Department of Neurobiology, Life Science Institute and Center for Neural Computation, The Hebrew University, Jerusalem 91904, Israel
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ABSTRACT |
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Cohen, Dana and Yosef Yarom. Cerebellar On-Beam and Lateral Inhibition: Two Functionally Distinct Circuits. J. Neurophysiol. 83: 1932-1940, 2000. Optical imaging of voltage-sensitive dyes in an isolated cerebellum preparation was used to study the spatiotemporal functional organization of the inhibitory systems in the cerebellar cortex. Responses to surface stimulation of the cortex reveal two physiologically distinct inhibitory systems, which we refer to as lateral and on-beam inhibition following classical terminology. Lateral inhibition occurs throughout the area responding to a stimulus, whereas on-beam inhibition is confined to the area directly excited by parallel fibers. The time course of the lateral inhibition is twice as long as that of the on-beam inhibition. Both inhibitory responses increase with stimulus intensity, but the lateral inhibition has a lower threshold, and it saturates at lower stimulus intensity. The amplitude of the on-beam inhibition is linearly related to the excitation at the same location, whereas that of the lateral inhibition is linearly related to the excitation at the center of the beam. Repetitive stimulation is required to activate on-beam inhibition, whereas the same stimulus paradigm reveals prolonged depression of the lateral inhibition. We conclude that lateral inhibition reflects the activation of molecular layer interneurons, and its major role is to increase the excitability of the activated area by disinhibition. The on-beam inhibition most likely reflects Golgi cell inhibition of granule cells. However, Purkinje cell collateral inhibition of Golgi cells cannot be excluded. Both possibilities suggest that the role of the on-beam inhibition is to efficiently modulate, in time and space, the mossy fiber input to the cerebellar cortex.
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INTRODUCTION |
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The cerebellum is built up of basically similar
units: the cerebellar modules. Each module receives inputs from the
mossy fibers and the climbing fibers, whereas a Purkinje cell axon, the
only output from the cortical modules, transmits the processed information to the deep cerebellar nuclei. The climbing fibers activate
the Purkinje cells directly, whereas the mossy fibers excite them
indirectly via the granule cells (Ito 1984;
Llinas 1981
; Palay and Chan-Palay 1974
).
This excitatory circuit is modulated by a comprehensive inhibitory
system, composed of the molecular layer interneurons, the Purkinje
cell collaterals, and the Golgi cells. A thorough exploration of the
inhibitory system is essential to elucidate the computational process
that is performed in each module. Here we have used optical imaging of
voltage-sensitive dyes in an isolated preparation to study the
functional spatiotemporal organization of the inhibitory system of the
cerebellar cortex.
The molecular layer interneurons, which are classically divided into
basket and stellate cells, operate within the molecular layer
(Palay and Chan-Palay 1974; Sultan and Bower
1998
). They are activated by the parallel fibers and inhibited
both by Purkinje cell collaterals (Llinas and Precht
1969
) and by other molecular layer interneurons (Hausser
and Clarck 1997
; Llano and Gerschenfeld 1993
).
Their axons either remain in the immediate vicinity of their cell body
or run up to 500 µm perpendicular to the parallel fibers (Ito
1984
). The short axons of stellate cells may inhibit each
Purkinje cell proportional to its excitation (De Schutter and
Bower 1994
; Marr 1969
). The long-range axons of
the stellate and basket cells are thought to induce lateral inhibition
that sharpens the borders of the area activated by the parallel fibers (Eccles et al. 1967
). However, in view of our recent
demonstration that beams of Purkinje cells activity are unlikely to be
generated by mossy fiber activation (Cohen and Yarom
1998
), the role of the long-range axons of the molecular layer
interneurons remains to be elucidated.
The Golgi cells are the largest inhibitory interneuron in the
cerebellar cortex. Their cell bodies are located in the upper part of
the granular layer, and their dendrites ascend to the molecular layer,
where they receive excitatory input from the parallel fibers. Their
axons ramify within the granular layer in a limited space underneath
their cell bodies, preventing the activation of granule cells by mossy
fiber input. The spatial distribution of the inhibitory output of Golgi
cells is therefore limited to the area activated by the parallel
fibers. The Golgi cells provide the only feedback inhibition to the
cerebellar modules, serving as a gain control of their mossy fiber
input (Marr 1969). Recent theoretical work has
demonstrated that Golgi cells may induce synchronized rhythmic activity
in granule cells, thereby increasing the parallel fiber input to the
Purkinje cells (Maex and De Schutter 1998
).
