Median eminence myelin continuously turns over in adult mice

Objective Oligodendrocyte progenitor cell differentiation is regulated by nutritional signals in the adult median eminence (ME), but the consequences on local myelination are unknown. The aim of this study was to characterize myelin plasticity in the ME of adult mice in health or in response to chronic nutritional challenge and determine its relevance to the regulation of energy balance. Methods We assessed new oligodendrocyte (OL) and myelin generation and stability in the ME of healthy adult male mice using bromodeoxyuridine labelling and genetic fate mapping tools. We evaluated the contribution of microglia to ME myelin plasticity in PLX5622-treated C57BL/6J mice and in Pdgfra-Cre/ERT2;R26R-eYFP;Myrffl/fl mice, where adult oligodendrogenesis is blunted. Next, we investigated how high-fat feeding or caloric restriction impact ME OL lineage progression and myelination. Finally, we characterized the functional relevance of adult oligodendrogenesis on energy balance regulation. Results We show that myelinating OLs are continuously and rapidly generated in the adult ME. Paradoxically, OL number and myelin amounts remain remarkably stable in the adult ME. In fact, the high rate of new OL and myelin generation in the ME is offset by continuous turnover of both. We show that microglia are required for continuous OL and myelin production, and that ME myelin plasticity regulates the recruitment of local immune cells. Finally, we provide evidence that ME myelination is regulated by the body’s energetic status and demonstrate that ME OL and myelin plasticity are required for the regulation of energy balance and hypothalamic leptin sensitivity. Conclusions This study identifies a new mechanism modulating leptin sensitivity and the central control of energy balance and uncovers a previously unappreciated form of structural plasticity in the ME.


INTRODUCTION
Oligodendrocytes (OLs) are the myelin forming cells of the central nervous system (CNS). Once thought to be exclusively generated during early postnatal life in rodents, it is now evident that new myelinating OLs continue to differentiate from a pool of oligodendrocyte progenitor cells (OPCs) in various white matter tracts of the adult brain long after developmental myelination is complete [1e6]. In addition to facilitating brain repair after injury [7], new OL and myelin generation in the adult brain optimizes physiological processes including motor skill learning [8,9] and memory processing [10,11]. Previous studies have demonstrated that new OLs generated during adulthood are long-lived and stably integrate into existing circuits [12] where they either contribute towards the modification of existing myelinated circuits, termed myelin remodeling, or myelinate previously naked axons, termed de novo myelination [13e15]. However, the rate of OPC proliferation and differentiation, and therefore new OL generation, varies by brain region with, for example, adult oligodendrogenesis occurring at a greater rate in the corpus callosum (CC) than motor cortex [2]. The median eminence (ME) is a circumventricular organ located adjacent to the arcuate nucleus (ARC) at the base of the hypothalamus, a region critical for homeostatic functions including the regulation of energy balance. Due to its unique fenestrated vasculature, the ME maintains open communication with the circulation, allowing the release of hypophysiotropic hormones directly into the portal system, and unbuffered exposure to circulating factors for optimal monitoring of circulating cues [16]. The ME is highly plastic, undergoing cellular and structural remodelling that is critical for adaptive physiological responses to stimuli such as energy deficit [17] and the regulation of the neuroendocrine axes [18]. Although the hypothalamus is largely devoid of myelin, myelinating OLs are present in the adult ME. Here, myelin can be observed in a dense band that extends laterally across the dorsal ME, directly below the tanycytes lining the wall of the third ventricle, where it ensheathes axons of magnocellular neurones [19,20]. In the ME, OPCs proliferate more rapidly than in adjacent hypothalamic nuclei in vivo [6,21], and the proportion of differentiating OPCs is higher and regulated by nutritional signals [19]. However, the consequences on local myelination are unknown. The initial goal of this study was to quantify adult oligodendrogenesis in the ME compared to the CC, a well-characterised white matter tract where continuous OL generation occurs in adulthood [2]. Using bromodeoxyuridine (BrdU) and genetic fate mapping approaches, we demonstrate that new myelinating OLs are rapidly and continuously produced in the healthy adult ME, and that new OL and myelin generation are offset by the continuous turnover of myelinating OLs which maintains stable OL and myelin density in the ME over time. Mechanistically, we show that microglia are essential for the maintenance of ME OL and myelin production and that OL plasticity is required for the maintenance of the local immune cell populations. Through the assessment of myelination and OL lineage plasticity in the ME of mice exposed to a high fat diet (HFD) or caloric restriction (CR), we provide evidence that ME myelin amounts are regulated by peripheral energy availability. Finally, we provide evidence that adult generated OLs contribute to the regulation of energy balance and leptin action.

