|Home | About | Journals | Submit | Contact Us | Français|
Although cortico-striato-thalamo-cortical (CSTC) circuit dysregulation is correlated with obsessive compulsive disorder (OCD), causation cannot be tested in humans. We used optogenetics in mice to simulate CSTC hyperactivation observed in OCD patients. Whereas acute orbitofrontal cortex (OFC)–ventromedial striatum (VMS) stimulation did not produce repetitive behaviors, repeated hyperactivation over multiple days generated a progressive increase in grooming, a mouse behavior related to OCD. Increased grooming persisted for 2 weeks after stimulation cessation. The grooming increase was temporally coupled with a progressive increase in light-evoked firing of postsynaptic VMS cells. Both increased grooming and evoked firing were reversed by chronic fluoxetine, a first-line OCD treatment. Brief but repeated episodes of abnormal circuit activity may thus set the stage for the development of persistent psychopathology.
OCD is characterized by intrusive distressing thoughts (obsessions) and/or repetitive mental or behavioral acts (compulsions) and is a leading cause of illness-related disability (1, 2). Although the pathophysiology underlying OCD is unclear, multiple lines of evidence implicate dysregulation within cortico-striato-thalamo-cortical (CSTC) circuits (3-6). Specifically, functional imaging studies suggest that hyperactivity in orbitofrontal cortex (OFC) and ventromedial striatum (VMS) is associated with OCD pathology (5, 7, 8). Furthermore, successful treatments are associated with reductions in hyperactivity (9, 10). However, it is not known if OFC-VMS hyperactivity can directly cause OCD symptoms, because increased activity could represent adaptive, homeostatic, or unrelated processes compensating for other primary abnormalities. We therefore used an optogenetic strategy to directly test whether hyperstimulation of glutamatergic OFC-VMS projections leads to OCD-like behaviors in mice.
A Cre-inducible adenovirus-associated vector (AAV) carrying the gene encoding channelrhodopsin (ChR2) fused to enhanced yellow fluorescent protein (EYFP) [pAAV-Ef1a-DIO-ChR2 (H134R)-EYFP; referred to as DIO-ChR2] (11) was stereotactically injected into OFC of EMX-Cre transgenic mice to ensure specific ChR2 expression in cortical glutamatergic neurons (Fig. 1A) (12). Cortical Cre expression led to sustained expression of ChR2-EYFP (Fig. 1B). Unilateral 473-nm stimulation through chronic fiber-optic implants in OFC yielded lateralized increased activation of the immediate early gene c-fos (P < 0.009) (Fig. 1, C and D), which demonstrated in vivo cellular activation by laser stimulation. Two weeks postinjection, EYFP staining was seen in OFC cell bodies and axons projecting to VMS (Fig. 1E), which indicated targeting of OFC-VMS projections. In vitro recordings in cortico-striatal slices demonstrated VMS field responses after 473-nm laser stimulation of OFC axon terminals in striatum (Fig. 1F). To verify adequate stimulation of ChR2-expressing OFC-VMS terminals in vivo, we implanted stereo opto-electrodes (optrodes) into VMS that permit combined fiber-optic stimulation and 32-channel simultaneous recording of multiple single units (Fig. 1G). In awake behaving mice, in vivo recordings demonstrated robust VMS field responses after 473-nm laser stimulation of OFC axon terminals in striatum (Fig. 1, H and I), which showed feasibility of activation of specific cortical-VMS projections.
