Role of the dorsal medial habenula in the regulation of voluntary activity, motor function, hedonic state, and primary reinforcement

Abstract

The habenular complex in the epithalamus consists of distinct regions with diverse neuronal populations. Past studies have suggested a role for the habenula in voluntary exercise motivation and reinforcement of intracranial self-stimulation but have not assigned these effects to specific habenula subnuclei. Here, we have developed a genetic model in which neurons of the dorsal medial habenula (dMHb) are developmentally eliminated, via tissue-specific deletion of the transcription factor Pou4f1 (Brn3a). Mice with dMHb lesions perform poorly in motivation-based locomotor behaviors, such as voluntary wheel running and the accelerating rotarod, but show only minor abnormalities in gait and balance and exhibit normal levels of basal locomotion. These mice also show deficits in sucrose preference, but not in the forced swim test, two measures of depression-related phenotypes in rodents. We have also used Cre recombinase-mediated expression of channelrhodopsin-2 and halorhodopsin to activate dMHb neurons or silence their output in freely moving mice, respectively. Optical activation of the dMHb in vivo supports intracranial self-stimulation, showing that dMHb activity is intrinsically reinforcing, whereas optical silencing of dMHb outputs is aversive. Together, our findings demonstrate that the dMHb is involved in exercise motivation and the regulation of hedonic state, and is part of an intrinsic reinforcement circuit.

Keywords: exercise motivation; interpeduncular nucleus; intracranial self-stimulation; medial habenula; optogenetics.

Figure 1.

Structure of the Pou4f1^flox allele and expression of Brn3a and a Syt6^Cre-induced transgene in Brn3a-expressing brainstem nuclei. A, Schematic of the Pou4f1^flox allele structure. B, Habenula expression of ZsGreen from Ai6, an inducible reporter line, driven by Syt6^Cre. Syt6^Cre mice were interbred with mice bearing the Ai6 allele, which allows conditional expression of the fluorescent reporter ZsGreen from the Gt(Rosa)26^Sor locus (Madisen et al., 2010). Neurons in the vMHb express acetylcholine and its synthesizing enzyme, ChAT, is used to distinguish this subnucleus from the dMHb. C–I, Syt6^Cre/Ai6 mice were examined for the expression of Brn3a protein and ZsGreen fluorescence in brain nuclei known to express Brn3a (Fedtsova and Turner, 1995). Brn3a immunofluorescence (red) is nuclear, whereas ZsGreen fluorescence is cytoplasmic, and also labels efferent fibers from areas of strong expression. Cellular colocalization thus produces a green periphery and yellow center. C, Syt6^Cre-induced ZsGreen is strongly coexpressed with Brn3a in the dMHb but not the vMHb. A subset of cells in the medial part of the LHb also express ZsGreen, and occasional coexpression with Brn3a is observed (example, arrow). D, Rostral IP. Numerous cells express ZsGreen and Brn3a, and sometimes overlap, but cellular colocalization is rarely observed. A rare example of possible colocalization is marked by the arrow. Note that the entire cell soma of most cells expressing ZsGreen is smaller than the nucleus of most cells expressing Brn3a. E, Caudal IP. Few Brn3a-immunoreactive neurons are seen at this level. ZsGreen labeling is seen in afferent IPL fibers from the dMHb. Specificity of this afferent projection for IPL (not IPC) confirms that Syt6^Cre activated gene expression specific for the dMHb, as defined by its principal target of innervation, IPL. F, Midbrain, ventral expression: colocalization of ZsGreen and Brn3a is observed in <10% of the cells of the Edinger–Westphal nucleus; no colocalization is observed in the red nucleus. G, Midbrain, superior colliculus expression. No colocalization of Brn3a and ZsGreen is observed in this region. H, Hindbrain. A single neuron with colocalized signal appears in the nucleus ambiguus (arrow). I, Hindbrain. No colocalization appears in the inferior olive. Amb, Nucleus ambiguus; EW, Edinger–Westphal nucleus; fr, fasciculus retroflexus; IO, inferior olive; IPC, interpeduncular nucleus, caudal; IPL, interpeduncular nucleus, lateral; IPR, interpeduncular nucleus, rostral; LHb, lateral habenula; mlf, medial longitudinal fasciculus; R, red nucleus; RLi, rostral linear nucleus raphe; SC, superior colliculus (of midbrain); SM, striae medularis; vMHb, medial habenula, ventral.

