Hypocretin‐1

JOURNAL OF NEUROCHEMISTRY | 2018 doi: 10.1111/jnc.14279

Department of Pharmacology, Physiology and Neuroscience, University of South Carolina School of Medicine, Columbia, South Carolina, USA

Abstract administration of OxA significantly increased c-Fos expres- Orexin/hypocretin neurons of the lateral hypothalamus and sion, a marker for neuronal activation, in the PFC and in perifornical area are integrators of physiological function. subpopulations of basal forebrain cholinergic neurons. Sub- Previous work from our laboratory and others has shown the sequently, we investigated the effects of acute intranasal OxA importance of orexin transmission in cognition. Age-related on neurotransmitter efflux in the PFC and found that intranasal reductions in markers of orexin function further suggest that OxA significantly increased both ACh and glutamate efflux in this neuropeptide may be a useful target for the treatment of this region. These findings were independent from any age-related cognitive dysfunction. Intranasal administration of changes in c-Fos expression in orexin neurons, suggesting orexin-A (OxA) has shown promise as a therapeutic option for that these effects are not resultant from direct activation of cognitive dysfunction. However, the neurochemical mecha- orexin neurons. In total, these data indicate that intranasal nisms of intranasal OxA administration are not fully under- OxA may enhance cognition through activation of distinct stood. Here, we use immunohistochemistry and in vivo neuronal populations in the cortex and basal forebrain and microdialysis to define the effects of acute intranasal OxA through increased neurotransmission of ACh and glutamate in administration on: (i) activation of neuronal populations in the the PFC. cortex, basal forebrain, and brainstem and (ii) acetylcholine Keywords: acetylcholine, cognition, hypocretin, intranasal, (ACh) and glutamate efflux in the prefrontal cortex (PFC) of microdialysis, orexin. Fischer 344/Brown Norway F1 rats. Acute intranasal J. Neurochem. (2018) https://doi.org/10.1111/jnc.14279

The hypothalamus is the primary central nervous system Received October 2, 2017; revised manuscript received December 1, locus for coordinating endocrine, autonomic, and behavioral 2017; accepted December 4, 2017. responses to peripheral cues indicative of homeostatic status. Address correspondence and reprint requests to Jim Fadel, Department Although the mammalian hypothalamus contains a divergent of Pharmacology, Physiology and Neuroscience, University of South Carolina School of Medicine, 6439 Garners Ferry Road, Columbia, SC array of nuclei and neural signaling molecules, the orexin 29208, USA. E-mail: [email protected] (hypocretin) system plays a particularly prominent role in Abbreviations used: ACh, acetylcholine; AChE, acetylcholinesterase; physiological regulation. The orexin system originates with aCSF, artificial cerebrospinal fluid; AIC, agranular insular cortex; BFCS, neurons largely restricted to a posterior hypothalamic level basal forebrain cholinergic system; ChAT, choline acetyltransferase; Cl, that includes the lateral hypothalamus, perifornical area, and claustrum; FBN/F1, Fischer 344-Brown Norway/F1 hybrid; HDBB, horizontal limb of the diagonal band of Broca; ILC, infralimbic cortex; parts of the dorsomedial nucleus, but gives rise to widespread LH/PFA, lateral hypothalamus/perifornical area; MOC, medial orbital projections that regulate limbic, cortical, and brain stem cortex; MS, medial septum; NaCl, sodium chloride; Ox1R, orexin-1- circuits (Peyron et al. 1998). This diverse array of projec- receptor; Ox2R, orexin-2-receptor; OxA, orexin-A; OxB, orexin-B; PBS, tions is consistent with the description of orexins as phosphate-buffered saline; PFA, paraformaldehyde; PFC, prefrontal ‘physiological integrators’ (Li et al. 2014), but also suggests cortex; PirC, piriform cortex; PPTg, pedunculopontine nucleus; PrLC, prelimbic cortex; PV, parvalbumin; RRID, research resource identifier; the potential to modulate cognition. For example, orexin-A SpTrN, spinal trigeminal nucleus; VDBB, vertical limb of the diagonal (OxA) administration modulates glutamatergic thalamocorti- band of Broca; VOC, ventral orbital cortex; VP/SI, ventral pallidum/ cal synapses and facilitates attention (Lambe et al. 2005; substantia innominata.

