Examination Of Diurnal Variation And Sex Differences in Hippocampal Neurophysiology And Spatial Memory Part 2

Whole-cell patch-clamp recordings

All data were collected from coronal hippocampal slices during ZT 2–6 (day) or ZT 13–17 (night) at 32°C in standard ACSF containing the following (in mM): 125 NaCl, 2.5 KCl, 1.25 Na2PO4, 2 CaCl2, 1 MgCl2, 25 NaHCO3, and 25 glucose, bubbled with 95% O2/5% CO2. 

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Whole-cell patch-clamp recordings of CA1 pyramidal neurons were obtained using the blind patch technique. Briefly, patch pipettes were placed at either the medial or lateral end of area CA1 (dependent on whether a slice was from the left or right hemisphere) at a depth of;50–150 mM, positive pressure was applied as the pipette was slowly advanced either medially or laterally through the pyramidal cell layer until a rapid increase in pipette resistance indicated contact with a neuron, at which point positive pressure was released, a tight seal (.1 GX) was obtained, and slight negative pressure was applied to achieve whole-cell patch configuration. 

Data were acquired using a Multiclamp 700B amplifier, Axon Digidata 1440A and 1550B digitizer, and pClamp10/11 software (Molecular Devices). Patch pipettes (BF150–086; Sutter Instruments) were pulled on a Sutter P-97 horizontal puller (Sutter Instruments) to a resistance between 2.5 and 5 MV. Cells were dialyzed for 5 min before experimental recordings. Cells used for analysis had access resistance,30 MV that did not increase by .20% for the duration of each 5-min experiment.

For voltage-clamp experiments, all cells were held at 70 mV, and signals were filtered at 5 kHz and digitized at 10 kHz. IPSCs experiments used a patch pipette solution containing (in mM): 140 CsCl, 10 EGTA, 5 MgCl2, 2 NaATP, 0.3 Na-GTP, 10 HEPES, 0.2% biocytin (pH 7.3, 290 mm), and 5 QX-314 (sodium channel antagonist) added at time of use. IPSCs were pharmacologically isolated with bath perfusion of 10 mM NBQX (AMPAR antagonist, Hello Bio) and 5 mM CPP (NDMAR antagonist, Hello Bio). 

EPSCs experiments used a patch pipette solution containing (in mM): 100 CsOH, 100 gluconic acid (50%), 0.6 EGTA, 5 MgCl2, 2 Na-ATP*3H2O, 0.3 Na-GTP, 40 HEPES, 7 phosphocreatine, biocytin (0.2%), and 5 QX-314 added at time of use. EPSCs were pharmacologically isolated with bath perfusion of 10 mM gabazine (GABAAR antagonist, Hello Bio). Separate experiments to measure miniature IPSC and miniature EPSCs (mIPSCs/mEPSCs) were recorded as above with the addition of 0.5 mM tetrodotoxin (TTX; voltage-gated sodium channel inhibitor, Tocris).

For current-clamp experiments, signals were filtered at 10 kHz and digitized at 20 kHz. Patch pipette solution contained (in mM): 135 K-Gluconate, 2 MgCl2, 0.1 EGTA, 10 HEPES, 4 KCl, 2 Mg-ATP, 0.5 Na-GTP, 10 phosphocreatine, and biocytin (0.2%; pH 7.3, 310 mOsm, and 2–4 MV). Neuronal excitability was assessed by injecting progressive steps of depolarizing current from rest (0–500 pA, D 20 pA) and counting the number of action potentials fired during each 1000 ms current step. 

The response slope was obtained by calculating the linear relationship between firing frequency and injected current across 160- to 400-pA steps. The maximum action potential (AP) firing frequency (max) and current at which max occurred (Imax) were also measured. Sag was measured as the amplitude (mV) of the peak voltage from a hyperpolarizing current injection that achieved a steady-state membrane potential of 90–93 mV. Input resistance (MX) was measured as the slope of the current response to a series of hyperpolarizing current injections (150 to 0 pA, D 50 pA). 

Rheobase was defined as the minimum current required to elicit a single AP. Single APs elicited by rheobase were used to analyze action potential properties (Tables 1, 2). AP amplitude was defined as the voltage difference between the AP threshold and its peak. The threshold was defined as the voltage (mV) at which the AP first derivative (dV/dt) exceeded 20 mV/s. AP rise time was the time (ms) for an AP to reach 90% of its peak amplitude from 10% of its peak. Decay time was the time between 90% and 10% of AP peak amplitude. Half-width was the time (ms) between the half amplitudes of the rise and decay of the AP waveform. Afterhyperpolarization (AHP) was the difference between baseline and the most hyperpolarized point occurring within 3 ms after the AP threshold for fast-AHP (fAHP) and 10–50 ms after the AP threshold for medium-AHP (mAHP). 

