Optogenetic inhibition or activation of vCA3-vCA1 projections with halorhodopsin (eNpHR3.0) or channelrhodopsin [hChR2(H134R)].

A. Schematics of stereotaxic bilateral injection of viral vectors into vCA3 and implantation of the fiberoptic ferrules into vCA1, including images of viral injection site, c-fos staining (coronal section). B. Schematics of behavioral analysis. C. Opsin viral vector immunofluorescence. The immunofluorescence images showing the fluorescently labelled viral injection site in vCA3, and the projections to vCA1 are largely terminating in the radial region (Rad) of vCA1, i.e., proximal to the vCA1 pyramidal cell bodies (Py). Last image is schematics of vCA3 projections to vCA1. D. c-fos staining images showing the neuronal activation (upper image) or inhibition (lower image) in vCA1 by optogenetic illumination of vCA3 projections. E. The level of c-fos positive cells in ventral and dorsal CA1 with illumination of channelrhodopsin (activation) or halorhodopsin (inhibition). n=8 each group. Data are presented as means ± SEM.

Illumination of vCA3 pyramidal neuronal projections at vCA1 modulates anxiety-like behavior.

A. Coronal schematics of the brain showing fiber optic laser illumination site at ventral CA1 with blue (activation) or yellow (inhibition) lasers. B.-D. An elevated plus maze (schematic in B.) was performed to assess anxiety-like behavior coupled with optogenetic laser illumination. The bar graph shows that activation or inhibition of vCA3 neuronal projections to vCA1 decreased or increased the open arm exploration time the in elevated plus maze, respectively (C), while the total distance travelled in the elevated plus maze was not altered by any optogenetic illuminations (D). E.-G. A novel open field exploration test (schematic in E.) was performed to assess anxiety-like behavior with optogenetic laser stimulation. The bar graph shows that the activation or inhibition of vCA3 neuronal projections to vCA1 decreased or increased the center exploration in novel open field test, respectively (F). The total distance travelled in the open field test was not altered by optogenetic activation or inhibition (G). H.-J. A Vogel conflict test (schematic in H.) was performed to assess anxiety-like behavior in an approach-avoidance conflict. The baseline (without shock) number of licks was similar in all groups (I). Vogel task coupled with optogenetic illuminations over the test period to assess anxiety-like behavior. The bar graph shows that activation or inhibition of vCA3 projections to vCA1 decreased or increased the percent number of licks performed by the mice, respectively (J). n=8-9 (4-5 males and 4 females). Data are presented as means ± SEM.

Projections from both vCA3 and lateral entorhinal cortex to vCA1 modulate fear memory retrieval.

A. Schematics show the trace fear conditioning procedure for optogenetic modulation of vCA3 or LEC projections at vCA1 during the context- and tone-related fear expression. B., D. Contextual fear expression testing in the same chamber that was used for conditioning. Activation or inhibition of vCA3 projections (B) or LEC projections (D) to vCA1 Increased or decreased, respectively, the freezing behavior or fear expression to context. C., E. Fear expression in response to the tone in a new context. Activation or inhibition of vCA3 projections to vCA1 increased or decreased, respectively, the freezing behavior or fear expression in response to the tone (C), while inhibition of the projections from LEC to vCA1 decreased the freezing behavior or fear expression in response to the tone (E). n=8-9 (4-5 males and 4 females). Data are presented as mean ± SEM.

Optogenetic inhibition or activation of lateral entorhinal cortex (LEC) to vCA1 projections with halorhodopsin (eNpHR3.0) or channelrhodopsin [hChR2(H134R)].

A. Schematics of bilateral injection of viral vectors into LEC, and implantation of the fiber optic ferrule into vCA1, including images of viral injection site- c-fos staining (coronal section). B. The behavioral experiments performed after surgical recovery include anxiety-like behaviors (elevated plus maze, novel open field, Vogel conflict test) and trace fear conditioning for fear expression. C. Top row: immunofluorescence images showing the opsin viral vector fluorescence in layer 2/3 LEC region. Bottom row: LEC projections to vCA1 are largely terminating in the lacunosum molecular region (LMol) of vCA1, i.e., distal to the vCA1 pyramidal cell bodies (Py). The right image in the top row is showing the target opsin viral injection site, and the right image in the lower row the schematics of LEC projections to vCA1. D. c-fos staining images showing neuronal activation (top image) or inhibition (bottom image) in vCA1 by optogenetic illumination of LEC projections. E. The level of c-fos positive cells in ventral and dorsal CA1 with illumination of channelrhodopsin (activation) or halorhodopsin (inhibition). n=8 (4 males and 4 females). Data are presented as means ± SEM.

Illumination of lateral entorhinal cortical projections in vCA1 modulates anxiety-like behavior.

A. Coronal schematics of the brain showing the fiber optic laser illumination site at ventral CA1 with blue (activation) or yellow (inhibition) lasers. B.-D. An Elevated plus maze (schematic in B.) was performed to assess the anxiety-like behavior coupled with optogenetic laser illumination. C. The bar graph shows that activation or inhibition of LEC projections to vCA1 does not alter the open arm exploration in elevated plus maze and the total distance travelled (D.) in the elevated plus maze. E. Novel open field exploration test performed to assess anxiety-like behavior with optogenetic laser illumination. F. The bar graph shows that the activation or inhibition of LEC projections to vCA1 does not alter the center exploration and the G. total distance travelled in novel open field test. H. Vogel conflict test in the schematics showing the experimental protocol in the optogenetic illumination coupled Vogel task. I. Vogel conflict test with baseline number of licks (without shock) was similar in all groups. J. Vogel task coupled with optogenetic stimulations over the test period to assess anxiety-like behavior. The bar graph showing that activation or inhibition of vCA3 projections at vCA1 does not alter the percent number of licks performed by the mice, respectively. n=8 (4 males and 4 females). Data are presented as means ± SEM.