Huang et al. 2006; Song et al. 2006). Administration of cognition and behavior remains extremely limited. Accord- OxA has also been shown to facilitate attentional processing ingly, the goal of this study was to use immunohistochemistry through activation of basal forebrain cholinergic neurons, and in vivo microdialysis to directly examine the effects of which in turn alter cortical acetylcholine release (Fadel et al. intranasal OxA on measures of neuronal activation and 2005; Fadel and Burk 2010; Zajo et al. 2016; (Villano et al. neurotransmitter efflux in a rodent model. 2017). Moreover, OxA also modulates synaptic plasticity, the putative correlate of learning and memory, in the Methods hippocampus through coordinated modulation of glutamatergic, GABAergic, noradrenergic, and cholinergic signaling Animals and surgery – (Selbach et al. 2004). Male Fisher 344/Brown Norway F1 hybrid rats (3 4 months; fi Prior work from our laboratory and from others has shown Harlan/NIA; research resource identi er (RRID): not registered) weighing approximately 250–300 g (upon arrival) were used for all a selective age-related loss of orexin neurons (Sawai et al. experiments. This strain of rats has been used extensively by our 2010; Kessler et al. 2011). In addition, previous studies have laboratory and others for neurobiology of aging studies, because revealed that aging also correlates with decreased expression they have reduced susceptibility to many of the non-neurological of orexin peptides and their receptors (Terao et al. 2002; age-related complications (e.g., intraperitoneal tumors) seen in other Zhang et al. 2002; Porkka-Heiskanen et al. 2004; Downs strains, even well into the thirrd year of life (Lipman et al. 1996; et al. 2007). Ultimately, these phenomena may serve as a Turturro et al. 1999). Although the current study focuses on young correlate to the cognitive dysfunction seen in aging and age- animals, we have previously studied the effects of aging on the related cognitive disorders such as Alzheimer’s disease. orexin system in this strain (Kessler et al. 2011; Stanley and Fadel Because of orexin’s role in important physiological and 2012; Hagar et al. 2017) and are currently examining the effects of behavioral phenomena such as stabilization of sleep/wake intranasal orexin in aged animals; therefore, we selected the Fischer states, food intake, addiction, and cognition, there is much 344-Brown Norway/F1 hybrid strain to facilitate comparisons with prior and ongoing work. All animals were housed in an environ- interest in orexin receptors as potential therapeutic targets mentally controlled animal facility and kept on a 12 : 12 light: dark (Messina et al. 2016; Chieffi et al. 2017). The two orexin cycle with lights on at 07:00 hours. Animals were allowed standard peptides, orexin-A (OxA) and orexin-B (OxB) act on two G rat chow and water ad libitum. All experiments were performed protein-coupled receptors, with the orexin 1 receptor (Ox1R) during the light phase with each experiment starting at least 1 h after binding OxA with high affinity and the orexin 2 receptor the start of the light phase and ending at least 1 h before the start of (Ox2R) binding both OxA and OxB with relatively high the dark phase. Animal care and use practices, including rationale affinity (Smart and Jerman 2002; Ammoun et al. 2003; Gotter for use and number of animal subjects, were performed in et al. 2012b; Leonard and Kukkonen 2014). Several single- accordance with protocols written under the guidelines of the and dual- orexin receptor antagonists have been developed National Institutes of Health Guide for the Care and Use of (Smart et al. 2001; Gotter et al. 2012a; Steiner et al. 2013; Laboratory Animals and approved by the Institutional Animal Care Roecker et al. 2016; Skudlarek et al. 2017), but there is a and Use Committee at the University of South Carolina (Animal Use Protocol #2248). Measures taken to minimize animals’ paucity of selective orexin agonists that might have experi- suffering include careful monitoring of anesthesia depth during mental or clinical utility (Mieda and Sakurai 2013; Turku surgery, administration of analgesics to ease post-operative pain (see et al. 2017). Administration of the native peptides, mean- below), and using a within-subjects multi-session design for in vivo while, is limited by potential peripheral side effects and microdialysis (one session with saline control; one session with limited ability to target the brain because of peripheral intranasal OxA), so each animal served as its own control in these degradation and the blood–brain barrier. One route that has experiments, thus reducing the numbers of animals required to been suggested for brain delivery of orexins and other achieve equivalent statistical power. neuropeptides is intranasal administration (Hallschmid and The first author (CBC) performed the methods herein, including Born 2008; Hanson and Frey 2008; Dhuria et al. 2009; group assignment, with the exception of the immunohistochemistry Lammers 2011; Spetter and Hallschmid 2015). Intranasal experiments within the brainstem, which were conducted by HF. OxA, for example, has been shown to ameliorate attentional The individual conducting the cell counts was unaware of the deficits produced by sleep deprivation in non-human primates treatment group of the animals (saline vs. OxA) during the counting process. Sample size calculations were not employed, but estimated (Deadwyler et al. 2007). Rodent studies have further shown sample sizes were based on previous studies from our laboratory that intranasal OxA increases food intake and locomotor using treatment groups of a similar size for immunohistochemistry activity (Dhuria et al. 2016). Finally, limited human trials and microdialysis experiments. have suggested that intranasal OxA can ameliorate wakeful- A general timeline for both the c-Fos and microdialysis exper- ness, olfactory and cognitive correlates of narcolepsy iments is depicted in Fig. 1. This timeline was repeated for each (Hallschmid and Born 2008; Baier et al. 2011; Weinhold batch of animals (see below) used for either type of experiment. et al. 2014). Despite these intriguing findings, however, our Under ketamine/xylazine anesthesia (90/10 mg/kg, ip), animals understanding of the anatomical and neurochemical mecha- used for in vivo microdialysis received a single guide cannula nisms that may underlie the effect of intranasal orexin on (BASi, West Lafayette, IN, USA) inserted into the prefrontal cortex

Fig. 1 General experimental timeline. Day 0 represents the day of used for c-Fos analysis. For the in vivo microdialysis experiments, arrival of each batch of animals at the University of South Carolina guide cannula surgery was performed during Days 8–10. After several School of Medicine vivarium. Handling and habituation of the rats days of recovery, animals received a week of daily habituation to began the following day. After 1 week, animals were assigned to either intranasal saline. The first microdialysis session was performed on Day the c-Fos expression or in vivo microdialysis experimental groups. For 19 and the second session on Day 21. Half of the rats received OxA in the c-Fos experiments, animals received a week of daily habituation to the first session and saline in the second session; the order was intranasal saline. On the test day (Day 15), each rat received intranasal reversed for the other half of the rats. On the day following the second orexin-A (OxA) or saline and was euthanized 2 h later. Processing of microdialysis session (Day 22), the rats were euthanized, their brains brain tissue, immunohistochemistry, cell counts and data analysis processed for histochemical verification of probe placement, and began on Day 16 and proceeded until complete for each batch of rats HPLC analysis of dialysate samples began.