Animals
Details of all reagents and animal models are detailed in Table 1. All animal experiments were performed in accordance with the UK Home Office regulations under the Animals (Scientific Procedures) Act (1986) and with the approval of the University of Cambridge Animal Welfare and Ethics Review Board. Animals were group-housed in a specific pathogen free facility and maintained on a standard 12-hour light/dark cycle (lights on 7:00e19:00) at 22 C with ad libitum access to water and standard laboratory chow (SAFE R105, SAFE Complete Care Competence, Rosenberg, Germany) unless otherwise stated. All experiments were performed on male mice starting from postnatal day 60 (P60). C57BL/6J mice were obtained from Charles Rivers Laboratories (Saffron Walden, UK). Pdgfra-Cre/ER T2 ;Rosa26-YFP;Myrf fl/fl mice were provided by William Richardson at University College London. Opalin-Cre/ER T2 ;Ai9 mice were provided by Professor Thora Karadottir at the University of Cambridge. Plp-Cre/ER T2 mice (stock number 005975) and B6; 129P2-Mapt tm2Arbr /J (tau-mGFP) mice (stock number 021162) were obtained from the Jackson Laboratories (Bar Harbour, Maine).

Tamoxifen preparation and administration
Tamoxifen (Sigma, St Louis, Missouri) was prepared in corn oil (Sigma) by sonication at 37 C at 30 mg/ml prior to administration by oral gavage at 300 mg/kg on 4 consecutive days. Where tamoxifen was administered by the intraperitoneal route, tamoxifen was prepared at 20 mg/ml for injection at 80 mg/kg for 8 consecutive days.

Cumulative bromodeoxyuridine (BrdU) paradigm
Mice were administered BrdU (Sigma), dissolved in drinking water at 1 mg/ml continuously for up to 60 h and concomitantly administered up to two intraperitoneal injections of BrdU (50 mg/kg; prepared in sterile saline at 5 mg/ml) in any 12-hour period. Mice were sacrificed 2, 12, 24 and 60 h after the onset of BrdU administration.
2.4. Caloric-restriction paradigm C57BL/6J mice were acclimatised to single-housing and randomly allocated to the ad libitum fed or caloric restricted (CR) group. For animals that were CR, 70% of the average amount of food eaten by the control group over the previous 24-hour period was provided at ZT11 daily. 7 days after the onset of CR, all animals were administered four intraperitoneal injections of BrdU (50 mg/kg) within a 24-hour period starting at ZT5 and ending at ZT4 on the eighth experimental day. Mice were sacrificed 1 h later, at ZT5, on experimental day 8.
2.5. 45% High fat diet studies C57BL/6J mice were fed a diet with 45% of calories from fat (45% HFD -D12451, Research Diets, New Brunswick, New Jersey) or a standard chow diet for 8 weeks from 7 to 8 weeks of age. Prior to culling, animals were administered 4 intraperitoneal injections of BrdU (50 mg/ kg) over 24 h as described above. For fate mapping studies in Plp-Cre/ER T2 ;R26R-eGFP mice, animals were fed a 45% HFD from P60 and were administered tamoxifen 1-or 8-weeks later. Mice were trans-cardially perfused 14 days after tamoxifen administration.

Leptin sensitivity test
Leptin sensitivity was assessed in single-housed mice in a crossover fashion. Mice were fasted for 24 h prior to the administration of mouse recombinant leptin (R&D Systems) prepared at 0.3 mg/ml in 20 mM TriseHCl pH 8.5 (Sigma) or vehicle by intraperitoneal injection at 10 ml/kg of body weight. Mice were subsequently refed standard chow and food intake and body weight gain recorded over the following 24 h. The experiment was repeated one week later, with animals being administered the alternative substance to which they received on the previous week.

