Hippocampal-Prefrontal Theta Transmission Regulates Avoidance Behavior

Abstract

Long-range synchronization of neural oscillations correlates with distinct behaviors, yet its causal role remains unproven. In mice, tests of avoidance behavior evoke increases in theta-frequency (∼8 Hz) oscillatory synchrony between the ventral hippocampus (vHPC) and medial prefrontal cortex (mPFC). To test the causal role of this synchrony, we dynamically modulated vHPC-mPFC terminal activity using optogenetic stimulation. Oscillatory stimulation at 8 Hz maximally increased avoidance behavior compared to 2, 4, and 20 Hz. Moreover, avoidance behavior was selectively increased when 8-Hz stimulation was delivered in an oscillatory, but not pulsatile, manner. Furthermore, 8-Hz oscillatory stimulation enhanced vHPC-mPFC neurotransmission and entrained neural activity in the vHPC-mPFC network, resulting in increased synchrony between vHPC theta activity and mPFC spiking. These data suggest a privileged role for vHPC-mPFC theta-frequency communication in generating avoidance behavior and provide direct evidence that synchronized oscillations play a role in facilitating neural transmission and behavior.

Keywords: anxiety; avoidance; hippocampus; medial prefrontal cortex; optogenetics; oscillations; synchrony; theta.

Figure 1:. Theta-frequency oscillatory stimulation of vHPC-mPFC inputs increases avoidance behavior.

A. Experimental schematic. AAV encoding ChR2 or a non-opsin fluorophore was injected into the vHPC and optical stimulation fibers were implanted over the mPFC. Eight weeks later, patterned light stimulation was delivered to vHPC terminals in the mPFC during exploration of the elevated plus maze (EPM) in 2-min light epochs. B. Example of CaMKIIa-ChR2(H134)-mCherry (left) and CaMKIIa-ChR2(H134)-eYFP (right; blue: neurotrace) viral expression and positioning of optical fibers (dashed lines) in the mPFC (top), and in vHPC (bottom). Scale bars, 100 μm (left) and 200 μm (right). C. Light was delivered to the mPFC in oscillatory vs pulsatile patterns at 8 Hz and at 2, 4 and 20 Hz with an oscillatory pattern. D. % open arm time in the EPM as a function of light pattern and virus type for 8 Hz stimulation (ChR2: 8 Hz sines n=10, 8 Hz pulses n=9; Non-opsin control: 8 Hz sines n=9, 8 Hz pulses n=7; Only 8 Hz sines group meets bonforenni-corrected significance with a paired comparison; Paired t-test for 8 Hz **p=0.0032). E. % open time as a function of virus type for 20 Hz oscillatory stimulation (ChR2, n=9; Non-opsin, n=6; two-way rmANOVA, no main effect of light, F_(1,13) = 1.854, p=0.1964; Bonferroni post-hoc, ChR2 20 Hz Sines ON vs OFF, multiplicity-adjusted p=0.4797). F. % entries into open arms as a function of light pattern and virus type for 8 Hz stimulation (ChR2: 8 Hz sines, n=10; 8 Hz pulses, n=9; eYFP: 8 Hz sines, n=9; 8 Hz pulses, n=7; Only 8 Hz sines group meets bonforenni-corrected significance with a paired comparison; Paired t-test for 8 Hz **p=0.0008). G. % entries into open arms as a function of pattern and virus type for 20 Hz stimulation (ChR2: n=9; mCherry n=6; 2 way rmANOVA no effect of light F_(1,13)=1.66, p=0.22 nor interaction between virus and light F_(1,13)=0.2345, p=0.63). Blue background: light ON H. % open arm time (left) and % open arm entries (right) in the EPM for 4 Hz oscillatory stimulation in ChR2-expressing mice (n=10; % open arm time: paired t-test p=0.4162; % open arm entries: paired t-test **p=0.0032). I. % open arm time (left) and % open arm entries (right) in the EPM for 2 Hz oscillatory stimulation in ChR2-expressing mice (n=10 mice; % open arm time: paired t-test p=0.5521; % open arm entries: paired t-test p=0.3527). See also Figures S1-2.

Figure 2:. Oscillatory stimulation of ChR2-expressing vHPC terminals in the mPFC enhances spontaneous-like activity in an ex vivo slice preparation.

