Diacylglycerol Lipase-Alpha Regulates Hippocampal-Dependent Learning and Memory Processes in Mice

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Research Articles: Behavioral/Cognitive

Diacylglycerol lipase-alpha regulates hippocampal-dependent learning and memory processes in mice

Lesley D Schurman1, Moriah C Carper1, Lauren V Moncayo1, Daisuke Ogasawara2, Karen Richardson1, Laikang Yu3, Xiaojie Liu3, Justin L. Poklis1, Qing-Song Liu3, Benjamin F Cravatt2 and Aron H Lichtman1,4

1Department of Pharmacology and Toxicology, Virginia Commonwealth University, 23298 2Department of Molecular Medicine, The Scripps Research Institute, 92037 3Department of Pharmacology and Toxicology, Medical College of Wisconsin, 53226 4Department of Medicinal Chemistry, Virginia Commonwealth University https://doi.org/10.1523/JNEUROSCI.1353-18.2019

Received: 27 May 2018

Revised: 24 April 2019

Accepted: 11 May 2019

Published: 24 May 2019

Author contributions: L.D.S., Q.-S.L., B.F.C., and A.H.L. designed research; L.D.S., M.C., L.V.M., K.R., L.Y., X.L., J.L.P., and Q.-S.L. performed research; L.D.S. and Q.-S.L. analyzed data; L.D.S. wrote the first draft of the paper; L.D.S., J.L.P., and Q.-S.L. wrote the paper; D.O. and B.F.C. contributed unpublished reagents/analytic tools; Q.-S.L., B.F.C., and A.H.L. edited the paper.

Conflict of Interest: The authors declare no competing financial interests.

This work was supported by the National Institutes of Health (NIH) [Grant NS093990 and DA039942] (to AHL), [Grant 1F31NS095628-01A1] (to LDS), [Grant DA035217] (to QSL), [Grant DA033760] (to BC), and Center grant [P30DA033934]. Start-up funds from the VCU School of Pharmacy also supported this work. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Corresponding Author: Aron H. Lichtman, Department of Pharmacology and Toxicology and Department of Medicinal Chemistry, Virginia Commonwealth University, 23298, USA. E-mail: [email protected]

Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.1353-18.2019

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1 Diacylglycerol lipase-alpha regulates hippocampal-dependent learning and memory processes

2 in mice

3 4 Lesley D Schurman1, Moriah C Carper1, Lauren V Moncayo1, Daisuke Ogasawara2, Karen

5 Richardson1, Laikang Yu3, Xiaojie Liu3, Justin L. Poklis1, Qing-Song Liu3, Benjamin F Cravatt2,

6 and Aron H Lichtman1,4

7 1Department of Pharmacology and Toxicology, Virginia Commonwealth University, 23298,

8 2Department of Molecular Medicine, The Scripps Research Institute, 92037,

9 3Department of Pharmacology and Toxicology, Medical College of Wisconsin, 53226,

10 4Department of Medicinal Chemistry, Virginia Commonwealth University, 23298.

11 12 Number: Pages 38, Figures 10

13 Number of words: Abstract 235, Introduction 650, Discussion 1500

14 Key Words: Diacylglycerol lipase-alpha, spatial memory, long-term potentiation,

15 endocannabinoid, 2-AG, hippocampus.

16 Corresponding Author: Aron H. Lichtman, Department of Pharmacology and Toxicology and

17 Department of Medicinal Chemistry, Virginia Commonwealth University, 23298, USA. E-mail:

18 [email protected].

19 Conflict of Interest: The authors declare no competing financial interests.

20 Acknowledgements: This work was supported by the National Institutes of Health (NIH) [Grant

21 NS093990 and DA039942] (to AHL), [Grant 1F31NS095628-01A1] (to LDS), [Grant DA035217]

22 (to QSL), [Grant DA033760] (to BC), and Center grant [P30DA033934]. Start-up funds from the

23 VCU School of Pharmacy also supported this work. The content is solely the responsibility of

24 the authors and does not necessarily represent the official views of the NIH.

25 Author Contributions: LDS, QSL, and AHL designed research; LDS, MCC, LVM, DO, KR, LY,

26 XL and JLP performed research; DO and BFC contributed unpublished reagents/analytic tools;

27 LDS analyzed data; and LDS and AHL wrote the paper.

DAGL-α regulates spatial memory

28 Abstract

29 Diacylglycerol lipase-α (DAGL-α), the principal biosynthetic enzyme of the endogenous

30 cannabinoid 2-arachidonylglycerol (2-AG) on neurons, plays a key role in CB1 receptor-

31 mediated synaptic plasticity and hippocampal neurogenesis, but its contribution to global

32 hippocampal-mediated processes remains unknown. Thus, the present study examines the role

33 that DAGL-α plays on long-term potentiation (LTP) in hippocampus, as well as in hippocampal-

34 dependent spatial learning and memory tasks, and on the production of endocannabinoid and

35 related lipids through the use of complementary pharmacologic and genetic approaches to

36 disrupt this enzyme in male mice. Here we show that DAGL-α gene deletion or pharmacological

37 inhibition disrupts LTP in CA1 of the hippocampus, but elicits varying magnitudes of behavioral

38 learning and memory deficits in mice. In particular, DAGL-α-/- mice display profound impairments

39 in the Object Location assay and Morris water maze (MWM) acquisition engaging in non-spatial

40 search strategies. In contrast, wild type mice administered the DAGL-α inhibitor DO34 show

41 delays in MWM acquisition and reversal learning, but no deficits in expression, extinction,

42 forgetting, or perseveration processes in this task, as well as no impairment in Object Location.

43 The deficits in synaptic plasticity and MWM performance occur in concert with decreased 2-AG

44 and its major lipid metabolite (arachidonic acid), but increases of a 2-AG diacylglycerol

45 precursor in hippocampus, prefrontal cortex, striatum, and cerebellum. These novel behavioral

46 and electrophysiological results implicate a direct and perhaps selective role of DAGL-α in the

47 integration of new spatial information.

DAGL-α regulates spatial memory

48 Significance Statement

49 Here we show that genetic deletion or pharmacologic inhibition of diacylglycerol lipase-α

50 (DAGL-α) impairs hippocampal CA1 LTP, differentially disrupts spatial learning and memory

51 performance in Morris water maze (MWM) and Object Location tasks, and alters brain levels of

52 endocannabinoids and related lipids. Whereas DAGL-α-/- mice exhibit profound phenotypic

53 spatial memory deficits, a DAGL inhibitor selectively impairs the integration of new information

54 in MWM acquisition and reversal tasks, but not memory processes of expression, extinction,

55 forgetting, or perseveration, and does not affect performance in the Objection Location task.

56 The findings that constitutive or short-term DAGL-α disruption impair learning and memory at

57 electrophysiological and selective in vivo levels implicate this enzyme as playing a key role in

58 the integration of new spatial information.

DAGL-α regulates spatial memory

74 Introduction

75 A growing body of evidence implicates the importance of 2-arachidonoyl glycerol (2-AG), the

76 most highly expressed endogenous cannabinoid in brain, in regulating learning and memory.

77 Diacylglycerol lipase (DAGL) forms 2-AG through the hydrolysis of diacylglycerols (Chau and

78 Tai, 1981; Okazaki et al., 1981) and exists as two distinct biosynthetic enzymes, DAGL-α and

79 DAGL-β (Bisogno et al., 2003b) that are expressed on distinct cell types, as well as in brain

80 areas important for learning and memory (Katona et al., 2006; Lafourcade et al., 2007). Within

81 the central nervous system, DAGL-β is highly expressed on microglia, while DAGL-α, expressed

82 on synapse-rich plasma membranes of dendritic spines (Yoshida et al., 2006), serves as the

83 principal synthetic enzyme of 2-AG on neurons (Viader et al., 2015).

