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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: J Chem Neuroanat. 2021 Jan 27;113:101920. doi: 10.1016/j.jchemneu.2021.101920

Seasonal changes in adenosine kinase in tanycytes of the Arctic ground squirrel (Urocitellus parryii)

C Frare a,b,1, KL Drew a,b,*
PMCID: PMC8091519  NIHMSID: NIHMS1695952  PMID: 33515665

Abstract

Hibernation is a seasonal strategy to conserve energy, characterized by modified thermoregulation, an increase in sleep pressure and drastic metabolic changes. Glial cells such as astrocytes and tanycytes are the brain metabolic sensors, but it remains unknown whether they contribute to seasonal expression of hibernation. The onset of hibernation is controlled by an undefined endogenous circannual rhythm in which adenosine plays a role through the activation of the A1 adenosine receptor (A1AR). Seasonal changes in brain levels of adenosine may contribute to an increase in A1AR sensitivity leading to the onset of hibernation. The primary regulator of extracellular adenosine concentration is adenosine kinase, which is located in astrocytes. Using immunohistochemistry to localize and quantify adenosine kinase in Arctic ground squirrels’ brain collected during different seasons, we report lower expression of adenosine kinase in the third ventricle tanycytes in winter compared to summer; a similar change was not seen in astrocytes. Moreover, for the first time, we describe adenosine kinase expression in tanycyte cell bodies in the hypothalamus and in the area postrema, both brain regions involved in energy homeostasis. Next we describe seasonal changes in tanycyte morphology in the hypothalamus. Although still speculative, our findings contribute to a model whereby adenosine kinase in tanycytes regulates seasonal changes in extracellular concentration of adenosine underling the seasonal expression of hibernation.

Keywords: ADK, Area postrema, Hibernation, Hypothalamus, Tanycytes

1. Introduction

Hibernation is a seasonal strategy of energy conservation (Carey et al., 2003) and, in obligate hibernators such as the Arctic ground squirrel (AGS), is regulated by a circannual rhythm (Pengelley et al., 1978; Frare et al., 2019). Recent studies highlight how the physiological transition between a summer phenotype to a winter phenotype occurs during the fall season (i.e. pre-hibernation season) (Sheriff et al., 2012; Frare et al., 2018, 2019; MacCannell et al., 2017). Evidence suggests that a seasonal modulation in thermogenesis leading to a decrease in euthermic body temperature (Tb), precedes the onset of hibernation (Sheriff et al., 2012; Frare et al., 2018, 2019) and persists throughout the hibernation season (Olson et al., 2013). The mechanisms that control hibernation are poorly understood, but A1 adenosine receptor (A1AR) signaling has emerged as a seasonal modulator of the phenomenon (Drew et al., 2017). A1AR agonist-induced hibernation in AGS is controlled by processes affecting sensitivity to A1AR signaling within the central nervous system (CNS) that is entrained to an endogenous circannual rhythm (Jinka et al., 2011; Frare et al., 2019). The nature of these processes are unknown, but are expected to include modulation of extracellular concentrations of adenosine in the CNS or the efficacy of A1AR agonist signaling.

Adenosine kinase (ADK), known to be expressed only in glial cells such as astrocytes, is the primary enzyme regulating extracellular levels of adenosine in the CNS (Boison, 2013). Recently, ADK has been suggested to be the connection between sleep and energy metabolism (Bjorness et al., 2016). Although the hibernation season is characterized by an increase in sleep pressure (Walker et al., 1980) and by a suppression in metabolism (Carey et al., 2003), it is unknown if ADK expression changes seasonally. Here we investigate if the levels of ADK change seasonally leading to the onset of the hibernation. Surprisingly, the seasonal change in the expression of ADK was found in tanycytes, but not in astrocytes. Tanycytes, ependymal glial cells with a distinct morphology, are metabolic sensors that deliver metabolic information from the periphery directly to the hypothalamic neurons regulating energy homeostasis (Frayling et al., 2011; Balland et al., 2014). Tanycytes have also been associated with phenotypic plasticity (Prevot et al., 2010; Bolborea and Dale, 2013; Herwig et al., 2013; Ebling, 2014). Our preliminary results suggest that tanycytes may regulate the transition from a summer to a winter phenotype, consistent with the previously described functions of tanycytes, and may modulate the sensitivity to A1AR signaling.