Although the extensive collaterals of Purkinje cell axons were
described by Cajal at the beginning of the century (Cajal
1955), knowledge about the target neurons of these collaterals
and the effects of their activity is rather limited. Recently, Bishop and colleagues (Bishop 1982
, 1988
;
Bishop and O'donoghue 1986
; O'donoghue and
Bishop 1990
) showed that each Purkinje cell axon gives rise to
one to three collaterals that form a plexus of varicose axonal
branches. With few exceptions, this plexus is formed in the immediate
vicinity of the parent cell, extending 300-400 µm in the transverse
plane (parallel fiber axis) and somewhat longer (500-600 µm) in the
sagittal plane. Most varicosities are asymmetrically distributed within
200-400 µm around the parent cell. The terminals of Purkinje cell
collaterals in the granular layer form a nestlike structure around
large cell bodies there, most likely Golgi cells. In the molecular
layer, they innervate the molecular layer interneurons and other
Purkinje cells. However, physiological studies have not succeeded in
demonstrating inhibition of Purkinje cells by other Purkinje cell
collaterals (Eccles et al. 1966
; Llinas and Precht 1969
). The spatial distribution of the inhibitory output of Purkinje cell collaterals, like that of Golgi cell inhibition, is
limited to the area activated by parallel fibers.
Although the individual components of the inhibitory systems in the
cerebellar cortex are well known, as are some of their properties,
little is known about their functional organization. Here we
demonstrate that the inhibitory systems of the cortex comprise at least
two physiologically distinct components. Although both types of
inhibitory responses are blocked by bicuculline (Cohen and Yarom
1998, 1999
), they differ in their mode of
activation, spatial distribution, time course, and dynamic properties.
These results shed new light on the role of the inhibitory interneurons in intermodule communication.
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METHODS |
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Optical measurement from the isolated guinea pig cerebellar
preparation has been described in detail elsewhere (Cohen and Yarom 1999). Briefly, the intact cerebellum and the brain stem are removed from the animal, and a cannula is inserted into one of the
vertebral arteries. Physiological solution is perfused via the
vertebral artery at a rate of 0.5 ml/min using a peristaltic pump. The
intravascular solution consists of (in mM) 124 NaCl, 5 KCl, 1.3 MgSO4, 1.2 KH2PO4, 26 NaHCO3, 10 glucose, 2.4 CaCl2, and 5% dextran. A similar solution
without dextran is used for the external solution. The preparation was
maintained at 28°C.
The voltage-sensitive dye RH-414 (2 µg/µl) was injected into one of the cerebellar folia using a glass micropipette. Optical signals were monitored by 128 photodiodes organized in a 12 × 12 array. Each element of the array detects light from a surface of 100 × 100 µm or 50 × 50 µm when using ×20 or ×40 objectives, respectively. The signals were amplified in two AC-coupled stages, with a time constant of 200 ms, and then sampled and digitized with 12-bit accuracy at maximal resolution of 50 µs (Microstar, DAP 3400a). Data were usually displayed as traces of absolute change in fluorescence as a function of time at each location. However, when the signals at two locations were compared, the relative fluorescence was used. To that end, the amplitude of the response was divided by the DC level measured at these locations. Concentric metal electrodes with a diameter of 200 µm were used to stimulate (0.1-ms pulses of 0.5-5 V) the cerebellar surface (parallel fibers).
When interpreting optical signals generated by voltage-sensitive dyes,
one should be aware that the signals represent a weighted sum of all
the changes in fluorescence detected by each photodiode. The weights
are determined by factors like membrane area, the depth of the
signals' source, and the amount of dye adhered to the membranes. Thus
the absence of a negative component does not necessarily imply lack of
inhibition. Furthermore, the size and shape of the signal cannot be
used to determine its functional importance. However, in a previous
study (Cohen and Yarom 1999) we have demonstrated that
in our system all the negative signals generated by surface stimulation
represent postsynaptic inhibitory signals, whereas positive responses
reflect excitatory postsynaptic activity, composed of synaptic and
regenerative responses. The parallel fiber action potential and the
glial cells do not contribute significantly to the optical responses.
