bioRxiv preprint doi: https://doi.org/10.1101/802405; this version posted October 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 1 2 3 4 5 6 7 8 Optogenetic mapping of feeding and self-stimulation within 9 the lateral hypothalamus of the rat 10 11 Short title: Optogenetic behavior mapping in the lateral hypothalamus 12 13 Kevin R. Urstadt#a *, Kent C. Berridge 14 Psychology Dept., University of Michigan 15 1004 East Hall, 530 Church Street 16 Ann Arbor, Michigan, U.S.A. 48109-1043 17 18 #a Current address: 19 Cognitive Science Dept., Occidental College 20 1600 Campus Road 21 Los Angeles, California, U.S.A. 90041 22 23 * Corresponding author 24 Email: [email protected] (KRU) 25 bioRxiv preprint doi: https://doi.org/10.1101/802405; this version posted October 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 2 26 27 Abstract 28 The lateral hypothalamus (LH) regulates eating and motivation, and includes several 29 anatomical subregions. This study explored localization of function across different LH 30 subregions in controlling food intake stimulated by optogenetic channelrhodopsin excitation, and 31 in supporting laser self-stimulation. We particularly compared the tuberal LH, the posterior LH, 32 and the lateral preoptic area. Local diameters of tissue optogenetically stimulated within LH 33 were assessed by measuring laser-induced Fos plumes and Jun plumes via 34 immunofluorescence surrounding optic fiber tips, and were used to map localization of function 35 for effects elicited by LH optogenetic stimulation. Optogenetic stimulation of the tuberal 36 subsection of the LH behaviorally produced the most robust food intake initially, but produced 37 only mild laser self-stimulation in the same rats. However, after repeated exposures to 38 optogenetic stimulation, tuberal LH behavioral profiles shifted toward more self-stimulation and 39 less food intake. By contrast, stimulation of the lateral preoptic area produced relatively little 40 food intake or self-stimulation, either initially or after extended stimulation experience. 41 Stimulation in the posterior LH subregion supported moderate self-stimulation, but not food 42 intake here, and at higher laser intensity shifted valence to evoke escape behaviors. We 43 conclude that the tuberal LH subregion may best mediate increases in food intake stimulated by 44 optogenetic excitation. However, incentive motivational effects of tuberal LH stimulation may 45 shift toward self-stimulation behavior after repeated stimulation. By contrast, the lateral preoptic 46 area and posterior LH do not as readily elicit either eating behavior or laser self-stimulation, and 47 may be more prone to higher-intensity aversive effects. 48 bioRxiv preprint doi: https://doi.org/10.1101/802405; this version posted October 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 3 49 Introduction 50 The lateral hypothalamus (LH) has been considered a powerful regulator of food 51 ingestion and reward-seeking motivation for over 60 years (1). Today the LH remains a prime 52 target of research into obesity, anorexia, and reward-related motivational dysfunctions (2). Early 53 decades of research used lesion, electrical stimulation, electrophysiological recording, and 54 intracranial microinjection techniques, whereas many contemporary studies have shifted to 55 optogenetic, DREADD, and optical imaging techniques. 56 The LH was first anatomically described nearly a century ago (3,4). Electrolytic LH 57 lesions were soon known to result in aphagia, adipsia, and sensory neglect behaviors (5–8). 58 Further, in early studies large LH lesions also produced pathological excessive ‘disgust’, evident 59 as gapes, headshakes and chin rubs that are normally elicited only by bitter or other unpalatable 60 tastes, becoming elicited by the taste of sucrose (7,9,10). However, subsequent excitotoxin 61 lesion studies showed that the site where neuron loss produced ‘disgust’ is actually in 62 caudolateral ventral pallidum, anterior to LH, and not in LH itself (11,12). LH lesions that do not 63 damage the ventral pallidum, as well as neurotoxic destruction of dopamine fibers of passage 64 through LH, do produce aphagia and sensory neglect, but not excessive ‘disgust’ ((7,8,11,13– 65 16). These lesion data paint a strong necessity role for the LH in intake, while neighboring 66 regions and fibers passing through the LH control a broader range of reward, disgust, and 67 motivation-related functions. 