2. Materials and methods

All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Alaska Fairbanks and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition). The objectives of this study were to identify seasonal changes in ADK expression in the hypothalamus, as the brain area involved in energy homeostasis, and to assess tanycytes morphology around the 3μV and 4μV.

2.1. Animals

Arctic Ground Squirrels (AGS, Urocitellus Parryii) were captured in the Brooks Range (68°07′46″N 149°28′33″W) under permit by Alaska Department of Fish and Game. After AGS were brought into the facility, they were housed individually in stainless steel ¼” wire mesh cages (dimensions 12”X19”X12”) and in an environmental chamber maintained at constant conditions, an ambient temperature (Ta) of 2©C and a photoperiod of 4L:20D until the time of tissue collection. The hibernation cycle persists under constant environmental conditions, which allows us to investigate the endogenous circannual rhythm without confounding variables such as Ta and photoperiod. We fed the animals approximately 47μg Mazuri rodent chow per day, and provided water ad libitum, even though AGS do not eat or drink during the hibernation season. We included both juvenile and adult animals of both sexes in the study as the age and sex of the animals depend on the availability at the time of capture.

For the purpose of this study, we processed brains from euthermic animals to assess adenosine-signaling status in summer, fall prior to onset of hibernation and winter during an induced arousal. Animals were housed for an entire hibernation season prior to collection of summer or fall samples.

Summer AGS (2 males and 3 females) were collected two months after the last day of hibernation, and Fall AGS (2 males and 3 females) were identified by the lower euthermic Tb that precedes the beginning of the hibernation season (Sheriff et al., 2012) (Fig. 1). During the hibernation season, we monitored torpor by the shavings added technique (Jameson, 1964); when the wood shavings placed on the animal’s back remain undisturbed the following day, the animal is considered to be hibernating (i.e., torpid). At least eight torpor bouts occurred before we collected brains from AGS during the interbout arousal (IBA) phase (3 males and 2 females), here referred to as Winter (Fig. 1). Interbout arousal was induced by tactile stimulation within the third day of the torpor bout, about 18μh prior to tissue collection. We measured rectal Tb in all AGS just prior to intracardial perfusion with a Digital Microprocessor Thermometer (model HH21 OMEGA Engineering, INC., Stamford, CT). We describe the physiological characteristics of each seasonal phenotype in Table 1.

Fig. 1. Timeline of tissue collection.

Fig. 1.

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AGS were housed at same environmental conditions from time of capture to the time of tissue collection. Fall AGS were collected in the pre-hibernation phase, characterized by a decrease in euthermic Tb. Winter (IBA) AGS were collected in the middle of the hibernation season. Summer AGS were collected two months after the last torpor bout. Summer and Fall samples were collected after an entire hibernation season.

Table 1:

Physiological characteristic of the animals in each group.

Weight (g) Tb (°C) Torpor Bouts
Summer 564± 18 37.2± 0.2a 0
Fall 645± 78 35.5± 0.3b 0
Winter (IBA) 486± 28 36.1± 0.3b ≥8
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Data are presented as mean ± SEM. Values with different letters are statistically different from each other (p<0.05, Tukey HSD test). Sample size n=5 animals per season.

2.2. Brain tissue processing

AGS were anesthetized via chamber induction with 5% isoflurane and maintained via facemask at 3% mixed with 100 % medical grade oxygen delivered at a flow rate of 1.5μL/min. AGS were intracardially perfused first with 0.9 % NaCl for 5μmin and then with 4% PFA in 0.1μM PB buffer pH 7.4 at a flow rate of 79.5μmL/min with a 18Ga needle through the left ventricle after the descending aorta was clamped to obtain a more efficient perfusion of the brain. Brains were removed and post fixed in 4%PFA overnight. Brains were blocked in three parts to allow better penetration of sucrose into the tissue. A gradient of sucrose solutions (5, 10, 15, 20 and 30 % w/v) was made in 0.1μM phosphate buffer (PB) pH 7.4 to cryoprotect the tissue. Brains were maintained in each sucrose solution for up to 3 days and in 30 % sucrose until brains sank. Brains covered with Tissue-Tek O.C.T. matrix (Electron Microscopy Sciences, Hatfield, PA) were rapidly frozen in a bath of n-Hexane 95 % cooled with dry ice to a temperature of −45©C. Coronal sections (40μμm) were cut with a cryostat (CM1850, Leica Biosystems, Buffalo Grove, IL).