Unstained preparation does not generate any signal, suggesting that
"intrinsic signals" are insignificant in our system.
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RESULTS |
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Surface stimulation of the cerebellar cortex activates, in addition to an excitatory beam of activity, two types of inhibitory responses: lateral inhibition and on-beam inhibition. The spatial distribution of these inhibitory responses is shown in Fig. 1. Lateral inhibition is evoked by a single surface stimulation and appears on both sides of the activated beam (Fig. 1A). Large depolarizing responses were recorded at the 8th to 10th photodiodes rows, whereas the response in other areas consisted of a depolarization followed by a prolonged hyperpolarization. The ratio between the depolarization and hyperpolarization gradually decreased with the distance from the center of activity. Prolonged hyperpolarization was observed only in the most distant locations (top 2 rows).
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The borders of the excitatory beam were determined by plotting the amplitude profiles of the excitation and inhibition measured in a randomly chosen column of diodes perpendicular to the direction of the parallel fibers. The largest excitatory response was measured at the 10th diode in the column, and it decreased monotonically on both sides (Fig. 1B, squares). The smallest inhibitory response was recorded at the ninth diode, and it increased on both sides until it reached a plateau starting at the sixth diode (Fig. 1B, circles). We therefore define the borders of the excitatory beam as the location where excitation equals inhibition. According to this definition, rows 7 to 11 in Fig. 1 compose the beam of activity (etched area).
On-beam inhibition is activated when a train of two or more stimuli is applied to the cerebellar surface (Fig. 1C) and is found within the borders of the excitatory beam (Fig. 1D). The amplitude profile of the excitation evoked by the train (Fig. 1D, squares) resembles that evoked by a single stimulus (Fig. 1B, squares). In contrast, the amplitude profile of the inhibition includes both the lateral and on-beam inhibitory responses. To reveal the contribution of the on-beam inhibition, we subtracted the inhibitory profile in Fig. 1B from the inhibitory profile in Fig. 1D. The result (Fig. 1D, triangles) demonstrates that the inhibition elicited by a train of stimuli is confined to the excitatory beam.
The two inhibitory responses have different time courses (Fig.
2A), the on-beam inhibition
decaying faster than the lateral inhibition. The linear behavior of the
logarithmic value of the decay (Fig. 2B) indicates that both
decays are described by a single time constant (Fig. 2, C
and D). In the example shown, the time constants were
calculated as 66 and 136 ms for the on-beam and the lateral inhibition,
respectively, and ranging in other experiments from 45 to 73 ms for the
on-beam inhibition and 100 to 140 ms for the lateral inhibition. The
variation in the values of time constants between different locations
in one experiment is much smaller than that between experiments. It is
not unlikely that variation between experiments reflects the
excitability of the inhibitory interneurons. It is well documented that
cortical inhibitory interneurons respond in a train of action
potentials to a single stimulus (Eccles et al. 1967).
The train duration will determine the time constant of the inhibitory
responses. In our preparation, the excitability of the interneurons may
vary between experiments, and therefore various durations of action potential will be generated.
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Dependence on stimulus intensity
LATERAL INHIBITION. As shown in Fig. 1, the lateral inhibition appears on both sides of the activated beam, the width of the inhibited area increasing with stimulus intensity and spread up to 800 µm from the center of the beam. The amplitude of the inhibitory response thus depends on both stimulus intensity and distance from the center of the activated beam (Fig. 3). Distally (Fig. 3A), where there were mainly prolonged negative signals, the response increased monotonically to a saturated level as stimulus intensity increased (Fig. 3D, squares). At the center of the beam (Fig. 3C), increasing the stimulus intensity resulted in an initial increase in amplitude of the negative component followed by a decrease (Fig. 3D, circles). At low stimulus intensity, the negative response dominated the center of the beam, although it was always preceded by a positive response (Fig. 3C, thick line). At distal areas, the positive component of the response was rather small and hardly changed with stimulus intensity (Fig. 3E, squares), but closer to the center of the beam (Fig. 3B) it increased monotonically with stimulus intensity (Fig. 3E, triangles). The steepest slope was observed at the center of the beam (Fig. 3E, circles).