84 DAGL-α modulates neurotransmission through distinct processes of neurogenesis and

85 endocannabinoid-mediated short-term synaptic plasticity, implicating it in learning and memory

86 processes. Adult neurogenesis represents a form of cellular plasticity in the developed brain.

87 Two prominent brain areas showing adult neurogenesis, the subventricular zone and the

88 hippocampus, that express high levels of DAGL-α (Goncalves et al., 2008) undergo marked

89 reductions in the proliferation of neuronal progenitor cells in DAGL-α-/- mice (Gao et al., 2010).

90 Similarly, the non-selective DAGL inhibitors RHC80267 and tetrahydrolipstatin reduce

91 proliferation of neuronal progenitor cells in the subventricular zone (Goncalves et al., 2008).

92 DAGL-α also plays an essential role in endocannabinoid-mediated retrograde synaptic

93 suppression in which 2-AG synthesized at post-synaptic terminals acts as a retrograde

94 messenger to activate the pre-synaptic cannabinoid receptor type 1 (CB1), which inhibits

95 synaptic transmission. Tanimura et al. (2010) reported that deletion of DAGL-α, but not DAGL-β,

96 annihilated two forms of endocannabinoid-mediated short-term synaptic plasticity,

97 depolarization-induced suppression of excitation (DSE) and inhibition (DSI) in hippocampus,

98 striatum, and cerebellum. Similarly, Gao et al. (2010) replicated these findings in hippocampus,

99 and Yoshino et al. (2011) reported that DAGL-α plays a necessary role in DSI in prefrontal

DAGL-α regulates spatial memory

100 cortex (PFC). Furthermore, the DAGL inhibitor DO34 blocked DSE in cerebellar slices and DSI

101 in hippocampal slices in a concentration dependent manner (Ogasawara et al., 2016).

102 The cell signaling events that occur after learning are frequently studied in terms of long-

103 term potentiation (LTP). Carlson et al. (2002) demonstrated that transient release of

104 endocannabinoids by depolarization facilitated the induction of LTP when preceded by DSI,

105 suggesting that endocannabinoids may enhance plasticity at Schaffer collateral-CA1 synapses

106 through a disinhibitory action. However, the role that the major 2-AG biosynthetic enzyme

107 DAGL-α plays in the induction of LTP remains unknown. Here we hypothesize that this enzyme

108 plays a necessary role in the induction of LTP. Accordingly, we predict that pharmacological

109 inhibition or genetic deletion of DAGL-α will disrupt LTP.

110 The present study also investigated the in vivo role of DAGL-α in spatial learning and

111 memory tasks. Specifically, we evaluated whether endocannabinoid-regulated short-term

112 synaptic plasticity disruption by DAGL-α deletion or inhibition translates to hippocampal-

113 dependent learning and memory task deficits in the Morris water maze (MWM) and Object

114 Location assays. Given the important role of hippocampal neurogenesis in the processing of

115 spatial memory (Lieberwirth et al., 2016), we investigated the consequences of DAGL-α

116 disruption on spatial memory processes of acquisition, expression, extinction, forgetting,

117 reversal, and perseveration in the MWM. To circumvent known pitfalls related to constitutive

118 genetic deletions, such as the role of DAGL-α in axonal guidance during development (Williams

119 et al., 2003), we utilized the DAGL-α/β inhibitor, DO34, and DAGL-α-/- mice. Because DO34 also

120 inhibits other serine hydrolases (i.e., ABHD2, ABHD6, CES1C, PLA2G7, PAFAH2), we

121 evaluated DO53, a structural analog of DO34 that cross-reacts with these off-targets, but does

122 not inhibit DAGL (Ogasawara et al., 2016).

123 In the present study, we ascertain the consequences of DAGL-α disruption at three

124 levels of investigation; cellular responses to LTP, in vivo spatial learning and memory tasks, and

125 alterations of endocannabinoid lipids, their substrates and metabolites, in hippocampus,

DAGL-α regulates spatial memory

126 prefrontal cortex, striatum, and cerebellum (brain areas important for learning and memory

127 showing high DAGL-α activity [Baggelaar et al., 2017]).

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152 Materials and Methods

153 Animals. Subjects consisted of adult male C57BL/6J mice (Jackson Laboratories, Bar Harbor,

154 Maine), and DAGL-α-/-,-α+/-, and -α+/+ mice on a mixed 99% C57BL/6 (30% J, 70% N) and 1%

155 129/SvEv background, as previously described (Hsu et al., 2012). DAGL-α+/- mouse breeding

156 pairs were originally generated in the Cravatt laboratory and transferred to Virginia

157 Commonwealth University. All mice (age 8-10 weeks) were pair-housed under a 12 h light/12 h

158 dark cycle (0600 to 1800 h), at a constant temperature (22ºC) and humidity (50-60%), with food

159 and water available ad libitum. All experiments were conducted in accordance with the National

160 Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publications

161 No. 8023, revised 1978), and were approved by the Virginia Commonwealth University and

162 Medical College of Wisconsin Institutional Animal Care and Use Committees.

163 Electrophysiology. For LTP experiments, DAGL-α+/+, -α+/-, -α-/- and C57BL/6J mice

164 were anaesthetized under isoflurane inhalation and decapitated. Hippocampi were dissected (in

165 C57BL/6J mice 2 h post vehicle [VEH], DAGL-α inhibitor DO34 (Cravatt Laboratory, La Jolla,

166 CA) [30 mg/kg], or its control analog and ABHD6 inhibitor DO53 [30 mg/kg] administration) and

167 were embedded in low-melting-point agarose (3%, Sigma-Aldrich A0701). Transverse

168 hippocampal slices (400 μm thick) were prepared using a vibrating slicer (Leica VT1200s) (Pan

169 et al., 2011; Zhang et al., 2015). Slices were prepared at 4-6°C in a solution containing (in mM):

170 68 sucrose, 2.5 KCl, 1.25 NaH2PO4, 5 MgSO4, 26 NaHCO3, and 25 glucose. The slices were

171 transferred to and stored in artificial cerebrospinal fluid containing (in mM): 119 NaCl, 3 KCl, 2

172 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, and 10 glucose at room temperature for at least 1

173 hour before use. All solutions were saturated with 95% O2 and 5% CO2.

174 Field excitatory postsynaptic potentials (fEPSPs) were recorded with patch clamp

175 amplifiers (Multiclamp 700B) under infrared-differential interference contrast microscopy. The

176 recordings were made blind to drug treatment and mouse genotype. Data acquisition and

177 analysis were performed using digitizers (DigiData 1440A and DigiData 1550B) and the analysis

DAGL-α regulates spatial memory

178 software pClamp 10 (Molecular Devices). Signals were filtered at 2 kHz and sampled at 10 kHz.

179 Field recordings were made using glass pipettes filled with 1 M NaCl (1-2 MΩ) placed in the

180 stratum radiatum of the CA1 region of the hippocampal slices, and fEPSPs were evoked by

181 stimulating the Schaffer collateral/commissural pathway at 0.033 Hz with a bipolar tungsten

182 electrode (WPI). Stable baseline fEPSPs were recorded for at least 20 minutes at an intensity

183 that induced ~40% of the maximal evoked response. LTP was induced by theta burst

184 stimulation (TBS), which consisted of a series of 15 bursts, with 4 pulses per burst at 100 Hz

185 with a 200 ms inter-burst interval. TBS is designed to mimic the in vivo firing patterns of

186 hippocampal neurons during exploratory behavior (Larson et al., 1986). All recordings were

187 performed at 32 ± 1°C by using an automatic temperature controller.