Slices were then stored until use at −20©C in cryo-protective (antifreeze) solution made in 0.1μM PB pH 7.4 with 60 %w/v sucrose, 2%w/v polyvinylpyrrolidone (PVP-40, Sigma, St. Louis, MO) and 60 %v/v ethylene glycol (VWR, Radnor, PA).

2.3. Immunohistochemistry

Upon tissue availability, we used five animals per experimental group, and two brain slices per single animal at Bregma −2.80μmm to −3.14μmm in the hypothalamus and Bregma −13.40μmm to −13.60μmm in the brainstem based on the Paxinos and Watson Rat Atlas Fourth Edition (Paxinos and Watson, 1998). Free floating sections were washed using PBS pH 7.2, treated with 0.5 % Triton X-100 for 20μmin at room temperature (RT), then blocked with 10 % normal goat serum (NGS) (Vector Laboratory, Burlingame, CA) in PBS for 30μmin at RT. After a second wash in PBS, the sections were incubated in a humidified chamber for 48μh at 2©C with a cocktail of antibodies diluted in PBS with 1% NGS and 0.1 % TritonX-100. The cocktail included mouse anti-vimentin (1:1000, Millipore, MA Cat# MAB3400, RRID:AB_94843), as a routinely marker used for tanycytes (Millán et al., 2010), and rabbit anti-adenosine kinase (ADK, 1:4000, Bethyl Laboratories, TX Cat# A304–280A, RRID:AB_2620476). Slices were washed in PBS and incubated with a mixture of goat anti-mouse Alexa 488 (1:500, Invitrogen, NY Cat# A28175, RRID:AB_2536161) and goat anti-rabbit Alexa 456 (1:750, Invitrogen, NY Cat# A-11035, RRID:AB_143051, AB_2534093) in PBS containing 1% NGS in a dark room for 2μh at RT. We mounted the brain slices on Superfrost Plus slides (VWR, Radnor, PA) with Vectashield mounting media (Vector Laboratories, CA).

2.4. Image analysis

Images were collected with Slidebook 6 software using the Olympus IX81 inverted confocal microscope (objective LUCPlanFLN 40X, NA 0.60), equipped with a CCD camera (Hamamatsu ORCA-ER,6.45μμmμxμ6.45μμm pixel size, Japan). To minimize exposure time, the images were first captured for ADK analysis (exposure time 3000μms, Gain150); next we collected images for tanycytes analysis as a Z-stack over 10μμm with a step of 0.5μμm (exposure time 400μms, Gain255). These images were analyzed using ImageJ software (version 1.52a, National Institute of Health, USA). During the image analysis, animal ID was not blinded.

We stained two consecutive brain slices per animal. In the hypothalamus (Fig. S1A), we analyzed six to eight fields of view per each brain slice, for a total of 12–16 fields of view (i.e. images) per animal. In the AP, we analyzed five to six fields of view, for a total of 10–12 fields of view (i.e. images) per animal.

The length of the tanycyte processes was measured adapting the standard Sholl analysis used for dendritic organizations (Sholl, 1953). A standard grid was superimposed to each acquired image and the grid number zero was assigned to the grid containing the cell body; then we traced the processes emanating from the cell body and counted the number of grid lines crossed by each process (Fig. S1B). We set the number of processes crossing the first line of the grid (grid1) as 100 % of the processes present in the field of view. We then calculated the percent of processes crossing the other lines of the grid as follows: % processes in gridn= (number of processes in gridn*100 %)/ (number of processes in grid1).