Because patients with OCD have hyperactivity in OFC-striatal circuits (3-6), we predicted that direct elevation of OFC-VMS activity would lead to increases in OCD-related behaviors including grooming, anxiety, and prepulse inhibition (PPI) deficits (13). We injected DIO-ChR2 into the left OFC of EMX-Cre mice and implanted fiber-optic probes unilaterally in left VMS (Fig. 2A). After waiting 3 to 4 weeks for surgical recovery and stable viral expression, we habituated mice to the open field and fiber-optic stimulation apparatus. We then repeatedly elevated activity in OFC-VMS projections by stimulating for 5 min at 10Hz for five consecutive days (10 ms, 1 to 5 mW) (14). Grooming behavior was recorded with digital video and scored by blind raters for 5 min before (Pre), during (Stim), and after (Post) stimulation (Fig. 2B). Whereas acute OFC-VMS stimulation did not produce grooming, a small but significant progressive increase in grooming time was noted during the prestimulation period on consecutive days (Fig. 2C) [repeated measures analysis of variance (ANOVA), main effect: P < 0.048; F = 4.43; Fisher’s protected least significant difference (PLSD): for day 3, P < 0.03; for day 5, P < 0.047]. Because the prestimulation measurement on days 2 to 5 served as a 24-hour time stamp for effects of stimulation the day before, this suggested that repeated stimulation led to chronic circuit changes that ultimately resulted in sustained, stimulation-independent OCD-like behavior. Although it is possible that stress from handling contributed to the grooming increase in the prestimulation period, stress was minimized by habituation to fiber-optic tethering daily for a week before data collection and was identical for controls and ChR2+ mice. To resolve the time-course of the grooming increase, we examined a new cohort an hour after stimulation (Groom1 hour post) (Fig. 2D). We observed a dramatic progressive increase in grooming over consecutive days using this measure (main effect: P < 0.02; F = 7.32) (Fig. 2E). Although total grooming time increased, there was not a significant increase in stereotyped syntactic grooming chains in ChR2+ animals on day 5 of stimulation (table S1). No differences in grooming time were observed between controls and ChR2+ animals on day 1 of stimulation, which indicated that ChR2 expression without laser stimulation did not lead to an increase in grooming. Notably, increased grooming persisted even in the absence of stimulation up to 2 weeks later (P < 0.03) (Fig. 2F).
Acute OFC-VMS stimulation led immediately to a large but transient increase in locomotion compared with controls; no differences were observed pre- or poststimulation (fig. S1A). To ensure that increased grooming was not simply a consequence of increased locomotion, we injected DIO-ChR2 and implanted fiber optics in motor cortex (M2) of EMX-Cre mice (fig. S1B). The 5-day stimulation paradigm described above led to increased locomotion (fig. S1C) but not to increased grooming (fig. S1D), which suggested that increased grooming was not simply a side effect of overall increased activity. To determine the specificity of OFC-VMS pathway hyperactivation in induction of persistent grooming, we injected AAV-ChR2 into infralimbic and prelimbic cortex (IL/PrL) (fig. S2A) and stimulated IL/PrL–VMS projections using our 5-day stimulation paradigm. Repeated stimulation of IL/PrL–VMS projections did not lead to a progressive increase in grooming behavior (fig. S2, B to D).
Other OCD-associated behavioral measures were tested after completion of the 5-day stimulation paradigm. No differences were seen in PPI (13) or anxiety levels (in open field and elevated plus maze) compared with testing before stimulation (fig. S3); in addition, no changes in open field anxiety were observed during acute stimulation (fig. S4). Together, these results suggest that repeated stimulation of OFC-VMS projections led to specific induction of repetitive behavior.
We next examined electrophysiologic changes correlated with the progressive grooming increase using VMS stereo-optrodes (Fig. 3A and fig. S5). In awake behaving mice, recordings of multiple single units in the VMS were obtained during the 15-min stimulation protocol and 1 hour post grooming assessment (Fig. 3B). Light-evoked responses were observed in individual units in response to 10 Hz stimuli (Stim) or 0.1 Hz probe pulses (used to measure light-evoked activity during pre-, post-, and 1 hour poststimulation) over 5 days of repeated stimulation. Cells displayed a range of responses to light pulses, including activation (Fig. 3C), suppression (Fig. 3D), and no effect (Fig. 3E); we therefore used each cell’s stimulation-induced change in firing rate (expressed as a Z-score; see supplementary methods) to assess responsiveness to afferent stimulation. Across 5 days of stimulation, mean responses increased both during stimulation and 1 hour poststimulation (Fig. 3F; R = 0.21 and 0.28; P < 0.002 and < 0.001, respectively). Z-scores were greater on day 5 than on day 1, which indicated increased evoked firing (at 10 Hz: P < 0.02; at 0.1 Hz: P < 0.004). Thus, repeated hyperstimulation led to a marked progressive increase in light-evoked firing paralleling the increase in repetitive behavior.
