Article Hypothalamic pregnenolone mediates recognition memory in the context of metabolic disorders Graphical abstract Authors Sara Ramı́rez, Roberta Haddad-Tóvolli, Marija Radosevic, ..., Amanda Jiménez, José C. Fernández-Checa, Marc Claret Correspondence ramirez@clinic.cat (S.R.), mclaret@clinic.cat (M.C.) In brief Ramı́rez et al. show that reduced hypothalamic pregnenolone levels, in the context of metabolic diseases, are associated with cognitive deterioration. POMC neuron-derived pregnenolone is a key mediator of cognition, but not metabolism, acting via hippocampal mechanisms. These results identify an unsuspected role for POMC neuron- derived pregnenolone in memory performance. Highlights d Acute western diet impairs memory performance, which is reversed by pregnenolone d Pregnenolone synthesis interference in POMC neurons causes cognitive dysfunction d POMC pregnenolone mediates memory function via hippocampal mechanisms d Pregnenolone in the CSF of individuals with unhealthy obesity correlates with cognitive score Ramı́rez et al., 2022, Cell Metabolism 34, 269–284 February 1, 2022 ª 2022 The Authors. Published by Elsevier Inc. https://doi.org/10.1016/j.cmet.2021.12.023 ll ll OPEN ACCESS Article Hypothalamic pregnenolone mediates recognition memory in the context of metabolic disorders Sara Ramı́rez,1,25,* Roberta Haddad-Tóvolli,1 Marija Radosevic,2 Miriam Toledo,1 Adriana Pané,3 Daniel Alcolea,4,5 Vicent Ribas,6,7,8 Maria Milà-Guasch,1 Macarena Pozo,1 Arnaud Obri,1 Elena Eyre,1 Alicia G. Gómez-Valadés,1 Iñigo Chivite,1 Tomas Van Eeckhout,1 Ioannis Zalachoras,9 Jordi Altirriba,10 Corinna Bauder,11 Mónica Imbernón,12 (Author list continued on next page) 1Neuronal Control of Metabolism (NeuCoMe) Laboratory, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain 2Neuroimmunology Laboratory, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clı́nic de Barcelona, University of Barcelona, Barcelona, Spain 3Obesity Unit, Hospital Clı́nic de Barcelona, Barcelona, Spain 4Memory Unit, Department of Neurology, Hospital de la Santa Creu i Sant Pau, Biomedical Research Institute Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain 5Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain 6Department of Cell Death and Proliferation, Institute of Biomedical Research of Barcelona (IIBB), CSIC, Barcelona, Spain 7Liver Unit, Hospital Clı́nic de Barcelona, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain 8Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain 9Laboratory of Behavioral Genetics, Brain Mind Institute, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland 10Laboratory of Metabolism, Department of Internal Medicine Specialties, Faculty of Medicine, University of Geneva, Geneva, Switzerland 11Department of Neuronal Control of Metabolism, Max Planck Institute for Metabolism Research, Cologne, Germany 12Department of Physiology, Centro de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS), University of Santiago de Compostela, Instituto de Investigación Sanitaria (IDIS), Santiago de Compostela, Spain 13Muscle Research and Mitochondrial Function Laboratory, CELLEX-IDIBAPS, Internal Medicine Department, Faculty of Medicine, University of Barcelona, Hospital Clı́nic de Barcelona, Barcelona, Spain 14Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain 15Center for ALPD, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 16Centro de Investigación Biomédica en Red de la Fisiopatologı́a de la Obesidad y Nutrición (CIBEROBN), Barcelona, Spain 17Neurophysiology Laboratory, Department of Biomedicine, Faculty of Medicine, Neuroscience Institute, University of Barcelona, Barcelona, Spain 18National Center for Diabetes Research (DZD), Neuherberg, Germany 19Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany (Affiliations continued on next page) SUMMARY Obesity and type 2 diabetes are associated with cognitive dysfunction. Because the hypothalamus is impli- cated in energy balance control and memory disorders, we hypothesized that specific neurons in this brain region are at the interface of metabolism and cognition. Acute obesogenic diet administration in mice impaired recognition memory due to defective production of the neurosteroid precursor pregnenolone in the hypothalamus. Genetic interference with pregnenolone synthesis by Star deletion in hypothalamic POMC, but not AgRP neurons, deteriorated recognition memory independently of metabolic disturbances. Our data suggest that pregnenolone’s effects on cognitive function were mediated via an autocrine mecha- nism on POMC neurons, influencing hippocampal long-term potentiation. The relevance of central pregnen- olone on cognition was also confirmed in metabolically unhealthy patients with obesity. Our data reveal an unsuspected role for POMC neuron-derived neurosteroids in cognition. These results provide the basis for a framework to investigate new facets of POMC neuron biology with implications for cognitive disorders. INTRODUCTION €her, 2019). Epidemiological and clinical studies denote that, (Blu among the several complications related to these metabolic dis- Obesity and type 2 diabetes mellitus (T2D) have reached orders, cognitive dysfunction is a prominent comorbidity. epidemic dimensions, thus becoming major global health issues Indeed, evidence shows that obesity and T2D are associated Cell Metabolism 34, 269–284, February 1, 2022 ª 2022 The Authors. Published by Elsevier Inc. 269 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ll OPEN ACCESS Article Gloria Garrabou,13,14 Carmen Garcia-Ruiz,6,7,8,15 Rubén Nogueiras,12,16 David Soto,17 Xavier Gasull,17 Carmen Sandi,9 € ning,11,18,19,20 Juan Fortea,4,5,21 Amanda Jiménez,3,16,22 José C. Fernández-Checa,6,7,8,15 and Jens C. Bru Marc Claret1,23,24,25,26,* 20Center for Endocrinology, Diabetes and Preventive Medicine (CEPD), University Hospital of Cologne, Cologne, Germany 21Barcelona Down Medical Center, Fundació Catalana de Sı́ndrome de Down, Barcelona, Spain 22Translational Research in Diabetes, Lipids and Obesity, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain 23Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain 24Faculty of Medicine, Universitat de Barcelona, Barcelona, Spain 25Senior author 26Lead contact *Correspondence: ramirez@clinic.cat (S.R.), mclaret@clinic.cat (M.C.) https://doi.org/10.1016/j.cmet.2021.12.023 with diverse forms of cognitive decline, including subtle cognitive (namely, POMC and AgRP neurons) play fundamental roles in decrements, mild cognitive impairment, and even severe de- the regulation of systemic energy balance and in the develop- mentia (Biessels and Despa, 2018; Tanaka et al., 2020). ment of obesity and T2D (Jais and Bru €ning, 2021). Second, An emerging concept posits brain insulin resistance as a rele- because the hypothalamus has been historically related to mem- vant condition linking metabolic disorders with cognitive deteri- ory disorders (Vercruysse et al., 2018), and third, because evi- oration (Biessels and Despa, 2018; Sripetchwandee et al., 2018; dence from recent studies shows that it may act as an interface Tanaka et al., 2020). In this context, multiple early risk factors for diverse types of cognition (Burdakov and Peleg-Raibstein, (poor glycemic control, hypertension, and dyslipidemia) have 2020). Despite this evidence, the role of the hypothalamus as a been proposed to participate in this pathological process (Bies- nexus connecting metabolism with cognitive disturbances and sels and Despa, 2018; Feinkohl et al., 2015). However, these the underlying molecular and cellular underpinnings remains studies were unable to dissociate confounding metabolic alter- unknown. ations or to dissect out their specific contribution to cognitive In the present study, we hypothesized that hypothalamic decline. Therefore, our current understanding of the initial events neuronal populations that are fundamental for energy homeosta- and mechanisms triggering cognitive dysfunction within the sis control are at the core of a functional axis that links meta- framework of metabolic abnormalities remains largely enigmatic. bolism and cognitive performance. In recent years, a complex bidirectional interplay between the disruption of brain cholesterol metabolism and insulin resistance RESULTS has been revealed. Indeed, murine models of diabetes and aging display a reduction in brain cholesterol biosynthesis associated Cognitive impairment caused by short-term obesogenic with cognitive phenotypes (Martı́n-Segura et al., 2019; Suzuki diet, in the absence of overt metabolic disturbances, is et al., 2010, 2013). In this regard, recent findings have disclosed reversed by central pregnenolone administration a role for the disruption of intracellular cholesterol trafficking in Obesity and T2D have emerged as risk factors for the develop- neurodegeneration, with the identification of steroidogenic acute ment of cognitive deficits, but the precise basis of this relation- regulatory protein (Stard1) as a potential preclinical marker asso- ship is masked by robust disease-related phenotypical traits ciated with early stages of Alzheimer disease (Arenas et al., 2020). (i.e., adiposity and hyperglycemia) (Biessels and Despa, 2018; Cholesterol is the precursor for the biogenesis of neurosteroids, Tanaka et al., 2020). To dissociate the impact of overt metabolic which are steroid hormone derivatives locally synthesized within alterations on cognitive dysfunction, we fed C57Bl/6J mice with the brain. In this process, Stard1 mediates the trafficking of high-fat high-sucrose western diet (WD) for just 4 days (4d-WD; cholesterol to the mitochondria, which is subsequently metabo- Figure S1A). This dietary regime did not cause noticeable meta- lized by CYP11A1 (P450Scc) into the neurosteroid precursor bolic perturbations (Figures S1B–S1E), thus excluding obesity pregnenolone (Arenas et al., 2017; Manna et al., 2016). and altered systemic glucose homeostasis per se as confound- Since its breakthrough description in the early 1990s, alter- ing factors. We next assessed spatial and recognition memory ations in neurosteroid production or action have been implicated via Barnes maze tests (BMTs; Figure 1A) and novel object recog- in the etiology of a wide range of neuropsychiatric and neurolog- nition test (NORT; Figure 1B), respectively. Short-term WD ical disorders: anxiety, depression, epilepsy, addiction, neuro- neither interfered with task learning (Figure S1F) nor caused degeneration, learning, and memory (Ratner et al., 2019). cognitive impairments in spatial memory (Figures 1C–1F). How- Numerous brain regions are able to synthetize and respond to ever, it significantly deteriorated recognition memory when neurosteroids, including the hypothalamus and classical mem- compared with chow-fed counterparts (Figures 1G–1J) in the ory-related structures (hippocampus, amygdala, or cortex), absence of exploration or locomotor alterations (Figures thus influencing the performance of different types of memory S1G–S1J). (Mellon et al., 2001; Ratner et al., 2019). The hippocampus plays a pivotal role in memory formation Despite receiving much less attention in the field of cognitive and flexibility. Long-term potentiation (LTP) is a form of synaptic science, the hypothalamus is crucially positioned to connect plasticity that is closely linked with memory function in mam- metabolic status with cognition for diverse reasons. First, mals. It has been reported that energy-dense diets reduce LTP because distinct hypothalamic nuclei and neuronal types magnitude in mice (Davis et al., 2021; Heyward et al., 2016). 