Patches of synchronized activity in the cerebellar cortex evoked by mossy-fiber stimulation: questioning the role of parallel fibers

Abstract

The discrepancy between the structural longitudinal organization of the parallel-fiber system in the cerebellar cortex and the functional mosaic-like organization of the cortex has provoked controversial theories about the flow of information in the cerebellum. We address this issue by characterizing the spatiotemporal organization of neuronal activity in the cerebellar cortex by using optical imaging of voltage-sensitive dyes in isolated guinea-pig cerebellum. Parallel-fiber stimulation evoked a narrow beam of activity, which propagated along the parallel fibers. Stimulation of the mossy fibers elicited a circular, nonpropagating patch of synchronized activity. These results strongly support the hypothesis that a beam of parallel fibers, activated by a focal group of granule cells, fails to activate the Purkinje cells along most of its length. It is thus the ascending axon of the granule cell, and not its parallel branches, that activates and defines the basic functional modules of the cerebellar cortex.

Figure 1

A single surface stimulus elicited a beam-like response. (A) The data are displayed as absolute change in fluorescence as a function of time in 128 sites in the cerebellar cortex. The calibration bar gives the scales for both time and space. The stimulating electrode was placed on the surface of the cerebellar cortex to the left of the recorded area. Each of the 128 traces is an average response of three identical stimuli repeated at a frequency of 0.3 Hz. The stimulus elicited a wave of positive responses (red), which propagated in a beam along the cerebellar folium. Negative signals (blue) were observed lateral to the positive beam. This well defined, narrow positive beam was always observed, but the negative signals were not detected in all experiments. (B) The traces marked with asterisks in A were superimposed to show that the signal propagated with a conduction velocity of 0.2 m/s. (C) The traces marked with triangles in A are superimposed on one another. Arrows denote the time of stimuli. The Insets in B and C are schematic representations of the experimental arrangement. A thick gray line represents the activated beam. The locations of the recording sites relative to the beam are marked by black rectangles.

Figure 2

A train of surface stimuli elicited on-beam inhibition. (A) Data presented as in Fig. 1. The stimulating electrode was placed on the surface of the cerebellar cortex to the left of the recorded area. The train of stimuli elicited a train of positive responses (red traces), followed by a prolonged negative wave. (B) Superimposed responses to three different stimulus intensities (recorded at the location marked by a triangle in A). The amplitude of the negative wave depends on stimulus intensity. (C) Superimposed responses to a train of one, two, and three stimuli recorded at the location marked by ∗. Note that the response to a single stimulus (blue trace) lacked the negative response. (Insets as in Fig. 1.)

Figure 3

Characterization of the optical signal. (A) The optical signal (red trace) and the field potential (blue trace) were recorded at the same location with the recording electrode close to the cerebellar surface. The negative peak of the electrical signal (inversely displayed) represents the parallel-fiber action potentials. It preceded the peak of the optical signal by 1 ms. The slow negative wave of the electrical response, which represents the postsynaptic excitation in Purkinje cells and other cortical cells, correlated with the slow positive decay of the optical response. The arrow denotes the time of stimulus. (B) DNQX blocked the optical response to surface stimulation. The three panels show responses recorded at three locations along the activated beam (see inset). The blue traces are the control, and the red traces are the responses after local application of DNQX at the location of the green rectangle (see inset). (C) Bicuculline blocked the on-beam negative signal. The blue and red traces were recorded before and after bath application of 50 μM bicuculline, respectively. (D) Lateral inhibition. Responses were recorded at three adjacent locations across two activated beams (see inset). The blue and the red traces are the responses to stimulation of the upper beam (stim 1) and the lower beam (stim 2), respectively. The green traces are the responses to simultaneous stimulation of both beams. The green trace (Middle; black rectangle) was smaller than the red trace, indicating that activity in the upper beam inhibited the lower-beam activity only in the region between the two beams. (E) On-beam inhibition. The response to a test stimulus (blue trace, stim 2 in the inset) was decreased by on-beam inhibition evoked by a train of stimuli (stim 1, red trace). Similar results were obtained with a shorter train of stimuli provided on-beam inhibition was generated.

Figure 4

Patch-like response evoked by white-matter stimulation. (A) The traces shown (obtained at a gain of 40× and displayed as in Fig. 1A) were obtained at high stimulus intensity. The red, blue, and green traces denote the activated areas at increasing stimulus intensities. Only the area marked in red was activated at the lowest stimulus intensity. At intermediate stimulus intensity, the activated area increased and is marked both by red and blue. (B) Superimposed responses to stimulation at the three intensities, at the location marked by a triangle in A. (C) The traces marked with ∗ in A were superimposed to show that response onsets and peaks occurred at the same time in all locations. (D) The patch of activity was reversibly blocked by Co²⁺. Each of the traces shown is the average response of seven diodes measured before (blue trace) and during (red trace) application of 5 mM Co²⁺. After washing, the response reappeared (green trace). (E) The patch-like response was blocked by on-beam inhibition. Responses in three locations across the activated beam (see inset) are shown. A train of stimuli (stim 1 in the inset) generated the on-beam inhibition. The response to white-matter stimulation (mf stim, red traces) and the response to both stimuli (mf stim and stim 1, blue traces; note that the positive responses to stim 1 were truncated) were superimposed. The response to mossy-fiber stimulation was completely blocked by the on-beam inhibition in the center of the activated beam (green rectangle; the response to mf stim seen in the red trace is missing in the blue trace). The response was partially blocked near the edge of the beam (black and yellow rectangles; the response to mf stim in the red traces is larger than the response to mf stim in the blue traces).

Figure 5

A schematic diagram of the components in the cerebellar cortex studied here, with Purkinje cells in blue and granular cells in green. The red–yellow circle marks the synapses formed by the ascending axon on Purkinje-cell dendrites, and the red circle marks the synapses formed by the parallel fibers on Purkinje cells along their path. The inhibitory network is not shown.