Acquisition, extinction, and reacquisition of a cerebellar cortical memory trace

Abstract

Associative learning in the cerebellum underlies motor memories and probably also cognitive associations. Pavlovian eyeblink conditioning, a widely used experimental model of such learning, depends on the cerebellum, but the memory locus within the cerebellum as well as the underlying mechanisms have remained controversial. To date, crucial information on how cerebellar Purkinje cells change their activity during learning has been ambiguous and contradictory, and there is no information at all about how they behave during extinction and reacquisition. We have now tracked the activity of single Purkinje cells with microelectrodes for up to 16 h in decerebrate ferrets during learning, extinction, and relearning. We demonstrate that paired peripheral forelimb and periocular stimulation, as well as paired direct stimulation of cerebellar afferent pathways (mossy and climbing fibers) consistently causes a gradual acquisition of an inhibitory response in Purkinje cell simple spike firing. This conditioned cell response has several properties that matches known features of the behavioral conditioned response. The response latency varies with the interstimulus interval, and the response maximum is adaptively timed to precede the unconditioned stimulus. Across training trials, it matches behavioral extinction to unpaired stimulation and also the substantial savings that occur when paired stimulation is reinstated. These data suggest that many of the basic behavioral phenomena in eyeblink conditioning can be explained at the level of the single Purkinje cell.

Figure 1.

Experimental setup. A, Wiring diagram and experimental setup showing the recording site and the different stimulation sites. PC, Purkinje cell; pf, parallel fiber, DCN, deep cerebellar nucleus; cf, climbing fiber; GrC, Granule cell; IC, Inferior colliculus. B, Sample eyelid EMG record of a typical CR on a CS-alone trial. The onset latency is ∼100 ms, and the duration is ∼350 ms. C, Hemispheral lobules III–VII of the cerebellar cortex. The blink-controlling area from which Purkinje cells were sampled is indicated in black.

Figure 2.

Responses to conditioned and unconditioned stimuli. A–D, Typical field potential responses recorded from the cerebellar surface (averages of 10 sweeps). Climbing fiber responses indicated by asterisks. E–H, Simple and complex spikes (asterisks) recorded from single Purkinje cells. A, E, Periocular stimulation (1 pulse, 3 mA) elicits two complex spikes after 11 and 23 ms, respectively, corresponding to the second and third positive field potentials in the surface recording (stimulus indicated by arrow). B, F, Stimulation of the inferior olive (1 pulse, 100 μA) elicits a complex spike response after 4 ms, corresponding to the first positive field potential response. C, G, Stimulation of climbing fibers in the inferior cerebellar peduncle (1 pulse, 100 μA) elicits a complex spike response after 2 ms, corresponding to the first positive field potential response but no simple spikes. D, H, Stimulation of mossy fibers in the middle cerebellar peduncle (1 pulse, 100 μA) elicits a positive field potential response after 1 ms, and presentation of 15 consecutive stimuli at 50 Hz (bar below graph) reliably causes an increase in simple spike activity but does not elicit any complex spikes.

Figure 3.

Acquisition of Purkinje cell CR. A, Two sample records from a Purkinje cell exposed to paired CS–US presentations (bars above graph), from trial 1 (top) and 640 trials later (bottom). The acquired Purkinje cell response had a latency of ∼60 ms. B, Raster plot of the simple spike activity recorded from the same cell during 640 trials of paired CS–US presentations. The inhibitory response during the CS period (framed) gradually developed as training progressed. C, Average raster plot based on 11 Purkinje cell records during acquisition. The plot is built up of squares, the shadings of which indicate average firing rate across all cells (for details, see Materials and Methods). The light area that gradually appears represents the Purkinje cell CR, i.e., an inhibitory response with a firing rate below background level (100%). The darker areas indicate increased simple spike activity. D, Simple spike activity in 11 individual Purkinje cell records shown as plots of firing frequency (percentage of background activity) during the whole 300 ms CS period. There is a consistent decrease in all cells regardless of whether periocular, olivary, or climbing fiber stimulation was used as US. In one cell, acquisition was observed twice: first using a mossy fiber CS and later using a forelimb CS (see Table 1). E, Temporal analysis of simple spike activity. The 300 ms CS period was divided into three 100 ms periods that are plotted individually. During acquisition, there was only a slight decrease in firing rate during the first 100 ms compared with the change observed in the rest of the CS period. F, Spontaneous simple spike activity during the 600 ms period before CS presentation in 11 individual Purkinje cell records, shown as percentage of background activity when recording started.