270 Cell Metabolism 34, 269–284, February 1, 2022 ll Article OPEN ACCESS Figure 1. Cognitive impairment caused by short-term obesogenic diet is reversed by central pregnenolone administration. (A) Schematic illustration of Barnes maze test (BMT) and experimental timeline. The white circle and the rectangle represent the scape hole and scape chamber location, respectively. The position of the scape chamber remained constant on each trial. On the test day, the scape hole was closed and the chamber removed. (legend continued on next page) Cell Metabolism 34, 269–284, February 1, 2022 271 ll OPEN ACCESS Article We conducted field potential recordings in the dendritic area of ures 2A and 2B) (hereafter referred to as AgRPStarKO and the CA1 region after stimulation of the Schaffer collaterals of POMCStarKO mice, respectively). This gene encodes for Stard1, CA3 neurons in hippocampal slices (Figure 1K) from C57Bl/6J a protein that mediates mitochondrial transfer of cholesterol, mice fed with either chow or WD for 4 days. Input-output rela- which is the rate-limiting step in neurosteroid production (Arenas tionship, at diverse stimuli intensities (Figure S1K), and paired- et al., 2017; Manna et al., 2016) (Figure S1M). pulse facilitation (Figure S1L) were comparable between both Tissue- and cell-specific deletion of Star was demonstrated by dietary conditions, suggesting analogous basal synaptic proper- recombination of the floxed alleles (Figures S2A and S2B), fluo- ties in terms of excitability and presynaptic calcium-dependent rescent in situ hybridization (Figures S2C and S2D), and qPCR neurotransmitter release. However, acute WD feeding signifi- (Figure S2E). The nucleus of the solitary tract (NTS) harbors a cantly reduced the field excitatory postsynaptic potential small population of POMC neurons, and consistently, Star (fEPSP) slope after theta-burst stimulation (TBS), indicating expression was also significantly reduced in this brain region impaired hippocampal LTP (Figures 1L–1N). (Figure S2E). Neuronal population size was unaltered in mutant Neurosteroids have been implicated in a broad spectrum of mice (Figures S2F and S2G), indicating that Stard1 deficiency neurological diseases (Ratner et al., 2019). The cholesterol did not affect neuron ontogeny or viability. metabolite pregnenolone is the first intermediate of the mito- Although the POMC promoter also recombines in the pituitary, chondrial steroid synthesis pathway and precursor of virtually the expression of genes involved in mitochondrial cholesterol all steroid hormones (Figure S1M). We sought to determine transport (Star, Cyp11a1, and Tspo) or in pituitary gland develop- whether acute exposure to WD was able to make an impact on ment and function (Crhr1, Gh, Pit1, Pomc, Tbx19, and Tshb) was neurosteroid concentration in brain regions classically involved analogous between controls and mutant mice (Figure S2H). in memory (perirhinal cortex and hippocampus) or metabolic Accordingly, pregnenolone concentration in the pituitary, as control (arcuate nucleus of the hypothalamus; ARC). Notably, well as basal and stress-induced corticosterone circulating we observed a significant reduction in pregnenolone content in levels, was comparable between that of control and POMC- ARC-enriched samples but not in the perirhinal cortex or hippo- StarKO mice (Figures S2I and S2J). Together, these results indi- campus of 4d-WD mice (Figures 1O–1Q). These results demon- cate that genetic recombination in the pituitary of POMCStarKO strate that overt manifestations of obesity/diabetes per se are mice did not hinder pituitary-adrenal axis function. not the primary cause of hypothalamic pregnenolone synthesis Hypothalamic AgRP and POMC neurons are fundamental shortage and cognition failure. nexus for the physiological control of energy balance and We next explored the potential causality of hypothalamic defi- glucose metabolism (Jais and Bru €ning, 2021). Thus, we conduct- ciency of pregnenolone biosynthesis upon cognitive impairment. ed detailed metabolic phenotyping of AgRPStarKO and POMC- To this end, we acutely administered pregnenolone intracerebro- StarKO mice. Interestingly, Stard1 deficiency in AgRP or POMC ventricularly (i.c.v.) (adjacent to the ARC) to 4d-WD mice after the neurons did not make an impact on energy balance and glucose NORT training phase. Notably, this strategy normalized memory metabolism (Figures S3A–S3J). (Figures 1R, 1S, S1N, and S1O). This evidence causally associ- Next, we tested whether genetic deletion of Star in AgRP or ates pregnenolone action in the hypothalamus with cognitive POMC neurons translated into reduced hypothalamic content function. of pregnenolone. The concentration of pregnenolone was not altered in any of the brain regions analyzed from AgRPStarKO Interference with pregnenolone biosynthesis in mice (Figures 2C–2E). In POMCStarKO animals, pregnenolone hypothalamic POMC neurons, but not AgRP neurons, content was unaltered in the perirhinal cortex, hippocampus, results in cognitive dysfunction or NTS (Figures 2F, 2G, and S3K), but it was significantly Intrigued by these findings, we next investigated the function of decreased in ARC-enriched samples (Figure 2H). This diver- pregnenolone in the ARC with regard to cognition. To this aim, gence was likely the consequence of the higher neurosteroido- we generated mice lacking Star in AgRP or POMC neurons (Fig- genic capacity of ARC POMC neurons, as suggested by the (B) Schematic illustration of the novel object recognition test (NORT) and experimental chronology. (C–F) Recorded parameters to assess BMT performance in mice fed with either chow or western diet for 4 days (4d-WD) during the test phase: (C) latency to target hole, (D) time spent in target quadrant, (E) total distance traveled, and (F) representative traces of mouse movement trajectories (n = 11/diet). (G–J) Recorded parameters to assess NORT performance in mice fed with either chow or 4d-WD during the test phase: (G) discrimination index (time exploring novel object time exploring familiar object)/(time exploring novel object + time exploring familiar object), (H) exploration time (time exploring novel object + time exploring familiar object), (I) total distance traveled, and (J) representative traces of mouse movement trajectories (n = 9–10/diet). (K–N) Hippocampal long-term potentiation (LTP) study in mice fed with chow or 4d-WD. (K) Schematic illustration of the experimental strategy. The Schaffer collateral pathway (SC, red) was stimulated (Stim), and field potentials were recorded (Rec) in the CA1 region of the hippocampus. LTP was induced by theta- burst stimulation (TBS). DG, dentate gyrus; CA, cornu ammonis. (L) Representative traces of individual recordings showing baseline fEPSPs before (black traces) and after (red traces) LTP induction. (M) Time course of fEPSP recordings demonstrating robust changes in fEPSP slope in chow-fed animals after TBS (arrow) (n = 5 recordings from 4 animals). In 4d- WD animals, LTP induction was markedly impaired (n = 7 recordings from 4 animals). (N) Quantification of fEPSP slope change over the last 45 min of recording period shown in (M). (O–Q) Quantification of pregnenolone concentration in the (O) perirhinal cortex, (P) hippocampus, and (Q) arcuate-enriched mediobasal hypothalamus in mice fed with either chow or 4d-WD (n = 7–8/diet). (R) Schematic illustration of central acute delivery of pregnenolone and subsequent NORT. Red arrow shows injection phase. (S) Discrimination index during the test phase of NORT in mice fed with either chow or 4d-WD treated with vehicle (Veh) or pregnenolone (Preg) (n = 6–8/group). All studies were performed on male mice at 8–9 weeks of age. Dots in panels represent individual samples. Data are presented as mean ± SEM. *p < 0.05. 272 Cell Metabolism 34, 269–284, February 1, 2022 ll Article OPEN ACCESS Figure 2. Genetic deletion of Star exclusively in POMC neurons impairs recognition memory (A and B) Schematic illustration of the generation of transgenic mice: (A) AgRPStarKO and (B) POMCStarKO mice. (C–E) Pregnenolone concentration in the (C) perirhinal cortex, (D) hippocampus, and (E) arcuate-enriched mediobasal hypothalamus from control and AgRPStarKO mice (n = 5–9/genotype). (legend continued on next page) Cell Metabolism 34, 269–284, February 1, 2022 273 ll OPEN ACCESS Article analysis of publicly available single-cell RNA-seq data (Campbell equivalent in the perirhinal cortex, hippocampus, and peripheral et al., 2017) (Figure S3L). Accordingly, while AgRPStarKO mice tissues, it was blunted in ARC-enriched samples from POMC- did not exhibit cognitive alterations (Figures 2I–2P and S3M), StarKO mice (Figures 3A–3E). POMCStarKO animals exhibited unaltered spatial memory (Fig- To further investigate the relevance of insulin signaling in ures 2Q–2T and S3N) but significant impairments in object POMC neurons upon cognition, we generated conditional mice recognition memory (Figures 2U–2X). This memory defect was lacking insulin receptors (POMCInsrKO; Figure 3F) (Könner corroborated in a second paradigm based on olfactory cues et al., 2007). No changes in pregnenolone content in the relevant (Figures S3O–S3Q). Evaluation of the neurosensory status (Fig- brain areas were observed (Figures 3G–3I). Overall, locomotor ures S3R and S3S), as well as anxiety- and depression-like activity, exploratory drive, and memory fitness of POMCInsrKO states (Figures S4A–S4Q), demonstrated the full capability of mice were indistinguishable from those of control counterparts POMCStarKO mice to perform the behavioral tests. (Figures 3J–3V). Together, these results indicate that insulin It has been reported that hypothalamic POMC neurons proj- signaling in POMC neurons is irrelevant for memory performance ect to the hippocampal subiculum (Wang et al., 2015). Using a maintenance and suggest that defective insulin signaling in tracing virogenetic approach (Figure S5A), we observed that POMCStarKO mice is likely a secondary effect. ARC POMC neurons (Figures S5B–S5E) established synapses in this brain region (Figures S5F–S5I), thus confirming previous POMC neuron-derived pregnenolone mediates findings. Next, we evaluated the impact of Stard1 deficiency cognitive performance likely via autocrine mechanisms on the synaptic plasticity of the hippocampus. Hippocampal To ascertain if reduced POMC neuron-derived neurosteroid pre- slices from control and POMCStarKO mice showed compara- cursor synthesis underlies the cognitive impairment observed in ble input-output relationship (Figure S5J) and paired-pulse our mutant mouse line, we i.c.v. injected pregnenolone into facilitation (Figures S5K–S5M). In contrast, LTP was signifi- POMCStarKO mice (Figure 4A). Remarkably, this strategy was cantly impaired in mutant mice as reflected by the reduced able to restore memory of POMCStarKO mice (Figures 4B–4D) fEPSP slope (Figures 2Y–2AA), confirming perturbed synaptic and to enhance hippocampal synaptic plasticity (Figures 4E– plasticity and congruent with the cognitive impairments 4G). Equivalent results were obtained when pregnenolone was observed. Hippocampal CREB-activated pathways are crucial directly infused into the ARC of mutant mice (Figures S6A– for learning, memory, and synaptic plasticity (Belgacem and S6D), thus confirming a causal link between ARC neurosteroid Borodinsky, 2017). Consistently, we observed a significant deficiency and cognitive dysfunction. These results also vali- reduction in the number of pCREB-positive cells in the dentate dated the i.c.v. route as an adequate experimental strategy in gyrus of cognitive-deficient POMCStarKO mice (Figures S5N the context of our research. and S5O). To identify the cellular targets of neurosteroids in the ARC, we Together, these results confirm a causal link between POMC i.c.v. injected C57Bl/6J mice with pregnenolone and analyzed neuron neurosteroid deficiency and memory dysfunction via hip- FOS positivity as a marker of neuronal activity (Figure 4H). Coloc- pocampal mechanisms. alization of POMC and FOS was increased upon pregnenolone administration, suggesting neurosteroid-mediated enhanced Insulin signaling in POMC neurons does not mediate activation of this neuronal population (Figures 4I and 4J). The cognitive performance proportion of activated non-POMC cells was similar in vehicle It has been reported that cognitive impairment is associated with and pregnenolone-injected mice (Figure 4K), denoting that in central insulin resistance (Biessels and Despa, 2018; Tanaka the ARC, pregnenolone mainly targets POMC neurons. et al., 2020). Thus, we next assessed the insulin signaling status Given that pregnenolone was able to activate POMC neurons, of our POMCStarKO mice. While AKT phosphorylation was we investigated whether stimulation of POMC neuron activity (F–H) Pregnenolone concentration in (F) perirhinal cortex, (G) hippocampus, and (H) arcuate-enriched mediobasal hypothalamus from control and POMCStarKO mice (n = 8–6/genotype). (I–L) Recorded parameters to assess Barnes maze performance in AgRPStarKO mice during the test phase: (I) latency to target hole, (J) time spent in target quadrant, (K) total distance traveled, and (L) representative traces of mouse movement trajectories (n = 8/genotype). (M–P) Recorded parameters to assess NORT performance in AgRPStarKO mice during the test phase: (M) discrimination index (time exploring novel object time exploring familiar object)/(time exploring novel object + time exploring familiar object), (N) exploration time (time exploring novel object + time exploring familiar object), (O) total distance traveled, and (P) representative traces of mouse movement trajectories (n = 8/genotype). (Q–T) Recorded parameters to assess Barnes maze performance in POMCStarKO mice during the test phase: (Q) latency to target hole, (R) time spent in target quadrant, (S) total distance traveled, and (T) representative traces of mouse movement trajectories (n = 9–10/genotype). (U–X) Recorded parameters to assess NORT performance in POMCStarKO mice during the test phase: (U) discrimination index (time exploring novel object time exploring familiar object)/(time exploring novel object + time exploring familiar object), (V) exploration time (time exploring novel object + time exploring familiar object), (W) total distance traveled, and (X) representative traces of mouse movement trajectories (n = 7–8/genotype). (Y–AA) Hippocampal long-term potentiation (LTP) study comparing control and POMCStarKO mice. (Y) Representative traces of individual recordings showing baseline fEPSPs before (black traces) and after (red traces) LTP induction. Slope and peak amplitude of fEPSPs increased after theta-burst stimulation (TBS) in control mice but was attenuated in POMCStarKO animals. (Z) Time course of fEPSP recordings demonstrating that LTP induction was markedly impaired in POMCStarKO animals (n = 6 recordings from 5 animals) when compared with controls (n = 9 recordings from 6 animals). (AA) Quantification of the fEPSP slope change over the last 45 min of recording period shown in (Z). All studies were performed in male mice between 10 and 16 weeks of age. Dots in panels represent individual samples. Data are presented as mean ± SEM. *p < 0.05; ***p < 0.001. 