188 Morris water maze (MWM) assessments. The MWM consisted of a circular,

189 galvanized steel tank (1.8 m in diameter, 0.6 m height). The tank was filled with water

190 (maintained at 20º C ± 2º C) with a white platform (10 cm diameter) submerged 1 cm below the

191 water’s surface. A sufficient volume of white paint (Valspar 4000 latex paint, Lowe’s Companies

192 Inc., Mooresville, NC) was added to render the water opaque and the platform invisible. In

193 addition to distal visual cues (shapes) on curtains surrounding the tank (30 cm from the tank

194 wall), five sheets of laminated paper with distinct black-and-white geometric designs were

195 attached to the sides of the tank serving as proximal cues. An automated tracking system (ANY-

196 maze, San Diego Instruments, Inc., San Diego, CA) analyzed the swim path of each subject and

197 calculated distance (path length between being placed in the water and finding the hidden

198 platform), average swim speed, number of platform crossings, and percentage of time spent in

199 each quadrant.

200 To assess MWM acquisition DAGL-α+/+, -α+/-, -α-/- mice received 10 Fixed Platform

201 training days (i.e. a submerged platform remained in the same location across days), and then

202 were assessed for expression of spatial memory on day 11 in a single MWM Fixed Platform

203 Probe Trial (i.e. the submerged platform was removed). C57BL6/J Mice received a 2 h

DAGL-α regulates spatial memory

204 pretreatment of the DAGL-α inhibitor DO34 (0.3, 3, and 30 mg/kg (Wilkerson et al., 2017)), or its

205 inactive analog and ABHD6 inhibitor DO53 (30 mg/kg), or VEH on each of 10 Fixed Platform

206 training days. A cued task test assessed sensorimotor/motivational confounds by placing a 10

207 cm high black cylinder on the submerged platform and mice were released from the farthest two

208 release points. In order to assess whether DO34 affects the expression of spatial memory,

209 separate groups of mice were given 10 days of drug-free Fixed Platform training. On day 11,

210 each subject was administered 30 mg/kg DO34 or VEH, and 2 h later underwent a single 2 min

211 MWM Fixed Platform Probe Trial. The extinction of spatial memory was assessed in drug-naïve

212 acquisition trained mice, which received injections of 30 mg/kg DO34 or VEH (2 h pretreatment)

213 at 1, 2, 3, 4 and 6 weeks post-acquisition and tested in 2 min probe trials. To assess forgetting,

214 a group of drug-naïve acquisition trained mice were administered 30 mg/kg DO34 or VEH on

215 day 11 and then returned to their home cage, given a second injection of 30 mg/kg DO34 or

216 VEH at 6 weeks post-acquisition, and 2 h later underwent a 2 min probe trial. Reversal learning

217 and perseverative behavior were assessed in drug-naïve acquisition trained mice, which

218 received injections of 30 mg/kg DO34 or VEH (2 h pretreatment) and trained in reversal task

219 trials in which the submerged platform was moved to the opposite side of the tank over five days

220 post-acquisition, followed by a 2 min drug-free probe trial on day 6 post-acquisition.

221 Object Location (OL) assessments. The Object Location (OL) task is a hippocampal-

222 dependent adaptation of the Object Recognition task (Assini et al., 2009). The apparatus

223 consisted of a clear acrylic plastic enclosure (40 x 40 x 40 cm) located inside a white sound

224 attenuating box (59 x 52 x 72 cm) with black geometric shapes (stripes vs circles) on two

225 opposing box walls. A red light illuminated each component of this task. For all experiments,

226 mice received 1 h acclimatization to the testing room, and then were placed in the OL

227 apparatus, without objects, to explore freely for 10 min across two habituation days (separated

228 by 24 h). An OL trial consisted of two phases (sample and choice) separated by 3 h (Figure 7A).

229 During the sample phase, mice were placed in the chamber for 10 min, with two identical

DAGL-α regulates spatial memory

230 objects placed on opposing corners of the same box wall. After the 3 h retention delay, each

231 subject was returned to the apparatus for a 2 min choice test, in which one object was moved to

232 the opposite side of the chamber (left and right objects counterbalanced across trials). During

233 pharmacological assessment of DAGL-α inhibition, 30 mg/kg DO34 was administered i.p. 2 h

234 prior to the sample phase (Figure 7E). An automated tracking system (ANY-maze, San Diego

235 Instruments, Inc., San Diego, CA) video recorded exploration, and each trial was subsequently

236 scored blind for sample and choice phase object exploration. Exploration of an object was

237 defined as directing the nose to the object at a distance of <2 cm and/or touching it with the

238 nose.

239 Lipid extraction and mass spectrometry. DAGL-α+/+, -α+/-, -α-/- mice, and C57BL6/J

240 mice receiving a 2 or 24 h pretreatment of DO34 (30 mg/kg) or VEH, were anaesthetized under

241 isoflurane inhalation and decapitated. Hippocampi, prefrontal cortex, striatum, and cerebellum

242 were dissected out from brain, flash frozen in liquid nitrogen, and stored at −80°C until assay.

243 On the day of lipid extraction, as previously described (Kwilasz et al., 2015), the pre-weighed

244 mouse brains were homogenized with 1.4 ml chloroform:methanol (containing 0.0348 g

245 PMSF/ml). Six point calibration curves ranged from 0.078 pmol to 10 pmol for AEA, 0.125 nmol

246 to 16 nmol for 2-AG, AA, and SAG, a negative control and blank control were also prepared.

247 Internal standards (Cayman Chemicals, Michigan, USA) (50 μl of each of 8 nmol SAG-d8, 1

248 pmol AEA-d8, 1 nmol 2-AG-d8, 1 nmol AA-d8) were added to each calibrator, control, and

249 sample, except the blank control. Each calibrator, control, and sample was then mixed with 0.3

250 ml of 0.73% w/v NaCl, vortexed, and centrifuged (10 min at 4000 × g and 4°C). The aqueous

251 phase plus debris were collected and extracted again twice with 0.8 ml chloroform, the organic

252 phases were pooled and organic solvents were evaporated under nitrogen gas. Dried samples

253 were reconstituted with 0.1 ml chloroform, mixed with 1 ml cold acetone, and centrifuged (10

254 min at 4000 × g and 4°C) to precipitate proteins. The upper layer of each sample was collected

DAGL-α regulates spatial memory

255 and evaporated to dryness and reconstituted with 0.1 ml methanol and placed in auto-sample

256 vials for analysis.

257 An Ultra performance liquid chromatography-tandem mass spectrometer (UPLC-

258 MS/MS) was used to identify and quantify the DAGL-α substrate 1-Stearoyl-2-Arachidonoyl-sn-

259 Glycerol (SAG, a nuclear diacyglycerol [Deacon et al., 2001] preferentially used by DAGLs

260 [Balsinde et al., 1991; Allen et al., 1992]), the DAGL-α metabolites 2-AG and arachidonic acid

261 (AA), and anandamide (AEA, a biosynthetically distinct endogenous cannabinoid ligand), in

262 brain. Sciex 6500 QTRAP system with an IonDrive Turbo V source for TurbolonSpray®

263 (Ontario, Canada) attached to a Shimadzu UPLC system (Kyoto, Japan) controlled by Analyst

264 software (Ontario, Canada). Chromatographic separation was performed on a Discovery® HS

265 C18 Column 15cm x 2.1mm, 3μm (Supelco: Bellefonte, PA) kept at 25°C with an injection

266 volume of 10 μL. The mobile phase consisted of A: acetonitrile and B: water with 1 g/L

267 ammonium acetate and 0.1% formic acid. The following gradient was used: 0.0 to 2.4 minutes

268 at 40% A, 2.5 to 6.0 minutes at 40% A, hold for 2.1 minutes at 40% A, then 8.1 to 9 min 100%

269 A, hold at 100% A for 3.1 min and return to 40% A at 12.1 min with a flow rate was 1.0 mL/min.

270 The source temperature was set at 600°C and had a curtain gas at a flow rate of 30 ml/min. The

271 ionspray voltage was 5000 V with ion source gases 1 and 2 flow rates of 60 and 50 ml/min,

272 respectively. The mass spectrometer was run in positive ionization mode for AEA and 2-AG and

273 in negative ionization mode for AA, and the acquisition mode used was multiple reaction

274 monitoring. The following transition ions (m/z) were monitored with their corresponding

275 collection energies (eV) in parentheses: AEA: 348>62 (13) and 348>91 (60); AEA-d8: 356>63

276 (13); 2-AG: 379>287 (26) and 379>296 (28); 2-AG-d8: 384>287 (26); AA: 303>259 (-25) and

277 303>59 (-60); AA-d8: 311>267 (-25). The total run time for the analytical method was 14 min.

