Memory trace and timing mechanism localized to cerebellar Purkinje cells

Abstract

The standard view of the mechanisms underlying learning is that they involve strengthening or weakening synaptic connections. Learned response timing is thought to combine such plasticity with temporally patterned inputs to the neuron. We show here that a cerebellar Purkinje cell in a ferret can learn to respond to a specific input with a temporal pattern of activity consisting of temporally specific increases and decreases in firing over hundreds of milliseconds without a temporally patterned input. Training Purkinje cells with direct stimulation of immediate afferents, the parallel fibers, and pharmacological blocking of interneurons shows that the timing mechanism is intrinsic to the cell itself. Purkinje cells can learn to respond not only with increased or decreased firing but also with an adaptively timed activity pattern.

Keywords: cerebellum; eyeblink conditioning; glutamate transmission; temporal control.

Fig. 1.

Experimental setup. (A) Blink-controlling area in cerebellar cortex. IC, inferior colliculus; Roman numerals indicate cerebellar lobules. (B) Periocular stimulation (1 pulse, 300 μA) elicits short-latency field potential responses on the cerebellar surface. Below are single-cell recordings of two complex spikes elicited by the periocular stimulation (1 mA) and simple spikes elicited by parallel fiber stimulation (4 μA). Arrows indicate stimulation; asterisks indicate responses. (C) Typical conditioned Purkinje cell response (CR). (D) Neuronal wiring diagram with stimulation, recording, and injection sites. AIN, anterior interpositus nucleus; CS, conditional stimulus; cf, climbing fiber; Gc; Golgi cell; Grc, granule cells; In, interneuron; IO, inferior olive; mf, mossy fibers; Pc, Purkinje cell; pf, parallel fibers; US, unconditional stimulus.

Fig. 2.

Conditioned Purkinje cell responses timed to interstimulus intervals. (A and B) Raster plots showing a typical Purkinje cell response to a 300-ms conditional stimulus before (A) and after (B) training with a 150-ms interstimulus interval (blue shading). (C and D) Responses of the cell in A and B to 17.5-ms (C) and 800-ms (D) conditional stimuli (CS) after training. (E) Response to a 300-ms conditional stimulus after extinction. (F–H) Raster plots and histograms illustrating responses of a Purkinje cell that was trained first with a 200-ms interstimulus interval and subsequently with a 350-ms interstimulus interval. Red vertical bars in F and H denote unconditional stimulus artifacts (data from paired conditional stimulus–unconditional stimulus trials). Purple horizontal bars indicate the conditional stimuli. ISI, interstimulus interval.

Fig. 3.

Time courses of conditioned responses after training with different interstimulus intervals and using conditional stimuli of different durations. (A–C) Smoothed and averaged simple spike activity after training with 150-ms (blue, n = 10) (A), 200-ms (red, n = 7) (B), and 300-ms (green, n = 6) (C) interstimulus intervals. Traces with lighter shading represent cells for which naive data are lacking. Colored horizontal bars indicate the interstimulus intervals. (D) Activity ± SEM for each interstimulus interval before training. Traces are truncated at the onset of the unconditional stimulus artifact, which prohibits identification of spikes. Abrupt downward inflections at the end of some traces reflect an effect of smoothing (0 identified spikes during the unconditional stimulus artifact). (E) Activity ± SEM for each interstimulus interval after training. (F) Activity ± SEM for cells trained with a 200-ms interstimulus interval and a coterminating conditional stimulus (cyan, n = 2) or an 800-ms conditional stimulus (magenta, n = 5).

Fig. 4.

Gabazine blocks interneuron inhibition of Purkinje cells but leaves conditioned responses to a 200-ms interstimulus interval intact. Orange horizontal bars indicate off-beam stimulation (800 ms, 81 pulses, 100 Hz). Purple horizontal bars indicate the conditional stimulus, and blue shading indicates the interstimulus interval (200 ms). (A and B) Purkinje cell responses to interneuron activation by off-beam parallel fiber stimulation (compare with Fig. 1D) before (A) and after (B) gabazine injection. Stimulation artifacts are masked. (C) Average (n = 4) responses ± SEM before (green) and after (cyan) gabazine injection. (D and E) Conditioned responses before (D) and after (E) gabazine injection in the same Purkinje cell shown in A and B. (F) The average response profile before (blue) and after (red) injection in the cells shown in C.

Fig. 5.

Gabazine blocks interneuron inhibition of Purkinje cells but leaves conditioned responses to a 300-ms interstimulus interval intact. (A) Average Purkinje cell responses (n = 3) ± SEM to interneuron activation by parallel fiber stimulation before (green) and after (cyan) injection of gabazine. The horizontal orange bar indicates off-beam stimulation (800 ms, 81 pulses, 100 Hz). (B) The average response profile to a 400-ms conditional stimulus before (blue) and after (red) in the cells shown in A. Black and purple horizontal bars indicate the interstimulus interval (300 ms) and conditional stimulus (CS, 400 ms), respectively.

Fig. 6.

Effects of different concentrations of gabazine on conditioned Purkinje cell responses to a parallel fiber conditional stimulus. Each row indicates one cell. (A, D, and G) Naive (black) and conditioned responses before (blue) and after (red) gabazine injection. Purple horizontal bars indicate a 300-ms conditional stimulus. Dashed lines indicate a 150-ms interstimulus interval. (B, E, and H) Magnification of the response during the interstimulus interval. (C, F, and I) Purkinje cell responses to interneuron activation by parallel fiber stimulation before (green) and after (cyan) gabazine injection. At 100 μM, gabazine distinctly blocks inhibition (C and F), whereas 10 μM gabazine blocks inhibition for the first 200 ms (I). Orange horizontal bars indicate off-beam stimulation (800 ms, 81 pulses, 100 Hz).