplitude of 0). The resulting distribution components are labeled successes and failures (dotted curves); their sum is the responses probability density function (solid line), which models the experimentally determined amplitude distribution. The best-fit scaled version of the noise distribution is shown also (estimated failures, shown by the dashed line). It overestimates the true failure probability by a factor of ~2. Variation of the CV of successes had little effect on the overall shape of the amplitude-estimated P relations that were obtained. The dotted and dashed curves in A were obtained with CVs of 0.15 and 0.5, respectively.
The curves showing the estimated failure probability for a true P(failure) of 0.1 give a remarkably good fit to the data. It is clear that a narrow range of failure probabilities (~0.1) could account for all of our data, which thus provide no evidence for the existence of low release probability connections.
Paired pulse facilitation
The parallel fiber connections displayed modest paired pulse facilitation at the 40 msec interval that was tested. Using a method analogous to that of Kim and Alger (2001)
We examined paired pulse facilitation of compound responses to extracellular stimulation in the molecular layer and in the granule cell layer (also at 40 msec intervals). Surprisingly, although stimulation in the granule cell layer produced similar facilitation (A2/A1, 1.37 ± 0.14; A3/A2, 1.08 ± 0.12; n = 8) to that reported above for unitary connections, stimulation in the molecular layer produced significantly greater facilitation (A2/A1, 1.70 ± 0.19; A3/A2, 1.30 ± 0.09; n = 8; p < 0.01 for the difference of A1/A2; Mann-Whitney U Test). The similarity of the paired pulse ratios for the unitary connections and compound responses elicited by granule cell layer stimulation strongly suggest that our sample unitary connections are representative of the population.
Undetected connections
When selecting granule cells to screen, we ensured their location within the beam connecting the recorded Purkinje cell by previous extracellular stimulation. Under these circumstances most of the parallel fibers are expected to intersect with the dendritic tree of the Purkinje cell. We confirmed this with a series of paired whole-cell recordings in which both granule cell and Purkinje cell were labeled (Fig. 6; no responses were detected in these whole-cell pairs). The separation between the cell somata was 50-100 µm, and the granule cells were simply guessed by the experimenter to be on-beam. Even so, in 17 of 20 successfully labeled pairs the parallel fiber passed within the perimeter of the Purkinje cell dendritic tree; i.e., at the point of intersection with the dendritic plane, the parallel fiber had dendrite to its left, its right, and above and below it. Because it was rarely possible to distinguish the parallel fiber in the dendritic tree (because of shadowing by the dendrites), these criteria usually were applied to the parallel fiber just either side of the dendritic tree.
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Figure 6. Morphology of a typical intersection of a parallel fiber and a Purkinje cell dendritic tree. Shown are a granule cell (GC) and Purkinje cell (PC) pair filled with biocytin during the whole-cell recording (without a detectable connection) and subsequently labeled. A, Somatic level. The plane of Purkinje cell dendritic tree extends down into the slice in a bottom left to top right plane. B, Near the level of intersection. The parallel fiber (PF) is seen shortly after it has traversed the dendritic tree near its middle. The parallel fiber is an enhanced montage of two images separated by 10 µm vertically. Only a tiny section of the ascending axon (AA) is captured in the image, although it could be followed easily when the focus is adjusted continuously. Scale bar, 50 µm.
We are therefore confident that the parallel fibers of most of the granule cells we screened would have intersected with the Purkinje cell dendritic tree. The careful anatomical work of Napper and Harvey (Harvey and Napper, 1988
The possibility that action potentials in parallel fibers fail to propagate reliably is raised often. A favorite site for the block of propagation is the "T" at which the ascending granule cell axon bifurcates into the two halves of the parallel fiber. A more prosaic problem that also needs to be addressed in our experiments is the fraction of granule cells for which the axons are cut near the slice surface. We were able to rule out important contributions from either of these mechanisms by combining loose cell-attached recording with antidromic stimulation of parallel fibers at the exposed surface of the molecular layer. It was usually possible (11 of a series of 14 granule cells tested) to demonstrate the ability of the parallel fiber to support antidromic conduction and, moreover, to confirm collision between the antidromic action potentials and orthodromic action potentials elicited at the soma. This suggests that few granule cell axons are cut in our preparation and that most can conduct antidromic action potentials along their length and orthodromic action potentials along at least some of their length.