278 Chromatographic analysis of SAG was performed on a Hypersil Gold® 3 X 50mm, 5 μ column

279 (Thermo Scientific, Waltham, MA) kept at 25°C with an injection volume of 2 μL. The mobile

280 phase consisted 10:90 mM ammonium formate: methanol mobile with a flow rate of 0.5 mL/min.

DAGL-α regulates spatial memory

281 The source temperature, curtain gas at a flow rate, ionspray voltage and source gas flow rates

282 were the same as indicated above. The mass spectrometer was run in positive ionization mode.

283 The following transition ions (m/z) were monitored with their corresponding collection energies

284 (eV) in parentheses: SAG: 646>341 (34) and 646>287 (38); SAG-d8: 654>341 (34). The total

285 run time for the analytical method was 5 min. Calibration curves were analyzed with each

286 analytical batch for each analyte. A linear regression ratio of the peak area analyte counts with

287 the corresponding deuterated internal standard versus concentration was used to construct the

288 calibration curves.

289 Experimental design and statistical analysis. Electrophysiological data are presented

290 as mean ± SEM. The magnitude of LTP (%) was calculated as follows: 100 × [mean fEPSP

291 slope during the final 10 min of recording/mean baseline fEPSP slope]. Post-tetanic potentiation

292 was calculated as the magnitude of the fEPSP slope relative to baseline for the first 5 min after

293 TBS. Results were analyzed with one-way ANOVA followed by Tukey’s post hoc analysis.

294 Behavioral data are presented as mean + SEM. All MWM acquisition, extinction,

295 forgetting, reversal, perseveration and swim speed data, and OL sample phase % exploration

296 were analyzed using a mixed factor ANOVA (distance, percent time in outer ring, and cm/s

297 measures). All probe trial analyses focused on the first minute of exploration, with all gene

298 deletion measures analyzed by one-way ANOVA and Sidak post hoc test, and DO34 measures

299 analyzed by independent groups t-test. All MWM cued task data were analyzed by one-way

300 ANOVA. MWM swim path selection metrics were assigned per Wagner et al., 2013 into three

301 categories; spatial (direct, indirect, and self-orienting), non-spatial (scanning, circling, and

302 random) and thigmotaxic, and were analyzed for gene deletion using a Chi Square analysis with

303 pairwise comparisons and Bonferroni corrections (MacDonald and Gardner, 2000), and for

304 drug-treatments using a Kruskall Wallis analysis. All OL sample phase total exploration and

305 choice phase measures were analyzed for gene deletion using a one-way ANOVA, and drug

306 studies using independent groups t-tests. The discrimination ratio was calculated by dividing the

DAGL-α regulates spatial memory

307 difference in time spent exploring the novel location object and familiar location object by the

308 total object exploration during the 2 min choice phase. From the sample sizes, power was

309 calculated using G*power 3 (Faul et al., 2007) and was found, with alpha at .05, to range from

310 0.67 to 0.84 for mixed factor ANOVAs, from 0.45 to 0.99 for one-way ANOVAs, and from 0.34 to

311 0.51 for independent group t-tests.

312 Prior to data analysis, brain level data were transformed to pmol/g of brain tissue for

313 AEA, and nmol/g of brain tissue for SAG, 2-AG and AA, and AEA. Drug and gene manipulations

314 on brain endocannabinoids were evaluated by one-way ANOVA, for each lipid in each brain

315 area, followed by a Sidak post hoc test. The criterion for significance in all experiments was set

316 at p < 0.05, and all analyses were conducted using IBM SPSS Statistics 22 for Windows (IBM

317 Software, New York, NY).

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333 Results

334 Experiment 1: Genetic deletion or pharmacological blockade of DAGL-α

335 attenuates LTP in CA1 region of the hippocampus. In hippocampal slices prepared from

336 DAGL-α+/+ and DAGL-α+/- mice (n = 9-11), TBS induced robust LTP that remained stable during

337 the 60 min recording period (Figure 1A). The fEPSP slope in hippocampal slices from DAGL-α-/-

338 mice (n = 8) subjected to the same TBS stimulation slowly decayed toward baseline. The

339 magnitude of LTP as measured between 50-60 min after TBS stimulation significantly differed

340 among the three groups (F(2,27) = 5.37, p = 0.011, ANOVA). Post hoc analyses indicated that

341 TBS-induced LTP was significantly greater in DAGL-α+/+ and DAGL-α+/- mice than in DAGL-α-/-

342 mice (DAGL-α+/+ vs. DAGL-α-/-, p = 0.039; DAGL-α+/- vs. DAGL-α-/-, p = 0.013). However, the

343 magnitude of LTP did not differ between DAGL-α+/+ mice and DAGL-α+/- mice (p = 0.926).

344 Hippocampal slices from C57BL/6J mice treated with VEH, DO34 (30 mg/kg), or DO53

345 (30 mg/kg) showed significant differences in the magnitude of LTP (F(2,23) = 7.90, p = 0.003,

346 ANOVA; Figure 1B). Post hoc tests indicated that TBS induced similar LTP in hippocampal

347 slices prepared from VEH-treated (n = 9) and DO53-treated mice (n = 8; p = 0.514 vs. vehicle),

348 but slices prepared from DO34-treated mice displayed a significantly decreased magnitude of

349 LTP (n = 7; p = 0.002 vs. vehicle; p = 0.031 vs. DO53). Moreover, the magnitude of post-tetanic

350 potentiation (PTP) during the first 5 min of LTP induction significantly differed among the three

351 treatment groups (F(2,23) = 7.17, p = 0.004, ANOVA), in which DO34 administration produced a

352 significant decrease in the PTP compared with vehicle (p = 0.007) and DO53 (p = 0.011).