274 Cell Metabolism 34, 269–284, February 1, 2022 ll Article OPEN ACCESS Figure 3. Insulin signaling in POMC neurons does not mediate memory performance (A–E) Assessment of insulin signaling via AKT phosphorylation (pS473-AKT) in the (A) perirhinal cortex, (B) hippocampus, (C) arcuate-enriched mediobasal hy- pothalamus, (D) skeletal muscle, and (E) liver from control and POMCStarKO mice. Representative blots and quantification (normalized to total AKT) are shown (n = 3–7/genotype). (legend continued on next page) Cell Metabolism 34, 269–284, February 1, 2022 275 ll OPEN ACCESS Article was sufficient to mediate recognition memory. With this aim, we mance without causing metabolic alterations. Thus, we next bilaterally delivered activatory (AAV-hM3Dq) designer receptors questioned the reverse, that is, whether established metabolic exclusively activated by designer drugs (DREADDs) into the conditions associated with cognitive impairments progressed ARC of control POMCCre/+ and POMCStarKO mice (Figures 4L with inadequate central pregnenolone levels. To examine this, and S6E). This permits Cre-mediated specific activation of we conducted studies on obese mice and human patients. We POMC neurons by clozapine N-oxide (CNO) injection (Fig- fed C57Bl/6J mice with either regular chow or WD for 16 weeks ure S6F). Notably, no changes in the cognitive capacity, explor- (16w-WD; Figure 5A). Predictably, this long-term diet regime re- atory drive, or distance traveled were promoted by chemogenetic sulted in dramatic metabolic perturbations, including obesity activation of POMC neurons in control or mutant mice (Figures (Figure 5B) and defective glucose metabolism (Figures 5C–5E). 4M, 4N, and S6G–S6J). Cognitive scrutiny revealed that chronic WD feeding did not alter In view of these results, together with the observation that cen- the latency to localize the target hole in the BMT (Figures 5F–5J). tral administration of neurosteroids did not improve the other- Nevertheless, 16w-WD mice spent more time in the target quad- wise normal cognitive function of control mice (Figures 1S, 4B, rant, but the interpretation of this result is intriguing (Figure 5H). and 4M), we reasoned that inhibition of POMC neurons should The NORT evidenced a significant reduction in the discrimination impair cognitive performance. For this purpose, we chemoge- index of 16w-WD mice, denoting an impairment in recognition netically inhibited POMC neurons via AAV-hM4Di DREADDs memory (Figures 5K–5N). The increased exploration time during (Figure 4L). As anticipated, acute repression of POMC neurons the test phase of NORT (Figure 5L) was attributed to enhanced caused a significant reduction in recognition memory of control exploration of the familiar object rather than the novel one, as it POMCCre/+ mice (Figures 4O, S6K, and S6L). can be inferred from the discrimination index data (Figure 5K). It has been speculated that neurosteroids largely exert their ef- Locomotor or exploration drive in an open field was not affected fects via autocrine mechanisms (Ratner et al., 2019), and our by the diet, ruling out physical inability to perform the behavioral study assessing POMC neuron activity after central pregneno- paradigms (Figures 5O–5R). lone treatment (Figures 4H–4K) is consistent with this idea. To Next, we aimed to evaluate whether diet-induced cognitive further explore the potential autocrine effects of neurosteroids deterioration was related to deficient pregnenolone concentra- in POMC neurons upon cognition, we injected pregnenolone tion in the relevant brain regions. Interestingly, similar to our into the third ventricle of POMCCre/+ or POMCStarKO mice previous findings, pregnenolone content was reduced in ARC- with prior chemogenetic repression of POMC neurons. Earlier in- enriched samples but not in perirhinal cortex or hippocampus hibition of POMC neuronal activity negated the beneficial effects of 16w-WD mice (Figures 5S–5U). These results indicate that of pregnenolone with regard to cognitive performance in both diet-induced metabolic perturbations are associated with models (Figures 4O, 4P, and S6K–S6N), suggesting that these cognitive impairments and reduced pregnenolone content in effects are mediated via an autocrine axis on POMC neurons. the hypothalamus. The collective interpretation of these results indicates that POMC neuron activity is necessary for adequate memory func- Pregnenolone concentration in the cerebrospinal fluid tion and for the beneficial effects of pregnenolone. Furthermore, of metabolically unhealthy patients with obesity POMC neuron stimulation fails to improve memory in the context positively correlates with cognitive score of genetic deficiency of pregnenolone, but it is rescued by its To examine the relevance of central neurosteroids upon cogni- central delivery. Hence, it is likely that adequate memory perfor- tion in the context of human biology, we determined the concen- mance requires both responsive POMC neurons and the release tration of pregnenolone in the cerebrospinal fluid (CSF) from a of pregnenolone. clinically characterized cohort of metabolically healthy (MHO) and unhealthy (MUO) patients with obesity for which cognitive Obesity-related metabolic complications associate with status was assessed by the Mini-Mental Status Examination impaired cognitive function and reduced hypothalamic (MMSE) (Figure 6A; Table S1). The MMSE is a commonly used neurosteroid concentration in mice screening test for the evaluation of cognitive impairments in Our observations in POMCStarKO mice indicated that primary adults that assesses several domains: orientation, registration, alterations in pregnenolone signaling affected cognitive perfor- attention and calculation, recall, and language. In MHO subjects, (F) Schematic illustration of the generation of POMCInsrKO mice. (G–I) Pregnenolone content in the (G) perirhinal cortex, (H) hippocampus, and (I) arcuate nucleus-enriched mediobasal hypothalamus from control and POMCInsrKO mice (n = 9/genotype). (J–M) Locomotor and exploratory activity of control and POMCInsrKO mice in an open-field paradigm: (J) time spent in the center, (K) entries in the center, (L) total distance traveled, and (M) global activity (n = 9/genotype). (N) Task learning curve for the latency to reach the scape hole during the training phase of the Barnes maze test (BMT). Results show the average of 2 trials per day during 5 consecutive days (n = 8–9/genotype). (O–R) Recorded parameters to assess Barnes maze performance in control and POMCInsrKO mice during the test phase: (O) latency to target hole, (P) time spent in target quadrant, (Q) total distance traveled, and (R) representative traces of mouse movement trajectories (n = 8–9/genotype). (S–V) Recorded parameters to assess NORT performance in control and POMCInsrKO mice during the test phase: (S) discrimination index (time exploring novel object time exploring familiar object)/(time exploring novel object + time exploring familiar object), (T) exploration time (time exploring novel object + time exploring familiar object), (U) total distance traveled, and (V) representative traces of mouse movement trajectories (n = 8/genotype). All studies were performed in male mice between 10 and 16 weeks of age. Dots in panels represent individual samples. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant. 276 Cell Metabolism 34, 269–284, February 1, 2022 ll Article OPEN ACCESS Figure 4. Central pregnenolone administration recovers cognitive function in POMCStarKO mice (A) Schematic illustration of central acute delivery of pregnenolone and subsequent NORT in POMCStarKO mice. Red arrow shows the injection phase. (B–D) Recorded parameters to assess NORT performance in POMCStarKO mice, after central administration of vehicle (Veh) or pregnenolone (Preg), during the test phase: (B) discrimination index (time exploring novel object time exploring familiar object)/(time exploring novel object + time exploring familiar object), (C) exploration time (time exploring novel object + time exploring familiar object), and (D) total distance traveled (n = 3–9/genotype). (legend continued on next page) Cell Metabolism 34, 269–284, February 1, 2022 277 ll OPEN ACCESS Article pregnenolone levels did not correlate with any of the diverse administration, which advanced without causing noticeable meta- anthropometric, clinical, or cognitive parameters (Figure 6B). bolic disturbances, was sufficient to specifically deteriorate recog- However, in MUO patients, CSF pregnenolone was negatively nition memory. In contrast to other reports, our dietary studies did associated with body mass index (BMI) and positively correlated not perturb spatial memory (Beilharz et al., 2016; Bocarsly et al., with MMSE (Figures 6C and 6D). These results evidenced a 2015). This discrepancy may be the consequence of variations mechanism linking central pregnenolone with cognitive function in the experimental procedures and nutrient composition of diets. in metabolically unhealthy obese individuals. Importantly, under both chronic and acute energy-dense dietary regimes, defective recognition memory was paralleled by a DISCUSSION reduced content of pregnenolone in the hypothalamus but not in memory-related regions such as the perirhinal cortex and hippo- Previous studies have placed brain cholesterol metabolism as campus. Nevertheless, we cannot exclude that western-style a bridge between metabolic and cognitive disorders (Martı́n- diet feeding also alters neurosteroid content in other brain struc- Segura et al., 2019; Suzuki et al., 2010, 2013). In light of this tures. This cognitive phenotype was rescued via central delivery evidence, we hypothesized that neurosteroids, which are syn- of pregnenolone, demonstrating the causality of the hypothalamic thesized from cholesterol and have been widely related to deficiency of this neurosteroid in the development of cognitive cognition and memory processes, could be implicated in the dysfunction. Another important conclusion of these findings, cognitive impairment observed in detrimental metabolic con- consistent with human data, is that overt manifestations of texts. In this study, we reveal that key appetite-regulating obesity/diabetes per se (i.e., overweight and hyperglycemia) are POMC neurons also mediate recognition memory via a preg- not the primary causes of hypothalamic pregnenolone synthesis nenolone-based mechanism. shortage and cognition decline. Instead, these observations Extensive evidence has demonstrated that obesity and T2D pointed toward rapid yet undetermined effects of nutrient excess are associated with cognitive dysfunction (Biessels and Despa, and/or their metabolites that attenuate hypothalamic pregneno- 2018; Tanaka et al., 2020). Our patient cohort study indicated lone production. It is reasonable to speculate that, similar to that pregnenolone concentration in the CSF positively correlated related biological responses (Sergi and Williams, 2019), an exces- with the MMSE and negatively correlated with BMI but solely in sive influx of saturated fatty acids in the hypothalamus might MUO patients. This observation suggests a neuroprotective participate in this context. role for neurosteroids and supports the notion that obesity itself To further understand the triangular connection between neu- is insufficient to perturb the pregnenolone-cognition axis. In this rosteroids, cognitive function, and metabolism, we generated regard, it is likely that the combination of additional metabolic mice lacking Stard1 in hypothalamic AgRP or POMC neurons. complications of obesity prompts such cognitive phenotypes. This strategy impinged upon cholesterol transport into the mito- Our data provide conceptual evidence that central pregnenolone chondria, thus disrupting the first and limiting step of neurosteroid may be relevant in cognitive impairments associated with meta- production in the aforementioned populations of neurons. Anal- bolic disorders in humans. ysis of publicly available single-cell sequencing data suggested Our studies in preclinical models confirmed that recognition a higher neurosteroidogenic potential for POMC neurons in com- memory was impaired after a chronic diet-induced obesity inter- parison with AgRP neurons. This notion was supported by the un- vention. Similarly, a brief period (4 days) of western-style diet altered hypothalamic pregnenolone content and normal cognitive (E–G) Hippocampal long-term potentiation (LTP) study after central administration of either