Examination Of Diurnal Variation And Sex Differences in Hippocampal Neurophysiology And Spatial Memory Part 3

To examine sex and time-of-day differences, we stratified our dataset based on the region of the anterior-posterior axis followed by three-way factorial ANOVA. We found that sex had no significant effect on several action potentials generated in response to depolarizing current injections in cells recorded from either anterior (p = 0.321, main effect of sex, three-way RM-ANOVA) or posterior slices (p = 0.568, main effect of sex, three-way RM-ANOVA); thus, data from both sexes were pooled for final analysis. 

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Anterior cells recorded at night fired more action potentials than those recorded during the day (p = 0.046, the main effect of time-of-day, two-way RMANOVA; Fig. 7B). However, posterior cells displayed no statistical day-night difference in the number of action potentials fired (p = 0.484, the main effect of time-of-day, two-way RM-ANOVA; Fig. 7E).

Examination of the baseline membrane potential revealed no differences in sex or time-of-day in cells recorded from anterior slices (p = 0.572 and 0.535, main effects of sex and time-of-day, respectively, two-way ANOVA; Fig. 7C). However, membrane potentials of cells recorded from posterior slices at night were more depolarized than those recorded during the day, regardless of sex (p= 0.044, the main effect of time-of-day, two-way ANOVA; Fig. 7F). 

We examined 15 additional membrane properties, including action potential properties of single action potentials elicited by rheobase current injection, sag, and input resistance (Table 1). There was a significant effect of sex on action potential rise time in anterior neurons (male: 0.295 6 0.01 ms, female: 0.275 6 0.01 ms; p = 0.047, two-way ANOVA). No other parameters reached statistical significance (Extended Data Table 1-1).

Given that action potential firing and membrane potential are both measures of neuronal excitability, we can conclude that CA1 pyramidal neurons across the hippocampal circuit are more excitable at night overall, but the mechanisms underlying this nighttime increase in excitability may vary across the hippocampal anterior-posterior axis.

Day-night differences in CA1 pyramidal neuron excitability are not intrinsic

As the observed day-night variation in excitability could be driven by synaptic and/or intrinsic factors, we again assessed excitability in a separate cohort of animals in the presence of synaptic antagonists (CPP, NBQX, GBZ) to isolate the CA1 pyramidal neuron from the circuit. Under these conditions, we found no significant effect of time-of-day in the number of action potentials generated in response to depolarizing current injections in cells recorded from both anterior and posterior slices (p = 0.933 for anterior and p = 0.569 for posterior, main effect of time-of-day, two-way RM-ANOVA; Fig. 8A, D). 

Furthermore, baseline potentials under these conditions, were not affected by time-of-day or sex in cells recorded from either anterior (p = 0.896 and 0.888, main effect of sex and time-of-day, respectively, two-way ANOVA; Fig. 8B) or posterior slices (p = 0.999 and 0.702, main effect of sex and time-of-day, respectively, two-way ANOVA; Fig. 8E). Additional membrane properties in the presence of synaptic antagonists were investigated, including action potential properties of single action potentials elicited by rheobase current injection, sag, and input resistance (Table 2). 

Both fast-action and medium-action potential hyperpolarization (fAHP and mAHP) were greater during the day (fAHP: 5.776 0.74mV, mAHP: 9.296 0.65mV), compared with night in anterior neurons (fAHP: 4.08 6 0.51 mV, mAHP: 7.06 6 0.59 mV; p = 0.044 and 0.024 for fAHP and mAHP, two-way ANOVA; Table 2). In anterior neurons, fAHP was greater in males (5.827 6 0.56 mV) compared with females (3.98 6 0.58 mV; p = 0.034, two-way ANOVA; Table 2). No other parameters in either anterior or posterior neurons reached statistical significance (Extended Data Table 1-1).

Overall, the absence of enhanced nighttime neuronal excitability (membrane potential and action potential firing rates) in the presence of synaptic antagonists suggests that diurnal differences in fast excitatory and/or inhibitory synaptic input at least partially contribute to the nighttime enhancement of excitability.

Discussion

Here, we examined the effects of sex and time of day on multiple facets of hippocampal physiology, from behavior to individual neuronal physiology. We demonstrate that time of day impacts spatial learning and memory, LTP magnitude, synaptic inhibition onto CA1 pyramidal neurons, and CA1 pyramidal neuronal excitability. We found that sex was most influential on day-night differences in OLM performance, while its effect on synaptic transmission, LTP magnitude, and neuronal excitability was subtle or absent completely. Additionally, we found that position along the anterior-posterior axis significantly impacts CA1 pyramidal neurons excitably. These findings illustrate the complexity of the hippocampal network and the importance of considering factors like sex and time of day in future studies.