353 Experiment 2.1: DAGL-α-/- mice display profound phenotypic MWM spatial memory

354 deficits and altered MWM search strategies. Per the experimental timeline (Figure 2A), the

355 first acquisition day of fixed platform training (data not shown) yielded no significant differences

356 among groups and across trials (F(6,114) = 1.67, p = 0.135, ANOVA). However, across the ten

357 days of MWM fixed platform acquisition training (Figure 2B), a significant interaction (F(18,342) =

358 2.48, p = 0.001, ANOVA) revealed DAGL-α-/- mice (n = 13) had longer distances to the platform

DAGL-α regulates spatial memory

359 than DAGL-α+/+ (n = 15) or DAGL-α+/- mice (n = 15) on acquisition days 2 (p = 0.001, p = 0.005),

360 3 (p = 0.000, p = 0.000), 4 (p = 0.000, p = 0.000), 5 (p = 0.000, p = 0.000), 6 (p = 0.000, p =

361 0.000), 7 (p = 0.000, p = 0.001), 8 (p = 0.001), 9 (p = 0.000, p = 0.001), and 10 (p = 0.000, p =

362 0.000), respectively. Also, DAGL-α+/+ mice and DAGL-α+/- mice, showed respective significant

363 reductions in distances to the platform on days 4 (p = 0.005, p = 0.014), 5 (p = 0.000, p =

364 0.025), 6 (p = 0.001, p = 0.004), 7 (p = 0.000, p = 0.000), 8 (α+/+; p = 0.000), 9 (p = 0.000, p =

365 0.007), and 10 (p = 0.000, p = 0.001) compared to day 1. In contrast, DAGL-α-/- mice showed no

366 significant difference in distance compared to day 1 across subsequent fixed platform

367 acquisition days (2; p = 1.000, 3; p = 1.000, 4; p = 1.000, 5; p = 1.000, 6; p = 1.000, 7; p =

368 0.625, 8; p = 0.399, 9; p = 0.373, and 10; p = 0.0670). Furthermore, during the fixed platform

369 probe trial (Figure 2C,D,E) DAGL-α-/- mice demonstrated significantly longer distances to the

+/+ 370 platform location (F(2,38) = 5.81, p = 0.006, ANOVA) than DAGL-α mice (p = 0.022) and DAGL-

+/- 371 α mice (p = 0.011), significantly fewer platform entries (F(2,38) = 7.40, p = 0.002, ANOVA) than

+/+ 372 DAGL-α mice (p = 0.001), and a lower spatial preference for the target quadrant (F(2,38) = 7.73,

373 p = 0.002, ANOVA) than DAGL-α+/+ mice (p = 0.002) and DAGL-α+/- mice (p = 0.016). The

374 absence of phenotypic deficits in cued task performance (Figure 2F; F(2,38) = 0.926, p = 0.405,

375 ANOVA) and no alterations in swim speed (Figure 2G; F(18,342) = 0.648, p = 0.860, ANOVA)

376 suggest that neither sensorimotor nor motivational impairments contribute to the impaired fixed

377 platform acquisition of DAGL-α-/- mice. Also, no significant differences in body weight at baseline

378 (F(2,38) = 3.08, p = 0.057, ANOVA; Figure 2H) or during fixed platform acquisition (F(2,38) = 2.84, p

379 = 0.071, ANOVA; Figure 2I) were evident among the genotypes.

380 On Fixed Platform day 10 (Figure 3A), DAGL-α-/- mice used significantly more non-

+/+ +/- 2 381 spatial and thigmotaxic swimming strategies than DAGL-α or DAGL-α mice, (X (4, N=41) =

382 21.9, p = 0.000, Chi Square). The change in DAGL-α-/- mice strategy proportion was indicated

383 by pairwise comparisons (p = 0.001). Furthermore, during Fixed Platform acquisition (Figure 3B)

-/- 384 a significant main effect of genotype (F(2,38) = 7.29, p = 0.002, ANOVA) indicated that DAGL-α

DAGL-α regulates spatial memory

385 mice spent more time in the MWM outer ring than DAGL-α+/+ mice (p = 0.002) and DAGL-α+/-

386 mice (p = 0.028). A significant main effect of day (F(9,342) = 13.9, p = 0.000, ANOVA) also

387 showed that collectively DAGL-α+/+, -α+/-, and -α-/- mice spent a decreased proportion of their

388 time in the outer ring on days 4 (p = 0.015), 5 (p = 0.034), 6 (p = 0.001), and 7-10 (p = 0.000)

389 compared to day 1.

390 Experiment 2.2: Pharmacological inhibition of DAGL-α delays MWM spatial

391 acquisition. Per the experimental timeline (Figure 4A), fixed platform day 1 (data not shown),

392 yielded no significant differences among groups and across trials (F(12,171) = 1.65, p = 0.082,

393 ANOVA). During MWM fixed platform acquisition (Figure 4B) a significant main effect of drug

394 (F(4,57) = 8.77, p = 0.000) revealed 30 mg/kg DO34-treated mice (n = 12) required longer swim

395 path distances to the platform than mice in the VEH (n = 15, p = 0.000), 0.3 mg/kg DO34 (n =

396 12, p = 0.002), and 30 mg/kg DO53 (n = 12, p = 0.000) conditions. However, these mice did not

397 statistically differ from mice receiving 3 mg/kg DO34 (n = 11, p = 0.103). Also, mice treated with

398 VEH (F(9,126) = 12.7, p < 0.001, repeated measures ANOVA), 30 mg/kg DO34 (F(9,99) = 12.3, p =

399 0.000, repeated measures ANOVA), 3 mg/kg DO34 (F(9,90) = 8.01, p = 0.000, repeated

400 measures ANOVA), 0.3 mg/kg DO34 (F(9,99) = 8.48, p = 0.000, repeated measures ANOVA),

401 and 30 mg/kg DO53 (F(9,99) = 15.8, p = 0.000, repeated measures ANOVA) showed significantly

402 reduced distances to the platform days 3 (VEH p = 0.022), 4 (VEH p = 0.000), 5 (VEH p =

403 0.005; 30 mg/kg DO34 p = 0.021; 3 mg/kg DO34 p = 0.019; 30 mg/kg DO53 p = 0.005), 6 (VEH

404 p = 0.001; 30 mg/kg DO34 p = 0.004; DO53 p = 0.003), 7 (VEH p < 0.001; 30 mg/kg DO34 p =

405 0.003; 3 mg/kg DO34 p = 0.008; DO53 p = 0.002), 8 (VEH p = 0.003; 30 mg/kg DO34 p = 0.034;

406 3 mg/kg DO34 p = 0.038; DO53 p = 0.001), 9 (VEH p = 0.001; 30 mg/kg DO34 p = 0.000; 3

407 mg/kg DO34 p = 0.002; DO53 p = 0.000), and 10 (VEH p = 0.000; 30 mg/kg DO34 p = 0.000; 3

408 mg/kg DO34 p = 0.024; 0.3 mg/kg DO34 p = 0.011; DO53 p = 0.000) compared to day 1.

409 In order to test whether DO34 affects memory of the platform location (Figure 4C), a

410 probe trial revealed similar distances to the platform location (t(15) = 0.0880, p = 0.931,

DAGL-α regulates spatial memory

411 independent t-test; Figure 4D) in DO34-treated and VEH-treated mice, similar number of

412 platform entries (t(15) = 1.17, p = 0.260, independent t-test; Figure 4E), and similar percentages

413 of time spent in the target quadrant (t(15) = 1.28, p = 0.220, independent t-test; Figure 4F). The

414 absence of drug-induced deficits in cued task performance (F(4,57) = 1.09, p = 0.368, ANOVA;

415 Figure 4G) suggests that DO34 does not elicit sensorimotor or motivational impairments.

416 During fixed platform acquisition, a significant swim speed interaction (F(36,513) = 1.60, p =

417 0.016, ANOVA; Figure 4H), revealed that mice administered 30 mg/kg DO34 swam faster than

418 VEH-treated mice on days 1 (p = 0.019), 2 (p = 0.010), 4 (p = 0.042), 5 (p = 0.009), and 8 (p =

419 0.003), but 0.3 mg/kg DO34-treated mice swam slower than VEH-treated mice on days 7 (p =

420 0.026), 9 (p = 0.044), and 10 (p = 0.022). Also, while no significant difference in body weight

421 was evident at baseline (F(4,57) = 2.03, p = 0.103, ANOVA; Figure 4I), a significant interaction

422 between drug and day for this measure occurred throughout acquisition training (F(56,798) = 7.06,

423 p = 0.000, ANOVA; Figure 4J). DO34 elicited body weight reductions compared to VEH at 30

424 mg/kg DO34 (days 2 through 15, p = 0.000), 3 mg/kg DO34 (days 4, p = 0.006; 8, p = 0.032;

425 and 13, p = 0.015), 0.3 mg/kg DO34 (day 4, p = 0.001), and 30 mg/kg DO53 (days 2, p = 0.000;

426 3, p = 0.000; days 4, p = 0.041; 5, p = 0.003; 7, p = 0.008; and 13, p = 0.000).