vehicle (Veh) or pregnenolone (Preg) in POMCStarKO mice. (E) Representative traces of individual recordings showing baseline fEPSPs before (black traces) and after (red traces) LTP induction. Slope and peak amplitude of fEPSPs improved after pregnenolone treatment. (F) Time course of the fEPSP slope change in vehicle-treated (n = 4 recordings from 4 animals) and pregnenolone-treated POMCStarKO mice (n = 5 recordings from 3 animals) after theta-burst stimulation (TBS; arrow). (G) Quantification of the fEPSP slope change over the last 45 min of recording period shown in (F). (H) Schematic illustration of central acute delivery of pregnenolone and subsequent analysis of FOS expression in control C57Bl/6J mice. Red arrow shows the injection phase. (I) Representative confocal images of FOS and POMC colocalization after central administration of vehicle or pregnenolone in control C57Bl/6J mice. White arrows show colocalization staining. Scale bar, 20 mm. (J) Quantification of FOS-positive POMC neurons in arcuate nucleus sections from control C57Bl/6J mice after central administration of vehicle or pregnenolone (n = 3/genotype). (K) Quantification of FOS positivity in non-POMC cells in arcuate nucleus sections from control C57Bl/6J mice after central administration of vehicle or preg- nenolone (n = 3/genotype). (L) Schematic illustration of the activatory and inhibitory chemogenetic strategy, coupled with central pregnenolone administration, in POMCcre/+ and POMCStarKO mice. Orange and red arrows show CNO and pregnenolone (Preg) injection, respectively. (M and N) Recorded parameters to assess NORT performance in mice after chemogenetic activation of POMC neurons, combined with central administration of vehicle (V) or pregnenolone (Preg), during the test phase: (M) discrimination index of control POMCcre/+ mice (n = 4–5/genotype) and (N) POMCStarKO mice (n = 4– 6/genotype). (O and P) Recorded parameters to assess NORT performance in mice after chemogenetic repression of POMC neuron activity, combined with central administration of vehicle (V) or pregnenolone (Preg), during the test phase: (O) discrimination index of control POMCcre/+ mice (n = 4–6/genotype) and (P) POMCStarKO mice (n = 5–6/genotype). All studies were performed in male mice between 10 and 16 weeks of age. Dots in panels represent individual samples. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. 278 Cell Metabolism 34, 269–284, February 1, 2022 ll Article OPEN ACCESS Figure 5. Obesity-related metabolic complications associate with impaired cognitive function and reduced hypothalamic neurosteroid con- centration in mice (A) Schematic illustration of the experimental strategy. Eight-week-old C57Bl/6J mice were fed with either chow or western diet for 16 consecutive weeks (16w-WD). (B) Body weight profile (n = 10/genotype). (C) Basal blood glucose concentration and (D) glucose tolerance test (n = 7/genotype). (E) Insulin sensitivity test (n=10/genotype). (legend continued on next page) Cell Metabolism 34, 269–284, February 1, 2022 279 ll OPEN ACCESS Article performance of AgRPStarKO mice. In contrast, loss of Stard1 in model cannot stimulate its release. These results reinforce the POMC neurons impaired recognition memory performance in importance of POMC neuron-derived pregnenolone as a key the face of normal metabolic and energy balance status. These mediator of memory performance. findings indicate that inadequate neurosteroid production by The beneficial effects of POMC neuron-derived pregnenolone POMC neurons is fundamentally implicated in cognition, but it is upon cognitive function are likely mediated by an autocrine axis, dispensable for energy homeostasis control. This suggests the as demonstrated by preferential activation of POMC neurons af- existence of divergent, yet coordinated, pathways influencing ter central pregnenolone delivery and a sequential combination memory and metabolism within POMC neurons. of chemogenetics and pharmacological rescue. Given the lipo- Brain insulin resistance has long been considered as one philic nature of neurosteroids, paracrine-autocrine signaling of the mechanisms underlying cognitive dysfunction. For has been postulated as a common mechanism underlying their example, it has been reported that insulin signaling is altered diverse neurobiological effects (Ratner et al., 2019). Neuroste- in preclinical models and patients with neurodegenerative dis- roids can act through multiple receptors, including N-methyl- eases (Kellar and Craft, 2020). Research has also evidenced D-aspartate glutamate receptors (NMDARs) (Tuem and Atey, that insulin and insulin sensitizers have neuroprotective effects 2017). The genes encoding the diverse NMDAR subunits under these pathological conditions (Kellar and Craft, 2020). (GRIN family) are expressed in POMC neurons (Lam et al., However, it is debatable whether brain insulin desensitization 2017), and thus, it is reasonable to speculate that neurosteroids is the cause or the consequence of cognitive dysfunction. The might exert autocrine effects on POMC neurons via NMDARs. cognitive deterioration observed in POMCStarKO mice pro- Nevertheless, we cannot rule out that pregnenolone also exerts gressed with attenuated insulin signaling exclusively in the hy- part of its effects on POMC neurons via indirect pathways. pothalamus. However, our results show that genetic blockage How this putative autocrine loop into POMC neurons influ- of insulin signaling in POMC neurons is insufficient to cause ences hippocampal LTP remains enigmatic. Despite POMC neu- detectable cognitive deficits in mice. While it is very likely rons projecting to specific regions of the hippocampus (Wang that brain insulin dysfunction can contribute to further deteri- et al., 2015; Figure S5A), we did not observe changes in hippo- oration of cognitive capacity once cognitive decline emerges campal pregnenolone content in any of the models investigated. or is established, our data do not support the concept of Hence, it seems unlikely that ARC POMC neurons release disturbed brain insulin signaling as a primary trigger of the pa- pregnenolone in this brain region. Instead, the involvement of thology in our mutant mice. POMC neuron-derived bioactive molecules is plausible. For DREADD-mediated inhibition of POMC neurons negatively example, the neuropeptide alpha-melanocyte-stimulating hor- influenced recognition memory in control mice, indicating that mone (a-MSH) influences synaptic plasticity in the hippocam- excitable POMC neurons are required for adequate memory pus. In this regard, it has been reported that the hippocampus function. Synaptic plasticity is an energy-demanding process expresses melanocortin 4 receptor (MC4R; the receptor for that is required for learning and memory formation. Consistent a-MSH) (Gantz et al., 1993; Liu et al., 2003). Furthermore, with this, recognition memory was repressed when a negative a-MSH has been shown to exert neuroprotective and neurogen- energy balance state was simulated through chemogenetic inhi- esis effects on the hippocampus to enhance LTP and improve bition of POMC neurons. From an energetic perspective, this cognitive function in models of neurodegenerative diseases could be part of a cost-effective program to limit an energy- (Giuliani et al., 2015; Gonzalez et al., 2009; Ma and McLaurin, consuming process (such as memory formation) or enhance 2014; Shen et al., 2013). permissiveness to certain food resources under conditions of Eating behavior is intimately connected with multiple and scarcity. In contrast, the activation of POMC neurons during complex neuro-cognitive processes. Diverse memory systems feeding conditions would support energy-consuming processes contribute to food-related decision-making to support nutritional related to the formation and storage of memories. The fact that needs. For example, it is crucial to locate, identify, and associate chemogenetic activation of POMC neurons did not make an food items with particular outcomes (taste, nutritional value, impact on cognition capacity in control mice may be a reflection toxicity, etc.) to sustain life (Higgs and Spetter, 2018). Given of the inability to further improve the otherwise normal cognition. the vital relationship between ingestive behavior and memory, Given the absence of pregnenolone synthesis in POMCStarKO it is evolutionarily plausible that fundamental biological systems mice, the chemogenetic activation of POMC neurons in this or neurons influencing appetite and energy balance may also (F) Task learning curve for the latency to reach the scape hole during the training phase of the Barnes maze test (BMT) in mice fed with either chow or 16w-WD. Results show the average of 2 trials per day during 5 consecutive days (n = 7–8/diet). (G–J) Parameters recorded to assess Barnes maze performance in mice, fed with either chow or 16w-WD, during the test phase: (G) latency to target hole, (H) time spent in target quadrant, (I) total distance traveled, and (J) representative traces of mouse movement trajectories (n = 7–8/genotype). (K–N) Parameters recorded to assess NORT performance in mice, fed with either chow or 16w-WD, during the test phase: (K) discrimination index (time exploring novel object time exploring familiar object)/(time exploring novel object + time exploring familiar object), (L) exploration time (time exploring novel object + time exploring familiar object), (M) total distance traveled, and (N) representative traces of mouse movement trajectories (n = 8–10/genotype). (O–R) Recorded parameters to assess open-field performance in mice fed with either chow or 16w-WD: (O) time spent in the center, (P) number of entries in the center, (Q) total distance traveled, and (R) global activity (n = 10/genotype). (S–U) Quantification of pregnenolone concentration in the (S) perirhinal cortex, (T) hippocampus, and (U) arcuate-enriched mediobasal hypothalamus in mice fed with either chow or 16w-WD (n = 9/genotype). All studies were performed on male mice at 24–26 weeks of age. Dots in panels represent individual samples. Data are presented as mean ± SEM. *p < 0.01; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. 280 Cell Metabolism 34, 269–284, February 1, 2022 ll Article OPEN ACCESS Figure 6. Pregnenolone concentration in the cerebrospinal fluid of metabolically unhealthy patients with obesity positively correlates with cognitive score (A) Schematic illustration of sample processing from the cohort of patients. (B) Analysis of Spearman’s rank correlation between cerebrospinal fluid (CSF) pregnenolone concentrations and diverse anthropometric, clinical, and cognitive parameters in a cohort of metabolically healthy (MHO) and unhealthy obese (MUO) patients. Preg, pregnenolone; MMSE, mini-mental state examination; BMI, body mass index; HOMA, homeostatic model assessment-insulin resistance; FPG, fasting plasma glucose; HbA1c, glycated hemoglobin; TG, triglyceride; SBP, systolic blood pressure; DBP, diastolic blood pressure; CHO, cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein. (C and D) Correlation between CSF pregnenolone concentrations and BMI (C) or MMSE (D) in MUO patients. Dots in panels represent individual samples. participate (directly or indirectly) in related cognitive processes In this study, we show that while metabolic disease is associ- (Kosse and Burdakov, 2019; Suarez et al., 2019; Zimmer et al., ated with reduced hypothalamic pregnenolone and cognitive 2019). Our results show that POMC neuron-derived neurosteroid deterioration, genetic interference with pregnenolone synthesis signaling principally mediates recognition memory, conceivably in POMC neurons impacts on cognition but not on metabolism. facilitating the identification and acquisition of food rather than This emphasizes the primary role of hypothalamic pregnenolone contributing to spatial navigation to locate food resources. in maintaining cognitive function. Our findings suggest that ARC Nevertheless, we cannot rule out that POMC neurons also POMC neurons integrate cognitive and metabolic health via modulate spatial memory via neurosteroid-independent routes. distinct pathways and advocate a wider role for this population It is noteworthy that POMC neurons also influence emotional of neurons beyond appetite and metabolic control. This novel states such as stress, anxiety, or fear (Greenman et al., 2013; facet of POMC neuron biology may have relevant implications Lee et al., 2020; Liu et al., 2007; Mandela et al., 2014; Qu et for cognitive disorders. al., 2020). These intermingled neurobiological mechanisms may represent complex adaptive responses to coordinate Limitations of study appetitive and consummatory behaviors, thus maximizing Our study defines a novel role for hypothalamic pregnenolone in survival in a constantly changing environment. This novel memory performance. However, we cannot rule out that a part POMC neuron functional axis at the interface of metabolism of these effects is mediated by downstream derivates as preg- and cognition lays the grounds for a conceptual framework to nenolone is the precursor for the biosynthesis