Graded Control of Climbing-Fiber-Mediated Plasticity and Learning by Inhibition in the Cerebellum

Abstract

Purkinje cell dendrites convert excitatory climbing fiber input into signals that instruct plasticity and motor learning. Modulation of instructive signaling may increase the range in which learning is encoded, yet the mechanisms that allow for this are poorly understood. We found that optogenetic activation of molecular layer interneurons (MLIs) that inhibit Purkinje cells suppressed climbing-fiber-evoked dendritic Ca²⁺ spiking. Inhibitory suppression of Ca²⁺ spiking depended on the level of MLI activation and influenced the induction of associative synaptic plasticity, converting climbing-fiber-mediated potentiation of parallel fiber-evoked responses into depression. In awake mice, optogenetic activation of floccular climbing fibers in association with head rotation produced an adaptive increase in the vestibulo-ocular reflex (VOR). However, when climbing fibers were co-activated with MLIs, adaptation occurred in the opposite direction, decreasing the VOR. Thus, MLIs can direct a continuous spectrum of plasticity and learning through their influence on Purkinje cell dendritic Ca²⁺ signaling.

Figure 1

MLIs suppress CF-evoked dendritic Ca²⁺ spiking in PCs. (A) dye-filled PC imaged with 2pLSM; MLIs expressing YFP-tagged ChR2 are in green. (B) Spiking responses in the soma and dendrite of a PC to CF stimulation, both in control and during coincident optogenetic activation of MLIs (timing of light stimulus in blue; electrical stimulus in black). (C) The subcellular effect of optogenetic-induced ML inhibition on CF-evoked spiking content in the dendrite (84.6 ± 11.6 μm from the axon hillock; range, 38.1 – 140.6 μm), soma, and axon (97.8 ± 36.4 μm from the axon hillock; range, 21.0 – 178.0 μm). Measurements from individual cells in gray, average in black. *P = 0.0002, paired t-test. (D) The change in amplitude of dendritic Ca²⁺ spikes during optogenetic excitation of MLIs. Data are normalized to control responses for each spike in the burst recorded in the absence of optogenetic stimulation (indicated by the gray dotted line in the plot). Example traces of spikes are shown above. *P < 0.05, 1-way ANOVA with Dunnett’s post-test. (E) Example traces of CF-evoked complex spikes, both in control and during persistent optogenetic excitation of MLIs. (F) CF-evoked dendritic Ca²⁺ spikes recorded in PCs during different levels of MLI activity (top trace is in the absence of MLI activity). The firing rate of MLIs during the optogenetic stimulus was an estimate based on a separate set of calibration experiments. (G) Changes in Ca²⁺ spike amplitude depended on the level of MLI activation. Data from individual cells are normalized to control responses for each spike as indicated by the gray dotted line (see Table S1 for statistics). (H) Across-cell comparison of the effect of MLI inhibition on dendritic Ca²⁺ spike content in the presence of SR 95531 or CPG 35348. Data are normalized to control trials without optogenetic-induced inhibition. *P = 0.04; paired t-tests. See also Figures S1, S2 and S3.