Figure 2.

Characterization of the Syt6^Cre/Pou4f1^flox dMHb^CKO mouse model. A, B, Brn3a nuclear immunostaining and a lacZ tracer targeted to the Pou4f1 locus are used to show the extent of the dMHb lesion in dMHb^CKO mice. Control mice have the genotype Pou4f1^flox/tlacZ and dMHb^CKO mice Syt6^Cre/Pou4f1^flox/tlacZ. The lacZ gene product β-gal allows the identification of neurons normally fated to express Brn3a in the absence of Brn3a protein. Nuclear Brn3a expression is reduced even in the surviving neurons in the dMHb of the dMHb^CKO mice (B′). No obvious change is observed in Brn3a expression in the LHb, consistent with the infrequent colocalization of Syt6^Cre and Brn3a in this part of the habenula (Fig. 1C). C, D, Neurons of the vMHb, distinguished by ChAT expression, are intact in both the control (C) and dMHb^CKO mice (D). E, Comparison of the area of the dMHb in the coronal plane of section along the entire rostrocaudal extent of the MHb in control and dMHb^CKO mice. Both the left and right dMHb is significantly reduced in the dMHb^CKO mice compared with control mice (n = 3 control and n = 7 dMHb^CKO mice). F, Measurement of the area/extent of ablation in the entire behavioral cohort of control and dMHb^CKO mice used in behavioral analyses. Sections were measured at two rostrocaudal levels: the rostral/central MHb at bregma −1.6 mm and the caudal MHb at bregma −1.9 mm, corresponding to sections 6 and 9 in E. Immunostaining was used to define the dMHb and vMHb compartments as in A–D. LR, Left/rostral; RR, right/rostral; LC, left/caudal; RC, right/caudal. The loss of dMHb neurons in dMHb^CKO mice was highly consistent and symmetrical (n = 9 control and n = 14 dMHb^CKO mice). G–J, Characteristic expression patterns of habenula-enriched transcripts. SP/Tac1 and Kcnip1 transcripts are restricted to the dMHb and vMHb, respectively, in control mice. The area of the region expressing SP/Tac1 mRNA is greatly reduced, whereas the area of Kcnip1 expression is not affected in the dMHb^CKO mice. LHb, Lateral habenula.

Figure 3.

WRA of dMHb^CKO mice. Voluntary WRA was measured under a 12:12 LD cycle. A, WRA in representative control and dMHb^CKO mice. WRA is displayed as double-plotted actograms with wheel turns as black bars on the vertical axis plotted against the 48 h period. White and gray areas represent time of lights-on and darkness, respectively. B, Summary of WRA for control and dMHb^CKO mice over a 24 h day. dMHb^CKO mice showed significantly less WRA throughout the day, especially during the first of the night (n = 11 and n = 14 for control and dMHb^CKO mice, respectively). Eleven days of WRA were averaged for each of the control and dMHb^CKO mice, with the exception for one control mouse where 7 d of averaged WRA was used. Zeitgeber time 12 (ZT12) corresponds to time of lights-off for a 12:12 LD cycle, and white and black bars below represent time of lights-on and darkness, respectively. Values for ZT0 are replotted at the end for easier visualization. N = 11 control and N = 14 dMHb^CKO mice. ****p < 0.0001 (ZT12 to 16), significant difference between genotypes. *p = 0.024 (ZT17), significant difference between genotypes.