of many neuro- understand the fundamental co-regulation of appetite and mem- steroids. The use of non-metabolizable pregnenolone (or other ory processes. neurosteroids) would provide further insights into this aspect Cell Metabolism 34, 269–284, February 1, 2022 281 ll OPEN ACCESS Article of the research. The microinjection of pregnenolone into the B NORT performance after i.c.v. or intra-ARC administra- ARC will inevitably spread, up to a certain extent, to neighboring tion of pregnenolone areas. Although we believe that the volume/injection rate used is B NORT performance after chemogenetic modulation of largely restricted to the ARC, we cannot fully assure the exclu- POMC neuron activity and i.c.v pregnenolone adminis- sive implication of the ARC in the observed pregnenolone tration effects. B Synaptophysin studies The combination of FOS, as a correlate of neuronal activity, B Electrophysiological studies and chemogenetic studies suggests that the beneficial effects d QUANTIFICATION AND STATISTICAL ANALYSIS of pregnenolone on memory are mediated, at least in part, by au- tocrine or paracrine effects on POMC neurons. This would be SUPPLEMENTAL INFORMATION consistent with the chemical nature of neurosteroids and other Supplemental information can be found online at https://doi.org/10.1016/j. reports suggesting this type of signaling (Ratner et al., 2019). cmet.2021.12.023. Nevertheless, it should be noted that our chemogenetic studies do not exclude potential alternative pathways that still converge ACKNOWLEDGMENTS on POMC neurons but via indirect routes. The analysis of pregnenolone in human patients has been per- We thank Josep Dalmau for financial support; Olga Jauregui, Albert Ferrés-Coy, formed in the CSF, which likely reflects overall biochemical and Analı́a Bortolozzi for scientific advice; and Servier Medical Art for illustra- changes in the brain. For translational significance, it would be tions. This work was supported by the Swiss National Science Foundation (no.176206; NCCR Synapsy grant no.185897) to C.S.; the European Research pertinent to measure pregnenolone content in human post-mor- Council (ERC) advanced grant SYNEME to J.C.B.; Instituto de Salud Carlos III tem hypothalamic samples under diverse pathophysiological (ISCIII)—Fondo Europeo de Desarrollo Regional (FEDER) (PI17/00296), RETICs conditions. Oftared (RD16/0008/0014), and Generalitat de Catalunya (2017SGR737) to X.G.; Ministerio de Ciencia e Innovación (BFU2017-83317-P) to D.S.; Ministerio de Economia, Industria y Competitividad, Maria de Maeztu (MDM-2017-0729) to STAR+METHODS Institut de Neurociencies, Universitat de Barcelona; ISCIII-FEDER (PI14/01126, PI17/01019), the National Institutes of Health (NIA grants 1R01AG056850- Detailed methods are provided in the online version of this paper 01A1, R21AG056974, and R01AG061566), Fundació La Marató de TV3 and include the following: (20141210), and Generalitat de Catalunya (SLT006/17/00119) to J.F.; ISCIII- FEDER (PI17/00279 and PI20/0042), Fundació La Marató de TV3 (201614.31), d KEY RESOURCES TABLE and Generalitat de Catalunya (SLT008/18/00127) to A.J.; Plan Nacional de I+D d RESOURCE AVAILABILITY funded by the Agencia Estatal de Investigación (AEI) and FEDER (PID2019- B Lead contact 111669RB-I00 and PID2020-115055RB-I00), CIBEREHD, the center grant B Materials availability P50AA011999 Southern California Research Center for ALPD and Cirrhosis funded by NIAAA/NIH, Generalitat de Catalunya (SGR-2017-1112), the Euro- B Data and code availability pean Cooperation in Science & Technology (COST) ACTION CA17112, FUNDA- d EXPERIMENTAL MODEL AND SUBJECT DETAILS CIÓN BBVA (‘‘ER stress-mitochondrial cholesterol axis in obesity-associated B Animal care and diets insulin resistance and comorbidities’’), and Red Nacional 2018-102799-T de En- B Mouse lines fermedades Metabólicas y Cáncer and Fundació La Marató de TV3 (201916/31) B Human subjects and cerebrospinal fluid samples to J.C.F.-C.; and ERC consolidator grant MITOSENSING (725004), ISCIII-FEDER d METHOD DETAILS (PI16/00963), ‘‘la Caixa’’ Foundation (ID100010434) under agreement LCF/PR/ HR19/52160016, and CERCA Programme/Generalitat de Catalunya to M.C. B Physiological tests D.A. is supported by ISCIII (INT19/00016) and Generalitat de Catalunya PERIS B Intracerebroventricular (i.c.v.) surgery and cannulation program (SLT006/17/125), A.P. is supported by Hospital Clı́nic de Barcelona B ARC surgery and cannulation (‘‘Ajut Josep Font’’), A.O. is supported by a Miguel Servet contract (CP19/ B Corticosterone measurements 00083) from ISCIII-FEDER, and R.H.-T. is supported by a Marie Sk1odowska-Cu- B Pregnenolone extraction and quantitative analysis rie Action fellowship (H2020-MSCA-IF) and NEUROPREG (891247). S.R. is a B In vivo insulin signaling analysis by immunoblot recipient of Juan de la Cierva Formación (FJCI-2016-28911) and Incorporación (IJC2018-037341-I) programs from the Spanish Ministry of Science and Innova- B Quantitative polymerase chain reaction (qPCR) tion. This work was carried out in part at Esther Koplowitz Centre. B Fluorescent in situ hybridization and quantification B Double POMC and FOS immunofluorescence after AUTHOR CONTRIBUTIONS pregnenolone delivery B POMC and AgRP neuron count S.R. conceived the study, designed and conducted experiments, analyzed results, and contributed to the manuscript preparation. R.H.-T., M.T., M.P., B pCREB analysis by immunofluorescence A.O., A.G.G.-V., I.C., T.V.E., I.Z., C.B., M.I., G.G., R.N., and C.S. contrib- B General behavioral procedures uted to the performance of experimental and behavioral procedures. B Neurosensory screen M.M.-G. contributed to the welfare of the mice and performed all the gen- B Barnes maze test otyping. J.A. and E.E. conducted biostatistical and bioinformatics analysis. B Novel object recognition test (NORT) M.R., D.S., and X.G. performed and analyzed electrophysiological studies. B Novel odor recognition test (NODR) C.B. and J.C.B. provided POMCIRKO animals. V.R., C.G.-R., and J.C.F.-C. generated Starflox/flox animals. A.P., D.A., J.F., and A.J. participated in the B Open field test recruitment and curation of the human cohort. M.C. conceived the study; B Light–Dark box test designed, coordinated, and supervised experiments; wrote the manuscript; B Elevated plus maze test and secured funding. All authors discussed the results, commented on the B Tail suspension test manuscript before submission, and agreed with the final submitted B Marble burying test manuscript. 282 Cell Metabolism 34, 269–284, February 1, 2022 ll Article OPEN ACCESS DECLARATION OF INTERESTS Feinberg, L.M., Allen, T.A., Ly, D., and Fortin, N.J. (2012). Recognition memory for social and non-social odors: differential effects of neurotoxic lesions to the The authors declare no competing interests. hippocampus and perirhinal cortex. Neurobiol. Learn. Mem. 97, 7–16. Feinkohl, I., Price, J.F., Strachan, M.W.J., and Frier, B.M. (2015). 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Neurosci. 33, 464–472. 141, 303–309. 284 Cell Metabolism 34, 269–284, February 1, 2022 ll Article OPEN ACCESS STAR+METHODS KEY RESOURCES TABLE REAGENT OR RESOURCE SOURCE IDENTIFIER Antibodies Rabbit Phospho-Akt (Ser473) Cell Signaling Cat# 9271S; RRID: AB_329825 Rabbit Akt (pan) mAB Cell Signaling Cat# C67E7; RRID: AB_915783 Rabbit polyclonal anti-c-FOS Santa Cruz Cat# Sc-52; RRID: AB_2106793 Rabbit polyclonal anti-POMC Phoenix Pharmaceuticals Cat# H-029-30; RRID: AB_2307442 Rabbit Phospho-CREB (Ser133) Cell Signaling Cat# 9198S; RRID: AB_2561044 (87G3) mAB Chicken Anti-GFP Aves Labs Cat#GFP-1010, RRID: AB_2307313 Rabbit Anti-RFP Rockland Cat#600-401-379, RRID: AB_2209751 Goat Anti-Rabbit IgG-Peroxidase GE Healthcare Cat# A0545; RRID: AB_257896 Alexa 488, Chicken anti-rabbit Life Technologies Cat# A21441; RRID: AB_2535859 Alexa 555, Donkey anti-rabbit A21207 Life Technologies Cat# A21207; RRID: AB_141637 Alexa 488, Goat anti-Chicken Life Technologies Cat# A-11039, RRID: AB_2534096 Alexa 594, Donkey anti-rabbit Life Technologies Cat# A-21207, RRIF: AB_141637 Bacterial and virus strains AAV8-hSYN-DIO-hM3D(Gq)-mCherry Addgene Cat# 44361 AAV8-hSYN-DIO-hM4D(Gi)-mCherry Addgene Cat# 44362 AAV1-phSyn1(S)-FLEX-tdTomato-T2A- Addgene Cat# 51509 SypEGFP Chemicals, peptides, and recombinant proteins Glucose 40% Fresenius kabi Cat# 620724 Human insulin (Humulin) Lilly Cat# C.N 710008.9 2-hydroxypropyl beta-cyclodextrin Sigma-Aldrich Cat# H5784 Pregnenolone Sigma-Aldrich Cat# P162 Artificial cerebrspinal fluid (aCSF) Tocris Bioscience Cat# 3525 Clozapine-N-oxide (CNO) Tocris Bioscience Cat# 4936 Trizol reagent Invitrogen Cat# 15596026 Gelatin Sigma-Aldrich Cat# G-2500 Triton X-100 Sigma-Aldrich Cat# X-100 Sodium Chloride (NaCl) 0.9% Merck Cat# 1064040500 N-hexane Sigma-Aldrich Cat# 296090 Paraformaldehyde aqueous solution 16% Electron Microscopy Sciences Cat# 15710 16 Ethylene glycol Sigma-Aldrich Cat# 1096211000 Bovine serum albumin Sigma-Aldrich Cat# A9647 Phosphate buffered saline 1x Sigma-Aldrich Cat# D8537 Ethanol VWR Cat# 1.009.831.000 Sucrose Sigma-Aldrich Cat# S0389 Acetonitrile Sigma-Aldrich Cat# 360457 Critical commercial assays Corticosterona EIA Immunodiagnostic systems Cat# AC-14F1 Pregnenolone ELISA LDN Cat# FR E-2700 Enhanced chemiluminescence reagent Pierce Cat# 32105 4–12% Pre-cast protein Gel Bio-Rad Cat# 345-0124 Protease and Phosphatase Inhibitor Sigma-Aldrich Cat# PPC1010 Cocktail Stripping buffer Thermo Fisher Cat# 21059 (Continued on next page) Cell Metabolism 34, 269–284.e1–e9, February 1, 2022 e1 ll OPEN ACCESS Article Continued REAGENT OR RESOURCE SOURCE IDENTIFIER High Capacity cDNA Reverse Thermo Fisher Cat# 4368813 Transcription Kit Premix Ex Taq mastermix Takara Cat# RR39LR RNAscope HybEz hybridization system Advanced Cell Diagnostics Cat# 321720 Bradford Reagent Sigma-Aldrich Cat# B6916 Experimental models: Organisms/strains POMC-Cre mouse Xu et al., 2005 N/A AgRP-Cre mouse Tong et al., 2008 N/A Starflox/flox mouse Torres et al., 2019 N/A Insrflox/flox mouse Könner et al., 2007 N/A C57BL/6J mouse Janvier Labs N/A Oligonucleotides RNAscope probe Pomc Advanced Cell Diagnostics Cat# 314081-C1 RNAscope probe Agrp Advanced Cell Diagnostics Cat# 400711-C2 RNAscope probe Star Advanced Cell Diagnostics Cat# 591531-C3 Taqman probe Star Thermo Fisher Mm00441558_m1 Taqman probe Cyp11a1 Thermo Fisher Mm00490735_m1 Taqman probe Tpso Thermo Fisher Mm00437828_m1 Taqman probe Crhr1 Thermo Fisher Mm00432670_m1 Taqman probe Gh Thermo Fisher Mm01258409_g1 Taqman probe Pit1 Thermo Fisher Mm00476852_m1 Taqman probe Pomc Thermo Fisher Mm00435874_m1 Taqman probe Tbx19 Thermo Fisher Mm00453377_m1 Taqman probe Tshb Thermo Fisher Mm00437190_m1 Software and algorithms Image J software NIH, Open source http://imagej-nih-gov.sire.ub.edu/ij/ Prism GraphPad Softwate http://www.graphpad.com/scientific- software/prism/ Axon pClamp 10.6 Molecular Devices https://www.moleculardevices.com/ products/axon-patch-clamp-system/ acquisition-and-analysis-software/pclamp- software-suite#gref SMART v3.0, video tracking software Panlab https://www.panlab.com/en/products/ smart-video-tracking-software-panlab Other Regular chow diet Tecklad, Envigo Cat# 2014C RD Western diet 43% Kcal from CH and Research Diets Cat# D12079B 40% Kcal from fat Microvette CB-300 Sarstedt Cat# NC9141704 Glucometer Arkray Cat# GT-1910 SuperFrost UltraPlus Adhesion slides Thermo Fisher Cat# 322000 ImmEdge hydrophobic barrier pen Thermo Fisher Cat# 10417002 ProLong Gold Antifade Mountant Thermo Fisher Cat# P36930 Vetbond 3M Cat# 1469SB RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact: Marc Claret (mclaret@clinic.cat). e2 Cell Metabolism 34, 269–284.e1–e9, February 1, 2022 ll Article OPEN ACCESS Materials availability All newly generated materials are available from the corresponding author upon reasonable request. Data and code availability d The data generated in this study will be shared by the lead contact upon reasonable request. d This study did not generate new code. d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. EXPERIMENTAL MODEL AND SUBJECT DETAILS Animal care and diets All animal studies were performed with approval of the University of Barcelona Ethics Committee, complying with current Catalan, Spanish and European legislation. Male mice were maintained on a temperature controlled (20-24 C), 12-hour light/dark cycle with free access to water and standard chow (Teklad maintenance diet 14% protein; Envigo). In particular studies, Western diet (40% Kcal from fat and 43% Kcal from carbohydrates; Research Diets) was provided ad libitum for 4 days or 16 weeks (starting at 8 weeks of age). Health status of the mice were checked regularly. The age and number of mice analyzed for each experiment is detailed in the figure legends. Mouse lines C57BL/6J mice were purchased from Janvier Labs. Mice with a floxed allele for Star (Starflox/flox) (Torres et al., 2019) were crossed with POMC-Cre (Xu et al., 2005) or AgRP-Cre (Tong et al., 2008) mice to generate animals selectively lacking Stard1 in either POMC neurons (POMCStarKO) or AgRP neurons (AgRPStarKO). Mice with a floxed allele for Insuslin