Involvement of cortical input to the rostromedial tegmental nucleus in aversion to foot shock

Abstract

The rostromedial tegmental nucleus (RMTg) encodes negative reward prediction error (RPE) and plays an important role in guiding behavioral responding to aversive stimuli. Previous research has focused on regulation of RMTg activity by the lateral habenula despite studies revealing RMTg afferents from other regions including the frontal cortex. The current study provides a detailed anatomical and functional analysis of cortical input to the RMTg of male rats. Retrograde tracing uncovered dense cortical input to the RMTg spanning the medial prefrontal cortex, the orbitofrontal cortex and anterior insular cortex. Afferents were most dense in the dorsomedial subregion of the PFC (dmPFC), an area that is also implicated in both RPE signaling and aversive responding. RMTg-projecting dmPFC neurons originate in layer V, are glutamatergic, and collateralize to select brain regions. In-situ mRNA hybridization revealed that neurons in this circuit are predominantly D1 receptor-expressing with a high degree of D2 receptor colocalization. Consistent with cFos induction in this neural circuit during exposure to foot shock and shock-predictive cues, optogenetic stimulation of dmPFC terminals in the RMTg drove avoidance. Lastly, acute slice electrophysiology and morphological studies revealed that exposure to repeated foot shock resulted in significant physiological and structural changes consistent with a loss of top-down modulation of RMTg-mediated signaling. Altogether, these data reveal the presence of a prominent cortico-subcortical projection involved in adaptive behavioral responding to aversive stimuli such as foot shock and provide a foundation for future work aimed at exploring alterations in circuit function in diseases characterized by deficits in cognitive control over reward and aversion.

Fig. 1. Anatomical characterization of cortical inputs to the RMTg.

A Map of cholera toxin B (CtB) retrograde tracer injection sites for all animals included in quantification. B Representative injection site image. C Representative high magnification image showing that inputs to the RMTg arise primarily from layer V of the mPFC. D The percent of CtB+ neurons relative to all layer V NeuN+ neurons is relatively consistent across ACC, PL, and IL subregions of the mPFC whereas the density of RMTg-projecting DP mPFC neurons increases substantially at more caudal levels. E Contralateral cortical afferents are substantially less dense than ipsilateral inputs as exemplified by a comparison of RMTg-projecting PL mPFC neurons in both hemispheres. F The density of layer V OFC neurons projecting to the RMTg is similar to that observed in the mPFC with LO inputs diminishing at more caudal levels. G CtB labeling is consistently observed in the AIC, albeit to a lesser degree than that observed in mPFC and OFC. H Quantification of punctate labeling indicative of collateral input from ROIs placed within each brain region in rats prepared using an intersectional dual-virus approach (inset) to fill RMTg-projecting dmPFC neurons with yellow fluorescent protein (YFP). I Representative images showing RMTg-projecting dmPFC neurons filled with YFP following amplification using standard immunohistochemistry. J Representative YFP staining in the amygdala shows relatively sparse collateralization of RMTg-projecting dmPFC neurons in the basolateral nucleus. K Representative YFP staining in the striatum shows dense collateralization in the dorsomedial but not dorsolateral striatum. Scale bar = 100 μm. ACC anterior cingulate cortex, AID dorsal agranular insular cortex, AIV ventral agranular insular cortex, DI dysgranular insular cortex, DLO dorsolateral orbitofrontal cortex, DP dorsopeduncular cortex, GI granular insular cortex, IL infralimbic cortex, LO lateral orbitofrontal cortex, MO medial orbitofrontal cortex, PL prelimbic cortex, VO ventral orbitofrontal cortex.

Fig. 2. RMTg-projecting dmPFC neurons express glutamatergic markers and are positive for D1 and D2 receptor mRNA.