To measure the width of the tanycyte processes, we traced a vertical line parallel to the 3μV, across the tanycytes’ processes and plotted the intensities across the line, a method previously used to measure filaments thickness (Reimold et al., 2013). We used the function “plot profile” in ImageJ, which displays the intensity peaks for each process crossing the traced line. Then, we set the threshold to 500 grey value and manually measured the process’s width at the base of each peak (Fig. S2). We averaged 12–16 plots for each AGS. We displayed the data as the percentage of tanycytes with processes’ width smaller than 5μμm and greater than 5μμm. We primarily investigated the α tanycytes, because in free-floating IHC the median eminence tends to fold over on itself which interferes with analysis of β tanycytes.

The ADK intensity in the layer of the 3μV was measured tracing the region of interest (ROI) around the tanycyte cell bodies (Fig. S1C). For the remainder of the hypothalamus, we traced the ROI excluding the ventricle layer ROI, in an area including part of the ventromedial nucleus and the arcuate nucleus of the hypothalamus, referred as mediobasal hypothlamaus. In the AP and 4μV, the same technique was applied. Since the ROI were different within fields of view, we used the image J measure of the mean grey value that allows comparison of different size ROI. The mean grey value measures the average of grey value within the ROI (the sum of all the grey value of all the ROI pixels divided by the total number of pixels in the ROI).

2.5. Statistical analysis

Data are presented as meanμéμSEM and a pμ≤μ0.05 was considered significant. Data analyses were performed using R (version 1.1.423). First, we tested the data for the assumption of normality using Shapiro test and the assumption of homogeneity of variance using the Bartlett test. If these assumptions were met, we compared the groups using a one way ANOVA followed by Tukey’s honestly significant difference (HSD) test. When the data did not meet the assumption of homogeneity of variance, even after being transformed to the square root, we performed a Welch’s ANOVA test followed by multiple comparison t-tests with the Bonferroni’s correction.

3. Results

3.1. Tanycytes express ADK

ADK is the primary regulator of extracellular adenosine concentration in the brain prompting us to ask if ADK expression changes seasonally in a manner that could influence extracellular adenosine concentration. No significant differences were detected between groups in ADK intensity in the astrocytes within the mediobasal hypothalamus (F(2,12)μ=μ1.456, pμ=μ0.27 one way ANOVA, Fig. 2A–B). We did, however, find ADK within the cell bodies of the tanycytes lining the 3μV (Fig. 2A, SI Figs. 3 and 4). We next measured ADK intensity in the α tanycytes and found that ADK intensity changed between the seasons (F (2,6.52)μ=μ12.287, pμ<μ0.01 Welch’s ANOVA). ADK intensity significantly decreased in the Winter group compared to the Summer (pμ<μ0.01 t-test with Bonferroni’s correction, Fig. 2C). It is also interesting to report that qualitative observations show two layers of tanycytes in two of the five Fall AGS (Fig. 2A), but in none of the other groups.

Fig. 2. ADK levels are lower in Winter compared to Summer in tanycyte cell bodies.

Fig. 2.

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Photomicrographs show ADK (red) present in the cell body of the tanycytes (immunolabeled for vimentin; green) adjacent to the 3 V in all groups (indicated by a white arrow). ADK is seen in astrocyte cell bodies located in the center of the slice away from the 3 V; this area corresponds to the mediobasal hypothalamus. Scale bar 25 μm (40x objective). 3 V: third ventricle (A). ADK intensity in the astrocytes, in the mediobasal hypothalamus, does not change within groups (B). ADK intensity, measured within the cell bodies of tanycytes lining the 3 V, is higher in Summer compared to Winter; solid line represents p < 0.01, t-test with Bonferroni’s correction (C). Sample size n = 5 animals per season, values in the y-axis are the average of 12 to 16 fields of view analyzed in two consecutive brain slices per each animal. In the whisker-box plot, the line in the middle of the box represents the median separating the first and the third quartile, the vertical lines (i. e., the whiskers) show the 1.5*interquartile range above and below the first and third quartile. Data points located outside of the 1.5*interquartile range boundaries are shown as unfilled dots (B and C).