receptor (Insrflox/flox) (Könner et al., 2007) were crossed with POMC-Cre mice to generate animals selectively lacking Insr in POMC neurons (POMCInsrKO). As control mice we used Cre negative, floxed littermates. Mice were on a C57BL/6 background. Human subjects and cerebrospinal fluid samples We studied 40 patients with obesity (BMI>35) aged R 40 years, with a sex distribution of 45% female and 55% male, belonging to an ongoing multicentric longitudinal cohort study (Hospital de la Santa Creu i Sant Pau and Hospital Clinic de Barcelona-IDIBAPS) aimed at assessing the effects of bariatric surgery on biochemical and neuroimaging Alzheimer’s disease biomarkers. Anthropometric, clin- ical and cognitive parameters were recorded from all individuals. Cerebrospinal fluid (CSF) samples were obtained by lumbar punc- ture and stored at -80 C until analysis. Pregnenolone concentration in CSF was determined using a commercially available ELISA kit (LDN) following manufacturer’s instructions. The reliability of the kit has been previously validated by liquid chromatography tandem mass spectrometry (Mahata et al., 2014). Patients were divided into metabolically healthy obese (MHO) or metabolically unhealthy obese (MUO) based on the criteria established by Aguilar-Salinas and collaborators (Aguilar-Salinas et al., 2008). According to these criteria, MHO patients exhibited a BMI >30 kg/m2 together with plasma HDL cholesterol of at least 40 mg/dl and the absence of type- 2 diabetes and arterial hypertension. Conversely, MUO patients were defined by the presence of one or more of the following meta- bolic abnormalities: plasma HDL cholesterol R40 mg/dl, type 2 diabetes, and arterial hypertension. Type 2 diabetes was considered present in subjects with a random glucose concentration of R200 mg/dl, a fasting glucose of R126 mg/dl, or those receiving glucose-lowering medication. Arterial hypertension was defined as the use of antihypertensive treatment and/or blood pressure >140 mm Hg systolic and/or >90 mm Hg diastolic. Glucose levels 2 hours after an oral glucose tolerance test were not included in the criteria. The local Ethical Committee approved the studies, and all study participants provided informed consent to donate blood and CSF samples. METHOD DETAILS Physiological tests Body weights were monitored using a precision scale. For feeding studies, mice were singly-housed and acclimatized for 1 week prior to study. Daily food intake was manually measured for 5 consecutive days using a precision scale. Blood samples were collected via tail vein using a capillary collection system with EDTA (Sarstedt). Blood glucose concentration was measured using a glucometer (Glucocard X-meter, Arkray). Glucose tolerance tests (2 g/Kg) were performed on overnight (16 hour) fasted mice. Insulin sensitivity tests (0.2 IU/Kg; Humulin, Lilly) were conducted on 6-hour food-deprived mice. Intracerebroventricular (i.c.v.) surgery and cannulation I.c.v. surgery was performed as described (Schneeberger et al., 2013). Briefly, adult (8-10 weeks old) mice were anaesthetized with a Ketamine/xylazine cocktail (i.p., 100 mg/Kg and 10 mg/Kg) and positioned in a stereotaxic frame (Kopf Instruments). The skull was exposed and a 26-gauge stainless steel guide cannula (PlasticsOne) was implanted into the third ventricle (midline 0 mm, 0.82 mm posterior from bregma, depth 4.8 mm from skull surface). The cannula was secured to the skull with screws and dental cement and temporarily occluded with a dummy cannula (PlasticsOne). After surgery, the mice were singly-housed and given at least 1 week to Cell Metabolism 34, 269–284.e1–e9, February 1, 2022 e3 ll OPEN ACCESS Article recover. I.c.v. injections were performed using a 30-gauge needle that extended 0.5 mm below the guide cannula (PlasticsOne), con- nected by cannula connector to a 5 ml Hamilton syringe and infused using a microinfusion pump (Harvard Apparatus). ARC surgery and cannulation Adult (8-10 weeks old) mice were anaesthetized with a Ketamine/xylazine cocktail (i.p., 100 mg/Kg and 10 mg/Kg) and positioned in a stereotaxic frame (Kopf Instruments). The skull was exposed and 26-gauge stainless-steel bilateral cannula (center-to-center dis- tance 0.8 mm; PlasticsOne) was implanted into the ARC parenchyma using stereotaxic procedures (coordinates from bregma 1.6 mm, lateral ±0.4 mm, ventral 5.0 mm from skull surface). The cannula was secured to the skull with stainless steel screws and dental cement, and temporarily occluded with a 33-gauge stainless-steel obturator. Animals were allowed to recover for at least 7 days before being studied. Treatments were given over 5 minutes via a 33-gauge needle extending 0.8 mm beyond the tip of the cannula (5.8 mm from dura) using a microinfusion pump (Harvard Apparatus), and followed by a 2-minute delay before needle withdrawal. Corticosterone measurements A standard restraint protocol was used to measure corticosterone-mediated stress response in control and POMCStarKO mice. The study was performed between 10:00 A.M. and 11:00 A.M. to minimize circadian corticosterone fluctuations. Immediately after col- lecting the first blood sample (baseline levels, time=0), mice were placed into a restrainer for 30 minutes. Afterwards, a second blood sample was taken (stress levels). Blood samples were collected via tail vein using a capillary collection system with EDTA (Sarstedt). Plasma corticosterone was measured using a commercially available EIA kit (Immunodiagnostic systems) following manufacturer’s instructions. Pregnenolone extraction and quantitative analysis Animals were killed by decapitation and the perirhinial cortex, hippocampus, arcuate-enriched mediobasal hypothalamus, nucleus of the solitary tract and pituitary were dissected and stored at 80 C until analyzed. Tissues were homogenized in 150 ml of acetonitrile using an electric hand-held homogenizer. Tissue lysates were centrifuged for 10 minutes at 10200 g at 4 C and the pellet was dis- carded. Next, we performed a liquid-liquid extraction with 300 ml of n-hexane in order to separate the neurosteroid fraction from the other components of the brain tissue. During this step, homogenates were vigorously shaken during 30 minutes at room temperature. Then we allowed the two phases to separate and recovered the neurosteroid fraction. Samples were dried in a preheated vacuum centrifuge (SpeedVac Savant SPD1010, Thermo Scientific) at 40 C. The dried steroid residues were kept at -20 C until they were resuspended (in PBS + 0.1% gelatin + 5% ethanol) for quantification with a validated pregnenolone ELISA test (LDN) (Mahata et al., 2014) following vendor’s instructions. The readings from the optical densities at 450 nm were compared to a standard curve that was also diluted in resuspension buffer in order to reduce the interference of the matrix. In vivo insulin signaling analysis by immunoblot Over-night (16 hour) fasted mice were anesthetized with ketamine/xylacine (i.p., 100 mg/Kg and 10 mg/Kg) and subsequently in- jected with 5U of insulin (humulin, Lilly) via the inferior cava vein. Relevant tissues were collected at specific time-points: 30 seconds (liver), 1 minute (skeletal muscle), 10 minutes (mediobasal hypothalamus), 11 minutes (perirhinal cortex) and 12 minutes (hippocam- pus). Tissues were immediately frozen in liquid nitrogen and stored at -80 C. Tissue samples were homogenized in a buffer containing 25 mM Tris-HCl (pH 7.4), 10 mM Na3VO4, 100 mM NaF, 50 mM Na4P2O7, 10 mM EGTA, 10 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, and 1% nonidet-P40 supplemented with a protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Tissue lysates were centrifuged for 15 minutes at 12000 rpm. Supernatants were collected and quantified for protein concentration using Bradford re- agent (Bio-Rad) and 10 mg of protein were subjected to gradient 4-12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad). Proteins were electrotransferred onto PVDF membranes (Millipore), blocked in 5% BSA buffer for 1 hour at room temper- ature and probed over-night (4 C) with pAKT (Ser473; 1:1000) or after 30 minutes of stripping (Thermo Scientific) with total AKT (1:1000). Both antibodies were from Cell Signaling Technology. After washing, membranes were incubated 1hour at room temper- ature with a secondary HPRT antibody (anti-rabbit IgG HPRT linked; 1:5000; GE Healthcare). Detection was performed by enhanced chemiluminescence (Pierce) and images were obtained using a LAS4000 imaging system (GE Healthcare). Protein expression was quantified by densitometry using ImageJ software (NIH). Quantitative polymerase chain reaction (qPCR) Tissues were harvested, immediately frozen in liquid nitrogen and stored at -80 C until use. Tissues were homogenized and total mRNA was isolated using Trizol (Invitrogen) using standard protocols. Retrotranscription was performed using reagents from Applied Biosystems. Quantitative PCR was conducted using Premix Ex Taq mastermix (Takara) in an ABI Prism 7900 HT system (Applied Bio- systems). Proprietary Taqman Gene Expression assay FAM/TAMRA probes (Applied Biosystems) used for qPCR analysis are listed in the key resources table. Gene expression was expressed relative to Hprt levels. Data was analyzed using the standard curve method. Fluorescent in situ hybridization and quantification Animals were transcardially perfused with saline followed by ice-cold 4% phosphate-buffered paraformaldehyde (PFA; pH 7.4). The brains were dissected and post-fixed in 4% PFA at 4 C for 24 hours, and cryoprotected by a sequence of 10-30% sucrose e4 Cell Metabolism 34, 269–284.e1–e9, February 1, 2022 ll Article OPEN ACCESS in 1x phosphate buffered saline (PBS, pH 7.4) at 4 C until the tissue sunk to the bottom of the tube. Brains were cut at 25 mm-thick on a freezing microtome and collected in 24-well plate containing cryoprotectant solution (30% ethylene glycol; 30% sucrose in PBS) and subsequently stored at -80 C to preserve RNA until further processing. Fluorescent in situ hybridization for the simultaneous detection of Pomc and Star or Agrp and Star mRNA was performed using RNAScope. All reagents were purchased from Advanced Cell Diagnostics . The Pomc probe contained 10 oligo pairs and targeted region 19-995 (Acc. No. NM_008895.3) of the Pomc transcript. The Agrp probe constituted 16 oligo pairs and targeted region 11-764 of the Agrp transcript (Acc. No. NM_001271806.1). The Star probe constituted 12 oligo pairs and targeted region 199-726 of the Star transcript (Acc. No. NM_011485.5). All incubation steps were performed at either at 60 C or 40 C using the ACD HybEz hybridization system. On the day before the assay, one every 4th sections throughout the ARC was mounted on SuperFrost UltraPlus Adhesion slides (ThermoFisher), air-dried at room temperature and stored at -20 C overnight. From each animal, one section from the same region of the brain was also mounted for assessment of negative control probe to allow subsequent calculation of background. On the day of the assay, one series section throughout the ARC was selected and equilibrated to room temperature. From each animal, one section from the same region of the brain was also mounted to assess the negative control probe to enable subsequent calculation of background. Slides were washed in PBS, baked at 60 C for 30 minutes and post-fixed with 4%PFA for 15 minutes. Sections were then dehydrated, baked for 30 minutes at 60 C and subsequently submerged into boiling (98.5 - 100 C) Target Retrieval reagent for 5 minutes, followed by two brief rinses in MilliQ-purified water. The slides were quickly dehydrated in 100% ethanol and allowed to air dry for 5 minutes. A hydrophobic barrier was then created around the sections using an ImmEdge hydro- phobic barrier pen (Vector laboratories). Slides were then placed into RNAScope holder and sections were treated with Protease III for 30 minutes at 40 C. The subsequent steps, i.e., hybridization of the probes and the amplification and detection steps, were performed according to the manufacturer’s protocol for RNAscope Multiplex Fluorescent Assay. The color module chosen for each experiment finally labeled the Pomc probe with Atto 550, the Agrp probe with Atto 647, and the Star probe with Atto 488. Sections were counterstained with DAPI and coverslipped with ProLong Gold Antifade Mountant (ThermoFisher) and stored in the dark at 4 C until imaging. Images were captured using a confocal Leica DM 2500 microscope, equipped with a 40x/1.15 oil objective and using a zoom of 2x. Z-stacks of 1 mm of the ARC were captured bilaterally from 4 rostral to caudal sections per animal. Laser intensities for the probe channels were kept constant throughout the entire image acquisition. Images were imported into Fijii (NIH) where maximum intensity projections were made. In order to acquire the minimum intensity value for analyzing the expression of Star, the threshold for probe recognition was calculated as the mean cell intensity present in the negative control sections +3xSD. All labeling above this value was considered to be true signal as published by previous reports (Timper