Figure 4.

Rotarod performance, locomotion, and gait analysis in dMHb^CKO mice. A, Accelerating rotarod latency to fall times for dMHb^CKO mice over four trials on day 1 of testing compared with control mice, showing a significant relative deficit for dMHb^CKO mice in this test. *p = 0.02, significant difference between genotypes for those trials. **p = 0.0013, significant difference between genotypes for those trials. ****p < 0.0001, significant difference between genotypes for those trials. B, Average latency to fall time in the rotarod for day 1 of testing (the lowest of the four values for each mouse was discarded before averaging). ****p < 0.0001. C, Correlation between latency to fall in the rotarod for four trials on day 1 versus day 2 of testing. Performance on day 1 is predicative of that on day 2. r = 0.66, p = 0.0003. D, Total distance traveled in the open field. No difference was observed for control and dMHb^CKO mice during the 30 min testing period. E, Distance traveled in the home cage (PhenoTyper; Noldus Information Technology) tracked over a 24 h day, following 2 d of acclimatization. Both genotypes were more active during the night, as expected, and no differences were observed between genotypes. White and black bars below represent time of lights-on and darkness, respectively. The same cohort of mice was used for experiments in A–E (n = 11 control and n = 14 dMHb^CKO mice). F, G, Balance beam test. The test consisted of five trials on each of four successively smaller round beams, which were video recorded for analysis. The last three completed trials for each beam diameter were used to compute the average transit time (F), and the average number of faults in which a rear paw lost contact with the beam (G) for each subject. Overall, the dMHb^CKO mice took longer to traverse the bars and committed more faults. N = 9 control and N = 10 dMHb^CKO mice.

Figure 5.

dMHb^CKO mice in models of hedonic state and depression. A, Results for the sucrose preference test for each mouse averaged over 4 d of testing. dMHb^CKO mice showed a significantly less preference for sucrose solution compared with control mice. *p = 0.043. B, Correlation of the results for individual subjects for the two trials of the sucrose preference test. The average of 4 d of testing for each mouse is shown. Individual preferences on Trial 1 were retained on Trial 2, and there was a high test–retest correlation (r = 0.82, p < 0.0001). N = 11 control and N = 14 dMHb^CKO mice. C, FST results for dMHb^CKO and control mice. Both genotypes were immobile for a similar amount of time during the 15 min test. N = 7 control and N = 9 dMHb^CKO mice.

Figure 6.