(PFC) at AP +2.8 mm, L Æ 0.5 mm, and DV À2.8 mm relative to individual administering the drug was not blinded to treatment. No bregma. The guide cannula was anchored in place using two to three additional randomization procedures were used to determine skull screws and dental cement. Guide cannula placement was treatment condition or order. All solutions were administered in counterbalanced, so that left and right hemispheres were equally four 12.5 lL increments over a 2-min period. Each animal was represented. Upon completion of the surgery, animals were given a habituated with intranasal saline treatment for 7 days before single dose of buprenorphine (0.01 mg/kg, sc) to ease post-operative experimentation. Beginning on day 8, animals received their pain and monitored until complete recovery. All animals were assigned treatment and were sacrificed 2 h later, the time at which allowed at least two full recovery days before habituation in the c-Fos expression peaks (Kaczmarek 1992). Rats were anesthetized microdialysis bowls. with isoflurane and perfused with phosphate buffered saline and 4% paraformaldehyde. After removal and overnight post-fixation, the Immunohistochemistry brains were coronally sectioned at a 50 lm thickness using a Upon arrival, animals were arbitrarily assigned to receive intranasal vibratome. A 1 : 4 serial sectioning method was utilized, allowing administration of either vehicle (50 lL of 0.9% saline) or OxA for 200 lm between each representative section collected. Sections (50 lL of a 100 lM solution; Enzo Life Sciences, Farmingdale, were stored in a 30% sucrose anti-freezing solution at À20°C until NY, USA). Animals used for immunohistochemistry were received use. For single-label immunohistochemistry, free floating sections in three separate batches from NIA (Batch 1, n = 6; Batch 2, n = 6; were incubated with a rabbit-anti c-Fos antibody (1 : 5000; Batch 3, n = 4). Both treatment conditions were equally represented Millipore, Billerica, MA, USA; catalog No. ABE457; RRID in each batch (e.g., for Batch 1, three animals received OxA and AB_2631318) for 48 h at 4°C followed by a biotinylated donkey three animals received saline); however, the order of treatment anti-rabbit secondary antibody (1 : 1000; Jackson ImmunoResearch within a batch of animals was counterbalanced (e.g., for Batch 1, Laboratories Inc.; West Grove, PA, USA; code No. 711-065-152; animal 1 received saline, animal 2 received OxA, etc., whereas for RRID AB_2340593) for 1.5 h at 23°C (RT), and horseradish Batch 2 animal 1 received OxA, animal 2 received saline, etc.). The peroxidase conjugated streptavidin (1 : 1600; Jackson