A. Experimental schematic. Voltage clamp recordings were obtained from mPFC pyramidal cells held at −70 mV while an optical fiber stimulated vHPC ChR2-containing terminals ex vivo. B. Representative postsynaptic current responses (EPSC) to the various stimulation patterns. Bottom trace shows postsynaptic current during oscillatory stimulation following application of the glutamate receptor blockers CNQX and APV. C. Top, average EPSC frequency (top) and amplitude (bottom) at baseline versus during light stimulation (8 Hz pulses n=11 cells; 8 Hz sines n=9 cells; 20 Hz sines n=9 cells; two-way rmANOVA, main effect of light F_(1,26)=57.24, p<0.0001; Bonferroni post-hoc, 8 Hz pulses ON vs OFF, ****multiplicity-adjusted p<0.0001, 8 Hz sines ON vs OFF, ***multiplicity-adjusted p=0.0005, 20 Hz sines ON vs OFF, *multiplicity-adjusted p=0.02597). Bottom, average EPSC amplitude at baseline versus during light stimulation (8 Hz pulses n=11 cells; 8 Hz sines n=9 cells; 20 Hz sines n=9 cells; two-way rmANOVA, light by stimulation interaction, F_(2,26)=4.572, *p=0.0199; Bonferroni post-hoc, 8 Hz pulses ON vs OFF, ***multiplicity-adjusted p=0.0010). D. Average frequency of EPSCs across the duration of the light stimulation (Two-way rmANOVA no main effect of time F_(9,234)=1.89, p=0.0537). E. Representative phase-locking of EPSCs to the pulsatile 8 Hz light vs oscillatory 8 Hz light. F. Left, phase-locking of EPSCs to the various optical stimulation patterns as measured by pairwise phase consistency (8 Hz pulses n=11 cells; 8 Hz sines n=9 cells; 20 Hz sines n=9 cells; Wilcoxon rank-sum 8 Hz pulses vs 8 Hz sines ****p=0.00048; Wilcoxon rank-sum 8 Hz pulses vs 20 Hz sines ****p=0.00052). Right, % cells with EPSCs that are significantly phase-locked to the different optical stimulation patterns (Chi-square 8 Hz pulses vs 8 Hz sines ****p<0.0001; Chi-square 8 Hz pulses vs 20 Hz sines ****p<0.0001). G. Coherence between the continuous amplitude of the current trace and the different optical stimulation patterns (rank-sum 8 Hz pulses vs 8 Hz sines, ***p=0.00019 rank-sum 8 Hz pulses vs 20 Hz sines, ***p=0.00019). Red dashed line indicates chance coherence levels obtained from shuffled data.

Figure 3:. Oscillatory stimulation of vHPC terminals at a theta frequency facilitates ongoing vHPC input to mPFC in vivo.

A. Experimental schematic. In vHPC ChR2-expressing mice, a stimulating electrode was implanted in the vHPC, and an optrode in the ipsilateral mPFC. Electrical stimulation (200-400 μA square wave pulse, 0.1 ms) was delivered during patterned optical stimulation of the terminals in the mPFC. B. Representation of the light patterns used to stimulate vHPC terminals. Note that the peak light power across stimuli was the same. C. Example mPFC single unit showing strong evoked responses to vHPC electric stimulation with and without patterned optical stimulation in the mPFC. D. Left, averaged mPFC single unit responses to vHPC electrical stimulation as a function of optical stimulation. Right, average peak evoked response (10-40 ms post-electrical stimulation) in all mPFC single units recorded (n=50 single units; non-parametric one-way rmFriedman test across groups ****p<0.0001; Dunn’s post-hoc, 8 Hz sines vs stim alone, ****multiplicity-adjusted p<0.0001, 20 Hz sines vs stim alone, ***multiplicity-adjusted p=0.0005, 8 Hz pulses vs stim alone, multiplicity-adjusted p=0.1208). Non-opsin control (n=34 single units; non-parametric one-way rmFriedman test across groups p=0.5112). E. Top, peak evoked firing rates in mPFC across phases of light stimulation (8 Hz sines rmFriedman test across phases ****p<0.0001; 20 Hz sines rmFriedman test ***p=0.0002). Black overlay of light stimulus indicates the phase information. Bottom, normalized evoked firing across phases of the light stimulation. Firing is normalized by subtracting the mean evoked firing for 10-40 ms post-electrical stimulation in the absence of light (n=50 single units; Wilcoxon signed-rank **p<0.01 ***p<0.001). F. Normalized evoked firing rates for 20 Hz vs 8 Hz oscillatory stimulation at 90°<phase<180°(n=50 single units; Wilcoxon signed-rank **p=0.01). See also Figure S3.