Using an extension of this approach, we were able to demonstrate that orthodromic action potentials are conducted very close to the site of stimulation in the molecular layer. This is illustrated in Figure 7. By increasing the separation between the stimulation site and the granule cell, we recorded five further granule cells with long antidromic conduction times, ranging from 4 to 8 msec, to enable clear observation of the conduction process. The refractory period in all of these cells for both orthodromic and antidromic stimulation was of the order of 1 msec (the slightly surprising form of the antidromic actions potentials recorded in loose cell-attached mode is explained in the legend). We then performed a collision experiment to determine how long after orthodromic stimulation an antidromic action potential would still collide with the orthodromic action potential. In each cell the behavior shown in Figure 7 was observed; a delay of one conduction time plus a refractory period had to elapse before the antidromic stimulation was successful. We conclude that orthodromic action potentials usually are able to propagate very near to the cut ends of the parallel fibers in the exposed molecular layer, beyond the sites of the synapses we expected to record.
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Figure 7. Action potential propagation along parallel fibers is not compromised. A, Loose cell-attached recording of a granule cell soma during molecular layer stimulation. The latency between the stimulus and arrival of the antidromic action potential (mostly conduction) in this cell was 5.3 msec (top trace, from left triangle to asterisk marking the antidromic action potential). Paired stimuli show that the refractory period was ~1 msec. The intervals between the two stimuli are 0.9 msec (top trace, one action potential) and 1.0 msec (bottom trace, two action potentials). B, Paired pulse somatic stimuli similarly reveal an orthodromic refractory period of 0.9 msec. The intervals between the two stimuli are 0.8 msec (top trace, one action potential) and 0.9 msec (bottom trace, two action potentials). C, So that annihilation by collision with the orthodromic action potential (somatic stimulation) can be avoided, an antidromic action potential (molecular layer stimulation) must be elicited >6.3 msec later. The intervals between the two stimuli in the panel are 6.3 msec (top trace, antidromic action potential absent) and 6.4 msec (bottom trace, antidromic action potential present). This interval indicates that the orthodromic action potential is conducted to the site of antidromic stimulation, beyond the point at which Purkinje cells would have been recorded. The surprising form of the loose cell-attached action potential signal (e.g., in A) corresponds to an intracellular action potential with an inflection on the rising phase. This is because the loose cell-attached signal is essentially a capacity current, proportional to the first derivative of the membrane potential (Barbour and Isope, 2000
If connections had a very low release probability (Dittman et al., 2000
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Figure 8. Whole-cell paired recordings reveal no strong parallel fiber connections with very low release probabilities. In each panel the top traces represent the average (black line) of >100 high-frequency bursts of action potentials in a granule cell (gray lines). The bottom traces show the average responses recorded in a Purkinje cell. A, A pair showing no detectable response. The averages were formed after alignment on approximately the fifth action potential of the train (n = 191). B, Another pair in which a response was detected. In the panel the averages were calculated after alignment on the first action potential of the train (n = 137). A and B share calibrations. C, A detail (expanded time and current scales) of the averages in B shows that a clear response onset (arrow) can be observed some 2 msec after the first action potential (timing indicated by the dashed vertical line).
Proximal granule cells
A long-running controversy in cerebellar physiology concerns the role of granule cell ascending axons. Parallel fibers, because they run orthogonally to the planes of Purkinje cell dendritic trees, usually make zero, one, or two synaptic contacts with each Purkinje cell. The situation is more confused for the short segments of granule cell axon that rise vertically through the molecular layer. Because these can be parallel to the Purkinje cell dendritic planes, they potentially could make numerous contacts with a given Purkinje cell. This possibility has led a number of workers to suggest that ascending axons are an important, even the dominant, input from granule cells to Purkinje cells. However, a detailed comparison of parallel fiber and ascending axon synapses has not been performed.