ImmunoResearch Laboratories Inc.; code No. 016-030-084; RRID results section were adapted from the third edition of The Rat Brain AB_2337238) for 1 h at 23°C. c-Fos staining was developed with by Paxinos and Watson (Paxinos and Watson 1998). 0.3% hydrogen peroxide and nickel-cobalt enhanced diaminobenzidine to yield blue-black immunopositive nuclei. For dual-label Microdialysis and HPLC immunohistochemistry, the above steps were followed with incu- Three separate batches of animals were used for the microdialysis = = = bation in either a goat anti-choline acetyltransferase (ChAT, experiments (Batch 1, n 2; Batch 2, n 4; Batch 3, n 4). 1 : 3000; Millipore, Temecula, CA, USA; catalog No. AB144; Beginning 3 days after cannula implantation, animals were habit- – RRID AB_90650) or mouse anti-parvalbumin (PV, 1 : 4000; uated in microdialysis bowls for 3 4 days and habituated with Sigma, St. Louis, MO, USA; catalog No. P3088; RRID intranasal saline for 7 days. On microdialysis days, guide cannula AB_477329) primary antibody for 48 h at 4°C. Secondary and stylets were removed and substituted with a microdialysis probe tertiary antibody incubation followed using either unlabeled donkey (BASi) that extended 2 mm past the guide cannula. Probes were l fl fi anti-goat (1 : 200; Jackson ImmunoResearch Laboratories Inc.; perfused at a 2 L/min ow rate with arti cial CSF (pH 7.4) code No. 705-005-003; RRID AB_2340384) or unlabeled donkey containing: 150 mM NaCl, 3 mM KCl, 1.7 mM CaCl2, 0.9 mM anti-mouse (1 : 200; Jackson ImmunoResearch Laboratories Inc.; MgCl2, and 4.9 mM D-glucose. Neostigmine bromide (50 nM; fi code No. 715-005-150; RRID AB_2340759) for 2 h at 23°C, and Sigma) was added to the arti cial CSF to increase recovery of either goat peroxidase anti-peroxidase (1 : 500; Jackson ImmunoR- acetylcholine in collected dialysates. Dialysate collection started esearch Laboratories Inc.; code No. 123-005-024; RRID after a 3 h discard period. Microdialysis sessions consisted of 1 h 9 AB_2338953) or mouse peroxidase anti-peroxidase (1 : 500; Jack- (4 15 min collections) of baseline collections followed by l son ImmunoResearch Laboratories Inc.; code No. 223-005-024; intranasal vehicle (0.9% saline) or OxA (100 M; Enzo Life l l RRID AB_2339261) for 1.5 h at 23°C. ChAT and PV immunore- Sciences), administered in a total volume of 50 L in 12.5 L activity were developed with 3% hydrogen peroxide and increments over a 2-min period. The individual performing diaminobenzidine to yield brown immunopositive cell bodies. intranasal administration was not blinded to treatment condition. 9 Sections were mounted onto slides with 0.15% gelatin, dried Dialysate collection then continued for 2 h (8 15 min collection) À ° overnight, dehydrated, delipidated and cover-slipped using DEPEX post-treatment. Upon collection, dialysates were stored at 80 C mounting medium. until analysis. All animals underwent two separate microdialysis In order to perform a qualitative examination of where intranasal sessions with an off day in-between and experiments were OxA distributed within the brain, a subset of animals (n = 4) counterbalanced, so that half of the animals received vehicle during received intranasal administration of either saline or a modified OxA session one, while the other half received OxA in session one. The peptide labeled with a green-fluorescent fluorophore (5(6)-FAM- day following the last microdialysis session, rats were euthanized fi (Glu1)-OxA trifluoroacetate salt; BACHEM, Bubendorf, Switzer- and their brains processed for probe placement veri cation using an fi land). These animals were received in a separate batch from the AChE background stain. Probe placement veri cation was per- other immunohistochemistry and microdialysis studies. As a result formed after all HPLC samples were run for each respective animal. of the descriptive and qualitative nature of this component of the Any probe placement visualized outside the medial PFC excluded study, no special selection or randomization procedure was used to the animal from the study. l select these animals. Animals receiving the fluorescein-tagged OxA Each 30 L dialysate was split prior to analysis by high peptide received 50 lL of a 500 lM dose split into four 12.5 lL performance liquid chromatography with electrochemical detection l l increments over a 2-min period and were sacrificed 30 min post- (HPLC-ECD), with 20 L analyzed for ACh and 10 L analyzed treatment. This time point was chosen based upon a previous study for glutamate. ACh was analyzed using an HTEC-510 HPLC-ECD fl l showing peak appearance of 125I–OxA in the CNS 30 min after (EicomUSA, San Diego, CA, USA). Brie y, 20 L of each intranasal delivery (Dhuria et al. 2009). Perfusion and brain dialysate was loaded into the AC-GEL separation column 9 ° sectioning was as described above. Sections were mounted onto (2.0 ID 150 mm; EicomUSA) maintained at a constant 33 Cin slides with 0.15% gelatin, dried overnight, dehydrated only and combination with mobile phase (pH 8.5) containing 49.4 mM l cover-slipped using Permount mounting medium. potassium bicarbonate (KHCO3), 134.3 M ethylenediaminete- traacetic acid disodium (EDTA-2Na), and 1.23 mM sodium Imaging 1- decanesulfonate. After analyte separation, post-column derivati- Histological experiments for single-labeled (c-Fos) cells, double- zation of ACh was attained through use of an AC-ENYM II enzyme labeled (c-Fos + ChAT or PARV) cells, and acetylcholinesterase reactor (1.0 ID 9 4 mm; EicomUSA) containing acetyl- (AChE) background staining were visualized using a Nikon E600 cholinesterase and choline oxidase, which generates hydrogen microscope fitted with a CoolSNAP digital camera (Roper Scien- peroxide (H2O2) proportional to the amount of ACh present. The tific, Trenton, NJ, USA). Fluorescence images were visualized using H2O2 is further broken down and detected on a platinum working a Nikon E600 microscope or a Leica SP8 multiphoton confocal electrode with an applied potential of +450 mV. The amount of microscope (Leica Microsystems, Wetzlar, Germany) equipped with ACh in each sample was measured by comparison with a three-point LAS AF 3 analysis software (Leica Microsystems). Immunoperox- external standard curve with values predicted to be in range of the idase photomicrographs were captured using IP Lab Software collected dialysates. The limit of detection for this analysis was (Scanalytics, Trenton, NJ, USA). Images were imported into Adobe approximately 5 fmol/injection. Photoshop 6.0 (Adobe Systems, San Jose, CA, USA) to adjust the Glutamate was analyzed using a CC-32 HPLC-ECD (BASi) with image size and to make minor alterations to contrast and brightness. modifications. First, 10 lL of each dialysate was loaded into the To indicate the approximate brain region where photomicrographs GU-GEL separation column (4.6 ID 9 150 mm; EicomUSA) in were obtained, diagrams and schematics of brain regions in the conjunction with a mobile phase (pH 7.2) containing 60 mM