Peak AP rise and fall were defined as the maximum slope (DmV/Dms) for AP rise and decay, respectively. Baseline membrane potential was calculated as the mean voltage over the 1400-ms sweep during the 0-pA step. Initial experiments were done in the absence of synaptic blockers to determine how sex and time of day contribute to CA1 pyramidal neuron excitability in the intact circuit. To begin to assess the influence of synaptic transmission on enhanced nighttime excitability, a separate, follow-up experiment was conducted in the presence of the GABAA antagonist, gabazine (10 mM), and the glutamatergic antagonists, NBQX (10 mM) and CPP (5 mM).

Immunohistochemistry

To confirm that cells recorded to measure postsynaptic currents were CA1 pyramidal cells, all cells were filled with biocytin for at least 20 min. Slices containing filled cells were fixed in 4% paraformaldehyde for at least 24 h, then washed for 3
10 min in PBS, and incubated for 2–3 h at RT in a TBS solution containing 10% NDS, 3% BSA, 1% glycine, 0.4% Triton X-100, and streptavidin-488 (1:1000). 

Slices were then washed for 3
10 min in PBS and mounted on glass slides and coverslips with ProLong Gold Antifade mounting media containing DAPI. Slides were visualized on a BZ-X700 fluorescence microscope (Keyence). Any cells that could not be classified as CA1 pyramidal cells based on location and morphology were excluded from the analysis.

Analysis and statistics
Data were analyzed and visualized using SPSS (version 27/28) and Prism-GraphPad software. Assumptions of parametric tests, including normality and homogeneity of variance were assessed, and if violated, data were transformed, or nonparametric tests were used. Unless otherwise stated, significance was ascribed at p, 0.05. A summary of all statistical tests is provided in Extended Data Table 1-1.

Object location memory

OLM data were analyzed using an independent two-way ANOVA with time-of-day and sex as independent variables and discrimination index as the dependent variable (Fig. 1D). A Pearson's correlation was used to assess the relationship between total exploration time and discrimination index scores and a contingency analysis was used to determine the distribution of high versus low exploration times across sex and time-of-day (Extended Data Fig. 1-1B).

Field recordings

Input-output data were analyzed using a linear mixed model with fEPSP slope as a function of Time-of-day, Sex, and Stimulation Intensity. For LTP experiments, the fEPSP slopes were normalized to the baseline responses, and responses obtained during the last 10 minutes of the 40-minute post-HFS recording period were analyzed using a three-way ANOVA with repeated measures (RM-ANOVA).

Whole-cell patch-clamp electrophysiology

Postsynaptic currents (inhibitory and excitatory) were automatically detected from a 5-minute recording using pClamp's event template search and then manually inspected for false event detection. The amplitudes and interevent interval (IEI) were analyzed using a generalized estimating equation (GEE) that allowed parameter estimates with population-averaged models while taking into account correlations between repeated measures within subjects (Reed and Kaas, 2010; Cook et al., 2016). 

The GEE model specified an unstructured working correlation matrix structure, a subject effect of the cell, and a within-subject effect of postsynaptic events. The raw data had a significant positive skew with extreme values and thus, were trimmed of upper and lower outliers (10%) followed by either a log transformation in the case of the amplitude data or a log 1 1 transformation in the case of IEI data, to meet assumptions of normal distributions before analysis.

All current-clamp data were analyzed with Easy Electrophysiology (Easy Electrophysiology, RRID: SCR_ 021190), a software package that utilizes Neo (Garcia et al., 2014). Action potentials were counted using the Action Potential Counting module with the default, AutoThreshold Spike algorithm. An RM-ANOVA was used to analyze action potentials across current steps in which data did not violate the assumptions of linearity and normality: 160– 400 pA. All other membrane properties (Tables 1, 2) were analyzed using a two-way ANOVA with independent variables of time of day and sex. All current-clamp data were stratified by position along the anterior-posterior axis before final statistical analysis.

Results

Day-night differences in OLM performance depend on sex

To examine the effect of sex on circadian rhythms of learning and memory, we used the object location memory (OLM) assay, which relies on a mouse's tendency to explore objects in novel locations, to assess hippocampal spatial memory (Barker and Warburton, 2011; Takahashi et al., 2013; Chao et al., 2016). Although circadian and diurnal differences in performance on OLM have been reported (Takahashi et al., 2013; Snider et al., 2016), the effect of sex on diurnal variation in OLM performance remains poorly understood. 

We found that OLM performance varies across time of day; however, the pattern of diurnal difference in performance differed between sexes (p = 0.023, two-way ANOVA interaction). 