We sought to record from granule cells making ascending axon connections. Because we have no method of identifying such cells a priori and any difference in synaptic properties appeared to be subtle, we developed a method that allowed us to determine a posteriori which granule cells were those most likely to have made an ascending axon connection and to pool results from several experiments. During an experiment we would take a video still of each granule cell that was screened. The Purkinje cell was labeled. After fixation and treatment of the slice, the plane of the Purkinje cell dendritic tree was reconstructed by drawing the intersection of the dendrites with each of a z-series of images and calculating the "best-fit plane" to these line segments. Comparison of the video stills and the reconstructed Purkinje cell allowed us to determine the position of each recorded granule cell with respect to the Purkinje cell. We then calculated the orthogonal distance of each granule cell to the Purkinje cell dendritic plane. When significant portions of the Purkinje cell dendritic tree were on different planes (even double dendritic trees occasionally are observed), the distance to the nearest dendritic plane was calculated. In this way we could pool results from many experiments by examining synaptic properties as a function of distance from the dendritic plane. We assume that granule cells in this plane are those most likely to form ascending axon synapses.
Figure 9A shows a reconstructed experiment. The positions of 10 screened granule cells are indicated on the surface plane. Most of those for which synaptic connections were detected appear to be clustered around the dendritic plane. Pooling the results from a number of Purkinje cells showed that this was not an accident (Fig. 9B). A remarkable peak, reaching ~50%, in the probability of detecting a connection is observed in the dendritic plane. The probability of connection falls off fairly rapidly with distance from the dendritic plane, and beyond 20 µm it is not significantly different from the probability obtained for parallel fiber connections from distant (300-500 µm) granule cells.
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Figure 9. Local granule cells constitute a privileged input, but powerful ascending axon connections are rare. A, Two image planes (top, slice surface; bottom, 10 µm below the surface) showing a labeled Purkinje cell. The intersections of the best-fit plane for the dendritic tree and the image planes are indicated by the dotted lines. The reconstructed positions of connected granule cells are indicated by a filled circle and nonconnected granule cells by a cross. Scale bar, 50 µm. B, Apparent connection rate (number of detectable responses/total number of granule cells tested) as a function of distance of the granule cell soma from the dendritic plane of the Purkinje cell. Each bin except the first and last represents 30 tested granule cells. The first (left-most) bin contains 15 granule cells and the last (for granule cells at 300-500 µm) contains 477. The significance of the differences between the apparent connection probabilities near to the Purkinje cell and that for parallel fibers is indicated over the relevant bins by asterisks (**p < 10 5; *p < 0.05; Fisher's Exact Test). The dotted horizontal line indicates the expected probability (0.54) of an anatomically defined synapse existing (see Results). C, Histogram of mean amplitudes for connections from granule cells within 35 µm of the dendritic plane of the Purkinje cell (light lines, shaded bars). Note two large responses of putative ascending axon connections. The distribution parameters were mean = 12.4 pA and SD = 15.7 pA (n = 28 of 166). A similar histogram of a series of connections for which reconstructions were not performed but the granule cell was close to the Purkinje cell is superimposed (heavy lines, open bars). Large responses were also infrequent in this sample. The distribution parameters were mean = 12.4 pA and SD = 14.1 pA (n = 40).
Synaptic connections made by ascending axons have been proposed to differ from parallel fiber synapses in a number of ways. First, they generally are thought to form relatively powerful connections by virtue of making multiple synaptic contacts on a Purkinje cell. Figure 9C shows the amplitude distribution for granule cells located within 35 µm of the dendritic plane. Overall, the EPSC amplitude distributions of connections from proximal and distal granule cells were remarkably similar. Only two proximal granule cells gave amplitudes that were clearly greater than those attainable by parallel fiber connections (based on our small sample). However, even these two large connections, which presumably were from ascending axons making multiple contacts, were no more than approximately twice as large as the largest parallel fiber connections. A similar distribution was obtained when we attempted to record from granule cells on or near the dendritic plane but for which no reconstruction was available (Fig. 9C). This suggests that even moderately large connections are quite rare.