© 2017 International Society for Neurochemistry, J. Neurochem. (2018) 10.1111/jnc.14279 Neuronal activation by intranasal orexin 5 ammonium chloride-ammonium hydroxide, 134.3 nM EDTA-2Na, Table 1 Comparison of c-Fos densities (nuclei/mm2) for each brain and 686 lM hexadecyltrimethylammonium bromide. After separa- region listed after intranasal vehicle (saline) or intranasal orexin-A tion, post-column derivatization of glutamate was attained through (OxA) (50 lL, 100 lM) administration the use of an E-ENZ enzyme reactor (3.0 ID 9 40 mm; Eico- 2 mUSA) containing glutamate oxidase, which generates H2O2 c-Fos density (nuclei/mm ) proportional to the amount of glutamate present. The H2O2 is further broken down and detected on a 3.0 mm glassy carbon Brain region Vehicle 100 lM OxA electrode (BASi) coated with a horseradish peroxidase osmium Piriform cortex* 582 Æ 38.24, n = 8 777.3 Æ 77.55, n = 8 polyvinylpyridine solution (0 mV applied potential). The amount of Agranular 125 Æ 10.23, n = 8 277.3 Æ 45.15, n = 8 glutamate in each dialysate was measured by comparison with a insular cortex** three-point standard curve using external standards expected to be in Prelimbic cortex** 859.4 Æ 86.19, n = 8 1234 Æ 81.4, n = 8 range of the collected dialysates. The limit of detection for this Ventral 703.1 Æ 80.97, n = 8 959.8 Æ 70.45, n = 7 method was approximately 3 fmol/injection. orbital cortex* Infralimibic 890.6 Æ 38.27, n = 8 714.8 Æ 74.8, n = 8 Statistics and analysis cortex (p = 0.0551) For the immunohistochemistry experiments, single-labeled c-Fos Medial orbital cortex 609.4 Æ 81.62, n = 8 660.7 Æ 56, n = 7 + and double-labeled (c-Fos ChAT/PARV) cells were counted Perirhinal cortex 334.7 Æ 78.12, n = 8 394.9 Æ 47.22, n = 8 within the bounds of a reticle embedded into the eyepiece of the Claustrum 605.5 Æ 45.34, n = 8 722.7 Æ 69.23, n = 8 microscope. Staining from each animal was counted using two Hippocampus (CA3) 17.35 Æ 4.1, n = 8 23.47 Æ 3.085, n = 8 representative sections at different rostro-caudal levels. Single- Hippocampus (CA1) 14.54 Æ 3.437, n = 8 19.39 Æ 3.876, n = 8 2 labeled c-Fos data were expressed as a density (c-Fos nuclei/mm ) Hippocampus 73.98 Æ 3.734, n = 8 85.71 Æ 10.09, n = 8 and analyzed by a two-tailed unpaired t-test (GraphPad Prism 5; (Dentate Gyrus) GraphPad Software, La Jolla, CA, USA). Double-labeled cells were Pedunculopontine 4.571 Æ 1.043, n = 7 8.125 Æ 0.8332, n = 8 expressed as the percentage of the total number of ChAT/PARV nucleus* neurons positive for c-Fos within the reticle and were analyzed by a two-tailed unpaired t-test. A significance cutoff of p < 0.05 was In the cerebral cortex, OxA significantly increased c-Fos expression in used for all analyses. the piriform, agranular cortex, prelimbic, and ventral orbital cortices For the microdialysis experiments, baseline neurotransmitter compared to intranasal saline. There was also a strong trend efflux was obtained during the first four sample collections (i.e., (p = 0.0551) for decreased c-Fos expression in the infralimbic cortex timepoints 1–4) and averaged to yield mean basal efflux. For each after intranasal OxA administration compared to vehicle. The medial sample analyzed, a raw value was obtained and expressed as orbital cortex, perirhinal cortex, and hippocampal brain regions yielded pmol/20 lL for ACh and lmol/l0 lL for glutamate. The micro- no significant differences between the two treatment groups. Among dialysis data presented in the graphs are expressed as percent of the brainstem regions, the pedunculopontine nucleus was the only area average baseline to account for individual variation in basal where intranasal OxA administration significantly increased c-Fos neurotransmitter efflux. Data were analyzed using two-way expression. Counts for c-Fos-positive nuclei in the pedunculopontine repeated-measure ANOVAs(SPSS; IBM, Armonk, NY, USA) with nucleus are reported as the total number in the entire region. There treatment as a within-subjects variable and time as a repeated were n = 8 animals per treatment group with the exception of the measure, using a significance cutoff of p < 0.05. A simple-effects vehicle group for the PPTg and the OxA group for the ventral and test with Bonferonni corrections was used to probe the source of medial orbital cortices. These three treatment groups are represented significant main effects and/or interactions. by n = 7 rats/group. *p < 0.05; **p < 0.01.

OxA administration. We also considered the possibility that Results intranasal OxA delivery may alter c-Fos expression within Intranasal OxA increases c-Fos expression in select brain brainstem regions involved in arousal. Indeed, intranasal regions OxA administration signiﬁcantly increased c-Fos expression

Intranasal administration of OxA signiﬁcantly increased within the pedunculopontine nucleus (PPTg; t13 = 2.692, c-Fos expression in multiple brain regions (Table 1; Fig. 2a). p = 0.0185) (Table 1). Below, we also provide corroborating In the cortex, intranasal OxA increased c-Fos expression in evidence that intranasal OxA delivery reaches more caudal the prelimbic cortex (t14 = 3.163, p = 0.0069), agranular regions of the brainstem, including the spinal trigeminal insular cortex (t14 = 3.291, p = 0.0054), ventral orbital nucleus (Fig. 6c). As an example, density measurements for cortex (VOC; t13 = 2.357, p = 0.0348), and piriform areas c-Fos were made using a 0.179 9 0.179 mm area in the (t14 = 2.259, p = 0.0404). Importantly, this effect was not a center of the prelimbic cortex (Fig. 2b). Photomicrographs global phenomenon as intranasal OxA failed to signiﬁcantly depict increased c-Fos expression in the prelimbic cortex of alter c-Fos expression within the claustrum, medial orbital animals treated with OxA compared to vehicle treatment cortex, and all regions of the hippocampus (Table 1). In fact, (Fig. 2c and d). For brainstem c-Fos counts, measurements there was a strong trend for decreased c-Fos expression in the represent the total number of c-Fos-positive nuclei within the infralimbic cortex (t14 = 2.092, p = 0.0551) after intranasal entire brain region.

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Fig. 2 Neuronal activation (c-Fos expression density) in cortical brain immunohistochemistry for c-Fos shown as blue/black punctate nuclei regions following intranasal OxA administration. (a) Single-labeled following acute treatment with intranasal saline. (d) Single-label c-Fos density for animals treated with intranasal saline (vehicle; PrLC, immunohistochemistry for c-Fos following acute treatment with AIC, VOC, n = 8 rats) or intranasal OxA (50 lL, 100 lM; PrLC and intranasal OxA. A clear increase in c-Fos expression density is seen AIC, n = 8 rats; VOC, n = 7 rats). Treatment with intranasal OxA in the OxA treatment group (d) compared to vehicle controls (c). resulted in increased c-Fos expression in neurons of PrLC, AIC, and Abbreviations: OxA, orexin-A; PrLC, prelimbic prefrontal cortex; AIC, VOC compared to vehicle-treated controls (b) Diagram indicating the agranular insular cortex; VOC, ventral orbital cortex. Scale bar approximate location (black-outlined square) within the PrLC for which represents approximately 100 lm (c and d). Error bars represent c-Fos counts and photomicrographs were obtained. (c) Single-label SEM. **p < 0.01, *p < 0.05.