3.2. Season alters tanycyte process width, but not length in the 3μV

Morphological change in tanycytes processes occurs in seasonal reproductive species. Therefore, we asked if tanycytes morphology changes across seasons in AGS. Qualitative observations showed a difference in tanycytes morphology between the Summer and the Winter groups along the vertical wall of the 3μV. The Winter group was characterized by tanycytes with consistently thinner processes compared to the Summer group, but the distinction was not as evident in the Fall group, which included both thin winter-like and wide summer-like processes (Fig. 3A). We did not find any difference in process length between seasons in the tanycytes of the 3μV (Fig. 3B). Since ADK has been used as a proxy for astrocyte proliferation, we asked if the number of tanycytes were different between groups. The mean number of tanycyte cell bodies present in grid 1 for each animal was analyzed by ANOVA to test if the number of tanycytes was different between groups. The number of tanycytes did not differ (F(2,12)μ=μ2.5, pμ=μ0.48 one way ANOVA, SI Fig. 5), thus changes in ADK do not reflect tanycytes proliferation.

Fig. 3. Tanycyte process width, but not length changes between seasons.

Fig. 3.

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Qualitative observations showed a distinct morphology in the tanycytes of the Summer and Winter, but not the Fall animals as illustrated by the photomicrographs of tanycytes stained with vimentin (green). Scale bar 25 μm (40x objective). 3 V: third ventricle (A). Morphological analysis of tanycytes showed that process length did not change between seasons; percent of processes refers to the percent of processes that reach the grid line indicated on the x-axis (B). Consistent with our qualitative observations, we measured a significantly higher number of tanycyte processes with width smaller than 5 μm in Winter AGS compared to Summer; the percent of processes represents the ratio between the thinner processes over the total number of processes measured, solid line represents p < 0.05, t-test with Bonferroni’s correction (C). In the whisker-box plot, the line in the middle of the box represents the median separating the first and the third quartile, the vertical lines (i.e., the whiskers) show the 1.5*interquartile range above and below the first and third quartile. Data points located outside of the 1.5*interquartile range boundaries are shown as unfilled dots (C).

We followed our qualitative assessment with a measure of the processes’ width in the 3μV. We found that Winter AGS showed a significantly higher number of thin tanycytes compared to Summer, reported as percentage of tanycytes with processes’ width less than 5μμm (F(2, 5.7)μ=μ15.396, pμ<μ0.01 Welch’s ANOVA, followed by t-tests with Bonferroni’s correction, Fig. 3C). This quantitative analysis supports our qualitative observation of thinner tanycyte processes in Winter AGS.

3.3. ADK expression in the tanycyte-like cells in the AP and 4μV

Next, we looked at the tanycyte-like cells in the AP and the ADK intensity in astrocytes and tanycyte-like cells. We observed ADK immunoreactivity in the AP and in the cell bodies of tanycyte-like cells lining the 4μV (Fig. 4A). We did not find a significant difference in ADK intensity between the groups in the AP (F(2,12)μ=μ0.22 pμ=μ0.806 one way ANOVA, Fig. 4B) and in the tanycyte-like cells (F(2,12)μ=μ0.15, pμ=μ0.86 one way ANOVA, Fig. 4C). We next examined the morphology of tanycyte-like cells around the central canal and compared them to the tanycytes lining the 3μV; the cell body is cubical with short processes converging towards the ventral part of the brain (Fig. 4D). The lack of long processes in 4μV tanycyte-like cells prevented us from quantifying process width and length.

Fig. 4. Tanycyte-like cells and ADK are present in the AP, but do not show seasonal changes.

Fig. 4.

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Vimentin (green) stained tanycyte-like cells in the AP and in the area contacting the 4 V. ADK (red) is visible within the tanycyte-like cell bodies lining the 4 V and in the astrocytes present in the AP shown in the representative photomicrographs of a Summer, Fall and a Winter AGS (A). Intensity of ADK did not change between seasons in the astrocytes within the AP (B) or within the cell bodies of tanycyte-like cells in the 4 V (C). The central canal is characterized by tanycyte-like cells with very short processes and a cubical cell body (for better resolution the image was constructed from two images taken under 40x objective) (D). Scale bar 25 μm (40x objective). 4 V: fourth ventricle, cc: central canal, AP: area postrema. Sample size n = 5 animals per season, values in the y-axis are the average of 10 to 12 fields of view analyzed in two consecutive brain slices per each animal. In the whisker-box plot, the line in the middle of the box represents the median separating the first and the third quartile, the vertical lines (i.e., the whiskers) show the 1.5*interquartile range above and below the first and third quartile. Data points located outside of the 1.5*interquartile range boundaries are shown as unfilled dots (B).

