Optogenetic Stimulation of Novel Tph2-Cre Rats Advances Insight into Serotonin's Role in Locomotion, Reinforcement, and Compulsivity

Abstract

Serotonin critically modulates the activity of many brain networks, including circuits that control motivation and responses to rewarding and aversive stimuli. Furthermore, the serotonin system is targeted by first-line pharmacological treatments for several psychiatric disorders, including obsessive-compulsive disorder. However, understanding the behavioral function of serotonin is hampered by methodological limitations: the (brainstem) location of serotonergic neuron cell-bodies is difficult to access, their innervation of the brain is diffuse, and they release serotonin in relatively low concentrations. Here, we advance this effort by developing novel Tph2-Cre rats, which we utilized to study serotonin in the context of motor, compulsive, and reinforced behaviors using optogenetics in both male and female rats. Specificity and sensitivity of Cre recombinase expression and Cre-dependent processes were validated immunohistochemically, and optogenetic induction of in vivo serotonin release was validated with fast-scan cyclic voltammetry. Optogenetic stimulation of serotonin neurons in the dorsal raphe nucleus did not initiate locomotion or alter aversion-induced locomotion, nor did it elicit (real-time) place preference, and it had no measurable effect on compulsive behavior in the schedule-induced polydipsia task. In contrast, this optogenetic stimulation moderately sustained ongoing spontaneous locomotion and robustly reinforced operant lever pressing for self-stimulation of serotonin neurons, which was exacerbated by food restriction. Together, this work both introduces a novel rat Cre line to study serotonin and advances our understanding of serotonin's behavioral functions. Complementing previous findings, we find that brainwide serotonin release has an overall relatively mild effect on behavior, which manifested only in the absence of natural reinforcers and was modulated by physiological state.

Keywords: compulsive behavior; dorsal raphe nucleus; intracranial self-stimulation; optogenetics; serotonin; transgenic rats.

Figure 1.

Validation of Tph2-Cre rat line and optogenetically induced serotonin release. A, Mouse PAC-based construct for the specific expression of Cre in rat serotonergic neurons. B, Immunohistochemical detection of Cre recombinase demonstrates extensive Cre staining in serotonin neurons located in both the anterior (left) and caudal (right) raphe nuclei (dorsal/median raphe nucleus, DRN/MRN). C, Cre expression (red) is restricted to TPH2-positive (green) cells, indicated by colocalization (yellow). D, Top, Tph2-Cre rats were bred with CAG-loxP.EGFP rats to generate double-transgenic Tph2-Cre/CAG-loxP.EGFP rats. Upon Cre-mediated recombination, lacZ is replaced with the second reporter gene, enhanced green fluorescent protein (EGFP). Bottom, Merged expression of EGFP (Cre reporter; green) and TPH2 (red) demonstrate selective expression in DRN serotonin neurons via Cre-mediated recombination (EGFP+/TPH2+). E, Viral-mediated transduction of ChR2 (EGFP, green) into serotonin neurons (TPH2, red) with AAV-5 (top) and AAV-DJ (bottom) shows colocalization (merged, yellow). F, Unilateral virus injection (colocalization of ChR2 and TPH2 in yellow) and optic fiber implantation (white, dotted outline) targeting the DRN were performed at a 30° angle. G, Both FSCV recording electrodes and optical fibers were positioned unilaterally in the amygdala and DRN, respectively. H, Histological verification of FSCV-electrode placement via lesion in the amygdala (left) and optical-fiber placement in the DRN (right). I, Example of an FSCV color plot displaying photostimulation-induced serotonin release (red bar; white-dotted line marks stimulation onset). N-shaped waveform used to record serotonin release superimposed on the image in white. J, Serotonin concentration in the amygdala, before and after administration of SSRI escitalopram (escit) in EYFP (n = 5 animals; left) and ChR2 (n = 4 animals; right) animals. ***p < 0.001.

Figure 2.