Two other differences have been reported between parallel fiber and ascending axon synapses, based on electron microscopic reconstructions (Gundappa-Sulur et al., 1999
Although we could not identify granule cells making ascending axon connections, any such cells are likely to have been among those contributing to the high connection probability near the Purkinje cell dendritic plane. How do the synaptic properties of this population of granule cells "enriched" in ascending axon connections compare with those for pure parallel fiber synapses? Figure 10 shows plots of connection properties expected to differ between ascending axons and parallel fibers as a function of distance from the dendritic plane. If the two strong connections were excluded, the amplitudes of the remaining proximal granule cell connections showed no trend toward greater values at the dendritic plane. Plots of estimated release probability and paired pulse facilitation against distance from the dendritic plane show a similar lack of any detectable trend (Fig. 10), and the average values were very close to those for more distal granule cells. Thus most proximal granule cell connections appear to be indistinguishable from those of parallel fibers. Note that the wide spread of individual paired pulse ratios results at least in part from the low signal-to-noise ratio of the amplitude measurements of the smaller connections.
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Figure 10. The properties of connections from proximal connections are similar to those of parallel fiber connections. Shown are plots of connection amplitude (A), estimated failure probability (B), and paired pulse ratio (C) as a function of distance from the Purkinje cell dendritic plane. Dashed lines represent the mean values for parallel fiber connections, and the solid lines are the best-fit lines. The two large amplitude connections (open circles) were excluded from the regression calculation for the amplitudes. Note that the mean P(failure) estimate (B) represented by the dashed line is for the parallel fiber connections shown in Figure 5A.
Summary of granule cell
Figure 11 summarizes the kinetics analysis of the connections we recorded. Average EPSCs were fit with bi-exponential functions. Because of the sensitivity of the fits to the levels of noise, Figure 11 contains only the parameters of kinetics from connections for which the EPSC amplitude exceeded 5 pA. By integrating the (extrapolated) bi-exponential fits, we estimated the EPSC charge transfer. This is probably a reasonable estimate of the synaptic charge. This can be argued as follows. First, inspection of the EPSP kinetics of Figure 3 indicates that charge loss via the membrane conductance is relatively slow. Such loss is governed by the membrane time constant, which can be estimated from the decay of the EPSP. This is clearly too slow to affect the response peaks of the EPSC and EPSP. It is also fairly slow compared with the rate of EPSC charge recovery in voltage clamp, suggesting that integration of the EPSCs will provide a reasonable indication of the total synaptic charge (Carnevale and Johnston, 1982
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Figure 11. Kinetic properties for all granule cell
Varying synaptic charge naturally will cause a proportional variation of EPSC amplitude. Similarly, the stronger dendritic filtering of more distal synapses might be expected to attenuate their somatic responses more than occurs for proximal inputs. To compare the importance of these two factors, charge and filtering, we examined to what extent they were correlated with EPSC amplitude. If we assume that
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Figure 12. EPSC amplitude is correlated with synaptic charge, but not kinetics. A, Plot of mean EPSC amplitude against reciprocal of the decay time constant from the bi-exponential fit of the EPSC. Small and large EPSCs (open circles below and above the dashed lines, respectively) were not included in the regression calculation (see Results). The best-fit regression (solid line) is shown with the 95% confidence limits (dashed curves). B, A similar analysis shows that a significant correlation exists between EPSC charge and amplitude.