While males performed better at night compared with the day, as expected (p = 0.028, simple main effects comparing day vs night in males; Fig. 1D), female mice performed better during the day compared with the night (p = 0.004, simple main effects comparing day vs night in females; Fig. 1D). There was no effect of time-of-day or sex on total exploration time (p = 0.926 and 0.936, two-way ANOVA main effects; Extended Data Fig. 1-1A). There was no relationship between total exploration and DI scores (r(52) = 0.053, ns p = 0.704, Pearson's correlation; Extended Data Fig. 1-1B).

LTP magnitude at night is greater than day, regardless of sex
Long-term potentiation (LTP) is considered a cellular correlate of learning and memory. LTP at CA3-CA1 synapses is higher at night compared with the day in male mice (Chaudhury et al., 2005; Besing et al., 2017; Davis et al., 2020), but to our knowledge, there are no published reports of the effects of time-of-day on LTP magnitude in adult female mice. Given our finding that diurnal differences in performance on a hippocampal-dependent memory assay are dependent on sex, we next sought to determine whether sex affects diurnal differences in LTP.

First, to assess the strength of basal synaptic transmission at CA3-CA1 synapses, we generated I/O curves by measuring the fEPSP slope from CA1 stratum radiatum in response to Schaffer collateral stimulation over a series of increasing stimulation intensities (0.2–200 mA, D 10 mA) during the day and night in male and female mice (Fig. 2A, B). While neither sex nor time-of-day had a significant effect on basal synaptic transmission over the stimulus range tested (p = 0.552 and 0.981, respectively, LMM main effect), there was significant sex-by-stimulation intensity interaction (p, 0.001, LMM; Fig. 2A, B). 

Males had larger fEPSP slopes compared with females only at 180,190, and 200mA, regardless of time-of-day (p =0.041, 0.043, and 0.035, respectively; simple main effects comparing males and females across all stim intensities; Fig. 2A). Overall, these results indicate that time-of-day does not affect basal synaptic transmission and that sex affects responses only at the highest stimulation intensities.

Next, we assessed synaptic plasticity at the CA3-CA1 synapse by measuring LTP in response to a brief, high-frequency stimulation (HFS; Fig. 2C, D). As previously reported, the magnitude of LTP was greater at night compared with the day in both male and female mice (p = 0.003, three-way RM-ANOVA; Fig. 2C, D); however, there was no significant effect of or interaction with sex. Together, these findings suggest that time-of-day affects synaptic plasticity in male and female mice, without influencing basal synaptic strength.

Synaptic inhibition onto CA1 pyramidal cells during the day is greater than at night, regardless of sex

Changes in LTP can be attributed to synaptic mechanisms and/or intrinsic changes in excitability. Therefore, we first sought to determine whether time of day and sex affect inhibitory and excitatory synaptic transmission onto CA1 pyramidal neurons. To examine synaptic inhibition onto CA1 pyramidal cells, we measured the amplitude and frequency of spontaneous IPSCs (sIPSCs) using whole-cell voltage clamp in male and female mice during the day and night (Fig. 3A). We found that sIPSC interevent interval (IEI) during the day was shorter than at night, regardless of sex (time-of-day: p = 0.033, GEE; Fig. 3A, C), indicating a greater frequency of inhibitory events during the day. 

The amplitude of sIPSCs during the day was larger than at night in both males and females (time-of-day: p = 0.008, GEE; Fig. 3A, E). This increased day-time frequency and amplitude of sIPSCs suggest stronger inhibition of CA1 pyramidal neurons during the day compared with night.

Stronger synaptic inhibition during the day could arise from an increase in presynaptic GABA release, or from increased postsynaptic GABAAR function. To distinguish between these possibilities, we measured miniature IPSCS (mIPSCs) in the presence of the voltage-gated sodium channel blocker tetrodotoxin (TTX) in both male and female mice during the day and night (Fig. 4A). While we found that neither sex (p = 0.392, GEE) nor time-of-day (p = 0.760, GEE) had a statistically significant effect on mIPSC IEI, events from males trended toward exhibiting a day-night difference (p= 0.068, sex
time-of-day interaction, GEE; Fig. 3C). 

The lack of day-night differences in females indicates that the time-of-day effects on sIPSCs are likely driven by local interneuron action potential firing. In males, the mean values between day and night differed by;12 ms (mean and SEM: male day, 98.24 6 1.05 ms; male night, 85.78 6 1.04 ms), suggesting that action potential-independent inhibitory vesicle release may be more frequent at night (Fig. 3C). When we examined mIPSC amplitude, we unexpectedly found a significant interaction between time-of-day and sex (p = 0.038, GEE), with amplitudes in females being larger than males only during the day (p = 0.006, Wald x2 pairwise comparisons; Fig. 4E); however, this;2-pA difference is likely not biologically relevant (female-day: 34.68 6 1.01 pA; male-day: 32.67 6 1.02 pA, mean 6 SEM).