No effect of optogenetic stimulation of DRN serotonin neurons on “naturally” reinforced behavior. A, For all behavioral experiments, virus injections and bilateral optic fiber implantation (white-dotted outlines) targeted the DRN (at a 30° angle). Viral expression of ChR2 is shown in green, TPH2 neurons in red, and colocalization in yellow. B, Histological verification of optical-fiber position in the DRN of EYFP (n = 34 on-target fibers; 20 rats) and ChR2 (n = 47 on-target fibers; 27 rats) animals. C, WN is an aversive stimulus as validated using an approach–avoidance task where rats (n = 11) foraged for food pellets in a WN-paired quadrant of an OF (left): with increasing WN intensity, rats spent significantly less time foraging (middle). This behavioral response was stable across sessions (right). D, A 90 dB WN (EYFP, 7 animals; ChR2, 8 animals) administered for 6 s induces an increase in locomotion speed (left). A 15 Hz DRN photostimulation at 15–20 mW did not change WN-induced speed in ChR2 and EYFP animals (right). AUC during the 6 s epoch (0–6 s; between dotted lines) is shown in insets. E, Left, SIP was induced by an FI30 reinforcement schedule in food-restricted animals that had free access to a water spout. Right, After water intake stabilized, animals underwent two 3 d blocks of photostimulation (flanked by 3 d blocks of regular SIP). During the first photostimulation block, 15 Hz of 15–20 mW optogenetic stimulation was delivered every trial for 5 s (starting 2 s after reward delivery). During the second block, 5 Hz of 1–5 mW stimulation was delivered throughout the entire session. F, Rats were divided into high- (ChR2: 5 animals; EYFP: 8 animals) and low-drinkers (ChR2: 6 animals; EYFP: 8 animals) based on a median split of prestimulation (Pre stim) water intake (3 d average). G, The 15 Hz stimulation block (3 d average) did not alter water intake (left) or number of licks (right). H, The 5 Hz stimulation block resulted in no change in water intake (left) or licks (right). I, The 15 Hz stimulation blocks (3 d average) increased food-magazine entries in ChR2 animals (both high and low drinkers). J, The increase in food-magazine entries in ChR2 animals does not occur when the stimulation is delivered, but during an anticipatory period before the next reward delivery. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Figure 3.

Self-stimulation of DRN serotonin neurons exclusively reinforces discrete operant behavior. A, Locomotion in an OF: 3 s of 15 Hz stimulation was applied, while animals were in motion or at rest. B, Photostimulation (EYFP, 15 animals; ChR2, 13 animals) did not differentiate speed in resting rats (left) but did sustain locomotion speed of ChR2 rats that were in motion during stimulation onset (right), relative to EYFP controls. Bar graph insets depict average change in speed during stimulation (0–3 s; gray area). C, The RT-PP task in which entry into one unmarked quadrant (shaded) induced either no (0 Hz), 5 Hz of 1–5 mW, or 15 Hz of 15–20 mW DRN photostimulation. D, The percentage of time stimulated (left) and (E) the average speed within the photostimulation-paired quadrant (right) was unaffected by DRN photostimulation in both ad libitum fed (EYFP, 13 rats; ChR2, 18 rats) and food-restricted (EYFP, 13 rats; ChR2, 9 rats) animals. F, The ICSS task in which an active-lever press triggered 3 s of 15 Hz DRN photostimulation (EYFP, 17 animals; ChR2, 21 animals). G, Left, ChR2 but not EYFP rats pressed the active lever more than the inactive one (across sessions). Right, Active-lever presses (averaged across 5 sessions) in ad libitum fed and food-restricted conditions. H, The average duration of self-photostimulation was ∼3 s (equivalent to one lever press; left) and the maximum duration of self-photostimulation bouts was ∼7.5 s (2.5 lever presses; right). Neither variable differed between EYFP and ChR2 animals. I, ChR2 animals spent significantly more time self-stimulating than EYFP animals (even more so in the food-restricted state; on average across 5 sessions), on average up to 18% of total time. J, ChR2 animals pressed the active lever consistently throughout the 30 min session in both ad libitum (left) and food-restricted states (right). **p < 0.001; ns, not significant.