DISCUSSION
Determinants of the synaptic weights of parallel fiber
The present recordings of parallel fiber connections, obtained at 32°C in slices from adult rats, have confirmed the existence of a wide range of unitary EPSC amplitudes (Barbour, 1993
In general, presynaptic mechanisms are not favored by our data. Action potential propagation appears to be reliable. Our release probability estimates are consistent with overall release probabilities falling within a fairly narrow range of elevated values (~0.9), and these values are accurate for the strongest connections. Our estimates of release probability for small connections, although consistent with a similar value of underlying probability, are more uncertain because of the experimental noise. Nevertheless, we still can conclude that connections >5 pA have failure probabilities below 0.5. We were unable to demonstrate the existence of very-low-probability connections by using action potential trains. It should be noted that, depending on synaptic parameters such as receptor saturation and the existence of multivesicular release, it is possible for small variations of a low failure probability to be associated with quite significant alterations of response amplitude. Our data are not of sufficient quality to rule out a contribution of release probability to determining the distribution of synaptic weights, but we derive no support for this hypothesis from our data.
The apparently high release probability in our recordings is awkward to reconcile with the conclusion that it is <5% (Dittman et al., 2000
Several postsynaptic mechanisms could contribute to the spread of synaptic weights. Somewhat surprisingly, the degree of filtering (estimated from the EPSC kinetics) had no predictive power for EPSC amplitude. This can be explained in part by the modest variations (less than ~2.5-fold) of dendrite-soma attenuation of synaptic responses with dendritic location (Roth and Hausser, 2001
Not only is a wide range of synaptic charges observed, but the synaptic charge was correlated strongly with EPSC amplitude. One obvious source of variable synaptic charge is the number of synaptic contacts involved: one and two contacts occur frequently (Harvey and Napper, 1988
Finally, Purkinje cells possess several voltage-dependent conductances (Llinas and Sugimori, 1980
It has been postulated that only a small fraction of parallel fibers is active at a given time (Marr, 1969
Ascending axons
The importance of the ascending axon input to Purkinje cells has been a subject of long-running controversy in the field of cerebellar research. Overall, we found that proximal granule cells elicited EPSCs with very similar properties to connections mediated by parallel fibers, with one crucial difference: local granule cells had a much higher probability of generating a detectable EPSC. Proximal granule cells therefore constitute a privileged input to Purkinje cells, and this provides a mechanism that could explain the sensitivity of Purkinje cells to their subjacent mossy fiber inputs. However, the overall difference in granule cell efficacy appears to be less than some predictions, and the underlying mechanism is unexpected. If we calculate average granule cell efficacy within 35 µm of the dendritic plane and compare this with the efficacy for more distant granule cells, we obtain a factor of 3.2 greater effect for the proximal granule cells. Only a factor of 1.4 of this results from differences in EPSC amplitudes, whereas a factor of 2.3 results from the higher probability of generating a detectable response. The range of 35 µm was chosen because this corresponds to a recent estimate of the extent (in the parallel fiber direction) of a mossy fiber terminal field (Sultan, 2001
Although we do not know how many ascending axon connections we recorded, we sampled granule cells throughout the region near the Purkinje cell. Clearly, powerful ascending axon connections are uncommon. If numerous ascending axon connections are included within our sample, they appear to have similar properties to parallel fiber connections.
Undetected synapses
Our results, when compared with the careful and detailed stereological work of Napper and Harvey (Harvey and Napper, 1988
We propose that the potentially large fraction of very weak parallel fiber
The existence of such weak synapses would raise questions about the processes of learning in the cerebellar cortex. Current theory holds that, during learning, parallel fiber inputs to Purkinje cells are depressed and nuclear cells are excited indirectly (Ito, 1989
FOOTNOTES Received July 23, 2002; revised Aug. 27, 2002; accepted Sept. 3, 2002.
P.I. was supported by a Ministère de l'Éducation Nationale de la Recherche et de la Technologie fellowship and the Ecole Normale Supérieure. We thank Philippe Ascher and the Laboratoire de Neurobiologie (Centre National de la Recherche Scientifique Unité Mixte de Recherche 8544) for their generous support. We also thank Philippe Ascher, Mariano Casado, Stéphane Dieudonné, Régis Lambert, Jacques Neyton, and Stéphane Supplisson for helpful discussions.
Correspondence should be addressed to Boris Barbour, Laboratoire de Neurobiologie, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France. E-mail: barbour{at}ens.fr.
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