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ON-BEAM INHIBITION. A train of three stimuli elicited the superimposed responses shown in Fig. 4A. These responses, measured in two representative diodes located along the center of the beam, were elicited by different stimulus intensities. The amplitude of both the positive and negative components increased with stimulus intensity. To quantify this increase, we measured the amplitude of the first positive component and the amplitude of the last negative component and plotted them as a function of stimulus intensity. As shown in Fig. 4, B and C, both components depend similarly on stimulus intensity. As a result, the amplitude of the negative component is linearly related to the amplitude of the positive component measured at four stimulus intensities (Fig. 4D). The same linear relationship was found when the positive and negative responses were measured at all the diodes located within the beam, independent of the stimulus intensity (Fig. 4E).
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Temporal properties of the inhibitory responses
The most significant functional difference between the two inhibitory systems is that the on-beam inhibition increases with repetitive stimulation, whereas the lateral inhibition decreases. This difference is shown in Fig. 5, where responses to two consecutive stimuli delivered to the cerebellar surface at various interstimulus intervals were superimposed. Although the second stimulus elicited a depolarizing wavelet at lateral locations (Fig. 5A), it failed completely to elicit an inhibitory response when applied <50 ms after the first stimulus. The amplitude and duration of the depolarizing wavelet evoked by the second stimulus increased relatively to the wavelet generated by the first stimulus. At longer interstimulus intervals, the blockade of the inhibition was partially removed, and the amplitude of the depolarizing wavelet and the inhibitory response to the second stimulus gradually returned to the levels occurring in response to the first stimulus.
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Quantitative analysis of these observations is given in Fig. 5B, where the normalized amplitude of the two inhibitory responses and the normalized difference in the depolarizing wavelet were plotted as a function of interstimulus interval. The amplitude of the first inhibitory response remained constant (squares), whereas the inhibitory response following the second stimulus was completely blocked for at least 50 ms (circles). The amplitude of the second inhibitory response recovered almost completely after 300 ms. Concomitantly, the difference between the amplitudes of the depolarizing wavelets of the first and second responses decreased with a similar time course (triangles).
A different behavior was observed at the center of the excitatory beam (Fig. 5C). Here, the response to the first stimulus is characterized by a depolarizing response followed by a prolonged and shallow hyperpolarization. The second stimulus elicits a pronounced inhibitory response at interstimulus intervals shorter than 70 ms. This response is strongest at shorter intervals (10-20 ms) and decreases with increasing interstimulus interval (Fig. 5D, circles). At longer intervals, the second stimulus fails to produce an inhibitory response, whereas the duration of the depolarizing component increases (Fig. 5C). Almost complete recovery of both components was observed at intervals longer than 200 ms (Fig. 5D).
The inhibitory response at the center of the beam displays a different temporal behavior than that of the lateral inhibitory response. The on-beam inhibition undergoes a strong facilitation, whereas the lateral inhibition shows a robust and long-lasting depression. It should be noted, however, that the prolonged and shallow hyperpolarization elicited by the first stimulus (Fig. 5C) has the same properties as the lateral inhibition. First, it is completely absent in the response to the second stimulus (Fig. 5C), and second, it is activated by a low stimulus intensity (Fig. 3C; thicker trace). Therefore the term "lateral inhibition" is inaccurate, for this type of inhibition occurs all over the activated area.