An optogenetic model for dMHb function and the effect of dMHb stimulation on locomotion. A, Transgenic strategy for conditional expression of ChR2-EYFP in the dMHb. B, Syt6^Cre-driven expression of ChR2-EYFP from the Ai32 reporter in the dMHb (top) and IPL (bottom). EYFP expression is also seen in some scattered cells, which have the appearance of astrocytes (arrows). EYFP-labeled terminal fibers are also observed in the lateral posterior thalamic nucleus (LP); these are projections from layer 5/6 cortical neurons. No EYFP-labeled fibers or cell bodies are observed in the paraventricular thalamus immediately ventral to the IP. No labeled fibers are observed in the VTA or other tegmental areas adjacent to the IP. White rectangles represent the targeted position and size of the implanted fiber optic cannula. Scale bar: low-power views, 200 μm; high-power views, 100 μm. C, Loose-seal cell-attached recording showing light-induced action potentials in the dMHb in a dMHb^ChR2 mouse. Irregular spontaneous firing at ∼3 Hz is observed at baseline. Application of 447 nm blue light pulses (20 ms, 2.0 mW/mm²) at 20 Hz elicits action potentials entrained to the pulse frequency. The maximum stimulation rate for which the cell shown would generate 1:1 action potentials was ∼20 Hz; at higher stimulation frequencies, some light pulses failed to elicit spikes. At the end of a 10 s interval of pulsed light delivery, a period of suppressed firing was observed (∼7 s, data not shown), followed by a gradual resumption of firing at the baseline rate. D, Bilateral optical cannula for implantation in the dMHb. Cannula consists of a zirconia ferrule (1) and a 0.1 mm polyimide-coated optical fiber (2,5), a polyether ether ketone (PEEK) insert (3), and an acetal (Delrin) guide (4). The bracketed area is covered with a stainless steel protective sleeve before implantation. E, Optical fiber placement in the habenula for in vivo optogenetic-stimulated control and dMHb^ChR2 mice. Blue represents fiber placement in control mice; red represents placement in dMHb^ChR2 mice. Fiber termini are shown on the level of a standard anatomical map (Paxinos and Franklin, 2001) closest to their rostrocaudal position at bregma −1.46, 1.58, 1.70, or 1.82 mm. Nearly all of the cannulas thus were positioned within ±0.2 mm of the intended coordinates at bregma −1.6 mm. Connected dots indicate the probable ventral termini of the optical fibers from each case. In some cases, the right and left optical fibers mapped most accurately to different planes of section and are shown by disconnected dots. If the cannula track could not be followed for the entire length of the optical fiber, the most ventral position and the direction of the cannula track observed are indicated by an arrow. In all cases, the optical fibers were intact and transmitted light efficiently when examined postmortem after the experimental protocol. Scale bar, 0.5 mm. F, G, Distance traveled by control and dMHb^ChR2 mice during a 10 min open field trial with intermittent laser stimulation. + (shaded), 30 s laser-on periods; −, 30 s laser-off periods. Mice were exposed to the open field and to laser stimulation before testing began to minimize effects of novelty. H, Summary of open field locomotor data. The distance traveled is summed across all laser-on and laser-off epochs for each mouse. In a within-subjects comparison, the dMHb^ChR2 mice show significantly greater distance traveled during the laser-on than the laser-off periods, whereas control mice exhibit no difference. **p < 0.01, significant difference between laser on/off periods for dMHb^ChR2 mice. N = 7 control and N = 11 dMHb^ChR2 mice. cp, cerebral peduncle, basal part; DG, Dentate gyrus; fr, fasciculus retroflexus; IPC, interpeduncular nucleus, caudal; IPL, interpeduncular nucleus, lateral; IPR, interpeduncular nucleus, rostral; LHb, lateral habenula; LP, lateral posterior nucleus of thalamus; ml, medial lemniscus; PV, paraventricular nucleus of thalamus; VTA, ventral tegmental area.

Figure 7.