et al., 2018). Brightness and threshold were adjusted uniformly across the sections. For quantification, each POMC positive neuron per section was manually selected and the amount of Star par- ticles were analyzed. Double POMC and FOS immunofluorescence after pregnenolone delivery C57Bl/6J mice were i.c.v injected with 2 ml of either vehicle (20% of 2-hydroxypropyl beta- cyclodextrin in NaCl 0.9%, Sigma-Aldrich) or pregnenolone (5 mg/ml, Sigma-Aldrich). Ninety minutes later, mice were transcardially perfused with saline followed by ice-cold 4% phosphate-buffered PFA. Brains were cryoprotected, frozen and subsequently cut into 30 mm-thick slices. Brain sections were blocked with 2% donkey serum in KPBS + 0.1% Triton X-100 + 3% BSA and incubated with rabbit anti FOS antibody (1:100; Santa Cruz) in blocking solution for 16 hours at 4 C. As secondary antibody, a donkey anti-rabbit Alexa Fluor 555 (1:200; Life Technologies) in KPBS + 0.1% Triton X-100 + 3% BSA was used (90 minutes at room temperature). After extensive washing, slices were blocked with 2% chicken serum in KPBS + 0.4% Triton X-100 and incubated with rabbit anti-POMC precursor antibody (1:1000; Phoenix Pharmaceuticals) for 16 hours at 4 C. Subsequently, slices were incubated with chicken anti-rabbit Alexa Fluor 488 (1:200; Life Tech- nologies) for 90 minutes at room temperature. Imaging was performed using a Leica DMI 6000B microscope with a 20x objective. A minimum of 388 POMC neurons from 3 mice per genotype were included in the analysis. POMC and AgRP neuron count The number of neurons were determined as described (Ramı́rez et al., 2017). In brief, brains from 10 week-old control, POMCStarKO and AgRPStarKO mice were collected. Mouse perfusion and brain slicing was performed as described above. POMC and AgRP neu- rons were identified by immunofluorescence or fluorescent in situ hybridization (color module Atto 488) respectively, following the protocols detailed above. Sections throughout the ARC (–1.1 mm to –2.7 mm from bregma) were collected in 3 series. The number of POMC and AgRP neurons were determined in 1 series and multiplied the neuron count by 3 to estimate the total cell number. Im- aging was performed using confocal Leica DM 2500 microscope, equipped with a 20x objective. The number of cells from 3 animals per genotype was counted manually using ImageJ software. We analyzed at least 2,409 POMC and 912 AgRP neurons per mouse. pCREB analysis by immunofluorescence Control and POMCStarKO mice under random-fed conditions were anesthetized and transcardially perfused with saline followed by ice-cold 4% PFA (pH 7.4). The brains were dissected and post-fixed in 4% PFA at 4 C for 24 hours, and cryoprotected by a sequence of 10-30% sucrose in 1x PBS (pH 7.4) at 4 C until the tissue sunk to the bottom of the tube. Brains were cut at 30 mm-thickness on a freezing microtome and collected. Serial hippocampal sections were blocked with 2% chicken serum in KPBS + 0.4% Triton X-100 Cell Metabolism 34, 269–284.e1–e9, February 1, 2022 e5 ll OPEN ACCESS Article and 3% BSA and incubated with rabbit anti pCREB antibody (1:200; Cell signaling) in blocking solution overnight (16 hours) at 4 C. As secondary antibody, a chicken anti-rabbit Alexa Fluor 488 (1:250; Life Technologies) in KPBS + 0.4% Triton X-100 was used (2 hours at room temperature). Imaging was performed using a Leica DMI 6000B microscope with a 20x objective. Three mice per genotype were included for the analysis of the number of pCREB-positive cells in the dentate gyrus of the hippocampus. For each mouse, the mean number was calculated by averaging the number of pCREB-positive cells counted from four images. General behavioral procedures Before each test, mice were acclimatized to the behavioral room and arena for 1 hour (or otherwise stated). The arena was cleaned with 70% ethanol before and after every trial. Light intensity was adapted to each task. The investigator remained outside of the behavioral room during testing. A video camera, positioned directly above the arena, was used to track the movement of each animal. Videos were recorded on a computer and analyzed with a video-tracking software (SMART v3.0, Panlab). Neurosensory screen Prior to behavioral studies, control and mutant mice were submitted to a basic sensorimotor assessment to ensure equivalent ca- pacity to perform subsequent tasks. This consisted in a battery of 10 score-based standardized measures to evaluate several pa- rameters, including scope response, vision, motor coordination, balance, orientation, reflexes or nociception. This protocol was based upon the behavioral screen previously described by Rogers and collaborators (Rogers et al., 1997). The test was performed as follows. A) Balance. Assessed by placing the mice on a 1-meter-long wooden beam, with 15 mm of flat surface, resting 50 cm from the surface. The latency to fall was recorded. Score: 0=falls off; 1=holds immobile; 2=walks slowly; 3=walks across the beam. B) Tail elevation. Scoring: 0=flaccid; 1=horizontal; 2=elevated. C) Piloerection. Scoring: 0=absence; 1=presence. D) Paw withdrawal reflex. Pressure was applied to the interdigital area of the right hind limb using a modified forceps (to standardize the amount of pressure). The movement of the leg was assessed. Scoring: 0=no reaction; 1=soft retraction; 2=moderate retraction; 3=quick retraction; 4=quick and repeated retractions. E) Ear withdrawal reflex. The outer ear of the mouse was gently touched using a swab stick. The movement of the ear was assessed. Scoring: 0=no reaction; 1=moderate retraction; 2=multiple retractions. F) Corneal reflex. A swab stick was progressively approached to the eye of the mouse. Scoring: 0=no reaction; 1=active eye blinking; 2=multiple eye blinking. G) Negative geotaxis. Mice were placed on a wire mesh grid that was lifted vertically, with the subject facing down. The latency to fall and overall performance was scored: 0=turns and climbs up; 1=turns but does not climb; 2=moves but does not turn; 3=does not move for 30 seconds; 4=falls off. H) Twisting. Mice were held by the tail, 20 cm above the surface, for a few seconds. Scoring: 0=no twisting; 1=twisting. I) Visual acuity. Mice were held by the tail, 20 cm above the surface, while a pencil was progressively approached to the mouse’s face. Mice should expand their forepaws as soon as they see the pencil. Scoring: 0=forepaws are not extended; 1=forepaws are extended after contact with pencil; 2=forepaws are extended before contact with whiskers. J) Scope response. Mice were held by the tail, 20 cm above the surface, and progressively lowered. Mice should expand their forepaws as they approach the surface. Scoring: 0=paws not extended; 1=paws extended after contact with the surface; 2=paws extended after contact with the whiskers; 3=paws extended before contact with the whiskers; 4=paws extended after start- ing movement. In addition, we also examined the olfactory capacity of mice as described in previous studies (Yang and Crawley, 2009). It consisted in the sequential presentation of diverse odors: water, non-social odor #1 (high-fat diet), non-social odor #2 (green tea) and social odor #3 (bedding from other cages). Mice were acclimated for 30 minutes to the arena (35 x 35 x 35 cm). Each odor was presented in a non-accessible clean container (separated by a grid) for 2 minutes in three consecutive video recorded trials. In- ter-trial interval was 1 minute (time needed to change the odor stimulus). Sniffing time was scored when the mouse was directly oriented towards the odor. Typically, mice exhibit a decrease in sniffing time over repeated presentations of the odor (habituation) followed by increased sniffing when a new odor is offered (dishabituation). Barnes maze test The Barnes maze is a widely used paradigm for the measurement of hippocampal-dependent spatial memory. Our protocol was based on original studies (Bach et al., 1995; Barnes, 1979). In brief, the maze consisted of a white elevated circular platform (85 cm stand height and 92 cm in diameter) with 20 equidistant holes located around the circumference. A black Plexiglas escape chamber (17.5 x 7.5 x 8.0 cm), that serves as refuge, was placed underneath one of the holes (target hole). The reference cues (a thick green cable, a white sliding door and a black cross) were presented in the walls surrounding the maze. Animals were tested under high-intensity light (>30 lux) to create an aversive environment in the surface of the apparatus. The protocol consisted of five days of training (acquisition phase), two days resting period and the test day. On the first training day, a mouse was placed in the center of the maze and given 3 minutes to find the target hole and enter into the attached escape cage. If they did not enter on their own during that time, the researcher would gently place the mouse to the side of the hole and help them enter. By doing so, the mouse realizes that the escape cage exists and gives them practice in stepping down to the platform in the cage. The mice were allowed to stay in the escape cage for 1 minute before being returned to the home cage. All mice performed two trials per day, with a 1-hour inter-trial interval, for five consecutive days. The position of the escape hole remained constant for each mouse during the acquisition phase. After these trials, rodents typically remember which hole contains the escape chamber and quickly proceed in a direct path towards the hole. Improved performance over sessions or trials reflects adequate learning. Mice were allowed to rest on days 6 and 7. The test trial was performed on day 8, in which the escape chamber was removed and animals were allowed to explore the maze for 2 minutes. During the test phase we scored the following parameters: a) latency to target hole (time spent from the center of the circular platform to the e6 Cell Metabolism 34, 269–284.e1–e9, February 1, 2022 ll Article OPEN ACCESS precise hole where the escape chamber was located); b) time in quadrant (time spent in the quadrant of the platform where the escape hole was located); c) total distance (total distance travelled by the mouse during the 2-minute test). All trials were video re- corded. Analysis of the latency to find the target hole, time spent in the target quadrant and total distance were automatically measured by the video-tracking software. Novel object recognition test (NORT) The novel object recognition test (NORT) is a highly validated task for recognition memory. Our protocol was adapted from Leger and collaborators (Leger et al., 2013). The test was conducted in a methacrylate arena (35 x 35 x 35 cm) under a low-intensity light (20 lux) environment. NORT consisted in three phases: habituation, training and test phase. During habituation (days 1-3), mice were allowed to explore the arena for 10 minutes. We considered object exploration when the mice were directing the nose toward the object, while actively sniffing and/or pawing, at a distance <3 cm. Sitting on top of the object while sniffing the surrounding air or chewing the object were not considered exploration, unless that action was accompanied with a nose-directing behavior toward the object. In the training phase (day 4), mice were allowed to explore two identical objects (equidistantly spaced in the arena) for 10 minutes and re- turned to their home cages immediately after training. The test phase was conducted 2 hours after the training phase. At this time interval, between training and testing periods, the involvement of the hippocampus in memory retention is expected (Antunes and Biala, 2012). In the test phase, one of the objects was replaced by a new one (novel object) and the mouse was allowed to explore them for 10 minutes. We kept the position of the 2 objects constant across sessions. Discrimination indices were calculated as: (Time exploring novel object – Time exploring familiar object) / (Time exploring novel object + Time exploring familiar object). In addition, we also scored total exploration time (Time exploring novel object + Time exploring familiar object) and total distance travelled during the test session. Animals that showed freezing behavior or exhibited <5 seconds of exploration behavior were excluded from the ana- lyses. To reduce experimenter interference, the trial was video-recorded and off-line analyzed in a blind manner using SMART v3.0 software (Panlab). Total distance was automatically scored by the software. Novel odor recognition test (NODR) The protocol was adapted from previous studies (Feinberg et al., 2012; Flores-Montoya et al., 2015). Similar to NORT, the test con- sisted in three phases: habituation, training and test phase. During habituation (days 1-3), mice were allowed to explore the arena for 10 minutes. The arena contained two identical leaky containers equidistantly positioned. In the training phase (day 4), mice were al- lowed to sniff two identical non-social odors (high-fat diet) placed in the leaky containers for 10 minutes, and returned to their home cage immediately after training. The test phase was conducted 2 hours after the