Figure 4.

Timing of Purkinje cell responses. A–D, Peristimulus time histograms of conditioned Purkinje cell responses to CS-alone presentations (bars above graphs indicate CS duration). The CS–US interval is the darkened part each histogram. Bin width is 10 ms, and simple spike activity was averaged over 50 trials in A–C and 20 trials in D. The mark on the y-axis indicates 100 Hz simple spike frequency. A–C, Three examples of Purkinje cell responses to CS-alone stimulation in which CRs were acquired using a 300 ms mossy fiber CS and a 300 ms CS–US interval to different unconditioned stimuli (i.e., stimulation of the periorbital area, inferior olive, or climbing fibers). D, This cell was trained with a longer (600 ms) CS and a shorter (200 ms) CS–US interval. E, Average response profiles during CS period for Purkinje cells trained with 200 and 300 ms CS–US intervals, respectively, showing longer latency to both onset and maximum response (dotted lines) in the second group (for additional details, see Materials and Methods and Results).

Figure 5.

Purkinje cell activity during extinction. A, Two example records from a Purkinje cell exposed to unpaired CS–US stimulation (bar above graph indicates the 300 ms CS period), from trial 1 (top) and 440 trials later (bottom). The Purkinje cell CR had an onset latency of 60 ms and a duration of 300 ms. B, Raster plot of simple spike activity recorded from the same cell as above during 440 trials of unpaired CS–US stimulation. The inhibitory response during the CS period (framed) gradually extinguished as training progressed. C, Average raster plot of simple spikes recorded from nine Purkinje cells during paired CS–US stimulation. The plot is built of squares, the shadings of which indicate average firing rate across all cells (for details, see Materials and Methods). The light area that gradually disappears represents the Purkinje cell CR, i.e., an inhibitory response with firing rate below background level (100%). D, Simple spike activity in nine individual Purkinje cells shown as plots of firing frequency (percentage of background activity) during the whole 300 ms CS period. There is a consistent increase of simple spike activity in all cells. E, Temporal analysis of training induced changes of simple spike activity. The 300 ms CS period was divided into three 100 ms periods and plotted separately. F, Spontaneous simple spike activity during the 600 ms period before CS presentation in nine individual Purkinje cell records, shown as percentage of background activity when recording started.

Figure 6.

Purkinje cell activity during reacquisition. A, Two example records from a Purkinje cell reexposed to paired CS–US stimulation (bars above graph) after extinction of the CR. Trial 1 (top) and 40 trials later (bottom) are shown. B, Raster plot of simple spike activity (recorded from the same cell as above) during 40 trials of reintroduced pairing of CS–US stimulation. Notice the savings in the rate of reacquisition compared with acquisition (see Fig. 3). After only four trials, the CR was reacquired. C, Average raster plot based on five Purkinje cell records during paired CS–US stimulation. The plot is built of squares, the shadings of which indicate average firing rate across all cells (for details, see Materials and Methods). The light area that gradually appears represents the Purkinje cell CR, i.e., an inhibitory response with a firing rate below background level (100%). D, Simple spike activity in four individual Purkinje cells shown as plots of firing frequency (percentage of background activity) during the whole 300 ms CS period. In one cell, reacquisition was observed twice: first using a periocular US and later using a climbing fiber US (Table 1). There is a consistent decrease of simple spike activity in all cases. E, Temporal analysis of changes of simple spike activity. The 300 ms CS period was divided into three 100 ms periods and plotted separately. F, Spontaneous simple spike activity during the 600 ms period before CS presentation in five individual Purkinje cell records, shown as percentage of background activity when recording started.