An Inhibitory Medial Preoptic Circuit Mediates Innate Exploration

Abstract

Animals have an innate motivation to explore objects and environments with unknown values. To this end, they need to activate neural pathways that enable exploration. Here, we reveal that photostimulation of a subset of medial preoptic area (MPA) neurons expressing the vesicular-GABA transporter gene (vgat+) and sending axonal projections to the ventrolateral periaqueductal gray (vPAG) increases exploration in a chamber but causes no place preference when tested there without photostimulation. Photoinhibition of MPA^vgat-vPAG projections leads to no emotional changes as measured by normal activity in an open field assay. Electrophysiological recordings revealed that most GABAergic vPAG neurons are inhibited by MPA^vgat neurons. In contrast to a previous report that suggested that MPA^vgat-vPAG neurons may impart positive valence to induce place preference, our results suggest that these neurons can increase innate exploration.

Keywords: GABAergic neuron; exploration; medial preoptic area; periaqueductal gray; reinforcement.

FIGURE 1

Activation of MPA^vgat neurons induces exploration but does not encode value. (A) Photoactivation of MPA^vgat neurons. Viruses were injected into the MPA, and fiber-optic cannulas were implanted above the injection site. (B) Virally mediated expression of ChR2 in MPA neurons of vgat-ires-Cre mice. Scale bar, 250 μm. (C) Photostimulation of MPA^vgat neuron increases movement in ChR2-expressing mice (n = 6; n = 3 males; n = 3 females) compared to mCherry control mice (n = 4; n = 1 male; n = 3 females); ***p < 0.001. Error bars represent SD. (D) Number of rearing increases during photostimulation of ChR2-expressing mice (n = 6; n = 3 males; n = 3 females) compared to control mice (n = 4; n = 1 male; n = 3 females); **p < 0.01. Error bars represent SD. (E) Representative movement trace of ChR2-expressing (red) mice and mCherry control (gray) mice during RTPP. (F) ChR2-expressing (n = 6; n = 3 males; n = 3 females) mice show real-time preference toward the stim chamber; ***p < 0.001. Error bars represent SD. (G) mCherry control (n = 4; n = 1 male; n = 3 females) do not show real-time place preference toward the stim chamber; p = 0.36. Error bars represent SD. (H) Conditioned place preference test 24 h after photostimulation with one chamber. mCherry control (n = 4; n = 1 male; n = 3 females) and ChR2-expressing (n = 6; n = 3 males; n = 3 females) mice do not have preference toward the stim chamber; p = 0.753. Error bars represent SD.

FIGURE 2

GABAergic neurons in the MPA send inhibitory input to GABAergic neurons in the vPAG. (A) Mapping connections from MPA^vgat neurons onto vPAG neurons using ChR2-assisted circuit mapping (CRACM). (B) Percentage of IPSCs evoked in postsynaptic vPAG neurons exposed to photostimulation. (IPSC connection, n = 2 5 cells; no connection, n = 9 cells). (C) Top, representative IPSC traces of vPAG neurons. Bottom, trace of single photostimulation, resulting in an IPSC, which was abolished by bicuculline (10 μM). (D) Photostimulation of MPA^vgat axon termini in the vPAG induces IPSCs that are abolished by pharmacological blockade of GABA_A receptors with bicuculline (n = 12); ***p < 0.001. Error bars represent SD. (E) Schematic of experiment, injection of AAV.DIO.hChR2.mCherry into the MPA and injection of AAV.DIO.eYFP into the vPAG of vgat-ires-Cre mice, followed by voltage clamp recordings of eYFP-positive cells in the vPAG. (F) Percentage of IPSCs evoked in postsynaptic vPAG^vgat neurons exposed to photostimulation. (IPSC connection, n = 12 cells; no connection, n = 6 cells).

FIGURE 3

Activation of the MPA^vgat–vPAG circuit induces exploration without encoding value. (A) Top, MPA–vPAG circuit photostimulation. Viruses were injected into the MPA, and fiber-optic cannulas were implanted over the vPAG. Bottom, virally mediated expression of ChR2 in MPA neurons of vgat-ires-Cre mice and axon termini in the vPAG. Scale bar, 250 μm. (B) Photoactivation of MPA^vgat neuron axon termini causes increased movement in ChR2-expressing mice (n = 7; n = 4 males; n = 3 females) compared to mCherry control mice (n = 3; n = 1 male; n = 2 females); ***p < 0.001. Error bars represent SD. (C) Number of rearing significantly increases in ChR2-expressing mice (n = 7; n = 4 male; n = 3 females) compared to mCherry control mice (n = 3; n = 1 male; n = 2 females); *p < 0.05. Error bars represent SD. (D) ChR2-expressing (n = 7; n = 4 males; n = 3 females) mice show more time in the stimulation chamber during the conditioning session compared to the pretest session; ***p < 0.001. Error bars represent SD. (E) mCherry control (n = 3; n = 1 male; n = 2 females) do not show real-time preference toward the stim chamber; p = 0.562. Error bars represent SD. (F) Testing for conditioned place preference 24 h after the conditioning session. mCherry control (n = 3; n = 1 male; n = 2 females) and ChR2-expressing (n = 7; n = 4 males; n = 3 females) mice do not show preference toward the stim chamber; p = 0.270. Error bars represent SD.

FIGURE 4

Photoinhibition of GABAergic neurons in the MPA projecting to the vPAG does not alter exploration and does not encode value. (A) Top, MPA–vPAG circuit photoinhibition induced by a 589-nm laser. AAV.DIO.eNpHR3.0.eYFP was bilaterally injected into the MPA, and dual-fiber optic cannulas were implanted over the vPAG. Bottom, virally mediated expression of NpHR in the MPA of vgat-ires-Cre mice and axon termini in the vPAG. Scale bar, 250 μm. (B) NpHR-expressing mice (n = 7; n = 5 males; n = 2 females) have no significant increase in distance moved compared to eYFP control mice (n = 4; n = 1 male; n = 3 females); OFF_pre, p = 0.169; ON, p = 0.245; OFF_post, p = 0.380. Error bars represent SD. (C) No difference in the number of rearing between NpHR-expressing mice (n = 7; n = 5 males; n = 2 females) and eYFP-expressing mice (n = 4; n = 1 male; n = 3 females); p = 0.431. Error bars represent SD. (D) Representative movement trace of NpHR-expressing (green) mice and eYFP control mice (gray) during RTPP. (E) NpHR-expressing (n = 7; n = 5 males; n = 2 females) mice show no significant difference in time spent in the stim chamber during RTPP; p = 0.098. Error bars represent SD. (F) eYFP control (n = 4; n = 1 male; n = 3 females) have no difference in time spent in the stim chamber during RTPP; p = 0.923. Error bars represent SD. (G) Twenty-four hours after the conditioning session. eYFP control (n = 4; n = 1 male; n = 3 females) and NpHR-expressing (n = 7; n = 5 males; n = 2 females) mice do not have preference toward the stim chamber; p = 0.488. Error bars represent SD.