Examination Of Diurnal Variation And Sex Differences in Hippocampal Neurophysiology And Spatial Memory Part 1

Abstract

Circadian rhythms are biological processes that cycle across 24 hours and regulate many facets of neurophysiology, including learning and memory. Circadian variation in spatial memory task performance is well documented; however, the effect of sex across circadian time (CT) remains unclear. 

Neurophysiology is the study of the structure, function, and pathology of the nervous system. Memory refers to an individual's ability to preserve, reproduce, and utilize memory information. There is an inseparable relationship between neurophysiology and memory.

In neurophysiology, there is a structure called synapse, which is an integral part of the nervous system. Synapses are the place where information is transmitted between neurons, and memory is based on synapses. When a neuron receives information, it communicates with other neurons through synapses and transmits the information. The strengthening or weakening of these contacts forms the basis for memory formation and change.

Memory is just the result of multiple exchanges and calculations between different neurons in the human brain. The number of neurons in the body is fixed, that is, our memory reserve is fixed. Therefore, improving memory means improving the strength of connections between neurons, as well as the number and quality of connections between synapses, which require further knowledge of neurophysiology.

Therefore, neurophysiological research is very beneficial to improving individual memory. Learning about neurophysiology can help us better understand how the human brain works, allowing us to improve memory through exercise and training. Some methods include strengthening the connections between neurons by reading more and thinking more, and improving the quantity and quality of synapses by performing various mental exercises.

In short, neurophysiological research can not only help us better understand the relationship between the human brain and memory but also provide various methods and techniques to improve individual memory. Therefore, we should actively learn relevant knowledge about neurophysiology to create a better environment for our memory. It can be seen that we need to improve our memory. Cistanche deserticola can significantly improve memory because Cistanche deserticola is a traditional Chinese medicinal material with many unique effects, one of which is to improve memory. The efficacy of minced meat comes from the various active ingredients it contains, including acid, polysaccharides, flavonoids, etc. These ingredients can promote brain health in various ways.

Click know 10 ways to improve memory

Additionally, little is known regarding the impact of time-of-day on hippocampal neuronal physiology. Here, we investigated the influence of both sex and time of day on hippocampal neurophysiology and memory in mice. Performance on the object location memory (OLM) task depended on both circadian time and sex, with memory enhanced at night in males but during the day in females. Long-term synaptic potentiation (LTP) magnitude at CA3-CA1 synapses was greater at night compared with day in both sexes.
Next, we measured spontaneous synaptic excitation and inhibition onto CA1 pyramidal neurons. Frequency and amplitude of inhibition were greater during the day compared with night, regardless of sex. Frequency and amplitude of excitation were larger in females, compared with males, independent of time-of-day, although both time-of-day and sex influenced presynaptic release probability. 

At night, CA1 pyramidal neurons showed enhanced excitability (action potential firing and/or baseline potential) that was dependent on synaptic excitation and inhibition, regardless of sex. This study emphasizes the importance of sex and time of day in hippocampal physiology, especially given that many neurologic disorders impacting the hippocampus are linked to circadian disruption and present differently in men and women. Knowledge about how sex and circadian rhythms affect hippocampal physiology can improve the translational relevancy of therapeutics and inform the appropriate timing of existing treatments.

Keywords: 

circadian; hippocampus; memory; plasticity; rhythms; synaptic.

Significance Statement

Circadian rhythms regulate many aspects of neurophysiology, including cognition. However, the impact of time of day and sex on hippocampal neurophysiology and hippocampus-dependent memory remains largely unexplored. 

Here, we report that the circadian regulation of object location memory (OLM) is sex-dependent. Furthermore, examination of hippocampal physiology across time-of-day in both sexes revealed: enhanced long-term synaptic potentiation at night, greater daytime inhibitory synaptic transmission onto CA1 pyramidal neurons, effects of both sex and time-of-day on excitatory synaptic transmission onto CA1 pyramidal neurons, and enhanced nighttime excitability of CA1 pyramidal neurons that is dependent on both synaptic input and position along anterior-posterior hippocampal axis. These results underscore the importance of accounting for sex, regional location, and time of day in the study of hippocampal physiology.

Introduction

The hippocampus is the seat of learning and memory in the brain and its primary output is generated by the principal cells (i.e., pyramidal neurons) in area CA1. Action potential firing by a CA1 pyramidal neuron, like any other neuron, is a combined function of excitatory and inhibitory synaptic drive, intrinsic membrane properties regulating excitability, and neuromodulators (Spruston, 2008). 

A relatively unexplored facet in the hippocampus is how CA1 pyramidal neuron physiology is modulated by time of day. At the cellular level, time-of-day variations in biological function are generated by a transcriptional-translational feedback loop (Partch et al., 2014). Tissue clocks throughout the body are hierarchically organized in a system that drives the timing of 24-hour rhythms in physiology and behavior, enabling organisms to adapt to and anticipate regularly occurring events in their environment (Pilorz et al., 2020; Buijs et al., 2021). 