427 On Fixed Platform day 10, DO34-treated mice showed no significant alterations in swim

2 428 path strategy (X (3, N=49) = 7.0;97, p = 0.069, chi square; Figure 5A). However, a significant main

429 effect of drug was found for time spent in the outer ring of the MWM throughout the ten

430 acquisition sessions (F(4,57) = 2.96, p = 0.027, ANOVA; Figure 5B). DO34 (30 mg/kg) produced a

431 modest increase in the time spent in the MWM outer ring compared with VEH-treated mice (p =

432 0.035). A significant main effect of day (F(9,513) = 59.4, p = 0.000, ANOVA) also showed that

433 mice spent a smaller proportion of their time in the outer ring on all subsequent days, 2, 3, 4, 5,

434 6, 7, 8, 9, and 10 (p < 0.001) than on day 1.

435 Experiment 2.3: Pharmacological inhibition of DAGL-α impairs MWM reversal, but

436 not extinction. The next experiments examined the effects of DO34 (30 mg/kg; n = 8) vs. VEH

DAGL-α regulates spatial memory

437 (n = 9) on extinction (Figure 6A), forgetting (Figure 6B), reversal learning, and perseveration

438 (Figure 7A, 30 mg/kg; n = 15, VEH; n = 15). As shown in Figure 6C, repeated exposures to the

439 MWM with removal of the hidden platform following fixed platform acquisition day 10 led to

440 increased swim paths regardless of drug treatment (F(5,75) = 3.08, p = 0.014, ANOVA). Subjects

441 had significantly longer distances to the previous platform location on extinction weeks 4 (p =

442 0.047) and 6 (p = 0.027) compared to probe trial. The lack of a statistical interaction between

443 drug and day (F(5,75) = 0.292, p = 0.916, ANOVA) and lack of significant main effect of drug

444 (F(1,15) = 1.98, p = 0.180, ANOVA) indicate that DO34 did not affect extinction. In the probe trial

445 assessment of forgetting (Figure 6C), the lack of a statistically significant interaction between

446 drug and day (F(1,10) = 0.638, p = 0.443, ANOVA) or a main effect of drug (F(1,10) = 2.50, p =

447 0.145, ANOVA) indicates DO34 did not impact forgetting performance.

448 In the evaluation of reversal learning (Figure 7B), a statistically significant interaction

449 between drug and day (F(5,140) = 4.43, p = 0.001, ANOVA) indicates that DO34 delayed reversal

450 acquisition. Specifically, DO34 increased swim distances compared with VEH on days 1 (p =

451 0.007), 2 (p = 0.007), and 5 (p = 0.016). Yet, all subjects displayed evidence of reversal learning

452 after exposure to five reversal sessions (and probe trial) via a main effect of day in VEH (F(5,70) =

453 6.05, p = 0.000, repeated measures ANOVA) and DO34 (F(5,70) = 13, p = 0.000, repeated

454 measures ANOVA) mice. VEH-treated subjects had significantly reduced distances to the new

455 platform location on reversal days 2 (p = 0.011), 3 (p = 0.0005) and 5 (p = 0.000) compared to

456 day one. Similarly, DO34-treated subjects showed reduced swim distances to the platform

457 location on days 2 (p = 0.033), 3 (p = 0.000), 4 (p = 0.008), and 5 (p = 0.010) as well as during

458 the probe trial (p = 0.001) compared to day 1. Figure 7C depicts the percentage of time spent in

459 the quadrant containing the prior platform location. There was no statistical interaction between

460 drug and day (F(4,112) = 0.498, p = 0.737, ANOVA), but significant main effects of drug (F(1,28) =

461 10.29, p = 0.003, ANOVA) and day (F(4,112) = 34.63, p = 0.000, ANOVA) were found. Further

462 analysis showed that DO34-treated mice (F(4,56) = 15.72, p = 0.000) spent decreased

DAGL-α regulates spatial memory

463 proportions of time in the quadrant containing the prior platform location on days 2, 3, 4, and 5

464 (day 2; p < 0.020, day 3; p < 0.009, day 4; p < 0.000, day 5; p < 0.006) compared with day 1.

465 These results demonstrate that while DO34 increased the percentage of time mice spent in the

466 quadrant containing the prior platform location compared with VEH throughout reversal training,

467 mice showed a decrease in the percentage of time in the quadrant containing the prior platform

468 location on days, 2, 3, 4, and 5 (p < 0.000) compared with day 1 irrespective of drug treatment.

469 Figure 7D depicts the percentage of time spent in the quadrant containing the new platform

470 location. Similar to the data in Figure 7C, there was no statistical interaction between drug and

471 day (F(4,112) = 1.34, p = 0.260, ANOVA), but significant main effects of drug (F(1,28) = 7.37, p =

472 0.011, ANOVA) and day (F(4,112) = 28.37, p = 0.000, ANOVA) were found. Although DO34

473 delayed the magnitude of spatial preference for the quadrant containing the new platform

474 location compared to VEH, it did not affect their spending an increased proportion of their time

475 in the quadrant containing the new platform location on days, 2, 3, 4, and 5 (day 2; p < 0.004,

476 days 3 to 5; p < 0.000) compared with day 1.

477 Experiment 2.4: The Object Location task reveals a dissociation of spatial memory

478 deficits between pharmacological inhibition and genetic deletion of DAGL-α. During

479 sample phase, DAGL-α+/+ (n = 13), DAGL-α+/- (n = 15), and DAGL-α-/- (n = 7) mice showed

480 similar total exploration times (F(2,32) = 2.59, p = 0.091, ANOVA; Figure 8B). None of the

481 genotypes showed a preference for either object (pre-assigned stationary or moved) (F(2,32) =

482 1.61, p = 0.22, ANOVA; Figure 8C). During the choice phase, a significant effect in the

483 discrimination ratio was detected (F(2,32) = 4.58, p = 0.018, ANOVA; Figure 8D) in which DAGL-

484 α-/- (p = 0.049) and DAGL-α+/- (p = 0.042) mice showed significant spatial memory deficits

485 compared to DAGL-α+/+ mice.

486 In the evaluation of DO34 on OL spatial memory, sample phase DO34 treatment (n =

487 15) lowered the total exploration time compared to VEH (n = 14), (t(27) = 2.69, p = 0.012, t-test;

488 Figure 8F). However, neither DO34 nor VEH administration produced a preference for either

DAGL-α regulates spatial memory

489 object (pre-assigned stationary or moved), (F(1,27) = 2.97, p = 0.096, ANOVA; Figure 8G). During

490 the choice phase, DO34-treated mice and VEH-treated mice demonstrated evidence of spatial

491 memory, with similar discrimination ratios (t(27) = 0.62, p = 0.54, t-test; Figure 8H).

