Research Article

Median raphe controls acquisition of negative experience in the mouse

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Science  29 Nov 2019:
Vol. 366, Issue 6469, eaay8746
DOI: 10.1126/science.aay8746

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Keeping tabs on bad experiences

Identifying the neural basis underlying how we acquire, process, and store negative experiences could help the search for effective treatments for mood disorders. Szőnyi et al. used a range of neuroscientific tools to elucidate the role of a specific neural circuit that originates in the median raphe region of the murine brainstem (see the Perspective by Ikemoto). A subpopulation of excitatory neurons projected to aversive brain areas and received recurrent feedback from the lateral habenula and convergent feedback from a range of fear-related circuitry. These neurons were activated by aversive stimuli, and artificial stimulation promoted aversion or anxiety-related behavior. This group of cells thus plays a pivotal role in a network that helps to mediate aversive motivation.

Science, this issue p. eaay8746; see also p. 1071

Structured Abstract

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 (DA) neurons. Overexcitation of LHb neurons leads to depression-like symptoms, whereas their inactivation has an antidepressant effect. Coping with negative experience also requires the septohippocampal 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 the LHb does not project directly to the septohippocampal 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, it is still unclear how MRR neurons process these negative experiences. Using cell type–specific neuronal tract tracing, monosynaptic rabies tracing, block-face scanning immunoelectron microscopy, and 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 gives 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 the LHb, mVTA, and MS/VDB (see figure). MRR vGluT2 neurons mainly innervated MRR- or mVTA-projecting cells in the 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 (see figure), 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 three-dimensional 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 inducing 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.

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 septohippocampal system for immediate acquisition of episodic memories of the negative experience. Maladaptations in processing negative experience form 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.

(A) MRR vGluT2 (VG2) neurons process aversive events by activating LHb and mVTA neurons and hippocampus (HIPP)–projecting memory acquisition–promoting PV-positive cells in MS/VDB. PFC, prefrontal cortex; IN, interneuron; PC, pyramidal cell. After viruses made MRR vGluT2 neurons light-sensitive (B), mice were light-stimulated in a specific area (C), which caused significant avoidance of that area compared with response in control mice (D). Medians and interquartile ranges; ***P = 0.00034; Mann-Whitney U test. AAV5, adeno-associated virus serotype 5; CTRL, control; ChR2, channelrhodopsin 2; eYFP, enhanced yellow fluorescent protein.

Abstract

Adverse events need to be quickly evaluated and memorized, yet how these processes are coordinated is poorly understood. We discovered a large population of excitatory neurons in mouse median raphe region (MRR) expressing vesicular glutamate transporter 2 (vGluT2) that received inputs from several negative experience–related brain centers, projected to the main aversion centers, and activated the septohippocampal system pivotal for learning of adverse events. These neurons were selectively activated by aversive but not rewarding stimuli. Their stimulation induced place aversion, aggression, depression-related anhedonia, and suppression of reward-seeking behavior and memory acquisition–promoting hippocampal theta oscillations. By contrast, their suppression impaired both contextual and cued fear memory formation. These results suggest that MRR vGluT2 neurons are crucial for the acquisition of negative experiences and may play a central role in depression-related mood disorders.

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