4. Discussion

The mechanisms that control the seasonal expression of hibernation are poorly understood. Seasonal response to A1AR agonist is thought to underlie the seasonal regulation of hibernation. Here we show for the first time that tanycytes express ADK and that expression within the 3μV changes with season.

Adenosine is metabolized by both enzymatic deamination to form inosine, and by ADK phosphorylation to form AMP. However, under normal physiological conditions, astrocytic ADK (AdK; EC 2.7.1.20) is the primary regulator of adenosine metabolism (Lloyd and Fredholm, 1995; Latini and Pedata, 2001). Therefore, altered ADK expression is significant because it reflects changes in the extracellular adenosine levels (Boison et al., 2010; Etherington et al., 2009). Increased ADK activity decreases intracellular adenosine levels, which in turn increases clearance of extracellular adenosine via equilibrative nucleoside transporters leading to an overall reduction in extracellular levels of adenosine (Baldwin et al., 2004; Boison et al., 2010).

Previous work showing that hypothalamic adenosine is higher during the entrance phase compared to torpor (Tamura et al., 2008), motivated us to investigate adenosine signaling in hypothalamic astrocytes, focusing on ADK. Interestingly, we found no differences in ADK in the hypothalamic area investigated, but we found lower ADK levels in winter compared to summer in tanycytes cell bodies of the 3μV. As a model for further study, a decrease in ADK would support higher levels of extracellular adenosine. Lower ADK expression would decrease the rate of conversion of adenosine to AMP in tanycytes increasing intracellular adenosine accumulation. Increased intracellular adenosine would increase the extracellular concentration via equilibrative nucleoside transporter. Hence, adenosine concentration in the extracellular milieu would rise and could activate A1AR and drive the natural onset of hibernation and contribute to the seasonal sensitivity to A1AR agonist-induced hibernation in AGS (Jinka et al., 2011; Frare et al., 2019). In contrast to our finding, in the whole hypothalamus of 13-lined ground squirrel ADK mRNA was higher during IBA compared to the end of the hibernation season (April) (Schwartz et al., 2013). Whole hypothalamus ADK levels would reflect ADK in both tanycytes and astrocytes and dilute differences in ADK expression that we found was restricted to tanycytes. The disparity between studies could also be related to the differences between mRNA expression and protein levels, but also to the different time point in active euthermic ground squirrel tissue collection. A detailed time course is needed to identify changes in ADK protein expression within tanycytes and astrocytes within the torpor bouts and across the year.

We acknowledge some limitations in our proposed model, adenosine metabolism is not limited to ADK activity, but other enzymes participate in its intracellular metabolism. Adenosine can be converted into inosine by adenosine deaminase (ADA) or into S-adenosylhomocysteine (SAH) by S-adenosylhomocysteine hydrolase. Unfortunately, due to limited tissue availability, it was beyond the scope of the present study to measure the expression or activity of these enzymes. Therefore, some questions remain unanswered such as do seasonal changes occur in ADA and SAH expression? Little is known about the role of inosine and SAH in energy homeostasis, but inosine has a neuroprotective effect in ischemic injury (Shen Hui et al., 2005). Interestingly, AMP and adenosine are reciprocal precursors. AMP is converted into adenosine by the 5′nucleotidase and ecto-5′nucleotidase, respectively in the intracellular and the extracellular space, but we do not know if these enzymes increase in winter enhancing adenosine concentration promoted by low ADK expression. Whether AMP is converted to adenosine or not, the activation of A1AR leading to hibernation onset, could persist since AMP is an A1AR agonist (Drew et al., 2017; Rittiner et al., 2012). Overall, a more complete characterization of adenosine metabolic pathways will be necessary to understand if ADK plays a primary role in modulating adenosine levels in our seasonal model.

