Roles of mediodorsal thalamus in observational fear-related neural activity in mouse anterior cingulate cortex

Abstract

Observational fear (OF) is the ability to vicariously experience and learn from another's fearful situation, enabling adaptive responses crucial for survival. It has been shown that the anterior cingulate cortex (ACC) and basolateral amygdala (BLA) are crucial for OF. A subset of neurons in the ACC is activated when observing aversive events in the demonstrator, which elicits OF. However, the neural circuit mechanisms underlying the expression of OF-related activity in the ACC remain unexplored. Previous studies have shown that the mediodorsal thalamus (MD) is crucial for OF, and MD neurons project to the ACC. Therefore, we hypothesize that the projection from MD to ACC may facilitate the OF-related activity in the ACC. By utilizing in vivo calcium imaging combined with the optogenetic terminal inhibition of MD-ACC pathway, we found that a subset of ACC neurons was activated when observing demonstrator's fearful situation in male mice. Furthermore, the optogenetic inhibition of the MD-ACC projection during the demonstrator's aversive moments significantly suppressed the OF-related activity in the ACC. Our data suggests that the MD-ACC projection plays a role in OF-related activity in ACC neurons.

Keywords: Anterior cingulate cortex; Calcium imaging; Empathy; Mediodorsal thalamus; Observational fear; Optogenetic inhibition.

Fig. 1

Retrograde tracing of MD-ACC pathway and in vivo calcium imaging in ACC. (a) Coronal section of ACC. CTB-555 (red) injection into ACC. DAPI (blue). (b) Coronal section of MD. CTB-555 (red). DAPI (blue). (c) Magnified image of MD from (b). (d) Viral injections (AAV_2/5-Syn-GCaMP6f into ACC, AAV_2/5-hSyn-eNpHR3.0-mCherry into MD) and GRIN lens implantation into ACC. (e) Viral expression and lens placement site at ACC. Coronal section of GRIN lens implanted animal. DAPI (blue). GCaMP6f (green). eNpHR3.0-mCherry (red). Dotted rectangle represents the position of GRIN lens. (f) AAV injection site at MD. (g) Maximum projection image of GCaMP6f-expressing cells acquired through a micro-GRIN-lens over 20 min from the ACC during the OF protocol. (h) Sample calcium traces in 8 cells. Calcium transients correspond to neural activity of the cells

Fig. 2

Shock Responding Cell (SRC) in mouse ACC. (a) OF behavioral model and experimental schedule. The observer mouse is placed across a stranger demonstrator who is exposed to the shock grid. After a 5 min habituation (HB) period, the demonstrator receives 20 electrical foot-shocks separated by 30–60 s each. Red LED is activated during the second 10 shocks (light-ON condition) to inhibit MD-ACC pathway. (b) Representative data from two detected SRCs. Left top panel) Mean of GCaMP6f fluorescent activity traces over 10 red light-OFF trials. Yellow color bar indicates the shock period (2 s). Left bottom panel) Raster plots of calcium transients before and after the shock moment over 10 shocks during red light-OFF condition. Black line plots indicate the event timing of calcium transients. Yellow color bar indicates the shock period (2 s). Right panels) Frequency distribution of mean spike rate determined by 1000 times shuffle. Dashed line indicates 95th percentile of the frequency distribution. Blue line indicates mean spike rate observed during shock moment. (c) 27 cells (6.9%) of the total 391 cells detected over 3 mice were identified as SRCs

Fig. 3

Effects of optogenetic inhibition of MD-ACC pathway on shock-responding activity in observer’s ACC. (a) Representative data from Cell #36(B2). Top panel) Mean of GCaMP6f fluorescent activity traces over light-OFF condition (left) and light-ON (right) condition. Yellow color bar indicates the shock period (2 second). Red color bars indicate the red-LED ON period. Middle panel) Raster plots of calcium transients before and after the shock moment over 10 shocks during red light-OFF (left) and light-ON (right) condition. Black line plots indicate the event timing of calcium transients. Yellow color bar indicates the shock period (2 second). Red color bars indicate the red-LED ON period. Bottom panels) Frequency distribution of mean spike rate determined by 1000 times shuffle. Dashed line indicates 95th percentile of the frequency distribution. Blue line indicates mean spike rate observed during shock moment during red light-OFF and light-ON condition. (b) Percentages of 27 SRCs from 3 mice (Fig. 2c) during MD terminal inhibition in ACC (96.4%, p < 0.0001, X² = 23.148, df = 1)

Fig. 4

Effects of optogenetic inhibition for MD-ACC pathway on OF. (a) Averaged percentages of observer freezing behavior during red light-OFF and red light-ON condition (t = 0.7464, df = 4, P = 0.4969). (b) Averaged number of observer freezing bouts during OFF and ON condition (t = 0.6202, df = 4, P = 0.5687). (c) Averaged duration of observer freezing bouts during OFF and ON condition (t = 1.377, df = 4, P = 0.2407). (d) Averaged freezing duration of freezing bouts beginning in the shock moment during OFF and ON condition (t = 0.7468, df = 4, P = 0.4967)