Activation of a Habenulo-Raphe Circuit Is Critical for the Behavioral and Neurochemical Consequences of Uncontrollable Stress in the Male Rat

Abstract

Exposure to uncontrollable stress [inescapable tailshock (IS)] produces behavioral changes that do not occur if the stressor is controllable [escapable tailshock (ES)] an outcome that is mediated by greater IS-induced dorsal raphe nucleus (DRN) serotonin [5-hydroxytryptamine (5-HT)] activation. It has been proposed that this differential activation occurs because the presence of control leads to top-down inhibition of the DRN from medial prefrontal cortex (mPFC), not because uncontrollability produces greater excitatory input. Although mPFC inhibitory regulation over DRN 5-HT activation has received considerable attention, the relevant excitatory inputs that drive DRN 5-HT during stress have not. The lateral habenula (LHb) provides a major excitatory input to the DRN, but very little is known about the role of the LHb in regulating DRN-dependent behaviors. Here, optogenetic silencing of the LHb during IS blocked the typical anxiety-like behaviors produced by IS in male rats. Moreover, LHb silencing blocked the increase in extracellular basolateral amygdala 5-HT during IS and, surprisingly, during behavioral testing the following day. We also provide evidence that LHb-DRN pathway activation is not sensitive to the dimension of behavioral control. Overall, these experiments highlight a critical role for LHb in driving DRN activation and 5-HT release into downstream circuits that mediate anxiety-like behavioral outcomes of IS and further support the idea that behavioral control does not modulate excitatory inputs to the DRN.

Keywords: amygdala; habenula; optogenetics; raphe; serotonin; stress.

Figure 1.

Optical inhibition of the LHb during IS prevents later anxiety-like behavior. A, Top left, Changes in LHb firing rate in response to continuous (10 s) green light illumination of LHb in rats expressing NpHR. Middle, Raster plot and perievent histogram showing the firing rate of a representative LHb neuron in a rat expressing NpHR in the LHb (bin size, 100 ms). Green bar represents continuous laser illumination. Bottom, The z-normalized firing rate of all valid neurons recorded during green light illumination of LHb (n = 36), B, Coronal micrographs demonstrating NpHR expression in the LHb in a rat that received IS plus optical silencing (IS+NpHR). Top left, Bilateral NpHR 3.0-eYFP expression (4× mosaic; scale bar, 1000 μm) and (top right) unilateral NpHR3.0-eYFP expression (inverted arrow indicates tissue damage from optical fiber implant (10×; scale bar, 200 μm). Middle, JSI data expressed as the percentage of baseline for rats previously injected with either eYFP or NpHR and later exposed to IS or HC treatment with green light illumination of the LHb (n = 10-11/group). Bottom, Diagram indicating the location of optical fiber tips in the LHb of rats that received IS plus NpHR 3.0 silencing (green dots) or IS plus eYFP (black dots). Position relative to bregma is denoted above individual images. Hipp, Hippocampus.

Figure 2.

Optical inhibition of the LHb during IS prevents the neurochemical consequences of the stressor. A, Rats were injected with NpHR or eYFP, and implanted with LHb optical fibers and BLA microdialysis probes 3 weeks later. B, Left, BLA 5-HT levels measured during IS in rats that received IS plus NpHR or IS plus eYFP with green light delivery during tailshocks. Right, Area under the curve for BLA 5-HT levels measured during IS (arbitrary units, relative to average of baseline BLA 5-HT samples). C, Left, BLA 5-HT measured during JSI in rats that previously received IS plus NpHR or IS plus eYFP with green light delivery during tailshocks. Right, Area under the curve for BLA 5-HT measured during JSI (arbitrary units, relative to average of baseline BLA 5-HT samples). D, Left, JSI data for all rats that received IS plus NpHR or IS plus eYFP (n = 7-8/group). Middle, Correlation between BLA 5-HT levels measured during JSI and social investigation. Right, Schematic of BLA microdialysis probes for all rats included in statistical analysis. Position relative to bregma is denoted.

Figure 3.

Stress, per se, increases Fos-IR in the LHbM. A, Photomicrographs of Fos-IR in the habenula from rats that were killed 2 h after receiving HC (top), IS (middle), or ES (bottom). Images are shown at 10× (left), 20× (middle), and 40× (right) magnification. Scale bars: left, 200 μm; middle, 100 μm; and right, 50 μm. B, Fos-IR was examined in the habenula at 0, 2, and 4 h following HC, IS, or ES (left to right). n = 8/group, two habenula sections/rat at all time points.

Figure 4.

Stress, per se, increases Fos-IR in the LHb–DRN pathway. A, Schematic showing FG injected directly into the DRN prior HC, IS, or ES (left). Photomicrographs show the site of injection at 4× (top) and 10× (bottom) magnification. Scale bars, 200 μm. Fluoro-Gold traveled retrogradely to the LHb prior to rats receiving HC, IS, or ES (right). B, Two weeks after Fluoro-Gold injection, rats received HC, IS, or ES and were killed 2 h later. Total FG (left), Fos-IR (middle), and the percentage of FG cells also expressing Fos (FG+Fos; right) were quantified in the MHb, LHbM, and LHbL (top). The total number of FG-labeled cells in each habenula subregion (FG+Fos) did not differ across treatment groups (left). Total Fos-IR was increased in the LHbM and LHbL of rats that received either IS or ES (middle). Similarly, activation of the LHb–DRN pathway (% FG+Fos) was increased in rats that received either IS or ES (right). Photomicrographs (bottom) of representative LHbM tissue samples are shown for rats that received HC (top), IS (middle), or ES (bottom). Images in the first three panels are taken at 20× (scale bar, 50 um) and magnified to 40× at the far right (scale bar, 50 μm). FG reliably labeled cell bodies in the LHbM 2 weeks after injection (left). Increased Fos-IR is evident in rats that received IS or ES, relative to HC (middle). IS and ES also increased LHb–DRN pathway activation relative to HC, as measured by the colocalization of FG and Fos-IR (right). Arrows indicate colocalized FG and Fos-IR (magenta spot). N = 12/group, two habenula sections/rat.