training session. In this phase, one of the odors was replaced by a new one (Western diet) and the mouse was allowed to sniff for 10 minutes. We kept the position of the 2 leaky con- tainers constant across sessions. Discrimination indices were calculated as: (Time exploring novel odor – Time exploring familiar odor) / (Time exploring novel odor + Time exploring familiar odor). In addition, we also scored total exploration time (Time exploring novel odor + Time exploring familiar odor) and total distance travelled. The non-social odors selected were two types of novel diets for the mice (40% high fat diet and western diet) due to their low level of anxiety generation. To exclude a preference effect of the diets, we performed an odor preference test with these two diets prior to the actual experiment in a different cohort of mice. Animals that showed freezing behavior or exhibited <5 seconds of exploratory behavior were excluded from the analyses. To reduce experimenter interference, the trial was video-recorded and off-line analyzed in a blind manner using SMART v3.0 software (Panlab). Total distance was automatically scored by the software. Open field test Open field test is commonly used for measuring the exploratory behavior and general activity of animals. Our protocol was based on previous studies (Fan et al., 2019). Mice were placed in the center of a dark methacrylate arena (35 x 35 x 35 cm) and allowed to freely explore it for 10 minutes. Trials were video recorded. Animals were tested under a low-intensity light (< 30 lux) to create a safe envi- ronment and avoid stress. Total distance and the time spent in the center of the arena, compared with time spent in the edges, was scored using SMART v3.0 software (Panlab). Light–Dark box test Dark-light box test assesses anxiety-like behavior. It is based on the innate aversion of rodents to brightly illuminated areas. Our pro- tocol was based on previous studies (Fan et al., 2019). The test apparatus consisted in a methacrylate arena (35 x 35 x 35 cm) divided into a small dark (safe) compartment and a large strongly-illuminated (200 lux; aversive) compartment. A door connected the two compartments. Mice were placed in the dark compartment and allowed to freely move between the two chambers for 5 minutes. Trials were video recorded. Video tracking data was analyzed using SMART v3.0 software (Panlab) to calculate the time spent in each compartment and the latency to enter the illuminated compartment. Typically, anxiety models spent less time in the bright chamber. Elevated plus maze test Elevated plus maze test is also widely used for measuring anxiety-like behavior. This task takes advantage of the innate aversion of rodents to open spaces. Our protocol was based on previous studies (Fan et al., 2019). The apparatus used was a cross-shaped 4- arm maze, with 2 open arms and 2 closed (wall sheltered) arms (25 x 5 cm). This structure was elevated 60 cm above the floor. Mice Cell Metabolism 34, 269–284.e1–e9, February 1, 2022 e7 ll OPEN ACCESS Article were placed in the center of the apparatus, facing towards a closed arm, and their behavior was video recorded for 5 minutes. Time spent in the closed arms and open arms was analyzed via video-tracking software (SMART v3.0 software; Panlab). The preference for being in closed arms is an indicator of anxiety-like behavior. Tail suspension test Tail suspension test is commonly used for measuring depression-like behavior. It is based on the fact that rodents subjected to short- term inescapable stress eventually give up and take an immobile position. Our protocol was based on previous studies (Fan et al., 2019). Mice were suspended during 6 minutes by their tails using adhesive tape and behavior was video recorded. The periods of agitation and immobility during the test were scored off-line manually using SMART v3.0 software (Panlab). Longer immobility times is an indicator of depression-like behavior. Marble burying test The marble burying test is used to evaluate anxiety and/or obsessive-compulsive behaviors (Deacon, 2006). It is based on the innate proclivity of mice to bury objects in their bedding. The test was done under low-intensity lighting (<30 lux). The arena (35 x 35 x 35 cm) was filled with bedding to a depth of 5.5 cm. Each mouse was habituated to the arena for 15 minutes. After the habituation phase, mice were returned to their home cages and 25 pied glass marbles (Ø 1 cm) were placed equidistantly in a 5 x 5 arrangement. Mice were allowed to explore the arena and the marbles during 15 minutes. The number of marbles buried (at least 2/3 of its surface area covered by bedding) were scored manually just after the test. The number of marbles buried reflects anxiety-like and/or obsessive- compulsive behavior. NORT performance after i.c.v. or intra-ARC administration of pregnenolone NORT was essentially performed as described above. Cannulated mice into the third ventricle were infused with 2ml of either vehicle (20% of 2-hydroxypropyl beta-cyclodextrin in NaCl 0.9%, Sigma-Aldrich) or pregnenolone (5 mg/ml, Sigma-Aldrich) right after the training phase. Intra-ARC cannulated mice were bilaterally microinfused with 250 nl of either vehicle or pregnenolone (at a rate of 50 nl/min) followed by a 2-min delay before needle withdrawal. The test phase was conducted 2 hours later. NORT performance after chemogenetic modulation of POMC neuron activity and i.c.v pregnenolone administration Ten-week-old POMCCre/+ and POMCStarKO mice were anesthetized with a Ketamine/Xylazine cocktail (i.p., 100 mg/Kg and 10 mg/Kg) and placed in a stereotaxic frame (Kopf Instruments) for subsequent injection of AAV vectors encoding for activatory (AAV8-hSYN-DIO- hM3D(Gq)-mCherry; 7.53x1012 gc/ml, Addgene) or inhibitory (AAV8-hSYN-DIO-hM4D(Gi)-mCherry; 1.10x1013 gc/ml, Addgene) Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Viruses were bilaterally injected into the ARC (300 nl/injection site) using a 33-gauge needle connected to a 5 ml Neuro-Syringe (Hamilton). AAVs were delivered over 8 minutes according to the following coordinates: 1.5 mm posterior to the bregma, ± 0.3 mm lateral to midline, and 5.8 mm below the surface of the skull. Imme- diately after the procedure, the same mice were cannulated into the third ventricle as described above. The incision was sutured by VetBond tissue adhesive (3M) and mice were placed in a heated cage until they recovered from anesthesia. Three weeks later, mice were submitted to the NORT habituation phase (days 1-3). Right after the training phase on the fourth day, mice were i.p. injected with Clozapine-N-oxide (CNO; 1.5 mg/Kg for the activation and 1 mg/Kg for the inhibition; Tocris Bioscience) and returned to their home cage. Ninety minutes after CNO injection, 2 ml of vehicle (20% of 2-hydroxypropyl betacyclodextrin in NaCl 0.9%, Sigma-Aldrich) or pregnenolone (5 mg/ml, Sigma-Aldrich) was administered via the implanted cannula. The test phase was conducted 30 minutes later. Synaptophysin studies Ten-week-old POMCCre/+ mice were anesthetized with a Ketamine/Xylazine cocktail (i.p., 100 mg/Kg and 10 mg/Kg) and placed in a stereotaxic frame for subsequent injection of Cre-mediated AAV vectors that express cytoplasmic tdTomato and presynaptic (syn- aptophysin-fused) EGFP (AAV1-phSyn1(S)-FLEX-tdTomato-T2A-SypEGFP; 4x1012 gc/ml, Addgene). Viruses were bilaterally in- jected into the ARC (300 nl/injection site) using a 33-gauge needle connected to a 5 ml Neuro-Syringe (Hamilton). AAVs were delivered over 8 minutes according to the following coordinates: 1.5 mm posterior to the bregma, ± 0.3 mm lateral to midline, and 5.8 mm below the surface of the skull. After the procedure, the incision was sutured by 30 VetBond tissue adhesive (3M) and mice were placed in a heated cage until they recovered from anesthesia. Six weeks after the injections, mice were transcardially perfused with saline fol- lowed by ice-cold 4% phosphate-buffered PFA. Brains were cryoprotected, frozen and subsequently cut into 30 mm-thick sections. Brain sections were blocked in 2% donkey serum, 2% goat serum, 0.05% tween in 1xPBS. The EGFP signal was amplified by stain- ing with a chicken anti-GFP antibody (1:500; Aveslab). To amplify the tdTomato signal slices we incubated with a rabbit anti-RFP antibody (1:1000; Rockland). Some sections were not incubated with primary antibodies in order to assess the specificity of the stain- ing (negative control). After extensive washes, sections were then incubated with secondary antibodies: goat anti-chicken 488 (1:300; ThermoFisher) and donkey anti-rabbit 594 (1.300; ThermoFisher) during 2 hours at room temperature. Images were acquired in a Zeiss LSM700 confocal microscope. POMC neurons located in the ARC were identified with a 532nm laser. POMC projections into the hippocampal subiculum were recognized with a 488nm laser. For each section and area, image stacks with 1 mm distance interval were taken. Tile scans were acquired for whole section visualization. The representative images shown were taken with a 63x objective and maximum intensity stacks were made in FIJI (NIH) equally adjusted for brightness and contrast. e8 Cell Metabolism 34, 269–284.e1–e9, February 1, 2022 ll Article OPEN ACCESS Electrophysiological studies Electrophysiological studies were conducted in mice belonging to three experimental settings: a) chow-fed vs. 4d-WD; b) control vs. POMCStarKO; c) POMCStarKO + i.c.v. vehicle (2 ml of 20% 2-hydroxypropyl betacyclodextrin in NaCl 0.9%, Sigma-Aldrich) vs. POMCStarKO + pregnenolone (2 ml at 5 mg/ml, Sigma-Aldrich). Mice were deeply anesthetized with isofluorane and decapitated. In the case of vehicle vs. pregnenolone studies, mice were sacrificed 2 hours after the i.c.v injection. Brains were removed in ice- cold, high-sucrose extracellular artificial cerebrospinal fluid (aCSF1, in mM; 206 sucrose, 1.3 KCl, 1 CaCl2, 10 MgSO4, 26 NaHCO3, 11 glucose, 1.25 NH2PO4, purged with 95% CO2/5% O2, pH 7.4) and subdivided into the hemispheres. Thick (380 mm) coronal slices of hippocampus were obtained with a vibratome (VT1000S; Leica) and transferred into an incubation beaker with extracellular aCSF appropriate for neurophysiological recordings (aCSF2, in mM; 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.25 NaH2PO4, 1.5 MgSO4, 25 NaHCO3, 11 glucose, purged with 95 % CO2/5 % O2, pH 7.4). Slices were kept at 32 C for 1 hour and subsequently at room temperature for at least 1 additional hour. For field potential measurements, single slices were then transferred into a measurement chamber perfused with aCSF2 at 2 ml/minute at 28-30 C. A bipolar stimulation electrode (Platinum-Iridium stereotrode, PI2ST30.1A5, Science Prod- ucts) was placed in the Schaffer collateral pathway. Recording electrodes were made with a puller (P-1000, Shutter Instrument Com- pany) from thick-walled borosilicate glass with a diameter of 1.5 mm (Sutter Instruments). The recording electrode filled with aCSF2 was placed in the dendritic branching of the CA1 region for local field potential measurement (field excitatory postsynaptic potential, fEPSP). A stimulus isolation unit A385 (World Precision Instruments) was used to elicit stimulation currents between 25-700 mA. Before baseline recordings for long-term potentiation (LTP), input-output (IO) curves were recorded for each slice at 0.03 Hz. The stimulation current was then adjusted in each recording to evoke fEPSP at which the slope was at 50-60 % of maximally evoked fEPSP slope value. After baseline recording for 30 minutes with 0.03 Hz, LTP was induced by theta-burst stimulation (TBS; 10 theta bursts of 4 pulses of 100 Hz with an interstimulus interval of 200 ms repeated 7 times with 0.03 Hz). After LTP induction, fEPSPs were recorded for 1 additional hour with 0.03 Hz. Paired-pulse fEPSPs in the test pathway were measured before baseline recordings with an interstimulus interval of 50 ms. All recordings were amplified and stored using amplifier AxoClamp 2B (Molecular Devices). Traces were analyzed using Axon pClamp software (Molecular Devices, version 10.6). QUANTIFICATION AND STATISTICAL ANALYSIS Data are expressed as mean ± SEM. Statistical analysis were performed using GraphPad Prism software. Two group-one factor comparisons were performed using a two-tailed unpaired Student’s t test. Datasets with two factors-one dependent variable were analyzed using two-way ANOVA followed by Sidak’s post-hoc test, except for the IO curve data that was analyzed with a two-way ANOVA followed by Tukey’s post hoc test. One-sample t tests were performed to determine whether the NORT discrimi- nation indexes observed in control/vehicle groups were significantly different from chance/0. In all cases p < 0.05. Correlations were assessed by Spearman coefficient. p < 0.05 was considered statistically significant. Symbols used are: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Statistical parameters can be found in the Figures and Figure legends. No statistical methods were used to determine whether the data met assumptions of the statistical approach. Cell Metabolism 34, 269–284.e1–e9, February 1, 2022 e9