Median raphe controls acquisition of negative experience in the mouse

2019. november 27.

INTRODUCTION: Coping with negative experience is essential for survival. Animals must quickly recognize a harmful situation, produce an adequate response, and learn its context, so that they can predict the reoccurrences of similar experiences. This process requires the lateral habenula (LHb) and the medial ventral tegmental area (mVTA) for evaluating and predicting aversive stimuli. LHb neurons promote encoding of aversive behavior, learn to respond to cues that predict aversive stimuli and activate negative experience-processing mVTA dopaminergic neurons (DA). Over-excitation of LHb neurons lead to depression-like symptoms, whereas their inactivation has an anti-depressant effect. Coping with negative experience also requires the septo-hippocampal system to record and recall contextual memories of events. This process necessitates increased firing of pacemaker parvalbumin (PV)-positive neurons in the medial septum and the vertical limbs of the diagonal bands of Broca (MS/VDB) and subsequent theta-oscillations in the hippocampus. However, how all these brain centers coordinate their activity during adverse events is poorly understood.

RATIONALE: Because LHb does not project directly to the septo-hippocampal system, the brainstem median raphe region (MRR) has been proposed to coordinate their activity. Although MRR plays an important role in regulating mood, fear and anxiety and neuronal projections from it have been extensively studied for decades, yet it is still unclear how MRR neurons process these negative experiences. Using cell type-specific neuronal tract-tracing, monosynaptic rabies-tracing, block-face scanning immuno-electron microscopy, in vivo and in vitro electrophysiological methods, we investigated the neurons of mouse MRR that are responsible for these functions. We used in vivo optogenetics combined with behavioral experiments or electrophysiological recordings to explore the role of MRR neurons responsible for the acquisition of negative experience.

RESULTS: We discovered that the MRR harbors a vesicular glutamate transporter 2 (vGluT2)-positive cell population that gave rise to the largest ascending output of the MRR. These neurons received extensive inputs from negative sensory experience-related brain centers, whereas their excitatory fibers projected to LHb, mVTA and MS/VDB (Fig. A). MRR vGluT2-neurons mainly innervated MRR- or mVTA-projecting cells in medial (“limbic”) LHb, creating a direct feedback in the MRR-LHb-mVTA axis. MRR vGluT2-neurons were selectively activated by aversive but not rewarding stimuli in vivo. Stimulation of MRR vGluT2-neurons induced strong aversion (Fig. B-D), agitation and aggression and suppressed reward-seeking behavior, whereas their chronic activation induced depression-related anhedonia. The latter can at least partly be explained by our 3D electron microscopy data showing highly effective synaptic targeting of LHb neurons and by our in vitro data showing that MRR vGluT2-terminals can trigger depressive behavior-related bursting activity of LHb neurons. MRR vGluT2-neurons seem to be involved in active responses to negative experience, therefore they induced aggression or avoidance, classical fight or flight responses. Suppression of MRR vGluT2-neurons precisely at the moment of the aversive stimulus presentation strongly disrupted the expression of both contextual and cued fear memories and prevented fear generalization. MRR vGluT2-neurons could facilitate the learning of negative experience, because their LHb-projecting axons bifurcated and selectively innervated pacemaker MS/VDB PV-positive neurons that projected to the hippocampus. Consequently, in vivo stimulation of MRR vGluT2-neurons instantly evoked memory acquisition-promoting hippocampal theta-oscillations in mice, in vivo.

CONCLUSION: Our results revealed that the MRR harbors a previously unrecognized brainstem center that serves as a key hub for the acquisition of negative experience. MRR vGluT2-neurons could activate the aversion- and negative prediction-related LHb-mVTA axis and could swiftly transform the state of the septo-hippocampal system for immediate acquisition of episodic memories of the negative experience. Maladaptations in processing negative experience is the basis of several types of mood disorders, which have a huge social and economic impact on individuals and society. Selective targeting of this neural hub may form the basis of new therapies.

MRR vGluT2-neurons serve as a key hub for aversive behavior. MRR vGluT2 (VG2)-neurons process aversive events by activating neurons of LHb and mVTA, and hippocampus (HIPP)-projecting memory acquisition-promoting parvalbumin (PV)-positive cells in MS/VDB (A). After viruses made MRR vGluT2-neurons light-sensitive (B), mice were light-stimulated in a specific area (C) that caused significant avoidance of that area, compared to control mice (D). (PFC: prefrontal cortex)

András Szőnyi ‡, Krisztián Zichó ‡, Albert M. Barth, Roland T. Gönczi, Dániel Schlingloff, Bibiána Török, Eszter Sipos, Abel Major, Zsuzsanna Bardóczi, Katalin E. Sos, Attila I. Gulyás, Viktor Varga, Dóra Zelena, Tamás F. Freund and Gábor Nyiri * Science 29 Nov 2019: Vol. 366, Issue 6469, eaay8746 DOI: 10.1126/science.aay8746

‡ These authors contributed equally. * Corresponding author.