Distinct ensembles of medial prefrontal cortex neurons are activated by threatening stimuli that elicit excitation vs. inhibition of movement

Abstract

Neural circuits controlling defensive behavior were investigated by recording single units in medial prefrontal cortex (mPFC) and dorsolateral periaqueductal gray (dlPAG) while rats expressed conditioned fear responses to an auditory conditioned stimulus (CS; 20-s train of white noise pips) previously paired with an aversive unconditioned stimulus (US; 2-s train of periorbital shocks). The CS elicited conditioned movement inhibition (CMI; characterized by decreased movement speed and freezing) when rats had not recently encountered the US, whereas the CS elicited conditioned movement excitation (CME; characterized by increased movement speed and flight behavior) after recent US encounters. Many mPFC neurons were "strategy-selective" cells that changed their firing rates only when the CS elicited CME (15/71) or CMI (13/71) responses, whereas few mPFC cells (4/71) responded nonselectively to the CS during either response. By contrast, many dlPAG neurons (20/74) responded nonselectively to the CS, but most (40/74) were excited by the CS selectively during CME trials (and none during CMI trials). CME-selective neurons in dlPAG responded phasically after CS pips that elicited CME responses, whereas CME-selective neurons in mPFC showed tonically elevated activity before and after pips that evoked CME responses. These findings suggest that, at the time when the CS occurs, tonic firing rates of CME- and CMI-selective mPFC neurons may bias the rat's choice of whether to express CME vs. CMI responses, perhaps via projections to downstream structures (such as amygdala and PAG) that influence how sensory stimuli are mapped onto motor circuits that drive the expression of competing behaviors.

Keywords: fear conditioning; infralimbic; medial prefrontal cortex; periaqueductal gray; prelimbic.

Fig. 1.

Histological reconstruction of recording sites in dorsolateral periaqueductal gray (dlPAG) and medial prefrontal cortex (mPFC). Reconstructed recording sites of dlPAG neurons (A; n = 74) and mPFC neurons (B; n = 71) are overlaid on coronal templates (with coordinates in mm relative to bregma) from the atlas of Paxinos and Watson (1997). Symbol colors indicate the type classification of each cell (see results), and symbol shapes indicate how the cell responded to conditioned stimulus (CS) pips or shock pulses (*, excited by pip and shocks; ▲, excited by shocks but not pips; ■, excited by pips but not shocks; ⧫, excited by pips and inhibited by shocks, ▼, no pip response and inhibited by shocks; ●, no response to pips or shocks). Left and right sides of the midline correspond to hemispheres ipsilateral (IPSI) and contralateral (CONTRA) to the eyelid where periorbital shocks were delivered. dPAG, dorsal PAG; lPAG, lateral PAG; ACC, anterior cingulate; PL, prelimbic; IL, infralimbic; CMI, conditioned movement inhibition; CME, conditioned movement excitation; CSR, CS responsive; MOV, movement cell; NR, nonresponsive.

Fig. 2.

Behavioral and neural activity during fear-conditioning trials. A: each session consisted of 6 CS-alone trials followed by 16 CS-unconditioned stimulus (US) pairings, so there were 7 pre-shock and 15 post-shock trials per recording session; line graphs show mean movement speeds during the context (CX) and CS periods of pre- vs. post-shock trials. B: line graphs show percentage of trials (for all rats combined) that were classified as CMI, no conditioned response (NCR), or CME per session (session 1 is the first session after 5 days of initial training); pie charts show proportions of trial types from all sessions combined. C: movement speed (top) and turning bias (bottom) during the CX (unshaded area), CS (light gray shaded area), and US (dark gray shaded area) periods of each trial; data are averaged (AV) separately for CMI, NCR, and CME trials over all sessions. D: example of spike data from a dlPAG neuron recorded over 2 days; color of shading beneath spike rasters indicates the rat's movement speed during each trial, and rate histograms (1-s bins) show the mean firing rate of the cell averaged over all CMI and CME trials.

Fig. 3.