While circadian rhythms regulate LTP and hippocampal-dependent learning and memory, the role of sex on diurnal differences in these processes was previously unknown. Surprisingly, we found that circadian rhythms regulate hippocampal-dependent memory performance in a sex-dependent manner. Male mice performed better on the OLM task at night, as previously reported (Takahashi et al., 2013; Snider et al., 2016). Female mice, however, performed better during the day. It is unclear why female mice would perform better during their inactive period, but a possible explanation could be the estrous cycle, which was not controlled for in the present study. 

While more research in naturally cycling females is needed to make definitive conclusions about the specific impact of the estrous cycle on hippocampus-dependent spatial memory, studies in rats found that females in proestrus and estrous outperformed those in diestrus on object-recognition and object placement tasks (Frye et al., 2007; Paris and Frye, 2008). Indeed, estrogen levels do impact performance on hippocampus-dependent memory tasks and administration of exogenous estradiol enhances performance on hippocampus-dependent memory tasks (Luine et al., 2003; Li et al., 2004; Phan et al., 2012; Vedder et al., 2013, 2014; Tuscher et al., 2015). Understanding how the estrous cycle and the circadian cycle converge to modulate cognition in females will be an interesting and important topic for future research.

Interestingly, we found that these sex effects on diurnal differences in OLM performance did not extend to diurnal regulation of LTP, which is considered a cellular correlate of learning and memory. It is important to note that CT and ZT times used in the two experiments did not perfectly mirror one another. OLM assays were conducted in the middle of the subjective day and night periods in constant darkness (CT four and CT 16), as adapted from (Snider et al., 2016), while electrophysiology experiments examined early day and early night in an LD cycle (ZT 1–6 and ZT 12–18). 

Nevertheless, this somewhat unexpected finding could be an example of how the same physiological process (LTP) can be used to achieve different outcomes depending on context (males vs females). Additionally, while the OLM task we chose is hippocampus-dependent (Barker and Warburton, 2011); learning and memory are complex processes that can rely on multiple memory systems, and perhaps females rely more heavily on other circuits compared with males. Moreover, the estrous cycle can influence learning strategies and the relative contributions of different female memory circuits (Korol et al., 2004). 

Therefore, the sex-dependent effects observed in the OLM task may be mediated by a mechanism other than LTP at the CA3-CA1 synapse. It is also possible that the high-frequency stimulation used, as opposed to a more physiological stimulation (i.e., theta burst), may have occluded detection of sex-dependent regulation of LTP. An additional limitation of the OLM assay worth noting is a possible disruption to behavioral rhythms resulting from repeated handling (5 min/d) 4 d before training and testing. Regardless, these results exemplify the importance of accounting for both sex and time of day when designing research studies and interpreting results.

Previous work from our laboratory revealed that sIPSCs onto CA1 pyramidal neurons exhibit diurnal differences, which are lost in a mouse model of Alzheimer's disease (Fusilier et al., 2021). Here, we replicated and expanded on previous findings by examining sIPSCs in both sexes and conducting additional experiments in the presence of TTX (mIPSCs) to begin to identify presynaptic and postsynaptic mechanisms contributing to diurnal differences. The lack of diurnal differences in mIPSCs IEI suggests that action potential-dependent inhibition onto CA1 pyramidal cells is greater during the day than at night in both male and female mice. Given that these inhibitory currents were pharmacologically isolated with glutamate receptor antagonists, likely, increased daytime interneuron activity is spontaneously generated. Indeed, prior reports suggest that some interneurons in area CA1 are spontaneously active (Sik et al., 1995; Maccaferri and McBain, 1996; Oliva et al., 2000; Amilhon et al., 2015; Huh et al., 2016; Miri et al., 2018); however, definitive evidence of diurnal variation in spontaneous interneuron firing in the hippocampus is lacking and will be an important area of future study that could provide insight into circadian dysfunction associated with diseases involving hyperexcitability of the hippocampal network, including Alzheimer's disease and epilepsy.

In addition to receiving inhibitory input from local interneuron populations, major excitatory input onto CA1 pyramidal cells arrives from the axons of principal neurons of the downstream area CA3 (Schaffer collaterals; CA3 pyramidal cells) or the entorhinal cortex (temperoammonic pathway). Examination of sEPSCs revealed a trend for excitatory input onto CA1 pyramidal cells to be greater at night than during the day. In the absence of action potential-dependent neurotransmitter release, this phenomenon persists in males, but not in females, suggesting that increased nighttime excitation in females is likely action potential driven. CA3 pyramidal cells also exhibit diurnal differences in excitability, such that night cells exhibit larger calcium current, decreased afterhyperpolarization, and reduced spike frequency adaptation compared with day cells (Kole et al., 2001). This increased nighttime CA3 pyramidal cell excitability could translate to increased sEPSC onto CA1 pyramidal cells at night compared with day.

