Cerebellar Purkinje cell activity modulates aggressive behavior

Abstract

Although the cerebellum is traditionally associated with balance and motor function, it also plays wider roles in affective and cognitive behaviors. Evidence suggests that the cerebellar vermis may regulate aggressive behavior, though the cerebellar circuits and patterns of activity that influence aggression remain unclear. We used optogenetic methods to bidirectionally modulate the activity of spatially-delineated cerebellar Purkinje cells to evaluate the impact on aggression in mice. Increasing Purkinje cell activity in the vermis significantly reduced the frequency of attacks in a resident-intruder assay. Reduced aggression was not a consequence of impaired motor function, because optogenetic stimulation did not alter motor performance. In complementary experiments, optogenetic inhibition of Purkinje cells in the vermis increased the frequency of attacks. These results suggest Purkinje cell activity in the cerebellar vermis regulates aggression, and further support the importance of the cerebellum in driving affective behaviors that could contribute to neurological disorders.

Keywords: aggression; cerebellum; mouse; neuroscience; optogenetics.

Figure 1.. Optogenetic control of Purkinje cell activity.

(a) Recording schematic for in vitro recording and optogenetic stimulation. (b) Firing rates elicited by ChR2 stimulation at different intensities (0.5 ms flashes, 50 Hz, n = 6). (c) Inhibition of PCs at different light intensities (sustained illumination, n = 4). (d) Schematic for recording PC activity during in vivo stimulation through a chronic fiber optic implant. (e) Top: Representative single unit recording during ChR2 stimulation and (bottom) average firing rate (n = 6). (f) Single unit recordings during halorhodopsin stimulation (n = 6). Scale bars, 100 ms (horizontal), 20 mV (vertical, (b,c), 0.2 mV (vertical, (e,f). (g) Schematic for recording DCN activity during in vivo stimulation in awake animals. (h) Averaged normalized firing rate of DCN neurons during ChR2 stimulation of vermal PCs (n = 4). (i) Averaged firing of DCN neurons during halorhodopsin-mediated inhibition of vermal PCs (n = 11). Average data in all figures represents mean ± SEM.

Figure 1—figure supplement 1.. Fluorescent images of ChR2-YFP expression in (a) a whole brain and (b) a sagittal cerebellar section from a PCP2-cre::Ai32 mouse, with lobules V-X labeled.

Scale bar = 1 mm.

Figure 1—figure supplement 2.. Millisecond-scale analysis of firing rates induced by in vivo stimulation in PCP2-cre::Ai32 animals.

(a) Peristimulus time histogram (0.5 ms bins) of Purkinje cell firing induced by optical stimulation (20 Hz, 5 ms flashes). Inset shows PC responses from the beginning of light onset. (b) As in (a), but for DCN recordings. These recordings represent the same data shown in Figure 1. Individual units are in gray, the mean ± SEM is displayed in blue.

Figure 2.. Manipulating vermal Purkinje cell activity does not affect coordination, locomotion or anxiety.

(a) Time to fall for rotarod assays during ChR2-mediated excitation. Mice were tested in two consecutive trials, and randomly assigned to receive stimulation during either the first or second trial. (n = 13) (b) Same as in (a), but for halorhodopsin-mediated inhibition of vermal PC firing. (n = 16) (c) Schematic for open field assay with optogenetic stimulation. (d) Representative tracking data throughout alternating periods with stimulation (blue) and without (gray). (e) Total distance traveled and time spent in the center of the arena for mice during epochs with and without stimulation of vermal PCs (n = 13). (f, g), Same as d), (e) but for halorhodopsin-mediated inhibition of vermal PCs (n = 17).

Figure 2—figure supplement 1.. Manipulating vermal Purkinje cell firing does not affect locomotion.

Animal speed in an open field, averaged across three consecutive epochs of stimulation for (a) PCP2-cre:ChR2 (n = 13) and (b) PCP2-cre:Halorhodopsin (n = 17) animals.

Figure 3.. Bidirectional control of aggression by optogenetic modulation of vermal Purkinje cell activity.

(a) Schematic for resident-intruder assays with optogenetic stimulation. (b) Representative scoring of social and aggressive behaviors. (c) Average number of attacks and social encounters during ChR2 assays (31 assays from 12 residents). (d) Peristimulus time histogram of the probability of attacks (top) and social investigations (bottom) within 10 s bins during epochs with and without ChR2-mediated excitation of vermal PCs. (e–g), Same as in (b–d), but during Halorhodopsin-mediated inhibition of vermal PCs (34 assays from 15 residents).

Figure 3—figure supplement 1.. Statistical significance of behavioral effects.

(a) Comparison of light-induced behavioral changes between experimental groups, for attack rates (left) and social investigations (right). Pairwise comparisons that were not significant (p>0.05) have not been labeled. (b) P values for light on/light off differences within experimental groups for rates of attack and social interactions. Data was first assessed for normality (Shapiro-Wilk test). Significance was assessed using two-tailed Student’s t-tests for data that met the criteria of normality, or Wilcoxon-Mann-Whitney tests for data that did not (see Materials and methods for details).

Figure 3—figure supplement 2.. Effect of manipulating vermal Purkinje cell activity on grooming, tail-rattling, and aggressive lunging during resident-intruder assays.

(a) Optogenetic stimulation significantly increased grooming behaviors for both ChR2-expressing mice (31 assays in 12 mice, p=0.02, t-test) and (b) halorhodopsin-expressing mice (34 assays in 15 mice, p=0.01, t-test).

Figure 3—figure supplement 3.. Aggression is not affected by light alone or by stimulating Crus II Purkinje cells.

(a) (Top) Schematic for optical stimulation over vermis in wildtype mice lacking channelrhodopsin. (Middle) Representative sequence of social and aggressive behaviors. (Bottom) Average number of attacks and social investigations during ON/OFF epochs of laser stimulation. (b) Same as in (a), but for PCP2-Cre::ChR2-YFP mice implanted with optical fibers over the lateral Crus II region of the cerebellum.

Author response image 1.