Intranasal OxA decreases c-Fos expression in cortical 2005; Fadel and Frederick-Duus 2008; Arrigoni et al. 2010; GABAergic interneurons Fadel and Burk 2010), we investigated if intranasal admin- To analyze the effects of intranasal OxA on neuronal istration of OxA elicited a similar response. Indeed, intranasal subpopulations in the cortex, we stained cells for PV, a marker OxA increased expression of c-Fos in cholinergic neurons of for fast-spiking GABAergic interneurons in the cortex (Hu the vertical limb of the diagonal band of Broca (VDBB; et al. 2014). Interestingly, we found that intranasal OxA t14 = 5.788, p < 0.0001) and ventral pallidum/substantia decreased c-Fos expression in PV neurons of the prelimbic innominata (VP/SI; t14 = 3.45, p = 0.0039) compared to cortex (t14 = 3.176, p = 0.0067) and VOC (t13 = 4.258, vehicle. Additionally, there was a strong trend for increased p = 0.0009). This contrasts with the increase in overall c-Fos c-Fos expression within the cholinergic neurons of the medial expression within these brain regions after intranasal OxA septum (t14 = 2.133, p = 0.0511). Data are expressed as the administration (Fig. 2a). Data are expressed as the percentage percentage of ChAT-positive neurons that also express c-Fos of PV-positive neurons also expressing c-Fos (Fig. 3a). (Fig. 4a). Single- and double-labeled ChAT neurons were Single-labeled and double-labeled PV neurons were counted counted within the same 0.45 9 0.45 mm area for each basal within the same 0.179 9 0.179 mm reticle area as c-Fos, so forebrain region. As an example, the reticle area in the VDBB that direct comparisons could be established (Fig. 3b). Dou- is depicted (Fig. 4b). Double-labeled ChAT neurons of the ble-labeled PV neurons of the prelimbic cortex are depicted by VDBB are represented by arrows pointing to the blue-black arrows showing blue-black immunoreactivity for c-Fos and immunoreactivity for c-Fos and brown immunoreactivity for brown immunoreactivity for PV (Fig. 3c). ChAT (Fig. 4b and c).

Intranasal OxA increases c-Fos expression in basal forebrain Intranasal OxA increases ACh and glutamate efﬂux in the cholinergic neurons PFC Owing to evidence that OxA activates the basal forebrain After observing increased c-Fos expression in the PFC, we cholinergic system (Eggermann et al. 2001; Fadel et al. investigated the effects of intranasal OxA on PFC

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(b) (c)

Fig. 3 Activation of PV-positive GABAergic interneurons in cortical between treatment groups. (b) Schematic indicating the approximate brain regions after intranasal OxA administration. (a) Percentage of location (black-outlined square) within the PrLC where c-Fos/PV double-labeled (c-Fos/PV) neurons relative to the total number of PV counts and photomicrographs were obtained. (c) Typical dual-label neurons within the PrLC, VOC, AIC, ILC, MOC, PirC, and Cl after immunohistochemistry for c-Fos (blue/black) and PV (brown) neurons treatment with intranasal saline (vehicle; all regions, n = 8 rats) or within the PrLC (arrows). Abbreviations: PV, parvalbumin; OxA, orexin- intranasal OxA (50 lL, 100 lM; PrLC, AIC, ILC, PirC, and Cl, n = 8 A; PrLC, prelimbic prefrontal cortex; VOC, ventral orbital cortex; AIC, rats; VOC and MOC, n = 7 rats). Intranasal OxA significantly increased agranular insular cortex; ILC, infralimbic prefrontal cortex; MOC, the percentage of dual-labeled PV neurons in the PrLC and VOC. medial orbital cortex; PirC, piriform cortex; Cl, Claustrum. Scale bar Other cortical regions including the AIC, ILC, MOC, and PirC, as well represents approximately 100 lm (c). Error bars represent SEM. as the subcortical claustrum, did not show significant differences ***p < 0.001, **p < 0.01. neurotransmission using in vivo microdialysis. Intranasal remained significantly elevated for more than 1 h when OxA significantly increased ACh efflux in the PFC as compared with ACh efflux after treatment with intranasal indicated by a significant main effect of TIME saline (Fig. 5a).

(F11,99 = 9.058, p < 0.001), a significant main effect of Intranasal OxA also significantly increased glutamate TREATMENT (F1,9 = 14.381, p = 0.004), and a significant efflux in the PFC compared to vehicle as specified by a TIME 9 TREATMENT interaction (F11,99 = 8.595, significant TIME 9 TREATMENT interaction (F11,99 = p < 0.001). Simple-effects comparisons with Bonferonni 2.128, p = 0.025) although there were no significant main corrections revealed that 100 lM OxA significantly effects of TIME (F11,99 = 1.008, p = 0.445) or TREAT- increased ACh efflux compared to vehicle from collections MENT (F1,9 = 2.866, p = 0.125). Simple-effects compar- five through nine (all t9 > 2.93; all p < 0.05). Efflux of ACh isons with Bonferonni corrections indicated significantly after intranasal OxA treatment peaked during collection five increased levels of glutamate from time points five through

(15 min post-OxA) at approximately 233% of baseline and seven (all t9 > 2.370; all p < 0.05). After intranasal OxA