Figure 2

Graded suppression of CF-evoked dendritic Ca²⁺ signals in PCs by MLI-mediated inhibition. (A) 2pLSM Ca²⁺ imaging was performed in the dendrites of PCs. MLIs in c-kit::Cre mice were transduced by AAV containing Cre-dependent bReaChES. The position of the line scan used to record the CF-evoked Ca²⁺ response is demarcated by the dotted line in the dendrite image. (B) CF-evoked somatic complex spikes (top panels) and dendritic Ca²⁺ transients (bottom panels) in control (left) and during optogenetic stimulation of MLIs (right; timing of light stimulus in orange; electrical stimulus in black). (C) Dendritic Ca²⁺ transients measured in PCs during different levels of MLI excitation. (D) Relationship between the suppression of dendritic Ca²⁺ elevation and the level of MLI excitation. Measurements from many different dendritic recording sites were collected from 3 PCs. *P < 0.05, repeated measures 1-way ANOVA with Tukey’s post-test. (E) Average Ca²⁺ transients measured from the spine demarcated by the yellow arrow head in the fluorescence image in response to a PF tetanus (100 Hz, 60 ms), CF stimulation, or the conjunctive activation of both (120 ms inter-stimulus interval). On the right, magnified view of the CF-evoked Ca²⁺ transient following conjunctive PF activity in comparison to the estimated response from the linear sum of the individual PF and CF transients. (F) CF-evoked Ca²⁺ transients measured in a PC spine following conjunctive activation with PFs (left) and with coincident MLI-mediated inhibition (middle). Response in control and during bReaChES-induced MLI excitation coincident with the CF stimulus are superimposed (right). (G) Effect of optogenetic-induced MLI inhibition on the peak CF-evoked Ca²⁺ transient during conjunctive stimulation with PFs. Responses, collected from spine measurements from 6 PCs, were normalized to the peak amplitude of the Ca²⁺ transient elicited by CF stimulation alone for each recording site. *P < 0.05, repeated measures 1-way ANOVA with Tukey’s post-test. (H) Relationship between the PF enhancement of CF Ca²⁺ signaling in PC spines and the sensitivity of the peak Ca²⁺ transient to optogenetic-induced MLI excitation. Data from individual spine measurements, in gray, were fit with a linear function (P = 0.0004, 1 sample t-test); binned averages of group data in black.

Figure 3

Control of synaptic plasticity by MLIs. (A) Pairing protocol used to generate associative plasticity in floccular slices (100 Hz PF tetanus, 60 ms, followed by CF stimulation; 120 ms interval after the 3^rd PF stimulus); pairing was repeated 300 times (1 Hz). Below are averaged test PSPs evoked by PF stimulation from the same cell before and after (30–40 min) the pairing protocol. (B) Plot of the change in test PSP amplitudes across cells normalized to the baseline response for each PC. Paired stimulation led to a lasting decrease in PSP amplitudes (black). Without the conjunctive pairing protocol, PF-evoked PSP amplitudes were stable over time (gray). (C) Conjunctive CF stimulation occurring coincident with a moderate level of ChR2-induced MLI activation (λ = 461; 20 ms). Below are averaged test PSPs evoked by PF stimulation from the same cell before and after (30–40 min) the pairing protocol. (D) After a transient increase in PSP amplitude, test responses to PF stimulation returned to baseline levels following the pairing protocol. (E) CFs were stimulated coincident with intensive activation of MLIs during the pairing procedure. Below are averaged test PSPs evoked by PF stimulation from the same cell before and after (30–40 min) the pairing protocol. (F) CF stimulation during the intensive activation of MLIs led to a lasting increase in PF-evoked PSP amplitude (black). A stimulus protocol without CF activation (100 Hz PF tetanus; repeated 300 times, 1 Hz) induced a comparable potentiation of the test PF response (gray). (G) During the stimulus protocol, MLIs were activated following the PF tetanus (repeated 300 times, 1 Hz). (H) Delayed MLI activation did not affect the ability of the PF tetanus to induce PSP potentiation (black). Repeated optogenetic activation of MLIs alone did not change PSP amplitude (gray). (I) Comparison of results across stimulus conditions (individual cells in gray; group average in black; estimated MLI firing rates for optogenetic stimuli are indicated in blue) (see Table S2 for statistics). (J) The influence of increasing levels of MLI activity on the outcome of long-term plasticity stemming from conjunctive PF + CF stimulation during the pairing protocol. (K) PF-evoked PSPs before and after plasticity induction. MLIs were photostimulated during the CF conjunctive stimulus. Responses were fit with a monoexponential function (red traces). (L) Changes in PSP decay time depend on the plasticity-inducing stimulus. See also Figures S4.