Circadian regulation of physiological processes is advantageous, and dysregulation of circadian rhythms can promote and exacerbate disease onset and symptoms (Logan and McClung, 2019; Colwell, 2021). Therefore, understanding the circadian influence on physiology is crucial for designing interventions for diseases with circadian dysfunction, such as neurodegenerative diseases (Lee et al., 2021). 

Moreover, the majority of foundational knowledge concerning fundamental principles of hippocampal physiology is based on studies conducted in nocturnal, mostly male, rodents during their inactive phase (daytime). While the scientific community has begun to address the importance of sex as a factor in biomedical research, the importance of time-of-day is still relatively underemphasized. The overarching goal of this study was to begin to unveil how sex and time of day interact to influence daily variation in hippocampal physiology and function.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal orchestrator of the endogenous circadian network, and the electrical properties of SCN neurons vary across time of day. 

Circadian regulation of neuronal excitability is widespread in the mammalian brain (Paul et al., 2020) and has been observed in a range of species, including rodents (Snider et al., 2018), Drosophila (Cao and Nitabach, 2008; Sheeba, 2008), and zebrafish (Elbaz et al., 2013). Although the SCN is the principal clock, autonomous circadian clocks exist in other brain regions, including the hippocampus (Paul et al., 2020; Hartsock and Spencer, 2020). At the molecular level, subregions of the hippocampus rhythmically express core clock proteins, with the cell body layer of area CA1 having the strongest expression of PER2 (Jilg et al., 2010). Moreover, over 600 genes, including those encoding ion channels and synaptic proteins exhibit circadian expression in the hippocampus (Zhang et al., 2014; Renaud et al., 2015). 

At the cellular level, long-term potentiation (LTP), a form of plasticity in which specific patterns of synaptic stimulation result in a long-lasting increase in the strength of synaptic transmission, is expressed at a greater magnitude at night compared with day in nocturnal mice (Chaudhury et al., 2005; Besing et al., 2017; Davis et al., 2020). Cognitive function is also regulated by the circadian system (Wright et al., 2012) and circadian regulation of performance on hippocampus-dependent memory assays has been demonstrated across several species (Snider et al., 2018). 

However, our understanding of how sex affects circadian regulation of cognition is limited. Furthermore, evidence at the cellular level is lacking, including a detailed understanding of how time-of-day and sex regulate synaptic drive onto and membrane properties of CA1 pyramidal cells. Here, we sought to determine how sex and time of day modulate the hippocampal circuit: from the behavioral level down to individual neuronal physiology. 

We found that circadian regulation of hippocampus-dependent memory is dependent on sex, while day-night differences in hippocampal LTP are not. We also found that synaptic transmission and neuronal excitability vary as a function of the time of day and uncovered that some of these changes depend on sex.

Materials and Methods

Animals

All animal procedures followed the Guide for the Care and Use of Laboratory Animals, United States Public Health Service, and were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. All experiments used 6- to 12-week-old C57BL/6J mice of both sexes obtained from Jackson Laboratories (http://jaxmice. jax.org/strain/013636.html) or the C57BL/6J colony at the University of Alabama at Birmingham. 

Mice were maintained on a 12/12 h light/dark cycle with ad libitum access to food (LabDiet Rodent 5001 by Purina) and water. Mice were group housed in same-sex cages of four mice for behavior experiments. For all other experiments, mice were group housed in same-sex cages of two to seven mice per cage.

Object location memory (OLM)

The object location memory (OLM) task (Snider et al., 2016) was conducted under,10 lux dim red light. Four cohorts of mice were used, with each cohort consisting of eight males and eight females. Within each cohort, mice were assigned to undergo habituation, training, and testing either during the day or during the night. In cohorts 1 and 3, males were tested during the day, and females were tested at night. In cohorts 2 and 4, females were tested during the day and males were tested at night.

Mice were entrained to a 12/12 h light/dark (LD) cycle, and then habituated to the arena for 2 d at either Zeitgeber time (ZT) four or ZT 16 (where ZT 12 refers to lights off), for day and night, respectively (days 1–2; Fig. 1A, B). After day 2, mice were released into constant darkness (DD) and again habituated to the arena at projected circadian time (CT) 4 or 16 (days 3–4, where CT 12 refers to the projected time of lights off from the prior LD cycle; Fig. 1A, B). Mice were allowed to acclimate to the behavior room for 20 min each day immediately before habituation or training/test. 

Habituation consisted of 5 min of handling followed by 5 min of arena exploration with visual cues present. Visual cues consisted of vertical stripes on one wall and a large red X on another wall. The arenas were 35.5
25.4 cm with 20.3 cm-high walls. OLM training and testing occurred in DD, 24 h after the final day of habituation at projected CT four or CT 16. Objects were made with PRETEX Building Blocks (Item No. 8030-100) and had three possible positions within the arena, all at least 8.9 cm away from the walls.

