Cholinergic Dynamics in the Septo-hippocampal System Provide Phasic Multiplexed Signals for Spatial Novelty and Correlate with Behavioral States

Abstract

In the hippocampal formation, cholinergic modulation from the medial septum/diagonal band of Broca is known to correlate with the speed of an animal's movements at subsecond timescales and also supports spatial memory formation. Yet, the extent to which subsecond cholinergic dynamics, if at all, align with transient behavioral and cognitive states supporting the encoding of novel spatial information remains unknown. In this study, we used fiber photometry to record the temporal dynamics in the population activity of septo-hippocampal cholinergic neurons at subsecond resolution during a hippocampus-dependent object location memory task using ChAT-Cre mice of both sexes. Using a linear mixed-effects model, we quantified the extent to which cholinergic dynamics were explained by changes in movement speed; behavioral states such as locomotion, grooming, and rearing; and hippocampus-dependent cognitive states such as recognizing a novel location of a familiar object. The data show that cholinergic dynamics contain a multiplexed code of fast and slow signals (1) coding for the logarithm of movement speed at subsecond timescales, (2) providing a phasic spatial novelty signal during the brief periods of exploring a novel object location, and (3) coding for recency of environmental change at a seconds-long timescale. Furthermore, behavioral event-related phasic cholinergic activity demonstrates that fast cholinergic transients correlate with a switch in cognitive and behavioral states. These findings enhance understanding of the mechanisms by which cholinergic modulation contributes to the coding of movement speed and encoding of novel spatial information.

Keywords: acetylcholine; encoding and retrieval; fiber photometry; hippocampal formation; medial septum; novel object location.

Figure 1.

Experimental design. A, Schematic drawing of the surgical approach taken to perform fiber photometry of cholinergic septo-hippocampal projection neurons using jGCaMP7/8 in ad libitum behaving ChAT-Cre mice. B, Schematic drawing of the fiber photometry system. C, Design of the ObLoM task. During the sample phase (15 min), mice explored two identical objects in a 40 × 40 cm² square arena with 30 cm high walls. During the test phase (15 min), one of the two objects was moved to a novel location (nonstationary object). The sample and test phases were separated by a 1 h delay phase, during which the mouse was returned to its home cage (6 mice). The ObLoM task was repeated in a counterbalanced design after 4–6 d (4 mice). D–G, Immunohistological verification of the optical fiber track position and cell type-specific expression of jGCaMP7/8 in cholinergic neurons of the MSDB (Mouse ID #4). (D) The green color indicates jGCaMP7/8 fluorescence; (E) the magenta color indicates immunolabeling of ChAT, a marker for cholinergic neurons; (F) the white color indicates colocalization of jGCaMP7/8 fluorescence and ChAT immunostaining (Scale bar, 50 µm); (G) histological confirmation of the fiber track position (yellow arrow points to the tissue displaced by the implanted optical fiber) within the MSDB (Scale bar, 200 µm).

Figure 2.

Cholinergic activity is linearly correlated to the logarithm of the animal's movement speed during an ObLoM task. A, Correlation between cholinergic activity, quantified as ΔF/F, and the animal's movement speed, for one example session recorded during the test phase. Signals are smoothed with a 1 s window; R, Pearson's correlation coefficient. B, Scatter plot of cholinergic activity, quantified as ΔF/F, as a function of the animal's movement speed for an example session, with an exponential function fitted to the data (red). C, Z-scores of ΔF/F of cholinergic activity across all sessions as a function of the animal's movement speed (20 sessions). Speed is binned with a bin width of 1 cm/s. Data shown as mean ± SEM. D, Same data as in A but using the logarithm of the animal's movement speed. E, Same data as in B but using the logarithm of the animal's movement speed, with a linear function fitted to the data (red). F, Z-scores of ΔF/F of cholinergic activity across test sessions (red, 10 sessions) and sample sessions (blue, 10 sessions) as a function of the logarithm of the animal's movement speed. Speed is binned with a bin width of 0.2. Data shown as mean ± SEM. G, Mean ± SEM of time scale-dependent Pearson's correlation coefficient distributions between the smoothed cholinergic activity and the logarithm of the animal's movement speed (20 sessions). H, I, Time-series data on movement speed and cholinergic activity, quantified as ΔF/F, from one example session (H) and averaged across all sessions (I). Time-series data are smoothed with a 1 s window for better illustration. Thick curves show the exponential fit to the data; τ = time constant of exponential fit. Data in I shown as mean ± SEM; the red bar shows the length of the significant cluster (zero to 18.86 s); *p = 0.002, cluster-based permutation test; n = 20 sessions (10 sample and 10 test sessions).

Figure 3.

Mice explore the novel location of a nonstationary object in the ObLoM task. A, DI for sample (blue) and test (red) sessions computed for 3 min intervals using a sliding window. Data show mean ± SEM. B, Difference in DI between test and sample sessions computed for 3 min intervals using a sliding window; *p = 0.008; n.s., not significant; n = 10 sessions. C, Top, Illustration of the ObLoM task. Bottom, Data on DI for sample and test sessions, computed from data on the first 12 min and 3 min, respectively. *p = 0.016; n = 10 sessions. DI, discrimination Index.

Figure 4.

Phasic cholinergic activity signals novelty of object locations and are correlated to behavioral states. A, Top, Average distribution and co-occurrence of time spent by animals in different behavioral states during sample sessions (10 sessions). Bottom, Pie charts display the proportion of behaviors during sample sessions. B, Same as A but for test sessions (10 sessions). C, Summary data comparing the z-scored cholinergic activity associated with each behavioral state during sample (10 sessions) and test sessions (10 sessions); *p < 0.001; p = 0.045 (rearing); ^†p = 0.008 [the significance associated with locomotion refers to significance of the log₂(speed), Table 3]. D, Estimates ± SE of the LMM coefficients for exploring nonstationary and stationary objects during sample sessions (10 sessions). Coefficients of the LMM were calculated over 3 min intervals using a sliding window, including only periods with at least 1 s of object exploration. E, Same as D but for test sessions (10 sessions).

Figure 5.

Fast cholinergic transients across cognitive and behavioral states. A, Top row, Data on movement speed (black), observed cholinergic activity (green), and cholinergic activity predicted from movement speed (red) are shown for the 5 s before onset, during, and 5 s after the offset of locomotion events. Only events with no occurrence of the behavioral state in question during the 4 s before or after the onset and offset were analyzed. Between the onset and offset of a behavioral state, data are plotted on a relative timescale. Data show mean ± SEM; n = 47 locomotion events; data from six mice. Bottom row, Proportion of total behaviors (%) are shown for the 5 s before the onset, during, and 5 s after the offset of locomotion events. Different colors represent distinct behaviors: locomotion (blue), grooming (green), rearing (purple), exploratory behaviors associated with stationary objects (orange), exploratory behaviors associated with nonstationary objects (brown), and background (gray) indicating the absence of all other behaviors. B, Same as in A for grooming. n = 73 grooming events. C, Same as in A for rearing. n = 71 rearing events. D, Data on exploring the stationary and nonstationary objects in the sample session. Data are visualized in the same way as in A. n = 32 events of exploring the stationary object; n = 27 events of exploring the nonstationary object. C, Data on exploring the stationary and nonstationary objects in the test session. Data are visualized in the same way as in A. n = 20 events of exploring the stationary object; n = 17 events of exploring the nonstationary object.