Lower ADK in winter could regulate other aspects of the winter phenotype characterizing seasonal hibernators. Adenosine plays a role in sleep and energy metabolism, and ADK is emerging as a component in the somnogenic properties of adenosine. Overexpression of astrocytic ADK suppresses sleep (Palchykova et al., 2010; Diógenes et al., 2014), while the ablation of ADK increases homeostatic sleep drive highlighting the connection between sleep and energy metabolism (Bjorness et al., 2016). In ground squirrels, the winter phenotype shows an increase in sleep drive compared to the summer (Walker et al., 1980). Adenosine within the preoptic area of the hypothalamus modulates thermogenesis (Ticho and Radulovacki, 1991) and recently we found seasonal modulation of thermogenesis in AGS. Prior to the hibernation season in AGS, vasoconstriction increases and euthermic Tb decreases; both thermoregulatory changes are the requisite for adenosine-induced hibernation (Frare et al., 2019). Similarly, the seasonal remodeling of adenosine signaling seems to occur during the fall transition period (i.e. the pre-hibernation season) as shown by the lack of difference in ADK expression between Fall and the Summer or Winter. Overall, our exploratory observations support further study of a novel model whereby seasonal modulation of ADK expression in tanycytes contributes to seasonal changes in adenosinergic tone that underlies seasonal sensitivity to A1AR agonist, seasonal sleepiness, and downregulation of thermogenesis that would allow the spontaneous expression of hibernation.

Until now, astrocytes were the only cell type known to express ADK in the adult brain (Boison, 2013). Finding ADK in tanycytes may not be surprising, since tanycytes share some properties with astrocytes (Rodríguez et al., 2005). Astrocytes and tanycytes both use ATP as a signaling molecule. ATP is released from tanycytes in the parenchyma by metabolic cues, such as glucose and amino acids, and it activates the nearby neurons (Frayling et al., 2011; Lazutkaite et al., 2017). The presence of purinergic receptors (P2X, P2Y1) in the cell membrane and the expression of ecto-ATPase nucleoside triphosphate diphosphonydrolase 2 (NTPDase2) enzyme, which hydrolyzes ATP to ADP, suggest that purinergic signaling is present in tanycytes (Braun et al., 2003; Prevot et al., 2018). Through this work, we extend the role of purinergic signaling described in tanycytes and we propose tanycytes as an additional regulator of extracellular adenosine.

A feature of tanycytes is their ability to change morphology depending on the physiological state of the animals such as reproductive state regulating neurohormone secretion (Prevot et al., 2010; Pouchain Ribeiro Neto et al., 2017), and energy status modulating the access of circulating metabolites to the brain parenchyma (Langlet et al., 2013; Ramalho et al., 2018). As hibernation is characterized by seasonal changes in the reproductive system (Barnes et al., 1986; Darrow et al., 1988; Barnes and York, 1990) and by drastic metabolic suppression (Andrews et al., 2009), changes in tanycytes morphology are likely to occur. We were able to measure a decrease in processes’ width in winter compared to summer, which is consistent with previous data in hamsters showing a decrease in vimentin gene expression in winter compared to summer (Herwig et al., 2013; Petri et al., 2014). In addition, transcriptomic analysis in 13-lined ground squirrel shows a lower hypothalamic level of nestin in IBA compared to spring euthermia (Schwartz et al., 2013). Nestin is an intermediate filament of the cytoskeleton as is vimentin, and is known to be highly expressed in tanycytes (Langlet, 2019). Overall, our data and previous work suggest a seasonal remodeling of the cytoskeleton. This seasonal remodeling may in part contribute to a decrease in cellular energy consumption. Housekeeping (non-signaling) processes (e.g. cytoskeleton dynamics) are costly and have been estimated to be around 40 % of total brain energy (Bernstein and Bamburg, 2003; Engl and Attwell, 2015). A reduction during the hibernation season may be an additional strategy to conserve energy, which is acquired in the fall (as seen by the blend of thick and thinner processes) when thermoregulatory pathways are gradually adjusted (Frare et al., 2019). Although we did not find any difference in process length, we cannot exclude a change in the tanycyte endfeet that is known to play a role in the regulation of the reproductive cycle and energy status (Langlet, 2019). Tanycytes processes interdigitate with brain nuclei directing molecules in the CSF, such as glucose and fatty acids, to specific brain regions (Elizondo-Vega et al., 2019; Hofmann et al., 2017). It is tempting to speculate that structural changes in tanycytes processes may allow a wide delivery of these molecules to the hypothalamic parenchyma in particular to the ventromedial hypothalamus and the arcuate nucleus, the primary brain regions associated with the α tanycytes characterized here. The hypothalamus regulates energy homeostasis (Blouet and Schwartz, 2010; Williams et al., 2001). Thus, α tanycytes facilitating access of metabolites in the CSF to the hypothalamus during the winter (i.e. hibernation season), could mediate seasonal changes in the metabolic phenotypes of hibernators, i.e. the switch from glucose to fatty acid metabolism (Andrews et al., 2009), or be part of the neuronal pathways regulating this adaptive seasonal switch. An alternative possibility is that differences in vimentin staining are due to a decrease in vimentin expression rather than structural changes of tanycyte processes. Further studies are needed to provide the structural details necessary to answer this question.