Figure 4:. Oscillatory stimulation of vHPC terminal does not increase overall firing rate.

A. Average firing rate with and without 8Hz and 20Hz oscillatory stimulation during the EPM for all recorded mPFC single units (8 Hz n=66, Wilcoxon signed-rank test p=0.08; 20 Hz n=67, Wilcoxon signed-rank test p=0.15) and for significantly phase-locked units (8 Hz n=14, Wilcoxon signed-rank test p=0.19; 20 Hz n=8, Wilcoxon signed-rank test p=0.25). B. Scatter plot showing firing rate for all single units stimulated with 8 Hz sines during the EPM (Not phase-locked units n=52, Wilcoxon signed-rank test p=0.08; Phase-locked units n=14, Wilcoxon signed-rank test p=0.19). C. Scatter plot showing firing rate for all single units stimulated with 20 Hz sines during the EPM (Not phase-locked units n=59, Wilcoxon signed-rank test p=0.15; Phase-locked units n=8, Wilcoxon signed-rank test p=0.25). Open circles represent non-phase-locked units and closed circles are units significantly phase-locked to the oscillatory stimulus.

Figure 5:. Anxiety-like behavior enhances phase-locking specifically to the 8 Hz stimulus.

A. Cumulative distribution of the strength of phase-locking of mPFC single units to the oscillatory stimulus at 8 and 20 Hz in a baseline condition (left) and during the EPM test (right). B. Left, average mPFC phase-locking to optical stimulus in a baseline condition for 8 and 20 Hz oscillatory stimulation (8 Hz n=90 single units; 20 Hz n=112 single units; Wilcoxon rank-sum p=0.93). Right, average mPFC phase-locking to the optical stimulus in the EPM for 8 Hz sines and 20 Hz sines (left panel, 8 Hz n=66 single units; 20 Hz n=57 single units; Wilcoxon rank-sum *p=0.03). C. Phase-locking to 8 Hz oscillatory stimulation at baseline and phase-locking to shuffled phases of the 8 Hz sine for each individual unit. Pie chart shows % phase-locked units for both conditions (8 Hz vs shuffled two-sample chi-square **p<0.01). D. Left, phase-locking of example unit to 8 Hz oscillatory stimulus in the open vs closed arms. Right, phase-locking strength to the oscillatory stimulus as measured with pairwise phase consistency in the open or closed arms for 8 Hz (n=39) and 20 Hz stimulation (n=42) (Only units that met spike number criteria were included in this analysis, see details in methods; 8 Hz Open vs Closed p<0.0001; 20 Hz open vs closed p=0.03; Open arms 8 vs 20 Hz p<0.0001; Closed arms 8 vs 20 Hz p=0.40). See also Figure S4.

Figure 6:. vHPC-mPFC theta synchrony is enhanced and entrained by the 8 Hz oscillatory stimulation during avoidance behavior.

A. Change in phase-locking strength (light ON – light OFF) for mPFC units to vHPC theta (4-12 Hz) during exploration of open and closed arms of the EPM (8 Hz n=36; 20 Hz n=27; Wilcoxon signed-rank test for closed vs open for 8 Hz p=0.017). B. Left, mean coherence across frequencies for the vHPC LFP (ChR2 n=8 animals; Non-opsin n=6 animals). Right, average coherence for 7-9 Hz between the vHPC LFP and 8 Hz oscillatory stimulation (ChR2 n=8, eYFP n=6; Wilcoxon rank-sum, **p=0.01). C. Left, coherence between vHPC and sinusoid in closed vs open arms for the ChR2 group. Right, average coherence around 7-9 Hz for closed vs open arms (n=8; Wilcoxon signed-rank closed vs open arms p=0.015). D. Left, coherence between the vHPC LFP and the 8 Hz oscillatory optical stimulus over time. Right, average coherence at 1 and 6 seconds (rank-sum ChR2 vs eYFP at 6 sec, **p=.0016). E. Left, coherence between the vHPC LFP and the 8 Hz oscillatory optical stimulus and open arm probability for mice expressing ChR2 during the first 10 seconds of light stimulation (for coherence ChR2 n=8, eYFP n=6; for behavior ChR2 n=10, eYFP=9, two light ON epochs per mouse used). Right, average open arm probability at 1 and 6 seconds (two light ON epochs used per mouse; ChR2 n=10, eYFP=9; Wilcoxon rank-sum ChR2 vs eYFP at 6 sec, *p=0.023). See also Figures S5-6.