Optogenetic activation of dMHb neurons in vivo mediates primary reinforcement. A, Structure of the ICSS trials. Each ¼ turn of the active response wheel is scored as an event. When the reward condition is met, a 4 s train of 25 ms light pulses is delivered at a frequency of 20 Hz, followed by a 2 s time out during which events do not count toward a reward. Turns of the inactive wheel reset the event counter. B, Stepped fixed-ratio reinforcement structure of the ICSS sessions. Each 45 min session is initiated at a fixed event:reinforcement ratio of 1:1 until five rewards have been earned, then 5:1 until 10 total rewards have been earned, then 25:1 thereafter. C, Response data for a representative dMHb^ChR2 mouse across nine sessions; three sessions without laser stimulation (0), three sessions with laser stimulation evoked by turning the initially nonpreferred wheel (NP), and three crossover sessions of stimulation of the initially preferred wheel (P). D, Response data for a representative control mouse. E, Summary of total wheel turns per session (sum of both wheels) for cohorts of dMHb^ChR2 and control mice. A trend toward increased wheel turning by dMHb^ChR2 mice relative to controls is observed on the first stimulated session (session 4) and is significant by session 6. **p < 0.01, significant difference between genotypes. *p < 0.05, significant difference between genotypes. F, Average number of rewards (light pulse trains) earned per session by dMHb^ChR2 and control mice. A dip is observed in the rewards earned on session 7 because of a large number of incorrect responses following the crossover of the rewarded side. The plateau observed in the control mice at ∼10 rewards is due in part to the stepped FR schedule, in which additional effort is required for the 11th and subsequent rewards. ***p < 0.001, significant difference between genotypes. **p < 0.01, significant difference between genotypes. G, Within-subjects comparison for mean wheel turns on the stimulated wheel (NP during sessions 4–6 and P during sessions 7–9) and nonstimulated wheel (P during sessions 4–6 and NP during sessions 7–9) across the 6 stimulated sessions. The baseline sessions are not included in the analysis because no light stimulation was administered. During the first three stimulations on the NP wheel, a trend was observed toward greater turning of the reward-associated wheel in sessions 4 and 5, which became significant by session 6. In the crossover sessions (sessions 7–9), mice rapidly shifted their response to the P wheel. **p < 0.01, significant difference between stimulation conditions. *p < 0.05, significant difference between stimulation conditions. H, Relative preference for the NP wheel across nine trials, expressed as NP wheel turns/total wheel turns × 100. The dashed line indicates 50% or no preference. dMHb^ChR2 mice showed positive preference for NP wheel when it was the reward-associated wheel (>50%) and negative preference (<50%) for NP when it was inactive. Preference differences from control mice were significant by the last session with each reward-associated wheel (sessions 6 and 9). **p < 0.01, significant difference between genotypes. *p < 0.05, significant difference between genotypes. N = 10 control mice and N = 12 dMHb^ChR2 mice.

Figure 8.

Inhibition of basal dMHb signaling to the IP induces acute place aversion. For in vivo experiments, a fiber optic was placed adjacent to the IP of control and dMHb^NpHR mice, and 640 nm light was delivered when the mice appeared in one of the two main chambers of the three-compartment box. A, Loose-seal cell-attached recording of the dMHb in an acute slice preparation from a dMHb^NpHR mouse. Irregular tonic firing at ∼3 Hz is observed at baseline. Application of 640 nm red light (46 mW/mm²) for 1 s (red bars) produces effective silencing of action potentials. A single escaped action potential in the second period of inhibition is indicated by an arrow. Attempts at longer periods of continuous silencing were accompanied by more escape firing due to the strong pacemaker properties of these neurons (data not shown). B, Optical cannula placement in the IP. Single optical fibers (100 μm diameter, 5 mm length) were surgically implanted and secured to the skull as described in Materials and Methods. The targeted coordinates for the cannula tip were as follows: rostrocaudal, bregma −3.52 mm; dorsoventral, −4.29 mm ventral to lambda-bregma line; lateral, at midline. Blue represents the termini of the optical cannula tracks in control mice; red represents dMHb^NpHR mice (on a standard anatomical map; Paxinos and Franklin, 2001). One track with an indeterminate end is depicted with an arrow. C, Open field locomotion of control and dMHb^NpHR mice. After a 10 min recording of baseline locomotion, the IP was illuminated intermittently for 10 min, consisting of 30 s laser-on periods and 30 s laser-off periods (640 nm, 8 mW). A comparison of locomotion for the entire baseline and entire period of illumination is shown. Locomotion was not significantly different between the acute laser on and off periods in either genotype, but locomotion decreased over the course of testing in dMHb^NpHR mice. **p < 0.001, significant difference between baseline and testing for dMHb^NpHR mice. D, Examples of locomotor activity recordings in the place preference/aversion assay for a single control and dMHb^NpHR mouse. E, Time spent in the laser-active compartment across three time intervals of the 15 min trial, demonstrating the development of aversion in dMHb^NpHR mice. Dashed line indicates equal occupancy in the two large chambers. Significant aversion to the laser-active compartment was observed in dMHb^NpHR mice after 5 min. *p < 0.05, significant difference between genotypes. F, Distance traveled in the three-compartment box during preference/aversion assay. N = 4 control and N = 6 dMHb^NpHR mice.