Leptin-induced pSTAT3
Mice were fasted overnight from ZT10 until ZT2, after which mice were administered an intraperitoneal injection of mouse recombinant leptin (R&D Systems) prepared at 0.3 mg/ml in 20 mM TriseHCl pH 8.5 (Sigma) at a dose of 10 ml/kg of body weight. Mice were terminally anaesthetised 30 min later for perfusion fixation and tissue collection.

Confocal microscopy and image analysis
For all experiments, slides and images were blinded to experimental condition. Sections were imaged with a Leica SP8 confocal microscope using either a 40Â or 63Â oil objective. Sections were imaged as zstacks at intervals of 3.3 mm with tile scanning to obtain signal from the entire depth and area of the region of interest (ROI). Microscope settings were identical for image acquisition within each experiment. Images were analysed using Fiji software. For all analyses, Z stacks were projected into a single image and the area of ROIs were selected using the freehand tool and measured. For cell counts, the Fiji manual cell counter was used to count marker-positive cells. Where area of immunoreactivity was calculated, all images within an experiment were identically thresholded and the area fraction, limited to that threshold, measured. 3D reconstruction images were generated using Imaris software.
2.11. Oil red O staining PFA-fixed tissues were cryoprotected in 30% sucrose solution (w/v in distilled water; Sigma) overnight before sectioning at 8e10 mm on a Leica CM1950 cryostat onto electrostatically charged slides. A stock solution of ORO was made up by gently heating 0.5 g ORO (Sigma) in 100 ml absolute isopropyl alcohol (Sigma) in a water bath overnight. A working solution was prepared by mixing 60 ml ORO stock solution and 40 ml 1% dextrin (w/v; Fisher Scientific) in distilled water. The working solution was allowed to stand for one day and filtered prior to use. Slides were rinsed in PBS and incubated with the ORO working solution for 20 min. Excess stain was rinsed off with distilled water and sections counterstained with haematoxylin for 20 s and blued in tap water. Coverslips were mounted with Pertex mounting medium (Pioneer Research Chemicals Ltd.).
2.12. Brightfield microscopy Images were acquired with an Axioscan Z1 Slide Scanner (Zeiss) using a 20Â objective.
2.13. Quantitative polymerase chain reaction Fresh hypothalami were collected into RNALater solution (Thermo-Fisher) and stored at À20 C until processing. RNA was extracted from  Table 2. Details of primers and probes used for qPCR with TaqMan technology are detailed in Table 3. All primers and probes were obtained from SigmaeAldrich and commercially available TaqMan assays were obtained from Thermo Fisher.
2.14. Statistical analysis All data visualisation and statistical analysis was performed in Prism 9 Software (GraphPad). Details of statistical tests are found in figure legends. All data are presented as mean AE standard error of the mean (S.E.M).

New oligodendrocytes are rapidly and continuously produced in the adult median eminence
We first characterised the cell cycle dynamics of OPCs in vivo, using a BrdU pulse-chase paradigm and immunostaining against pan-OL marker SRY-box transcription factor 10 (Sox10) and OPC marker platelet derived growth factor receptor alpha (PDGFRa) to visualise proliferative OPCs (Sox10 þ /PDGFRa þ /BrdU þ ) in the ME, ARC and CC at postnatal day 60 (P60). In line with previous reports of rapid BrdU incorporation into ME OPCs [6,21], ME OPC total cell cycle time (T C ) was w3.4 days, two to three times shorter than OPC T c in the CC or ARC, (Supplementary Figure 1A,B), indicating rapid proliferation of ME OPCs. Unlike ARC and CC OPCs, ME BrdU þ OPCs rapidly lost PDGFRa expression (Supplementary Figure 1C) and gained expression of postmitotic OL marker adenomatous polyposis coli (APC -also known as CC1) following 24-or 60-hours BrdU administration (Figure 1AeC), suggesting that adult-born OLs are rapidly and continuously produced in the ME.
To further establish rapid oligodendrogenesis in the adult ME in vivo, we labelled adult OPCs using Pdgfra-Cre/ER T2 ;Rosa26-YFP mice administered tamoxifen at P60 and followed their fate over time by immunolabelling against OL markers and yellow fluorescent protein (YFP). Consistent with results from BrdU incorporation studies, labelled OPCs (Sox10 þ /PDGFRa þ /YFP þ ) lost PDGFRa expression (Supplementary Figure 1D,E) and gained APC expression faster in the ME than in the CC, indicating rapid new OL production. Strikingly, adult-born OLs (APC þ /YFP þ ) represented 43.0 AE 5.4% of the total OL population in the ME at P60 þ 40 ( Figure 1D,E) compared to just 9.4 AE 0.7% of the total OL population in the CC (Supplementary Figure 1F). Collectively, these data indicate that new OLs are rapidly and continuously produced in the adult ME.