492 Experiment 3.1: DAGL-α-/- mice show profound alterations in brain

493 endocannabinoids and related lipids. DAGL-α-/- mice (n = 8) possessed elevated levels of the

494 DAGL-α substrate, SAG, across all four brain regions; hippocampus, (F(2,21) = 174, p = 0.000,

495 ANOVA; Figure 9A), PFC (F(2,21) = 753, p = 0.000, ANOVA; Figure 9B), striatum (F(2,21) = 52, p =

496 0.000, ANOVA; Figure 9C), and cerebellum (F(2,21) = 32, p = 0.000, ANOVA; Figure 9D),

497 compared to both DAGL-α+/+ (n=8, p = 0.000) and DAGL-α+/- mice (n=8, p = 0.000). DAGL-α-/-

498 mice also showed profound reductions of 2-AG and its metabolite AA, respectively, across each

499 brain region: hippocampus (F(2,21) = 246, p = 0.000, ANOVA; Figure 9E; F(2,21) = 156, p = 0.000,

500 ANOVA; Figure 9M), PFC (F(2,21) = 29, p = 0.000, ANOVA; Figure 9F; F(2,21) = 207, p = 0.000,

501 ANOVA; Figure 9N), striatum (F(2,21) = 48, p = 0.000, ANOVA; Figure 9G; F(2,21) = 126, p = 0.000,

502 ANOVA; Figure 9O), and cerebellum (F(2,21) = 70, p = 0.000, ANOVA; Figure 9H; F(2,21) = 155, p

503 = 0.000, ANOVA; Figure 9P), compared to both DAGL-α+/+ (p = 0.000) and DAGL-α+/- mice (p =

504 0.000). DAGL-α+/- mice showed small, but significant reductions of 2-AG compared to DAGL-α+/+

505 mice in striatum (p = 0.04), hippocampus (p = 0.002), and cerebellum (p < 0.002). Also, region

-/- 506 selective reductions of AEA were found in DAGL-α mouse hippocampus (F(2,21) = 51, p =

507 0.000, ANOVA; Figure 9I) and cerebellum (F(2,21) = 10.2, p = 0.001, ANOVA; Figure 9L), but not

508 in PFC (Figure 9J) or striatum (Figure 9K) compared to both DAGL-α+/+ and DAGL-α+/- mice.

509 Experiment 3.2: DAGL inhibition produces brain region selective alterations in

510 endocannabinoids and related lipids. DO34-treated mice possessed elevated levels of SAG

511 at 2 h post-injection (n = 5) across all four brain regions; hippocampus (F(2,15) = 7.48, p = 0.006,

512 ANOVA; Figure 10A), PFC (F(2,15) = 30, p = 0.000, ANOVA; Figure 10B), striatum (F(2,15) = 5.46,

513 p = 0.017, ANOVA; Figure 10C), and cerebellum (F(2,15) = 6.83, p = 0.008, ANOVA; Figure 10D)

514 compared to VEH (n = 5, p = 0.005, p = 0.000, p = 0.038, and p = 0.006, respectively).

DAGL-α regulates spatial memory

515 However, at 24 h post-injection, SAG levels did not differ between DO34- and VEH-treated mice

516 in each respective brain region (p = 0.232, p = 0.992, p = 0.999, p = 0.145). DO34-treated mice

517 also showed consistent reductions of 2-AG in hippocampus (F(2,15) = 37, p = 0.000, ANOVA;

518 Figure 10E), PFC (F(2,15) = 11.6, p = 0.001, ANOVA; Figure 10F), striatum (F(2,15) = 4.82, p =

519 0.024, ANOVA; Figure 10G), and cerebellum (F(2,15) = 94, p = 0.000, ANOVA; Figure 10H) at 2 h

520 (p = 0.000, p = 0.001, p = 0.036, p = 0.000 respectively). At 24 h, DO34 continued to decrease

521 2-AG levels compared with VEH in hippocampus (p = 0.000) and cerebellum (p = 0.000), but

522 not in PFC or striatum. However, DO34 did not affect AEA levels in hippocampus (F(2,15) = 0.92,

523 p = 0.421, ANOVA; Figure 10I), PFC (F(2,15) = 3.04, p = 0.078, ANOVA; Figure 10J), striatum

524 (F(2,15) = 2.70, p = 0.099, ANOVA; Figure 10K), or cerebellum (F(2,15) = 2.44, p = 0.121, ANOVA;

525 Figure 10L). Finally, DO34 reduced AA levels in all brain regions; hippocampus (F(2,15) = 13.8, p

526 = 0.000, ANOVA; Figure 10M) at 2 h (p = 0.005) and 24 h (p = 0.000), PFC (F(2,15) = 65, p =

527 0.000, ANOVA; Figure 9N) at 2 h (p = 0.000) and 24 h (p = 0.000), striatum (F(2,15) = 145, p =

528 0.000, ANOVA; Figure 9O) at 2 h (p = 0.000) and 24 h (p = 0.000), and cerebellum (F(2,15) = 59,

529 p = 0.000, ANOVA; Figure 9P) at 2 h (p = 0.000) and 24 h (p = 0.000). AA levels of DO34-

530 treated mice remained significantly reduced in each brain region at 24 h, but were significantly

531 elevated at 24 h compared with 2 h in PFC (p = 0.000) and striatum (p = 0.000).

DAGL-α regulates spatial memory

532 Discussion

533 The present study makes the novel observations that genetic deletion or inhibition of

534 DAGL-α profoundly disrupts hippocampal LTP accompanied with varying magnitudes of spatial

535 learning deficits. Specifically, DAGL-α-/- mice display impaired Object Location and MWM Fixed

536 Platform acquisition accompanied by non-spatial search strategies. Whereas DO34-treated

537 mice show significantly delayed MWM acquisition and reversal learning, performance is spared

538 in probe trial memory, extinction, and forgetting tasks, and in the Object Location assay.

539 Additionally, these alterations in LTP and in vivo learning and memory paradigms occur in

540 concert with distinct altered patterns of endocannabinoids and related lipids, across brain areas

541 integral to learning and memory.

542 In addition to corroborating a study showing that non-selective DAGL inhibitors block

543 CA1 pairing-induced potentiation in rats (Xu et al., 2012), the present study found significant

544 reductions in hippocampal 2-AG. This pattern of findings in combination with other work

545 showing that inhibitors of 2-AG hydrolysis facilitates CA1 LTP (Silva-Cruz et al., 2017)

546 implicates 2-AG as a possible LTP facilitator. The finding that endogenously produced

547 endocannabinoids facilitate LTP at CA1 hippocampal synapses through stimulation of astrocyte-

548 neuron signaling (Gomez-Gonzalo et al., 2015), or when preceded by DSI (Carlson et al., 2002),

549 suggests that endocannabinoids enhance plasticity by disinhibition. 2-AG acts as an retrograde

550 modulator of short-term CB1 receptor-mediated synaptic plasticity (i.e., DSE/DSI), (Tanimura et

551 al., 2010; Yoshino et al., 2011). Furthermore, CB1 receptor activation correlates with enhanced

552 CA1 LTP (Chevaleyre and Castillo, 2004). Thus, reducing 2-AG production may disrupt LTP

553 because of decreased CB1 receptor signaling. However, the consequences of directly disrupting

554 CB1 receptor signaling on behavioral performance in hippocampal-dependent learning assays

555 differ from those resulting from DAGL-α disruption. Specifically, wild type mice administered a

-/- 556 CB1 receptor antagonist or CB1 mice displayed similar MWM Fixed Platform acquisition as

557 control mice, but showed impaired MWM extinction (Varvel and Lichtman, 2002; Varvel et al.,

DAGL-α regulates spatial memory

558 2005). The close spatial localization of DAGL-α to excitatory CB1 receptor synapses (also

559 adjacent to group I metabotropic glutamate receptors) and distance from inhibitory synapses

560 (Katona et al., 2006; Yoshida et al., 2006) may account for the resilience of DAGL-α

561 compromised mice in the MWM extinction task.