Taken together, these spontaneous and miniature IPSC data suggest that action potential-dependent inhibition, but not spontaneous vesicle fusion, onto CA1 pyramidal cells is greater during the day compared with night in both males and females.

Synaptic excitation onto CA1 pyramidal cells depends on the sex

We next wanted to determine whether spontaneous excitatory synaptic input onto CA1 pyramidal neurons was affected by sex and time of day. First, we measured spontaneous EPSCs (sEPSCs) using whole-cell voltage-clamp recordings (Fig. 5A). Although there was no significant main effect of time-of-day on sEPSC amplitude (Fig. 5E), regardless of sex (p = 0.371, GEE), a statistical trend for a significant main effect of the time-of-day indicated that sEPSC IEI recorded during the day may be greater than those recorded at night (p = 0.052, GEE), suggesting a greater frequency of excitatory events at night (Fig. 5C). Overall, we found that females had more excitatory synaptic input, with larger sEPSC amplitudes and shorter IEIs compared with males (p = 0.022 and 0.020, respectively, main effect of sex, GEE; Fig. 5C, E).

Next, we repeated these experiments in the presence of the voltage-gated sodium channel blocker tetrodotoxin (TTX) and measured miniature spontaneous excitatory synaptic currents (mEPSCs) onto CA1 pyramidal neurons (Fig. 6A). Amplitude of mEPSCs did not vary across sex or time-of-day (p= 0.227 and p= 0.150, main effect of sex and time-of-day, respectively, GEE; Fig. 6E); however, day-night variation in mEPSC IEIs was dependent on sex (p= 0.021, time-of-day by sex interaction, GEE; Fig. 6C). In males, mEPSC IEIs were shorter at night than during the day (p= 0.002, Wald x2 pairwise comparisons), indicating a greater frequency of excitatory events at night in male mice and therefore a likely increase in presynaptic release probability; however, there was no significant day-night difference in females (p= 0.765, Wald x2 pairwise comparisons; Fig. 6C).

Taken together, these results suggest that the trend toward increased nighttime sEPSC frequency (especially in females) is action potential dependent. However, in the males, blocking action potentials uncovers a nighttime increase in frequency that was not seen in the sEPSCs.

CA1 pyramidal neurons are more excitable at night

Broadly, our observations suggest that synaptic inhibition is greater at night and synaptic excitation is greater during the day; thus, we next wanted to determine whether this opposing diurnal variation in synaptic excitatory and inhibitory input results in diurnal variation in CA1 pyramidal neuron excitability. To this end, we patched CA1 pyramidal cells in current clamp mode with the circuit intact (i.e., in the absence of synaptic antagonists) and without clamping cell membrane potential. We injected increasing amounts of depolarizing current (0–500 pA, D 20 pA, 1000-ms duration) into pyramidal neurons and measured the number of action potentials elicited.

Data were collected from neurons throughout the anterior-posterior axis of the hippocampus. Previously published studies found electrophysiological diversity in CA1 pyramidal neurons that are dependent on position across axes (Spruston, 2008; 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); thus, we chose to account for this factor by classifying all neurons as either "anterior" or "posterior" based on the coronal section anatomy (Allen Reference Atlas from https://atlas.brain-map. org/; Fig. 7A). 

When we included the anterior-posterior axis as a factor in our initial ANOVA model assessing several action potentials per current step, we found that the largest contributing factor was region (p= 0.005, main effect, four-way RM-ANOVA). Additionally, significant regional differences were found for input resistance (A: 64.27 6 1.95 MX, P: 75.90 6 2.47 MX, p, 0.001, three-way ANOVA), rheobase (A: 146.69 6 7.50 pA, P: 112.32 6 6.10 pA, p, 0.001, threeway ANOVA), and Imax, or current at which neurons fired at maximum frequency (A: 417.27 6 9.29 pA. P: 386.90 6 10.82 pA, p= 0.047, three-way ANOVA). 

These differences between anterior and posterior neurons aligned with previously published studies that found diversity among dorsal and ventral CA1 pyramidal neurons. While our coronal slice preparation did not allow us to truly isolate ventral CA1, posterior sections are more likely to include some ventral CA1 pyramidal neurons. Indeed, we found that posterior neurons had properties consistent with previously published data in ventral CA1 pyramidal neurons, while anterior neurons were similar to dorsal pyramidal neurons (Dougherty et al., 2012; Malik et al., 2016).

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