Oligodendrocytes turn over in the adult median eminence
Despite continuous new OL production, histological assessments in both Pdgfra-Cre/ER T2 ;Rosa26-YFP mice ( Figure 1F) and a separate cohort of C57BL/6J mice (Supplementary Figure 1GeI) indicate that OL numbers are stable in the ME between P60 and P140. Intriguingly, the proportion of YFP-labelled adult-born OLs declines after P60 þ 85 in the ME ( Figure 1E), but not CC (Supplementary Figure 1F). This formed a rationale to directly characterise the stability of ME OLs using Plp1-Cre/ER T2 ;Rosa26-GFP mice, in which myelinating OLs are labelled with green fluorescent protein (GFP) following tamoxifen administration ( Figure 1G). In the ME, the proportion of GFP-labelled OLs present in the ME at the time of tamoxifen administration significantly decreased between P60 þ 9 and P60 þ 37 and remained stable thereafter ( Figure 1H) despite no differences in the total number of OLs over time ( Figure 1I). Thus, myelinating OLs are short-lived and continuously replaced in the adult ME. In the CC over the same period, no decline in the proportion of GFP-labelled OLs was observed, as previously reported [12], (Supplementary Figure 1J). Consistent results were obtained with Opalin-iCre/ER T2 ;Ai9 mice (alternative OL-specific Credriver; Supplementary Figure 1KeO), collectively supporting the conclusion that ME OLs turn over in adulthood.

Myelin is continuously replaced in the healthy adult median eminence
We next asked whether ME adult-born OLs become functionally myelinating using Pdgfra-Cre/ER T2 ;tau-mGFP mice, in which  Table 2 e Sequences of primers used in SYBR Green quantitative polymerase chain reaction analysis.
Gene Table 3 e Sequences of primers and probes used in TaqMan quantitative polymerase chain reaction analysis. tamoxifen administration at P60 induces the expression of membrane-targeted GFP (mGFP) upon OPC differentiation, resulting in the labelling of adult-generated myelin [23]. Tissues were immunolabelled for mGFP and myelin basic protein (MBP) to distinguish between adult-generated (mGFP þ /MBP þ ) and pre-existing (mGFP À / MBP þ ) myelin (Figure 2A). mGFP-labelled myelin rapidly populated the ME, indicating that high rates of local OL production are associated with new myelin formation ( Figure 2B), and appeared to be specific to the ME since scarce mGFP immunolabelling was detected in other hypothalamic nuclei including the dorso-medial hypothalamus (DMH), ventro-medial hypothalamus (VMH), lateral hypothalamus (LH) and ARC ( Figure 2D). At P60 þ 80, 73.8 AE 0.8% of ME myelin was adult-generated, but this proportion declined thereafter ( Figure 2B) despite total myelin amounts remaining stable over time ( Figure 2C). This suggests that in the ME, adult-generated myelin is eventually replaced by new myelin. To test this, we labelled pre-existing myelin at P60 using Plp1-Cre/ER T2 ;tau-mGFP mice ( Figure 2E) and followed its fate over time. In the ME, the amount of mGFP-labelled preexisting myelin decreased by w65% while total myelin amounts remained stable ( Figure 2F,G). By comparison, previous reports demonstrate that while new myelin is generated after P60 in the CC this occurs at a slower rate and without myelin turnover [2,12]. Further supporting local myelin turnover, we observed degraded myelin (dMBP) [24] immunolabelling interspersed within the myelin band in the adult ME ( Figure 2H), indicating myelin degradation. Active phagocytes (Iba1 þ /CD68 þ ) as well as myelin-phagocytosing foamy macrophages are also present in the adult healthy ME ( Figure 2I,J) [25]. Finally, consistent with data indicating ongoing myelin turnover in the healthy adult ME, we observed MBP immunolabelling inside Iba1þ cells in the ME of healthy adult mice