We next wanted to examine how diurnal variation in excitatory and inhibitory synaptic transmission might impact CA1 pyramidal neuron excitability. A previous study in rats found that membrane excitability oscillated across circadian time, and neurons were more depolarized during the subjective late night/subjective early day (Naseri Kouzehgarani et al., 2020). A recent study found increased nighttime excitability in mouse CA1 pyramidal neurons (Fusilier et al., 2021). Here, we replicated previous findings and expanded our study to account for potential sex differences and the influence of the anterior-posterior hippocampal axis. Overall, we found that, in an intact synaptic circuit (i.e., without synaptic antagonists), CA1 pyramidal neurons were more excitable at night compared with day, regardless of sex. Interestingly, nighttime enhancement of excitability was not uniform across the hippocampal anterior-posterior axis. While neurons recorded from anterior slices fired more action potentials in response to depolarizing current injections and displayed no diurnal difference in baseline membrane potential, posterior neurons were more depolarized at night but did not display a statistically significant nighttime increase in several action potentials fired. 

These findings suggest that the underlying mechanisms for nighttime enhancement of neuronal excitability may be different depending on location across the anterior-posterior axis. While our coronal slice preparation meant we were unable to truly isolate the ventral hippocampus from the dorsal hippocampus, we observed that neurons from posterior slices displayed characteristics consistent with previously published data collected from ventral CA1 pyramidal neurons, while neurons from anterior slices were consistent with data examining dorsal CA1 pyramidal neurons (Malik et al., 2016; Milior et al., 2016). Specifically, posterior (ventral-like) neurons were more excitable compared with anterior neurons, reaching max firing rate earlier than anterior neurons, had higher input resistance, and lower rheobase values compared with anterior (dorsal-like) neurons. Given known differences in dendritic morphology and ion channel expression in dorsal versus ventral hippocampal neurons (Bannerman et al., 2004; Fanselow and Dong, 2010; Marcelin et al., 2012; Dougherty et al., 2012, 2013; Hönigsperger et al., 2015; Kim and Johnston, 2015; Malik et al., 2016; Milior et al., 2016; Soltesz and Losonczy, 2018), it is unsurprising that mechanisms underlying diurnal differences in excitability may be different across these populations. The absence of day-night differences in neuronal excitability in the presence of synaptic antagonists suggests that synaptic inputs at least partially contribute to the nighttime enhancement of excitability. However, future experiments are needed to determine the specific role of both synaptic and intrinsic factors in regulating diurnal differences of physiology in CA1 pyramidal neurons. 

Additionally, the difference between anterior and posterior cells was lessened in the presence of synaptic antagonists, suggesting synaptic factors could be at least partially responsible for some of the regional differences we observed. It will be interesting to narrow down how the expression and function of various neurotransmitter receptors and ion channels are modulated across both time of day and location along the longitudinal axis. A benefit of the blind patch technique used in the present study is the ability to collect data from neurons located deep below the surface of the tissue, where cell health and viability are greatest but visualized targeting of neurons would be difficult. However, this approach prohibits targeting specific neuronal subpopulations; thus, pyramidal neurons throughout all areas of the CA1 pyramidal cell layer were included in the study. Future studies examining time-of-day and sex effects across different subpopulations of CA1 pyramidal cells (e.g., deep vs superficial neurons) will be informative for understanding cell type-specific physiology.

The hippocampus is one of the most studied and well-characterized circuits in the mammalian brain. However, most of the knowledge about how this circuit functions is based on studies conducted during the day in, mostly male, nocturnal rodents. Failing to account for factors like time of day and sex, leads to an incomplete picture of hippocampal physiology and how it dynamically functions across multiple contexts. Sex and time of day are especially important considerations for the translational relevancy of studying the hippocampus in models of diseases like Alzheimer's or epilepsy, which are influenced by circadian rhythms and can affect men and women differently. 

It is important to note that, except for experiments testing OLM, all experiments in this study were conducted in a light-dark cycle. Therefore, future studies in constant conditions are needed to determine the role of the circadian system on observed diurnal differences. While only two-time points were examined here, it will be interesting and helpful to determine how hippocampal physiology is dynamic across multiple time points in the circadian cycle in future studies. In conclusion, this study reveals diurnal variation in hippocampal synaptic and neuronal function and underscores the importance of considering sex, circadian rhythms, and neuronal heterogeneity within a brain region in the study of neural circuits.

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