Cell type classifications in dlPAG (A) and mPFC (B). A1 and B1: summary table provides a color-coded key for the number of cells of each type; labels denote which cell types belong to the strategy-, stimulus-, and speed-selective categories. A2 and B2: large pie chart shows proportion of cells from each region that was classified in each type category; small pie charts show proportions of cell classifications by subregion. A3 and B3: table shows distribution of recording days across which cells were held (vertical axis) and of which session cells were first encountered in (horizontal axis, session 1 is the first session after 5 days of initial training); type category is indicated by symbol color, and rat is indicated by symbol shape (table at upper right summarizes total number of cells in each category by rat). A4 and B4: distributions of waveform parameters for all cells, with symbol shapes denoting which subregion the cell was recorded from (NR cells are plotted as small dots).

Fig. 4.

Dominant cell types in dlPAG and mPFC neurons. A: each panel shows population-averaged firing rates (top) and a peristimulus time histogram (PSTH) for 1 example cell (bottom) for the 3 most prevalent cell types recorded in dlPAG: CME+ cells (n = 40), CSR+ cells (n = 20), and MOV+ cells (n = 8). B: population-averaged firing rates (top) and a PSTH for 1 example cell (bottom) for 3 prevalent cell types recorded in mPFC: CME+ cells (n = 14), CMI+ cells (n = 6), and MOV+ cells (n = 15). For all graphs, bin size is 1 s, and vertical gray bars indicate individual CS pips.

Fig. 5.

Pip-evoked responses of dlPAG and mPFC neurons. A: short-latency pip-evoked responses of CME+ neurons in dlPAG, Top: population-averaged firing rates (n = 40); bottom: rasters and PSTHs during CMI and CME trials for 1 example of a pip-responsive CME+ cell (bin size = 2 ms; gray shaded region indicates time window of the 250-ms pip presentation). B: same as A except data are shown for the population of CSR+ cells in dlPAG (n = 20, top) and 1 example of a pip-responsive CSR+ cell (bottom). C: mean movement speed calculated in 20-ms bins within a time window surrounding all freezing and flight pips (see materials and methods for definitions) during which neurons were recorded; gray shading indicates the 250-ms pip, and dashed line marks the time at which the mean speed first exceeds the pre-pip baseline during flight trials (Z test, P < 0.05 of individual time points against all samples from the 500-ms period before pip onset). D: population-averaged responses of CME+ and CSR+ cells in dlPAG during freezing vs. flight pips (20-ms bins); colored dots (green for freezing and orange for flight) mark time points at which the population Z score exceeded 0 (P < 0.05 against the mean pre-pip firing rate for freezing pips), whereas black dots mark time points at which the Z score for flight pips exceeded that for freezing pips (P < 0.05). E: same as D except data are shown for CME+ and CMI+ cells in mPFC; bottom: colored stars mark time points at which the population Z score fell significantly below 0 (P < 0.05 against the mean pre-pip firing rate for freezing pips).

Fig. 6.

Shock-evoked responses of dlPAG and mPFC neurons. A: spike rasters and PSTHs for 2 example cells recorded in dlPAG that were excited (left) and inhibited (right) by shock pulses; t = 0 marks onset of the 2-ms shock pulse, and gray shading indicates the ∼6-ms time window during which spike recording was occluded by stimulus artifact (see materials and methods). B: same as A except data are shown for 2 example cells recorded in mPFC.

Fig. 7.

Post-shock responses of dlPAG and mPFC neurons. A: mean turning velocity (top) and movement speed (bottom) averaged over all CS-US pairings (CMI and CME trials combined) during which neurons were recorded; blue and red shaded regions indicate the CS and US periods, respectively, whereas green dots indicate time points at which the plotted value is significantly (P < 0.05) above or below the mean from the CX baseline period. B: population-averaged firing rates during CS-US pairings for CME+, CSR+, and MOV+ cells recorded in dlPAG. C: population-averaged firing rates during CS-US pairings for CME+, CMI+, CMI-, and MOV+ cells recorded in mPFC.