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Fig. 4 Activation of cholinergic neurons in the basal forebrain by intranasal OxA administration. (a) Percentage of double- labeled (c-Fos/ChAT) neurons relative to the total number of ChAT-positive neurons within the VDBB, VP/SI, MS, and HDBB after treatment with intranasal saline (vehicle; all regions, n = 8 rats) or intranasal OxA (50 lL, 100 lM; all regions, n = 8rats).Intranasal OxA significantly increased the percentage of dual-labeled ChAT neurons in the VDBB and VP/SI compared to vehicle-treated controls. (b) Schematic indicating the approximate (b) (c) location (black-outlined square) within the VDBB where c-Fos/ChAT counts and photomicrographs were obtained. (c) Typical dual-label immunohistochemistry for c-Fos (blue/black) and ChAT (brown) neurons within the VDBB (arrows). Abbreviations: OxA, orexin-A; ChAT, choline acetyltransferase; VDBB, vertical limb of the diagonal band; VP/SI, ventral pallidum/ substantia innominata; MS, medial septum; HDBB, horizontal limb of the diagonal band. Scale bar represents approximately 100 lm (c). Error bars represent SEM. ***p < 0.001, **p < 0.01. administration, significantly increased glutamate efflux was sensory and spinal subdivisions of the trigeminal nucleus observed during collection five and peaked at collection (Fig. 6a and b), the sources of somatosensory innervation of seven (45 min post-OxA) with a maximum efflux around the olfactory mucosa. The fluorescein-tagged OxA peptide 133% of baseline (Fig. 5b). was also detected in cortical regions, including the medial Probe placement verification via acetylcholinesterase PFC (Fig. 6c and d). As a result of the qualitative nature of background staining stain revealed that the 2 mm active these experiments, a relatively modest treatment size was membrane portion of the dialysis probe was centered in the used (n = 4). This pattern of fluorescence was not observed prelimbic portion of the PFC with a small degree of overlap following intranasal vehicle (saline) administration (n = 4). dorsally into the anterior cingulate cortex or ventrally into infralimbic portion of the prefrontal cortex (Fig. 5c.) Discussion Intranasal 5(6)-FAM-(Glu1)-hypocretin-1 administration Collectively, these experiments demonstrate that intranasal To visually assess the possible modes by which intranasal OxA administration increased c-Fos expression in many, but OxA enters the brain and to provide qualitative supporting not all areas of cerebral cortex, basal forebrain, and evidence for the above results, we intranasally administered a brainstem. The selective nature of OxA-elicited neuronal fluorescein-tagged OxA peptide (5(6)-FAM-(Glu¹)-Hypocre- activation was further demonstrated by a limited phenotypic tin-1). This ultimately revealed a distribution of the peptide analysis of these effects, which revealed increased activation to both rostral and caudal brain regions. Prior work has of basal forebrain cholinergic neurons but decreases in Fos suggested that intranasally delivered peptides reach the brain expression in GABAergic, PV+ interneurons in prelimbic via diffusion along sensory trigeminal pathways (Hanson and and ventral orbital cortices. Additionally, our microdialysis Frey 2008; Chapman et al. 2013; Meredith et al. 2015; results demonstrate that intranasal OxA administration Spetter and Hallschmid 2015). Therefore, we visualized rapidly and significantly increases ACh and glutamate within delivery of the fluorescein-tagged peptide in the principal the PFC, an area important for multiple aspects of executive

and cognitive function. Thus, in our rodent model, intranasal OxA proved effective at reaching the brain and modulating the activity of multiple brain regions and neurotransmitter systems that mediate cognition and behavior.

Effects of intranasal OxA on c-Fos expression Orexin regulation of PFC activity has been well-documen- ted. Orexin neurons project robustly to the medial PFC where they enhance neurochemical, electrophysiological, and behavioral correlates of attention (Fadel et al. 2005; Lambe et al. 2005; Huang et al. 2006; Vittoz and Berridge 2006). The prelimbic and ventral orbital regions of PFC, where we observed the greatest OxA-elicited neuronal activation, have been implicated in attentional shifting and reversal learning, respectively (Dalley et al. 2004). We also observed robust activation in the agranular insular cortex, part of an area described as putative ‘interoceptive cortex’ because of its role in sensing the organism’s physiological status (Craig and Craig 2002; Avery et al. 2017; Hassan- pour et al. 2017). Given the role of orexin neurons in responding to peripheral cues indicative of physiological status, intranasal OxA may facilitate interoceptive processing within the insular region. Additionally, increased c-Fos expression was also observed in the piriform cortex, a brain region important for olfactory discrimination (Stettler and Axel 2009; Bekkers and Suzuki 2013). Interestingly, there was a (non-significant) trend for decreased c-Fos expression after intranasal OxA in the infralimbic PFC cortex compared to vehicle. This finding is not surprising as divergent functions between the prelimbic and infralimbic PFC have been described with respect to fear memory, reward processing, and appetitive learning (Vidal- Gonzalez et al. 2006; Ashwell and Ito 2014; Flores et al. 2014; Hayen et al. 2014). Neuronal activation by intranasal OxA was not limited to the forebrain as we detected increased c-Fos expression within the PPTg, a part of the reticular activating system that sends projections to PFC (Mesulam et al. 1983; Eckenstein et al. 1988). Importantly, this brain region plays an important Fig. 5 Effect of intranasal OxA administration on ACh and glutamate role in wakefulness/arousal and attention (Datta et al. 2001; efflux in the PFC. (a) Intranasal OxA (50 lL, 100 lM; n = 10 rats) Cyr et al. 2015). treatment after baseline collections (arrow) significantly increased ACh efflux compared to intranasal vehicle (saline; n = 10 rats). A significant Effects of intranasal OxA on specific neuronal phenotypes increase in ACh efflux was observed from time points five through nine While we observed an overall increase in c-Fos expression in the n = fi versus vehicle (b) Intranasal OxA (arrow; 10 rats) also signi cantly prelimbic and VOC, we detected the opposite response within fl increased glutamate ef ux within the prefrontal cortex compared to PV+ interneurons of these brain regions (Fig. 3a), suggesting treatment with intranasal saline. The significant effect lasted from time that intranasal OxA primarily activates glutamatergic neurons in points five through eight versus vehicle treatment. (c) Diagram indicating the approximate probe placement within the PFC for each of the PFC. While it is possible that the overall increases in c-Fos fl + animals that underwent microdialysis. Typical probe placement in the expression re ect activation of other (non-PV ) GABAergic PFC is indicated on AChE background-stained section from an animal populations, this is unlikely as PV+ GABAergic neurons are the that underwent two microdialysis sessions. Abbreviations: PFC, dominant phenotype within the cortex (Kawaguchi and Kubota prefrontal cortex; ACh, acetylcholine; in, intranasal; OxA, orexin-A. 1997; Xu et al. 2010; Kelsom and Lu 2013). One potential Error bars represent SEM. **p < 0.01, *p < 0.05. mechanism may be through OxA activation of PV+ GABAergic