Figure 4

Optogenetic activation of CFs during behavior. (A) AAV containing bReaChES was injected in the IO of WT mice to transduce CFs. Floccular PCs were transduced with GCaMP6f using a PC-specific AAV strategy. The confocal image shows the specificity of the viral approach to target PCs. (B) An implanted optical fiber was used to deliver and collect light for photoactivating CFs and measuring PC Ca²⁺ activity. (C) Average PC Ca²⁺ response evoked by optogenetic activation of CFs with amber light (3 pulses, λ = 589, 15 ms, 8 Hz, 8 mW). Laser pulse timing is indicated by the orange tic marks; the concurrent isosbestic measurement is also shown (purple dotted trace). (D) Relationship between the number and intensity of optogenetic CF stimuli and the evoked Ca²⁺ response in PCs. Data are normalized to the response measured from a single laser pulse at 8 mW. (E) On the left, Ca²⁺ activity in PCs in quiescent mice; traces are from two different time points. On the right, PC Ca²⁺ activity during different trials of vestibular stimulation to evoke the VOR. (F) Mice were intermittently subjected to vestibular stimuli with opposite-direction visual motion, a pairing procedure that, if repeated, results in gain-increase adaptation. On the top right, average Ca²⁺ responses in PCs to vestibular stimulation alone (elicited in darkness); concurrent isosbestic measurements (purple dotted trace) show the absence of motion artifacts. On the bottom right, responses with and without visual pairing during the vestibular stimulus are superimposed (solid and dotted green traces, respectively). Vestibular stimuli are indicated in gray. (G) Comparison of the phase (relative to the end ipsiversive head movements) and amplitude of peak PC Ca²⁺ activity during the vestibular stimulus and that induced with visual-vestibular pairing that eventually leads to gain-increase adaptation. *P < 0.05; paired t-test. (H) The same mice were also intermittently subjected to pairing of vestibular stimuli with optogenetic activation of CFs in association with completion of either ipsiversive or contraversive head turns (in darkness). Average PC Ca²⁺ activity measurements during CF pairing (green trace) are shown superimposed with responses evoked by the vestibular stimulus alone (green dotted trace). (I) Summary data showing the effect of phase-specific, optogenetic CF activation on modulation of PC Ca²⁺ activity evoked by vestibular stimulation. *P < 0.05, 1-way repeated measures ANOVA with Bonferroni post-test. See also Figures S5 and S6.

Figure 5

Dual-color optogenetic control of CFs and MLIs. (A) In nNOS-ChR2 mice, AAV containing bReaChES was injected in the IO resulting in expression in floccular CFs. (B) Somatic complex spike evoked in a PC following optogenetic activation of CFs with amber light. (C) Superimposed complex spikes in the same PC evoked by either optogenetic or electrical stimulation of the CF. (D) Probability of evoking a complex spike in PCs increased with the power of amber light. (E) In a slice recording from a nNOS-ChR2 mouse, the responsiveness of a PC to optogenetic activation of bReaChES-expressing CFs to blue light alone (top) or in combination with amber light at low (middle) or high (bottom), blue-light powers. (F) With high powers, blue-light excitation of bReaChES-expressing CFs evoked complex spikes in PCs. (G) Direct optogenetic excitation of a ChR2-expressing MLI by blue light; the cell was insensitive to amber light. (H) In the absence of synaptic blockers, CF excitation with amber light drove infrequent spiking in MLIs. *P = 0.02, unpaired t-test. (I) In a PC dendritic recording, Ca²⁺ spiking induced by optogenetic CF activation in control (black) or during the coincident photo-activation of ChR2-expressing MLIs (gray). (J) The effect of MLI activation (blue light) on optogenetic CF-evoked dendritic spiking (amber light). *P = 0.02, paired t-test. Within-cell comparison in gray; group average in black. (K) Suppression of bReaChES induced Ca²⁺ spiking by ChR2 activation of MLIs was abolished by the GABA_A receptor blocker SR 95531 (20 μM). See also Figures S5 and S7.