During training, each mouse was allowed to explore an arena with two objects for 5 min. Afterward, the mouse was returned to its home cage for 30 min, during which one of the objects from the original exploration was moved to a new position (the novel location) while one remained in its original position (the familiar location; Fig. 1C). A 30-min recall period was chosen based on previously published methods (Snider et al., 2016) and to avoid memory interference because of sleep deprivation or memory enhancement from an overnight sleep period. 

In the test phase, each mouse was placed back in the arena with the novel and familiar location objects and allowed to explore for 5 min. All habituation, training, and testing were recorded at 30 FPS (ELP Camera Model: ELP-USBFHD05MT-KL36IR). Exploration was tracked using a computer model made via DeepLabCut. Object interaction was then analyzed using custom MATLAB (MathWorks) scripts developed by Mary Phillips (https://github.com/PhillipsML/DLC-NovelObject#dlc-novel object). For data analysis, several exclusion criteria were applied: mice that exhibited a clear side preference, mice that spent most of their time exploring objects to try to escape the arena, and mice with a high preference for one object over the other during training were excluded. The discrimination index was calculated as: (time spent exploring novel object location time spent exploring familiar object location)/(time spent exploring novel object location 1 time spent exploring familiar object location).

Electrophysiology

Slice preparation

Mice were killed with cervical dislocation and rapid decapitation at ZT 0–1 or ZT 11–12 for day and night experiments, respectively. Both sex and time of day were interleaved. For extracellular field experiments, brains were removed and 350-mm coronal slices were prepared using a VT1200 S vibratome (Leica Biosystems) in an ice-cold solution containing the following (in mM): 85 NaCl, 2.5 KCl, 4 MgSO4 *7H2O, 0.5 CaCl2 * 2H2O, 1.25 NaH2PO4, 75 Sucrose, 25 NaHCO3, 25 Glucose saturated in 95% O2 and 5% CO2. Slices were allowed to rest for at least 1 h in a recovery solution of standard artificial CSF (ACSF) containing the following (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO4 * 7H2O, 2.5 CaCl2 * 2H2O, 1 NaH2PO4, 26 NaHCO3, and 11 glucose, bubbled with 95% O2/5% CO2. 

For whole-cell patch-clamp experiments, brains were removed and 300-mm thick coronal slices were prepared using a VT1200 S vibratome (Leica Biosystems) in an ice-cold solution containing the following (in mM): 110 choline chloride, 25 glucose, 7 MgCl2, 2.5 KCl, 1.25 Na2PO4, 0.5 CaCl2, 1.3 Na-ascorbate, 3 Na-pyruvate, and 25 NaHCO3, bubbled with 95% O2/5% CO2.
Slices were allowed to rest for at least 1 h at room temperature in a recovery solution 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. For experiments measuring inhibitory synaptic events, 2 mM kynurenic acid was added to the recovery solution.

Field recordings

Data were obtained from ZT 1–6 or ZT 12–18 for day and night recordings, respectively. Coronal hippocampal slices were placed in a submersion chamber and continuously perfused with standard ACSF at 3–5 ml/min and 26–28°C. Schaffer collateral axons were stimulated using a bipolar stimulating electrode placed in the stratum radiatum of area CA3. Field EPSPs (fEPSPs) were obtained with a recording electrode placed in the stratum radiatum of area CA1, within 200–300mm of the stimulating electrode. 

The initial slope of the fEPSPs (fEPSP slope) was measured at the linear region immediately following the fiber volley and preceding the fEPSP peak. Data were acquired and analyzed using pCLAMP10/11(Molecular Devices). Data were recorded using a Kerr Scientific S2 amplifier (Kerr Tissue Recording System, Kerr Scientific Instruments). Signals were digitized at 10 kHz (Digidata 1550B).

Input-output (I/O) curves were generated by measuring the slope of fEPSPs from CA1 stratum radiatum in response to a series of increasing stimulation intensities (0.2–200 mA, D 10 mA) at the Schaffer Collaterals. Baseline fEPSPs were obtained by delivering a 0.1-Hz stimulation to elicit fEPSPs of approximately 0.20 mV/ms for 20 min.

Long-term potentiation (LTP) experiments were conducted by obtaining and maintaining a stable baseline fEPSP response for 20 min, and then LTP was induced by delivering a high-frequency stimulation (HFS; 100 Hz;0.5 s duration; delivered 2
with 15-s interval). This weaker stimulation protocol was chosen to avoid masking a day/night difference in LTP magnitude (Besing et al., 2017; Davis et al., 2020). After HFS, fEPSP slopes were recorded for 40 minutes.

For more information:1950477648nn@gmail.com