Microglia regulate median eminence oligodendrocyte and myelin plasticity
In demyelinating pathologies, myelin debris clearance by phagocytic microglia is critical for new OL formation and myelin repair [26].
Consequently, we hypothesised that if myelin turnover in the ME requires myelin debris removal by local phagocytes, ablation of microglia would blunt OL and myelin plasticity in the ME. To test this, C57BL/6J mice were treated with PLX5622, a colony stimulating factor 1 receptor inhibitor, to ablate microglia ( Figure 3A,B). Two weeks PLX5622 treatment reduced MBP immunolabelling in the ME by w30% ( Figure 3C,D), suggesting that local microglia are required for ME myelin maintenance. This was associated with a decrease in OPC proliferation and differentiation, and a reduction in ME OL density (Figure 3EeG). Thus, consistent with a role for local microglia in myelin turnover in healthy mice, microglia ablation decreases new OL generation in the ME. On the contrary, while PLX5622 treatment decreased microglia density in the CC (Supplementary Figure 3A), microglia ablation did not alter the density of OL lineage cells or OPC proliferation and differentiation here (Supplementary Figure 3B). Infiltrated macrophages densely populate the ME in healthy mice [27], leading us to ask whether myelin turnover and the associated production of myelin debris is responsible for their recruitment. To test this, we used Pdgfra-Cre/ER T2 ; R26R-eYFP; Myrf fl/fl mice (Myrf fl/fl mice), where conditional deletion of myelin regulatory factor (Myrf), a transcription factor required for OPC terminal differentiation, blocks new OL formation in the adult brain (Supplementary Figure 2). In the ME, Myrf deletion rapidly reduced myelin density ( Figure 3H,I), and www.molecularmetabolism.com myelin debris accumulation ( Figure 3J,K) and was associated with a decrease in the density of Iba1 þ cells ( Figure 3L,M), suggesting changes in the microglial population. Since Iba1 labels both microglia and circulating macrophages, we quantified Iba1þ cells co-expressing Tmem119, a marker specific to inactivated, resting microglia. The density of both Iba1 þ /Tmem119 þ (resting microglia) and Iba1 þ / Tmem119 -(activated microglia and circulating macrophages recruited to the ME) cells was significantly decreased in Myrf fl/fl mice ( Figure 3L,M), indicating that adult OL formation in the ME regulates activation of microglia and/or recruitment of infiltrated macrophages.
Myrf deletion did not affect myeloid cell density in the CC (Supplementary Figure 3C). Collectively these data indicate that (1) microglia are required for ongoing ME myelin plasticity and (2) new OL formation is necessary for the maintenance of the local myeloid cell populations and the recruitment and/or activation of microglia and peripheral macrophages.  Data analysed by student's t-test or Welch's test, *p < 0.05, **p < 0.01, ***p < 0.001, n ¼ 6e10/group. Brief Communication 8 3.5. Energy availability regulates median eminence myelination Nutritional signals regulate ME OPC differentiation [19], but the longterm consequences on ME myelination are unknown. Here we tested whether energy excess or deficit produce changes in ME myelination. We first assessed the consequences of chronic energy excess on ME myelination using a model of diet-induced obesity (DIO) ( Figure 4A, Supplementary Figure 3A). DIO increased ME MBP immunolabelling ( Figure 4B,C) and OL density (Sox10 þ /PDGFRa À ; Figure 4D,E) but paradoxically decreased OPC proliferation (Sox10 