Figure 6

Alteration of CF-evoked motor learning by MLI activation. (A) Optical fibers targeting both flocculi were implanted into nNOS-ChR2 mice. CFs expressed bReaChES by AAV-mediated transduction of the IO. (B) Over the course of training, floccular CFs were activated with amber light in association with completion of ipsiversive head turns during sinusoidal vestibular stimulation. In alternative training sessions, CFs were activated coincident with MLIs by concomitant pulses of blue light. (C) Training paradigm with associative pairing of optogenetic activity with contraversive head movements. (D) Timing of laser pulses for CF activation relative to the vestibular stimulus (gray) for both flocculi during ipsiversive optogenetic training. (E) Average VOR-evoked eye movements measured before and after training. The traces were normalized to that of time-matched, control responses recorded during sessions of vestibular stimulation alone. (F) Dual-color optogenetic stimulation of CFs and MLIs during training. (G) Average VOR-evoked eye movements from the same mouse shown above but with CF activation coincident with that of MLIs during training. (H) Summary plots showing the effect of MLI inhibition on artificial motor learning produced by CF stimulation in association ipsiversive movements. VOR performance was measured in the dark to head rotation at the beginning and ending of training as well as after holding mice in the dark for 120 min during the post-training recovery period. A training session of vestibular stimulation alone controlled for darkness-induced habituation. In the first plot, motor learning is quantified as the change in VOR amplitude after training relative to baseline performance (ΔVOR Gain). In the second plot, the subtracted difference of sessions with and without optogenetic stimulation shows the isolated effect of CF activation on VOR performance. *P < 0.05, 2-way repeated measures ANOVA with Bonferroni post-test. (I) Results obtained for training with CF activation in association with the end of contraversive head turns. (J) Concomitant pulses of amber and blue light in association with the completion of ipsiversive movements had no effect on VOR performance, relative to control measurements, in c-kit::Cre mice injected with GFP-containing AAVs in place of optogenetic actuators. (K) Similarly, pairing optogenetic MLI activation with blue light pulses with the end of ipsiversive head movements did not change VOR performance beyond darkness-induced habituation. See also Figures S8 and S9.

Figure 7

Optogenetic activation of MLIs affects the outcome of VOR learning induced by retinal slip. (A) MLIs in c-kit::Cre mice were transduced with ChR2; optical fibers targeted both flocculi. Shown below, opposite-direction visual-vestibular pairing with optogenetic activation of MLIs. (B) Control sessions of training consisted of pairing head rotation (solid gray) and opposite-direction visual motion (dotted gray) without optogenetic activity. (C) Average VOR-evoked eye movements before and after training. (D) During opposite-direction visual-vestibular pairing that normally leads to gain-increase adaptation, laser pulses used to activate MLIs (in blue) occurred with the peak phase of ipsiversive head velocity during sinusoidal stimulation. (E) VOR-evoked eye movements from the same mouse as above, but with optogenetic activation of MLIs during the pairing procedure. (F) Mice were retrained using a lower power laser stimulus for activating MLIs. (G) Comparison of average VOR eye movements before and after visual-vestibular pairing with low-power laser stimuli. (H) The effect of optogenetic-induced MLI activity on opposite-direction visual-vestibular pairing on VOR learning. Two different stimulus intensities were used during pairing (HP and LP; high and low power, respectively; determined for each mouse, see STAR Methods). *P < 0.05, 2-way repeated measures ANOVA with Bonferroni post-test. (I) Photostimulation of floccular MLIs in c-kit::Cre mice injected with AAV containing GFP occurred following ipsiversive head movements during opposite-direction visual-vestibular pairing. (J) Summary results from a second cohort of animals showing the phase-specific influence of MLI activity on learning during gain-increase training. *P < 0.05; 2-way repeated measures ANOVA with Bonferroni post-test. (K) Optogenetic activation of MLIs had no effect on gain-decrease learning produced during vestibular pairing with same-direction visual motion. See also Figures S9.