The complete blockade of the lateral inhibitory response by a preceding stimulus (Fig. 5, A and B) can be attributed either to synaptic depression or inhibition of the inhibitory interneurons at the level of either their cell body (postsynaptic inhibition) or their axonal terminals (presynaptic inhibition). To distinguish between these two possibilities, we studied the reciprocal interactions between two lateral inhibitory responses elicited by different sources. Two stimulating electrodes were used to activate two excitatory beams 800 µm apart (stim 1 and stim 2, inset in Fig. 6). Both stimuli elicited an inhibitory response in the area between the two excitatory beams (top 2 traces in Fig. 6A). The two stimuli were applied sequentially with different interstimulus intervals. The second inhibitory response was always blocked by the first stimulus (Fig. 6A, bottom traces and 6B), regardless of the order of stimulation or the location within the activated area. To demonstrate the extent of the blockade, we superimposed, in Fig. 6A (bottom traces), the expected sum of the two top traces (thick lines). As shown in Fig. 6, B and C, a complete blockade occurred when the interval between the two stimuli was shorter than 70 ms, and gradual recovery was observed at longer intervals. Moreover, the time course of this recovery resembles the recovery time course of the second inhibitory response measured when a single beam elicited both responses (Fig. 5, A and B).
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We therefore conclude that the blockade of the inhibitory response by a preceding lateral inhibition is independent of the location or the source of the lateral inhibition, which supports the possibility of interneurons inhibition rather than synaptic depression.
Dynamics of the on-beam inhibition
The dynamics of the on-beam inhibition were studied by analyzing the behavior of the on-beam inhibition as a function of the intratrain frequency and train duration (i.e., the number of stimuli in the train). Figure 7 shows the buildup of the on-beam inhibition at different train durations and frequencies. Each panel shows six superimposed traces, each with a different train duration ranging from one to six stimuli. The intratrain time interval is 10, 20, and 30 ms in A, B, and C, respectively. It is clear that the amplitude of the on-beam inhibition increases with both the train duration and intratrain frequency. However, saturating states are reached at lower frequencies (Fig. 7, B and C), the level of this saturation increasing with intratrain frequency. At higher intratrain frequency (Fig. 7A), train duration of up to six stimuli did not reach saturation. This behavior suggests that each stimulus makes a constant contribution to the buildup of the inhibition. Occasionally, when a long train duration was used, the on-beam inhibition was followed by a prolonged depolarizing wave that lasted hundreds of milliseconds. The source and properties of this wave were not studied here.
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The buildup of the on-beam inhibition is quantitatively described in
Fig. 8. The increase in inhibition as a
function of the number of stimuli in the train (train duration) is
given in Fig. 8A. As expected, the amplitude of the
inhibitory response saturated and the steady-state level increased with
the frequency of the train (,
, and
). We have shown that the
amplitude of the on-beam inhibition is linearly related to the
amplitude of the first depolarizing response (Fig. 4, D and
E). Hence here we describe the maximum inhibition as a
function of the excitation in all locations for two frequencies (Fig.
8, B and C). Each of the panels shows this relation for three train durations (1, 3, and 6 pulses). The linear relationship is maintained with a similar level of significance in all
cases, the slope of this relationship increasing with the number of
stimuli in the train and with the intratrain frequency (cf. Fig. 8,
B and C).
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To start characterizing the process governing the buildup of the on-beam inhibition (Fig. 8D), we determined the contribution of the second stimulus in the train by subtracting the response to a single pulse from the response to twin pulses. The difference (Fig. 8E, bottom trace) gives the contribution of the response to the second stimulus to the on-beam inhibition. Similarly, we sequentially subtracted all consecutive responses, thus isolating the contribution of each stimulus in the train (Fig. 8E). The striking outcome is the identical contribution of each stimulus in the train to the buildup of the inhibition, except for the second stimulus whose contribution was smaller. Thus the facilitatory process that produces the on-beam inhibition has a rather short dynamic range that reaches saturation within two or three pulses.
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DISCUSSION |
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Several lines of evidence were brought here to demonstrate for the first time that the activity in the cerebellar cortex is governed by two distinct inhibitory systems, which differ in their physiological properties. The differences are summarized in four points. 1) Lateral inhibition occurs throughout the area responding to a stimulus, whereas on-beam inhibition is confined to the region activated directly by the parallel fibers. 2) Both inhibitory responses increase with stimulus intensity, yet the lateral inhibition has a lower threshold and saturates at lower stimulus intensity. In addition, the amplitude of the on-beam inhibition is linearly related to the excitation at the same location, whereas the amplitude of the lateral inhibition at distal locations is linearly related to the excitatory response closer to the center of the beam. 3) The time course of the lateral inhibition is twice as long as that of the on-beam inhibition. 4) Repetitive stimulation is required to activate the on-beam inhibition, whereas long-lasting depression of the lateral inhibition occurs on repetitive stimulation.