þ /PDGFRa þ /BrdU þ ) and differentiation (Sox10 þ /PDGFRa À /BrdU þ ; Figure 4F), suggesting that DIO blunts adult oligodendrogenesis in the ME. To test whether increased ME myelination might be the result of changes in OL turnover, we exposed Plp1-Cre/ER T2 ; R26R-GFP mice to the DIO paradigm. DIO significantly increased the density of YFP-labelled OLs in the ME ( Figure 4G,H). indicating reduced rate of OL turnover. Finally, we assessed ME myelination in mice exposed to a 7-day 70% caloric restriction (CR) paradigm ( Figure 4I, Supplementary Figure 3B). CR reduced ME myelin amounts ( Figure 4J,K) without impacting OL density (Sox10 þ /PDGFRa À ; Figure 4L,M). However, CR decreased OPC proliferation (Sox10 þ /PDGFRa þ /BrdU þ ) and differentiation (Sox10 þ /PDGFRa À /BrdU þ ; Figure 4N) in the ME. Thus, energy deficit reduces new OL production in the ME, resulting in local For all images, overview scale bars ¼ 100 mm, inset scale bars ¼ 10 or 20 mm. Data analysed by student's t-test or two-way ANOVA with Sidak's multiple comparisons test, *p < 0.05, **p < 0.01, n ¼ 3e6/group. hypomyelination. Collectively, these data demonstrate that energy availability regulates ME myelination.
3.6. Adult-born oligodendrocytes are required for the regulation of energy balance and hypothalamic leptin sensitivity OPCs have been previously implicated in the regulation of body weight and hypothalamic leptin sensing [28]. However, the contribution of adult born OLs to these processes is unknown. To determine whether adult oligodendrogenesis is required for the central control of energy balance, we assessed the metabolic phenotype of Myrf fl/fl mice between P60 and P130, a timeframe during which oligodendrogenesis is substantial in the ME but negligible by comparison in other white matter tracts [2]. Blockade of new OL production significantly reduced dark-phase food intake in Myrf fl/fl mice maintained on a chow diet ( Figure 5A). Consistently, ARC expression of orexigenic neuropeptide Y (Npy) and agouti-related peptide (Agrp) was decreased following Myrf deletion ( Figure 5B). Despite this, body weight ( Figure 5C) and composition ( Figure 5D) were not altered by Myrf deletion, presumably because energy expenditure ( Figure 5E, Supplementary Figure 5A) and ambulatory activity ( Figure 5F) were comparably reduced. Consistent with reduced energy expenditure, Myrf fl/fl mice defended a lower core body temperature during the dark phase ( Figure 5G, Supplementary Figure 5B) and the expression of thermogenic genes deiodinase 2 (Dio2) and uncoupling protein 1 (Ucp1) was significantly reduced in the brown adipose tissue (BAT) of Myrf fl/fl mice (Supplementary Figure 5C). Myrf deletion had no effect on the respiratory exchange ratio (RER; Supplementary Figure 5D). Consistent with unaltered body composition, plasma leptin levels were comparable between Myrf fl/fl mice and littermate controls ( Figure 5H). However, exogenous leptin did not suppress food intake in fasted Myrf fl/fl mice ( Figure 5I, Supplementary Figure 5E) and physiological leptin replacement during a fast ( Figure 5J) [22] failed to produce the expected decrease in ambulatory activity in Myrf fl/fl mice ( Figure 5K, Supplementary Figure 5F). Furthermore, leptin-induced pSTAT3 expression was significantly reduced in the ARC of Myrf fl/fl mice ( Figure 5L,M) indicating decreased sensitivity of hypothalamic neurocircuitry to exogenous leptin. Collectively, these data support a role for OL plasticity in the physiological action of leptin.

