Novel nose poke-based temporal discrimination tasks with concurrent in vivo calcium imaging in freely moving mice
The hippocampus has been known to process temporal information as part of memory formation. While time cells have been observed in the hippocampus and medial entorhinal cortex, a number of the behavioral tasks used present potential confounds that may cause some contamination of time cell observations due to animal movement. Here, we report the development of a novel nose poke-based temporal discrimination task designed to be used with in vivo calcium imaging for the analysis of hippocampal time cells in freely moving mice. First, we developed a ten second held nose poke paradigm for use in mice to deliver a purer time metric for the analysis of time cell activity in hippocampus CA1. Second, we developed a temporal discrimination task that involves the association of held nose poke durations of differing lengths with differential spatial cues presented in arms on a linear I-maze. Four of five mice achieved successful temporal discrimination within three weeks. Calcium imaging has been successfully performed in each of these tasks, with time cell activity being detected in the 10s nose poke task, and calcium waves being observed in discrete components of the temporal discrimination task. The newly developed behavior tasks in mice serve as novel tools to accelerate the study of time cell activity and examine the integration of time and space in episodic memory formation.
KeywordsTime Timing Temporal discrimination Space Memory Hippocampus Time cell Entorhinal cortex In vivo calcium imaging
Calcium/calmodulin-dependent protein kinase II alpha
- GRIN lens
While the hippocampus (HPC) is traditionally known for processing spatial information , it also processes temporal information which is important for the formation of episodic memories [2, 3, 4, 5]. Neurons that repeatedly fire at a specific moment during the delay period of behavior tasks have been found in the hippocampus and medial entorhinal cortex (EC), and are referred to as time cells [3, 4, 5, 6, 7]. Time cell activity has been observed during the delay periods of treadmill running tasks, trace eye blink conditioning, spatial working memory tasks, timing tasks in virtual reality and of delayed matching to sample tasks [3, 6, 7, 8, 9, 10, 11]. The activity of these time cells and their sequences during delay or interval periods is considered to link events in time, and remember specific timing in memory formation [3, 4, 5]. While time cells can be identified during the delay periods of timing-related behavior tasks even with animal body movement, it would be more desirable to analyze time cell activity in an immobile condition [8, 9, 10, 12].
After nose poke training, we then examined the somatic calcium activity (% ΔF/F) of individual hippocampal CA1 pyramidal cells during the nose poke task. Calcium activity was captured at 20 Hz on a miniature fluorescent microscope. Single unit calcium activity from hippocampal CA1 pyramidal cells was isolated by using Inscopix Data Processing Software v1.2 and ImageJ, as previously examined [13, 14, 15] (Fig. 1d,g). We obtained individual calcium activity from 302 cells across 3 mice (90, 50, and 162 = 302 cells in total) during the nose poke task, indicating that we successfully developed the nose poke task with in vivo calcium imaging in freely moving mice. Then, we analyzed individual calcium activity during successful nose poke periods to identify time cell activity. We observed 22% of hippocampal CA1 pyramidal cells that were repeatedly activated at a specific moment during successful nose poke periods in at least one fifth of the total trials (total of 90 cells were imaged from mouse "SST-Cre-9", Fig. 1g.). Some neurons had phasic responses within the first 2 s (6/20), and others activated later with responses at the middle (seconds 2–5, 5/20; seconds 5–8, 2/20) or end (seconds 8–10, 7/20) of that period (Fig. 1g, upper). We also characterized time cells as an ensemble of neurons imaged simultaneously in a single mouse, and observed that the mean peak of calcium activity for each time cell occurred at sequential moments and that the ensemble of time cells bridges the entire nose poke period (Fig. 1g, lower).
We then further developed a nose poke-based temporal discrimination task in mice. Mice which were previously trained for the 10s nose poke task were used in this task. Five mice were habituated to the new apparatus for two days. The apparatus consists of a nose poke port located between two linear arms which are gated with mechanical doors. Each of the arms had unique spatial features on the floor, with the left arm having vertical black and white stripes 3 cm thick, and the right having black polka dots 1.5 cm in diameter on a white background. The walls of the room had additional unique visual cues comprised of shapes and patterns made from vertical or horizontal strips of tape (Fig. 1h, Additional file 1: Figure S1). Mice were trained to associate a 2.5 s tone with one arm, and a 10s tone with the other for 7 days. After training, we started the temporal discrimination task in which mice were randomly given a short or long poke threshold (Fig. 1i). In the temporal discrimination task, upon reaching the threshold of the randomly assigned 2.5 or 10 s nose poke, both doors open and the mouse must choose which arm is associated with the poke duration it performed to open the doors. Each mouse was given 20 trials per day, with four training repetitions given prior to each testing period. The statistical threshold for considering successful acquisition of temporal discrimination was set at a 70% correct response rate for 20 trials (Binomial test, P < 0.05) on two consecutive days. Four of five mice were observed to successfully discriminate within 22 days (Fig. 1i). Furthermore, we successfully obtained individual calcium activity during the nose poke, running and reward periods in the temporal discrimination task (Fig. 1j).
In this study, we developed novel nose poke-based temporal discrimination task with in vivo calcium imaging in freely moving mice. While time cells have been observed using head-fixed animals [8, 9], we believe that our task in freely moving mice can be useful to study the neural mechanisms of temporal discrimination, and furthermore will provide added variety to the extant behavioral approaches for examining the integration of time and space with the application of transgenic strains and viral manipulations.
We thank all members of the Kitamura laboratory for their support. We thank Drs. Hiroshi Ito and Emilio Kropff for their discussion in developing the ideas that led to this project. We also thank Dr. Jason Paris and Dr. Shari Birnbaum for their helpful conversations on behavioral approaches, and Dr. Patrick Stemkowski for his advice on calcium imaging analysis.
WDM and TK conceived the study. WDM, JY, SKO, and TK designed experiments. WDM conducted experiments. WDM and HO analyzed data. WDM, HO, JY, SKO, and TK interpreted data. WDM and TK wrote the manuscript. All authors read and approved the final manuscript.
This work was supported by grants from Endowed Scholar Program (T.K), Human Frontier Science Program (T.K), Brain Research Foundation (T.K), Faculty Science and Technology Acquisition and Retention Program (T.K), Mary and John Osterhaus for their donation and to the Brain & Behavior Research Foundation (T.K), and The Whitehall Foundation (T.K).
All animal experiments were approved by the UT Southwestern Medical Center IACUC (Protocol# 2017–102301).
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The authors declare that they have no competing interests.
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