Origins of the inhibitory responses
Based on the specific properties of the two inhibitory responses
described above, we suggest that each response reflects the activity of
different neuronal elements. The properties of the lateral inhibition
suggest that it is generated by the molecular layer interneurons. The
spatial distribution of the lateral inhibition fits the distribution of
the axons of the molecular layer interneurons, which are either
restricted to the vicinity of their cell body or extend sagittally for
different distances. In addition, these neurons are small and have
high-input resistance, both imply a low threshold for activation as
shown (Fig. 3C). Indeed, it has been reported that a single
granule cell is sufficient to activate a stellate cell (Barbour
1993). Furthermore, the recent demonstration (Mann-Metzer and Yarom 1999
) that these neurons form
local inhibitory networks, whose neurons are electrotonically coupled,
suggests that an input to one interneuron can activate, via the gap
junctions, a group of interneurons, thereby inducing a relatively fast
saturation of the inhibitory system.
Based on morphological data, the most likely source of on-beam
inhibition is the Golgi cell inhibition of granule cells. The relatively large optical signals recorded during on-beam inhibition indicate that a large membrane area participates in the response. Although the granule cells are small and located deep below the surface, they outnumber Purkinje cells by 5 orders of magnitude and
therefore can contribute to the on-beam inhibition. An alternative possibility is that the on-beam inhibition reflects hyperpolarization of Golgi cells. Namely, Purkinje cells, activated by the parallel fiber
volley, induce, via their collaterals, hyperpolarization of Golgi cells
and other molecular layer interneurons. The Golgi cells are relatively
large and numerous (the Golgi cells to Purkinje cells ratio vary
between 1:1.5 and 3) (Ito 1984) and therefore can
produce a large optical signal without reaching saturation, whereas
small cells, like the molecular layer interneurons, can account for
only a small part of the response. The finding of Bishop and colleagues
(Bishop 1988
; O'donoghue and Bishop
1990
) that Purkinje cell collaterals extend more sagittally
than transversely cannot exclude this alternative possibility. In their
measurements most of the synapses are within a distance of <400 µm
from the parent Purkinje cell, and the difference between the sagittal and transverse distribution is <100 µm. It is difficult to detect such differences in our imaging system.
Both possibilities are in agreement with the linear relationship between the excitatory and inhibitory responses of the on-beam inhibition. If most of the excitation reflects the activity in Purkinje cells, then it should be linearly related to the inhibition produced by their collaterals. A linear relationship would also be predicted if the on-beam inhibition occurs at the granule cells level and is activated by the Golgi cells axons. The Golgi cells are of a similar size to Purkinje cells and are activated by the same beam of parallel fibers. Therefore their activity is expected to be linearly related to Purkinje cells activity, meaning that the inhibition produced by the Golgi cell axons will be linearly related to Purkinje cells activity.
Which cell type produces the on-beam inhibition is of considerable significance for our understanding of the functional organization of the cerebellar cortex. If on-beam inhibition reflects Golgi cell activity, then it would inhibit mossy fiber inputs. If, on the other hand, it reflects Purkinje cell collateral activity, then by inhibiting Golgi cells it would enhance mossy fiber inputs. The enhanced excitability could occur in the same module or in neighboring modules, depending on the extent of Purkinje cell collaterals.
Kinetics of the on-beam inhibition
Two physiological processes can account for the facilitation of the on-beam inhibition. The first is enhanced release of neurotransmitter, and the second is summation of synaptic potentials. Enhanced release of neurotransmitter may occur at the synapses formed by the parallel fibers on Purkinje cells and/or Golgi cells and also at the inhibitory terminals of Purkinje cell collaterals or Golgi cells axons. Summation of synaptic potentials may occur in Purkinje cells or in Golgi cells. Regardless of location, the pronounced facilitatory process has a rather short dynamic range; a single stimulus fails to evoke on-beam inhibition, whereas two stimuli at intervals shorter than 20 ms already reach saturation. Even at longer intervals of 30-40 ms, saturation is reached within few stimuli. The short dynamic range allows a complex temporal regulation of the activity in the cerebellar cortex, in which the first input signal is transferred unaffected, whereas the following inputs are either enhanced, if Purkinje cell collaterals are involved, or depressed, if Golgi cells are involved.