Qualitative observations lead to new studies and ideas; therefore, we reported the interesting observation that two Fall AGS showed two layers of tanycytes lining the 3μV that were not seen as distinctly in the Summer and Winter groups. When counted, the number of tanycytes did not differ significantly within groups, but Fall AGS tend to have a higher number of tanycytes compared to Winter that could become significant with larger sample sizes.

We also acknowledge that the limited number of animals available for this study did not allow us to analyze the experimental groups by sex, limiting us from identifying any sex-dependent changes. Previous work in hibernators showed changes in neuronal morphology between torpor and IBA, but the sex of the animals was not stated (Ohe et al., 2006, 2007). A later paper investigated gross brain morphology in male and female Richardson’s ground squirrels during the breeding and non-breeding seasons, and the sex-differences reported were associated with the reproductive phase (Keeley et al., 2015). In non-hibernators, changes in tanycytes morphology were originally found in female mice based on their estrous cycle (Prevot et al., 2010, 2018). A more recent study on sex differences in neurogenesis, found that high fat diet stimulates neurogenesis in female mice, but not in male mice and that this effect is likely mediated by tanycytes (Lee et al., 2014). Based on these papers, we did not expect sex-dependent changes in brain morphology and tanycytes because our animals were collected during euthermia (not torpor), in the non-reproductive season (Spring was purposely excluded from the study) and were fed regular rodent chow. However, sex-dependent changes cannot be fully ruled out.

Tanycyte-like cells are known to be present in the floor of the fourth ventricle (4μV) and in the area postrema (AP) and serve as a barrier between the periphery and the CNS (Felten et al., 1981; Langlet et al., 2013). Previous work showed that A1AR agonist, and potentially endogenous adenosine, acts on the Nucleus Tractus Solitarius (NTS) to promote torpor in AGS and hypothermia in rats (Frare et al., 2019; Tupone et al., 2013). Therefore, we examined tanycytes morphology and ADK expression near the 4μV suspecting that tanycytes in this region may modulate adenosine levels in the NTS. We observed morphological differences between tanycytes in the 4μV and the AP compared to the tanycytes in the 3μV, but we did not find any seasonal changes.

In conclusion, tanycytes express ADK and ADK expression changes with season in the hibernating AGS. The changes in ADK expression in the 3μV tanycytes warrants further study as a mechanism to influence extracellular adenosine concentrations in the CNS, in particular the hypothalamus, and play a role in the seasonal sensitivity to A1AR-induced hibernation.

Supplementary Material

Suppl. Material

Acknowledgments

We thank L. Smith, Dr. K. Hueffer, Dr. T. Kuhn, Dr. Bult-Ito and Dr. D. Boison for technical support.

Funding

Research reported in this publication was supported by NSF10S-1258179; the National Institutes of Health under Award Numbers NS081637; the National Institute Of General Medical Sciences of the National Institutes of Health under the Award Number TL4GM118992; Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant numbers P20GM103395 and P20GM130443. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Abbreviations:

A1AR

A1 adenosine receptor

ADK

adenosine kinase

AGS

Arctic ground squirrel

AP

area postrema

CNS

central nervous system

ROI

region of interest

Tb

body temperature

3V

third ventricle

4V

fourth ventricle

Footnotes

Ethical statement

All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Alaska Fairbanks and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition) and the ARRIVE guidelines.

Declaration of Competing Interest

KD has a financial interest in Be Cool Pharmaceutics.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jchemneu.2021.101920.

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