DISCUSSION
Adult oligodendrogenesis is emerging as a new form of brain plasticity observed in a variety of physiological contexts [8,10,11]. In the adult ME, oligodendrogenesis seems to happen within a rapid timeframe with one eighth of OLs being formed within the previous 60 hours, which is unlike what occurs in other brain regions such as the ARC and CC where no adult born OLs are detected within the same period. This high rate of new OL production in the adult ME would have little significance should these new OLs not survive and fully differentiate into myelinating OLs. However, 42 days after inducing the labelling OPCs with YFP at P60, 43% of ME OLs express YFP and hence have been formed from OPCs student's t-test or two-way ANOVA with Sidak's multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n ¼ 6e10/group. that differentiated after P60. This suggests that, unlike what occurs in other regions [13,29], a large fraction of ME adult born OLs survive and become myelinating OLs. Remarkably, the high rate of new OL formation in the healthy adult ME is offset by a comparably high turnover rate, with the proportion of OLs labelled with GFP at P60 decreasing by w40% over 4 weeks, from 75% at P60þ 9 to 49% at P60 þ 37. Collectively, using data obtained from Plp-CreER T2 ;R26R-eGFP and Opalin-CreER T2 ;Ai9 mice, we estimate that the half-life of adult ME OLs is between 6 and 10 weeks which is remarkably shorter than that previously reported for CC OLs (>10 years) and sharply contrasts with evidence for OL longevity in the adult brain [12].
Our study is consistent with work by Zilka-Falb and colleagues who reported a seven-fold difference in the density of BrdU þ OPCs between the ME and CC [21]. Here we found that the cell cycle time of ME OPCs is w3.4 days compared to w10.5 days in the adjacent ARC and w6.5 days in the CC. Of note, our estimate of OPC T C in the CC is w3 days shorter than that previously calculated by Young and colleagues [2], which may result from different paradigms for the administration of BrdU or EdU used in our respective studies. Nevertheless, our data imply that in the healthy adult ME, each OPC produces a new OL and is replaced by a new OPC every 3.4 days, assuming that all ME OPCs proliferate [2] and undergo asymmetric division [30]. We propose that the unique cycling properties of ME OPCs are the result of the unique local environment within the ME, with exclusive unbuffered access to circulating factors including hormones, growth factors and metabolites previously reported to regulate OPCs [31e33]. Thus, changes in the availability of these signals with dietary challenges alters ME OL lineage plasticity. How ME OL death is orchestrated and regulated remains to be determined but given the remarkable stability of the number of OLs in the ME, we propose that myelinating OLs may produce a homeostatic feedback signal that regulates both new OL formation and survival, allowing tight control of the OL population. Depletion of microglia impairs this homeostatic control, suggesting that they might form a necessary component of the process. In later adulthood, this homeostatic control seems to be dysregulated (starting P60 þ 120 AE 6 weeks of age), with increased numbers of OLs in the ME, but whether this is due to decreased rates of turnover or increased rates of OL formation remains to be determined. The differentiation potential of OPCs might decrease with age in the ME, as has been described in the cortex and CC [2,34]. Alternatively, changes in microglial phenotypes with aging [35] might also contribute. Interestingly, high fat feeding, which rapidly induces inflammation in this area of the hypothalamus [36], blunts OL turnover and the homeostatic control of the ME OL population. Thus, the pro-inflammatory phenotype of hypothalamic microglia might reduce their ability to contribute to OL turnover. Our work supports the functional relevance of ME OL/myelin plasticity in the regulation of energy balance. We find that in conditions of energy deficit or energy excess, OPC differentiation is blunted in the ME, albeit with opposite consequences on ME myelination. Genetically blocking adult OPC differentiation in Myrf fl/fl mice significantly changes energy intake and energy expenditure, decreases the responsiveness of ARC neurones to exogenous leptin and reduces the functional consequences of leptin. While the phenotype of Myrf fl/fl mice contrasts the hyperphagic obesity phenotype associated with OPC ablation [28], our study corroborates a role for ME OL lineage cells in hypothalamic leptin sensitivity and indicate that OPCs may play distinct roles from myelinating OLs in the regulation of energy balance. However, further work is needed to elucidate the specific role of ME myelin in these responses.
It should also be noted that reductions in energy expenditure, activity and leptin action observed in Myrf fl/fl mice here could contribute to other aspects of their phenotype e for example, their learning and memory deficits [8e10]. However, since learning produces activitydependent oligodendrogenesis in specific brain areas such as the subcortical white matter [8,9]; interruption of this local effect, rather than systemic effects originating in the ME, is perhaps more likely to underlie learning and memory dysfunctions Our work offers new insights into how the ME couples energy sensing to adaptive responses essential for normal fuel homeostasis. We show that unlike what occurs in other brain regions [12], rapid turnover of myelinating OLs is a feature of the healthy adult ME, and that this process is required for proper sensing of fuel-related signals such as leptin. What could be the function of this energetically demanding process? Such high turnover exists in epithelial barriers where external insults continuously compromise cellular integrity [37]. Likewise, the myelinating OL population of the ME sits at the ME-ARC interface and is exposed to gluco-and lipo-toxic components of the blood that might rapidly affect the composition and structure of local myelin, perhaps forming a trigger for OL apoptosis and turnover. However, the fact that nutrient availability regulates ME myelin plasticity suggests this might form a mechanism through which the hypothalamus adapts to changes in energy levels. Therefore, future studies could determine whether continuous turnover of myelinating OLs protects the mediobasal hypothalamus from metabolic or inflammatory insults associated with obesity, type 2 diabetes, and related disorders.

DATA AVAILABILITY
Data will be made available on request.