Median raphe region stimulation alone generates remote, but not recent fear memory traces

Abstract

The median raphe region (MRR) is believed to control the fear circuitry indirectly, by influencing the encoding and retrieval of fear memories by amygdala, hippocampus and prefrontal cortex. Here we show that in addition to this established role, MRR stimulation may alone elicit the emergence of remote but not recent fear memories. We substituted electric shocks with optic stimulation of MRR in C57BL/6N male mice in an optogenetic conditioning paradigm and found that stimulations produced agitation, but not fear, during the conditioning trial. Contextual fear, reflected by freezing was not present the next day, but appeared after a 7 days incubation. The optogenetic silencing of MRR during electric shocks ameliorated conditioned fear also seven, but not one day after conditioning. The optogenetic stimulation patterns (50Hz theta burst and 20Hz) used in our tests elicited serotonin release in vitro and lead to activation primarily in the periaqueductal gray examined by c-Fos immunohistochemistry. Earlier studies demonstrated that fear can be induced acutely by stimulation of several subcortical centers, which, however, do not generate persistent fear memories. Here we show that the MRR also elicits fear, but this develops slowly over time, likely by plastic changes induced by the area and its connections. These findings assign a specific role to the MRR in fear learning. Particularly, we suggest that this area is responsible for the durable sensitization of fear circuits towards aversive contexts, and by this, it contributes to the persistence of fear memories. This suggests the existence a bottom-up control of fear circuits by the MRR, which complements the top-down control exerted by the medial prefrontal cortex.

Fig 1. Channelrhodopsin (ChR2)-mediated optic stimulation robustly activated the MRR and altered behavior.

(A) Effects of in vitro stimulation on ^3H5-HT release from coronal brain slices including the MRR, which demonstrates the responsiveness of the area to photostimulation. The time-resolution of the curves is 1 min and covers the 10 min stimulation periods indicated by color. (B) The location of optic fibers in the in-vivo experiments, which are presented in panels C-E. All mice showed robust ChR2 expression in the MRR. Red and blue lines show iso-intensity lines of light penetration at 10% and 1% of release intensity, respectively (based on [18]). (C) MRR stimulation increased ambulation in freely moving animals. Mice were connected to the stimulation equipment by optic fibers, were transferred into a novel cage to mimic the conditions of the subsequent MRR-conditioning study and were stimulated at 50Hz theta-burst frequency by blue light (“stimulated (central)”). Controls were either stimulated in the absence of ChR2 expression (“no ChR2”), or ChR2 expression was induced, but light was not administered (“no light”). (D) Rhythmic decrease in exploration was induced by intermittent (rhythmic) stimulation of the MRR. (E) Rhythmic changes illustrated as ON-OFF responses, i.e. changes in behavior elicited by the onset of stimulations (ON responses) and those elicited by their halting (OFF responses). DRN: dorsal raphe; MRN: median raphe; MRR: median raphe region; LineX: line crossings; pMR: paramedian raphe; RtTg: reticulotegmental nucleus of the pons. * p<0.01 significant difference from “no ChR2” and from “no light”; # p<0.01 significant ON-OFF difference.

Fig 2. MRR stimulation selectively activates brain areas involved in emotional control.

Optic stimulation selectively increased the expression of the activity marker c-Fos in two sub-regions of the medial prefrontal cortex (A), the whole periaqueductal gray (B), and the paraventricular nucleus of the hypothalamus (C). PrL and the amygdala was activated by cage transfer, but not stimulation (D); the hippocampus showed no responses (E). Panel F is a 3D illustration of the Multiple Regression analysis presented in the text. BLA: basolateral amygdala; CA1: CA1 region of the hippocampus; CeA: central amygdala; Cg1: anterior cingulate cortex; DG: dentate girus of the hippocampus; dl-: dorsolateral; dm-: dorsomedial; IL: infralimbic cortex, l-: lateral; MeA: medial amygdala; PAG: periaqueductal gray; PrL: prelimbic cortex; PVN: paraventricular nucleus of the hypothalamus; vl-: ventrolateral. * p<0.05 significant difference from home-cage controls; # p<0.05 significant difference from “no ChR2”.

Fig 3. The acute effects of MRR- and shock-conditioning are differentiated by freezing.

(A) Photomicrographs illustrating the location of the tip of the optic fibers and the distribution of GFP-labeled ChR2 expression. For stimulation patterns see the right-hand side of the figure. (B) and (C) MRR stimulation decreased exploration and increased ambulation only when it targeted the dorso-central region of the MRR ("central stimulation"). Partial stimulations (that reached ventral, lateral, anterior or posterior aspects of the MRR) were ineffective. (D) The rhythmic delivery of 50Hz theta bursts induced a corresponding rhythm of behavioral changes as indicated here by ON-OFF responses. Actual behavioral rhythms were similar to which is shown in Fig 1D and were presented in S1 Fig. Note that behavior scoring was time-structured in a similar fashion in all groups to allow their comparison. (E) Central, but not partial MRR stimulations elicited “runs”, which were behaviorally similar to those observed in shocked mice. (F) Freezing was readily elicited by shock administration, but not by MRR-conditioning. Aq: aqueductus cerebri; DRN: dorsal raphe nucleus; MRR: median raphe region; LineX: line crossings; Of: optic fiber; ON-OFF responses: average changes in behavior elicited by the onset of stimulation (ON responses) and those elicited by their halting (OFF responses); stimulation/shock runs: episodes of rapid ambulation without exploration. * p<0.01 significant difference from stimulation controls (either light-stimulated without ChR2 expression, or ChR2 expression without stimulation); # p<0.01 significant ON-OFF differences; ‡ p<0.01 significant difference from shock controls.

Fig 4. MRR stimulation led to the late-onset development of conditioned fear.

A. Freezing by MRR-conditioned mice one and seven days after MRR-conditioning (day 2 and 8 of the study). The MRR was stimulated on day 1. B. Freezing by intact mice submitted to shock-conditioning on day 2 and 8. * p<0.05 significant difference from stimulation or location/no-shock controls.

Fig 5. Halorhodopsin-mediated silencing of the MRR ameliorates acute and remote, but not recent effects.

Mice were submitted to electric shock conditioning either in a regular way or while their MRR was silenced by halorhodopsin illumination by yellow light. Halorhodopsin was expressed in all mice by a viral vector that carried the NpHR gene, and all mice were connected to optic fibers. (A) and (B) The acute effects of electric shocks were partially ameliorated by MRR silencing during the conditioning trial. (C) Effects were not secondary to alterations in pain perception, which was studied in the hot-plate and expressed as the temperature that consistently elicited paw licking. (D) Halorhodopsin silencing did not affect recent conditioned fear 24h after shock-conditioning, but markedly ameliorated remote freezing 7 days later. Note that freezing was statistically similar in MRR-silenced and non-shocked groups. * p<0.05 significant difference from non-shocked; # p<0.05 significant difference from shocked.