68 Other electrical stimulation experiments demonstrated that LH activation elicits eating, 69 drinking and other natural motivated behaviors (17). Additionally, rats were typically willing to 70 self-stimulate or work to activate the same LH electrodes, implicating the LH in both hunger and 71 reward (17–21). However, for most LH stimulation effects, there was not clear localization of 72 function within the LH (17). Conceivably, stimulation of fibers of passage by an LH electrode 73 may also play a role in behavioral effects, which could possibly obscure localization of function bioRxiv preprint doi: https://doi.org/10.1101/802405; this version posted October 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 4 74 within subregional clusters of LH neurons (22,23), making it difficult to specify the relative 75 contribution of intrinsic LH neurons. The possibility of localization of function within LH for 76 neuronal stimulation effects could yet emerge, if explored with modern techniques, such as 77 optogenetic stimulation of neurons in particular LH subregions. 78 Beyond the question of subregional LH differences in localization of function, another 79 important issue is the permanence versus malleability of behavioral effects of LH stimulation. 80 For example, using electrode stimulation in the LH, Valenstein and colleagues (24) showed that 81 the behavior evoked from rats by LH stimulation could change over time, due to repeated 82 experiences with electrical LH stimulation (Cox and Valenstein, 1969). For example, some LH 83 stimulation sites did not initially evoke eating, but subsequently did after rats received prolonged 84 exposures to electrode stimulation overnight while in a food deprived state (25). For other rats, 85 the dominant type of behavior elicited by LH stimulation switched from LH-evoked eating to LH- 86 evoked drinking in the same rats, after repeated experiences with LH stimulation in which food 87 targets were removed but water was available (26). Later when food was returned and a choice 88 was available, those rats remained stimulation-bound drinkers. Such reports suggest the 89 possibility that repeated experience might also possibly change the type of behavior evoked by 90 LH optogenetic neuronal stimulation. 91 To address these issues, we utilized optogenetic channelrhodopsin (ChR2) stimulation 92 to excite neurons in specific LH subregions of different rats. Excitation of LH neurons in specific 93 subregions, without stimulating fibers of passage, was assessed both for laser self-stimulation 94 and for stimulation-bound increases in eating. We also mapped potential localization of function. 95 In order to identify how large a subregion of LH was activated by ChR2 stimulation, which is 96 information required to assign localization of function, we measured immunofluorescence of 97 local Fos plumes induced by laser illumination around an optic fiber tip, and compared their 98 sizes to those of corresponding Jun plumes. Additionally, we assessed if any laser-evoked 99 behaviors changed after a phase of extended experience with LH optogenetic stimulation. bioRxiv preprint doi: https://doi.org/10.1101/802405; this version posted October 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 5 100 101 Methods and materials 102 Subjects 103 Pair-housed adult Sprague-Dawley rats (Rattus norvegicus; N=32; male NChR2 = 10; 104 male NeYFP = 6; female NChR2 = 11; female NeYFP = 5) weighing 250-400 g at the time of surgery 105 were maintained on a reverse 12 hr light-dark cycle at 21°C. Rats were always provided ad 106 libitum access to standard Purina rat chow and to water in home cages unless otherwise 107 specified. Males and females were housed in separate rooms, tested separately, and test 108 chambers were cleaned between uses to prevent sexual odor cues from influencing behavior. 109 Animal use procedures were approved by the Institutional Animal Care and Use Committee at 110 the University of Michigan. 111 Surgery 112 Rats were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (7 mg/kg, i.p.) and 113 were given atropine (0.04 mg/kg, i.p.), carprofen (5 mg/kg, s.c.), cefazolin (60 mg/kg, s.c.), and 114 isotonic saline (2 mL, s.c.) intraoperatively to prevent respiratory occlusion, inflammation, 115 infection, and dehydration, respectively. Using a stereotaxic apparatus, rats received surgical 116 microinjections of either an AAV5 virus containing genes for channelrhodopsin (ChR2) for 117 optogenetic stimulation, an enhanced yellow fluorescence protein (eYFP) reporter, and hSyn 118 promoter (AAV5 hSyn-ChR2-eYFP), or of an optically-inactive control virus, containing genes 119 only for hSyn promoter and eYFP reporter (AAV5 hSyn-eYFP).