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Fig. 6 Localization of a green-fluorescent- tagged OxA peptide in the brain after intranasal administration. (a) Typical distribution of the fluorescent-tagged OxA peptide (50 lL, 500 lM) in the PrLC after intranasal administration. Green fluorescence indicates (c) (d) the appearance of the labeled peptide in the PrLC. (b) Schematic indicating the approximate location (black-outlined square) within the PrLC where fluorescence photomicrographs were obtained. (c) Typical distribution of the fluorescent-tagged OxA peptide in the spinal trigeminal nucleus (SpTrN). (d) Schematic indicating the approximate location (black-outlined square) within the SpTrN where fluorescence photomicrographs were obtained. Abbrevi- ations: OxA, orexin-A; PrLC, prelimbic prefrontal cortex; SpTrN, spinal trigeminal nucleus. Scale bar approximately 100 lm. neurons in the basal forebrain, which form inhibitory synapses Mechanistic considerations for neuronal activation and onto PV+ interneurons in the cortex (Henny and Jones 2008). In neurotransmission addition, current evidence suggests that orexin activation of The effects of intranasal OxA administration on c-Fos are PV+ GABAergic neurons in the basal forebrain may be corroborated by our effects seen through in vivo microdial- primarily an Ox2R-mediated phenomenon (Wu et al. 2002; ysis. In summary, intranasal OxA significantly increased Mieda et al. 2011). Whether this pattern of activation extends both acetylcholine and glutamate efflux within the PFC. The into other basal forebrain regions that contain more cortically Ox1R and Ox2R are both expressed in the basal forebrain projecting PV+ GABAergic neurons is unknown. Further (Marcus et al. 2001). While direct receptor mechanisms studies assessing the relative roles of each receptor in the basal involving intranasal OxA administration cannot be deter- forebrain will be vital to understanding orexin regulation of mined from this study, prior studies indicate that our effects wakefulness, arousal, and attention. may primarily be an Ox1R-mediated phenomenon. For As described above, intranasal OxA induced significant example, intrabasalis administration of OxA elicits a greater increases in c-Fos expression within the VDBB and VP/SI response on somatosensory cortical ACh release than OxB regions of the basal forebrain. Cholinergic and GABAergic (Dong et al. 2006). Studies from our laboratory corroborate neurons of the VDBB are vital for olfactory memory and this finding as the specific Ox1R antagonist SB-334867 spatial memory consolidation (Paolini and McKenzie 1993; block stimulated ACh release during feeding (Frederick- Lecourtier et al. 2011), while the collective VP/SI is Duus et al. 2007). Still, it is possible that the Ox2R plays a important for attention, in particular involving reward, large role in the effects mediated by intranasal OxA motivation, and feeding (Smith et al. 2009). Prior mecha- administration as previous work using in vitro electrophys- nistic descriptions of OxA-basal forebrain interactions have iology has reported that OxB is as effective as OxA at indicated that intrabasalis administration of OxA results in exciting basal forebrain cholinergic neurons (Eggermann robust increases in cortical ACh release (Fadel et al. 2005). et al. 2001). Importantly, the large increases in ACh efflux Furthermore, the basal forebrain appears to be an important (233% of baseline) we observed translate to previous work site for OxA modulation of cholinergic-dependent attentional showing that high levels of ACh in the hippocampus and functions (Boschen et al. 2009; Fadel and Burk 2010; Zajo neocortex set the dynamics for increased attention (Hasselmo et al. 2016). and McGaughy 2004).

We also observed significant, although less robust, intranasal OxA administration. Further experimentation increases in PFC glutamate efflux after intranasal OxA using intranasal OxB will help to define the roles for these administration. From a mechanistic standpoint, this effect can receptors more clearly. Together, these neurochemical cor- arise from a variety of sources. Directly, intranasal OxA may relates highlight key systems that underlie the cognitive be acting locally within the PFC as orexin receptors are effects of intranasal OxA and suggest that intranasal OxA expressed in this brain region (Hervieu et al. 2001; Marcus may be a viable treatment option for cognitive dysfunction. et al. 2001) and OxA has been shown stimulate presynaptic glutamate release in the PFC (Lambe et al. 2007). Visual- ization of (5(6)-FAM-(Glu¹)-Hypocretin-1) in the PFC Acknowledgments and conflict of interest supports this notion (Fig. 6c and d). Indirectly, the increases disclosure in cortical glutamate efflux that we observed could also come This work was supported by grants from the National Institutes of from excitation of glutamatergic thalamocortical synapses Health (R01AG050518; JRF), the University of South Carolina originating from the paraventricular nucleus (Lambe et al. SPARC Graduate Research Program (CBC) and the University of 2005; Huang et al. 2006) or possibly from the orexin South Carolina Magellan Scholars Program (HF). The authors thank neurons that colocalize glutamate (Rosin et al. 2003). The Meredith Calva for valuable assistance with artwork. All authors latter possibility seems unlikely, however, as we observed no declare no financial conflicts of interest. differences in c-Fos expression in orexin labeled neurons All experiments were conducted in compliance with the ARRIVE between intranasal vehicle and intranasal OxA-treated guidelines. animals (data not shown). 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