Finally, we determined whether a medication regimen used to treat OCD would reverse the increases in repetitive behavior and evoked VMS activity. After 7-day grooming induction, we initiated fluoxetine treatment (18mg/kg body weight per day) while continuing daily stimulation (Fig. 4A). Fluoxetine was chosen because serotonin reuptake inhibitors (SRIs) are the only proven monotherapy for OCD (15). Although there was no effect of fluoxetine after 1 week, 2 weeks of treatment led to a reversal of grooming behavior to control levels (Fig. 4B) (main effect: P < 0.009, F = 9.53; Fisher’s PLSD: baseline versus week 2, P < 0.003). This delayed response is consistent with the delayed onset of effective SRI treatment in OCD patients. We also repeated this experiment using a vehicle control group (Fig. 4C). Again, 2 weeks of fluoxetine led to reversal of induced grooming (Fig. 4D). Moreover, in a separate cohort of stereo-optrode–implanted animals, the increase in light-evoked activity induced by repeated 10 Hz stimulation was normalized after chronic fluoxetine (Fig. 4, E and F).
Repeated hyperactivation of OFC-VMS projections generates a progressive increase in grooming, temporally linked to a cumulative increase in VMS light-evoked firing. Acute stimulation of the OFC-VMS pathway was not sufficient to produce OCD-relevant excessive grooming (3, 16-18). The behavioral change was persistent, becoming stimulation-independent within 6 days. Although classic theories suggest that abnormal repetitive behaviors, including OCD symptoms, directly result from hyperactivity in CSTC loops (19-21), causation has been difficult to prove. Though genetic and pharmacologic manipulations of norepinephrine and dopamine can lead to transient increases in repetitive behaviors (22), the interventions were not limited to specific circuits, and associated electrophysiologic changes were observed in multiple brain regions. Our optogenetic system permits activation of specific cortico-striatal circuits and genetic definition of the activated cell-type as cortical glutamatergic projection neurons.
Our in vivo electrophysiology data suggest a circuit-based mechanism for establishment of repetitive behaviors. Repeated hyperstimulation led to a marked progressive increase in light-evoked firing paralleling the increase in grooming, suggesting plasticity at OFC-VMS synapses that builds over consecutive days. We speculate that brief episodes of light-induced activity lead to long-lasting changes that prime OFC-VMS synapses, decreasing the activation threshold during subsequent bouts of stimulation. In turn, increased activity at OFC-VMS synapses may transmit information through the CSTC circuit (23-25) and lead to multiple downstream events that ultimately reinforce repetitive behaviors, including (i) plasticity in downstream structures such as thalamus and prefrontal cortex (26), and (ii) increased motivational saliency mediated by the ventral tegmental area (22). This mechanism would be consistent with the observed fluoxetine effects, since selective SRIs have been shown to reduce primary reward processing (27, 28).
OCD is a heterogeneous disorder. Our study therefore may have greater relevance for particular OCD subtypes. For example, dimensional models of OCD have been proposed in which different types of obsessions and compulsions are associated with different circuits (29, 30). Because our results suggest that repeated stimulation of OFC-VMS projections led to specific induction of repetitive grooming, our model may be of particular importance for OCD patients with predominant contamination concerns.
Our findings yield new insight into how psychopathology could develop. Only 5 min of stimulation per day was sufficient to lead to sustained significant behavioral effects. This raises the possibility that pathological changes, including compulsions in OCD, may result from small but repeated bursts of abnormal neuronal activity and also offers suggestions for new treatment approaches or refinements of existing therapies for disorders characterized by repetitive behaviors. For example, our data are consistent with recent clinical studies demonstrating efficacy of ventral capsule–ventral striatum deep brain stimulation in OCD (31, 32), which is thought to act via inhibition of OFC hyperactivity. Optogenetic approaches could be used to dissect circuit mechanisms underlying deep brain stimulation and other treatments, with a goal of identifying new treatment targets.
We thank H. B. Simpson and C. Kellendonk for critical discussions and reading of the manuscript and D. Flicker and M. Cloidt for behavioral scoring assistance. S.E.A. is supported by National Institute of Mental Health (NIMH) K08MH087718; the Louis V. Gerstner, Jr., Scholars Program; the Irving Institute for Clinical and Translational Research; the Gray Matters Foundation; the Leon Levy Foundation; and a Brain and Behavior Research Foundation NARSAD Young Investigator Award. M.A.K. is supported by NIMH K01MH099371, the Sackler Institute, and a NARSAD Young Investigator Award. K.D. is supported by the Howard Hughes Medical Institute, NIH, California Institute for Regenerative Medicine, and the Defense Advance Research Projects Agency The Reorganization and Plasticity to Accelerate Injury Recovery (REPAIR) Program. J.G. is supported by NIH R01 MH096274, the Hope for Depression Research Foundation, and the International Mental Health Research Organization. R.H. is supported by the Hope for Depression Research Foundation.
Materials and Methods
Figs. S1 to S5