A Rats were injected with the retrograde tracer, cholera toxin B (CtB), into the RMTg and slices prepared for dual immunofluorescence. Representative dmPFC images co-labeled for the B_1–3 glutamatergic marker CaMKIIα (red) and CtB (blue) and the C_1–3 GABAergic marker GAD67 (green) and CtB (blue). Scale bar = 25 μm. D Quantification of co-labeling reveals that RMTg-projecting neurons are CaMKIIα⁺. E For in-situ hybridization, fluorescent retrobeads were injected into the RMTg and slices processed using RNAScope. F, G Quantification of D1 and D2 mRNA labeling in retrobead+ cells revealed that most RMTg-projecting dmPFC neurons express transcript for both D1 and D2 receptors. H_1–5 Representative dmPFC images co-labeled with retrobeads (green), D1 mRNA (red), and D2 mRNA (yellow), as well as an image of Imaris rendered 3D soma, which was used for defining neuronal labeling of the bead and mRNA transcripts. Scale bar = 20 μm.

Fig. 3. Optogenetic stimulation of RMTg-projecting dmPFC terminals drives avoidance.

A Representative ChR2 expression in dmPFC. B Rats spend significantly less time relative to chance in the light-paired side of a two-compartment chamber during initial testing (test 1) and when the light-paired compartment is reversed (test 2) when light delivery results in stimulation of dmPFC terminals in the RMTg. C A similar degree of avoidance of the light-paired chamber is observed upon stimulation of lateral habenula inputs to the RMTg. D Unlike stimulation of dmPFC terminals in the RMTg, stimulation of dmPFC terminals in the VTA fails to produce either preference for or avoidance of the light-paired compartment. E Direct comparison of circuit manipulations reveals significant avoidance when stimulating inputs to the RMTg relative to the VTA. Light-paired side indicated by blue bar in representative maps above each dataset. *p ≤ 0.01, scale bar = 1000 μm.

Fig. 4. cFos induction in RMTg-projecting dmPFC neurons following exposure to aversive stimuli.

A Experimental procedures. B Rats that had tone paired with foot shock delivery displayed significantly more freezing behavior in response to tone presentation than rats that were exposed to the same number of tone-shock presentations but in an unpaired manner. C Significantly greater cFos expression was observed in RMTg-projecting dmPFC neurons (CtB+) following exposure to either a series of foot shocks or a tone predictive of foot shock relative to a neutral tone or the testing context alone. D Representative images of CtB and cFos labeling in the dmPFC of a context-exposed rat and a rat exposed to foot shock. Representative CtB⁺/cFos⁻ neurons are indicated with a yellow arrowhead; Representative CtB⁻/cFos⁺ neurons are indicated with a black arrow; Representative CtB⁺/cFos⁺ neurons are indicated by a red asterisk. Inset shows representative injection site in RMTg. Scale bar = 200 μm.

Fig. 5. Physiological and structural neuroadaptations in RMTg-projecting dmPFC neurons following exposure to foot shock.

A For ex-vivo electrophysiology experiments, rats were injected with retrobeads into the RMTg. B Representative retrobead injection site in the RMTg. Scale bar = 1000 μm. C Significantly fewer spikes were observed in Shock-exposed rats relative to controls in current clamp recordings of retrobead-labeled dmPFC neurons. D Representative traces from a control and Shock-exposed rat. Decreased spiking was associated with a significant increase in E rheobase, H membrane capacitance, and I action potential height as well as a significant decrease in G membrane resistance and J action potential half-width. No significant difference was observed in F action potential threshold or K after hyperpolarization. L For structural analyses, an intersectional dual-virus approach was used to fill RMTg-projecting dmPFC neurons with yellow fluorescent protein (YFP). M Representative YFP-filled primary apical dendrites in the dmPFC and accompanying Imaris renderings for Context- and Shock-exposed rats. Scale bar = 5 μm. N Spine density did not differ between groups regardless of subclass. However, Shock-exposed rats exhibited significantly greater spine neck diameter O and shorter spine length P across all subtypes (main effect of shock) relative to rats exposed to the neutral testing context. *p ≤ 0.05.