Depression of the lateral inhibition
In contrast to the classical view, our results suggest that the major role of lateral inhibition is to remove inhibition throughout the activated area. This disinhibition is a robust and pronounced phenomenon that lasts for up to 300 ms (Figs. 5 and 6). Thus the spread of lateral inhibition increases, by disinhibition, the responsiveness to incoming information for a significant period. In addition to its effect of disinhibition, lateral inhibition actually inhibits Purkinje cells and other molecular layer interneurons. However, the inhibitory effect is rather short lasting (<20 ms), and, although mediated via a bisynaptic pathway, it is strong enough to decrease the amplitude and duration of the preceding excitatory response. The increase in amplitude and duration of the excitatory response during disinhibition (Fig. 5B) emphasizes the efficiency and strength of this inhibitory effect.
A similar blockade of the inhibitory response accompanied by an
increase in the excitatory response has been previously reported (Eccles et al. 1966). In this in situ set of
experiments, Purkinje cells were intracellularly recorded, and the
responses to surface stimulation, applied via two very close
electrodes, were analyzed. The stimulus intensity was adjusted so that
each electrode evoked similar inhibitory postsynaptic potentials
(IPSPs) in the recorded Purkinje cell. As in our experiments,
sequential activation of the two IPSPs resulted in a time-dependent
depression of the second response. Eccles and his colleagues proposed
that this was due either to a decrease in transmitter release,
desensitization of synaptic receptors, or the proximity to equilibrium
potential of the inhibitory synaptic currents.
In view of our results synaptic depression is less likely because
disinhibition is present even when two separate populations of
inhibitory interneurons are activated (Fig. 6). In addition, the
increase in the excitatory wavelet during disinhibition (Fig. 5) argues
against presynaptic inhibition at the parallel fiber synapses
(Dittman and Regher 1997). Therefore the most likely explanation is interactions among the molecular layer interneurons, either at the cell bodies of these interneurons or at the presynaptic terminals of their axons. The latter seems more probable because the
large distance between the two stimulating electrodes prevents axosomatic interactions. The presynaptic nature of the disinhibition is
further supported by its long time course. The disinhibition may be
mediated by GABAB receptors, which are known to have a slower time course relative to the activity in GABAA
receptors, and are mostly located on presynaptic terminals
(Misgeld et al. 1995
).
Conclusions
The patchlike organization of the response to mossy fiber input
(Cohen and Yarom 1998) and the powerful depression of
the lateral inhibition have significant consequences for our
understanding of the functional organization of the cerebellum. They
imply that the basic independent modules of the cerebellar cortex
consist of a localized group of granule cells and the overlaying group of Purkinje cells connected via excitatory synapses. The activity of
this module is modulated by Golgi cell feedback inhibition and stellate
cell feed-forward inhibition. Mutual interactions between modules occur
via the parallel fibers in the mediolateral axis and via the long-range
axons of the molecular layer interneurons in the sagittal axis.
However, the mediolateral interaction via the parallel fibers is rather
weak and occurs only during a narrow time window. Due to disinhibition,
the sagittal interaction is far more significant and has a prolonged
time window. Because of the large time tolerance provided by the
disinhibition, it is more likely that activity be organized in
parasagittal bands than in the mediolateral direction.
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ACKNOWLEDGMENTS |
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This study was supported by the US-Israel Binational Science Foundation, the Israel Academy of Sciences, and the European Commission. D. Cohen was the recipient of a Clore Fellowship.
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FOOTNOTES |
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Address for reprint requests: Y. Yarom, Dept. of Neurobiology, Life Science Institute, The Hebrew University, Jerusalem 91904, Israel.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 September 1999; accepted in final form 10 December 1999.
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REFERENCES |
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