562 Contrary to the present work, Sugaya et al., 2013 reported that DAGL-α-/- mice show no

563 change in CA1 LTP. As they measured LTP through in-dwelling electrodes, it is plausible that

564 the altered lipid profile disruption may have interacted with on-going milieu changes in an awake

565 brain. Thus, procedural differences may account for the different LTP outcomes following

566 DAGL-α deletion. DO34-induced impairment of post-tetanic potentiation within minutes of TBS

567 suggests reduced Ca2+ buildup in presynaptic axon terminals (Zucker and Regehr, 2002). It is

568 unlikely that DO34 off-targets (e.g. ABDH6) affected the measures given that DO53 did not

569 affect SC-CA1 LTP. Similarly, DO53 did not alter hippocampal DSI (Ogasawara et al., 2016). As

570 DO34 also inhibits DAGL-β, this 2-AG biosynthetic enzyme cannot be excluded. Yet, its low

571 neuronal expression and high expression on microglia (Hsu et al., 2012) argues against its

572 involvement in the present results.

573 The phenotypic MWM deficits displayed by DAGL-α-/- mice and impaired acquisition and

574 reversal learning performance in DO34-treated mice, represent the first evidence supporting a

575 role of DAGL-α in rodent spatial learning and memory models. Specifically, these novel findings

576 indicate that DAGL-α is required for the integration of new spatial information, but not for

577 memory retrieval, extinction, forgetting, or the promotion of perseverative behavior. The findings

578 that DAGL-α deletion profoundly disrupts MWM fixed platform learning are compatible with the

579 body of evidence highlighting the necessity of hippocampal neurogenesis for spatial memory

580 processing (Snyder et al., 2005; Sahay et al., 2011), which this enzyme significantly contributes

581 (Goncalves et al., 2008; Gao et al., 2010). DO34-impaired reversal learning is also consistent

582 with chronic stress-induced impairments in MWM reversal learning, which occur concomitantly

583 with lowered hippocampal 2-AG (Hill et al., 2005). Contrary to stress-induced perseveration (Hill

DAGL-α regulates spatial memory

584 et al., 2005), DAGL-α inhibition did not elicit perseverative behavior. Accordingly, this enzyme

585 may be required specifically for integrating new spatial information. The altered search strategy

586 may reflect a stress-induced shift from utilizing hippocampal-cognitive to striatal-habit learning

587 (Wirz et al., 2017). The anxiety-like phenotype of DAGL-α-/- mice (Shonesy et al., 2014;

588 Jenniches et al., 2016) as well as the stress-inducing component of the MWM may drive

589 reliance on non-spatial circular search strategies after DAGL-α disruption. Furthermore,

590 elevations in stress hormones, e.g. corticosterone, influence hippocampal-dependent task

591 performance (Kim et al., 2015), though additional studies are needed to ascertain whether

592 stress hormones contribute to DAGL-α-/- phenotypic deficits in hippocampal-dependent task

593 performance. Contrary to the present findings, extinction but not acquisition deficits occur in

594 DO34-treated mice (Cavener et al., 2018) or DAGL-α-/- mice (Jenniches et al., 2016; Cavener et

595 al., 2018) in conditioned fear tasks. While DAGL-α is expressed in regions mediating fear

596 conditioning, such as the basolateral amygdala (Yoshida et al., 2011), stress reduces

597 amygdala-hippocampal connectivity (Wirz et al., 2017). Accordingly, DAGL-α may differentially

598 modulate memory processes based on brain circuitry, region, or neuronal type.

599 The differential impact between genetic deletion and pharmacological inhibition of

600 DAGL-α on performance in spatial learning tasks may partly be a consequence of 2-AG lipid

601 profile differences. However, developmental alterations in the brains of the DAGL-α-/- mice

602 across ontogeny may also be a contributing factor. In the developing brain, DAGL-α is

603 expressed on presynaptic terminals (Bisogno et al., 2003a), participating in the control of axonal

604 growth and guidance (Williams et al., 1994; Brittis et al., 1996), but is expressed on post-

605 synaptic neurons in the adult brain. Accordingly, across ontogeny DAGL-α switches roles from

606 growth and guidance to a modulator of synaptic signaling. The finding that DAGL-α-/- mice, but

607 not DO34-treated mice, possessed significantly reduced AEA levels in hippocampus and

608 cerebellum, raises the possibility that diminished AEA levels may contribute to the DAGL-α-/-

609 phenotypic learning and memory deficits and altered search strategy.

DAGL-α regulates spatial memory

610 Profound lipid profile changes accompanied the spatial memory impairments of DAGL-α-

611 /- mice. As previously reported (Gao et al., 2010; Tanimura et al., 2010; Shonesy et al., 2014;

612 Jenniches et al., 2016), these mice displayed substantial reductions of 2-AG across all four

613 brain areas. In DAGL-α+/- mice, modest reductions of 2-AG in striatum, hippocampus, and

614 cerebellum may have contributed to Object Location deficits, though they displayed similar

615 performance as wild type mice in MWM tasks. Whereas previous studies reported DO34-

616 induced 2-AG decreases in whole brain (Ogasawara et al., 2016; Wilkerson et al., 2017), we

617 found reductions of 2-AG in each region evaluated. DAGL-α-/- mice showed reductions of AEA in

618 hippocampus and cerebellum, but not in cortex or striatum. Similarly Tanimura et al. (2010)

619 reported reduced striatal AEA levels, while Jenniches et al. (2016) found AEA reductions in

620 cortex and amygdala. DO34 did not affect AEA levels in any brain region investigated, despite

621 50% reductions of AEA in whole brain (Ogasawara et al., 2016; Wilkerson et al., 2017). Thus,

622 DAGL-α substrates and metabolites appear to play a complex role in AEA biosynthesis. Given

623 that inhibitors of the primary AEA hydrolytic enzyme, fatty acid amide hydrolase (FAAH), impair

624 LTP (Basavarajappa et al., 2014), yet enhance MWM acquisition and extinction (Varvel et al.,

625 2007), the consequences of reduced hippocampal AEA in DAGL-α-/- mice must be considered.

626 Examining whether a FAAH inhibitor rescues DAGL-α-/- phenotypes may provide insight into

627 whether AEA functionally contributes to the impairments observed in the present study.

628 Nonetheless, it should be noted that a FAAH inhibitor failed to reverse fear extinction deficits in

629 DAGL-α-/- mice (Cavener et al., 2018).

630 The impact of DAGL-α blockade on downstream and upstream lipid signaling molecules,

631 previously only evaluated in forebrain (Shonesy et al., 2014) or whole brain (Ogasawara et al.,

632 2016) merits discussion. The expression of DAGL in invertebrate species lacking cannabinoid

633 receptors (e.g. Drosophila; Elphick and Egertova, 2005) underscores the important role of this

634 enzyme in regulating lipid signaling molecules independent of substrate production for

635 cannabinoid receptor activation. As anticipated, both DAGL-α-/- mice and DO34-treated mice

DAGL-α regulates spatial memory

636 showed consistent elevations of the DAG substrate for DAGL-α, SAG, across all four brain

637 areas. SAG activates protein kinase C (PKC) (Hindenes et al., 2000), and while PKC activation

638 facilitates memory (Bonini et al., 2007), the effects of sustained SAG elevations are not known.

639 The dramatically reduced levels of AA across all four brain regions in DAGL-α-/- and DO34-

640 treated mice suggest DAGL-α is an important intermediate for the maintenance of basal AA

641 levels in brain. Given hippocampal neurons activate PKC through AA cascades (Hama et al.,

642 2004), the DAGL-α disruption-induced AA reductions may also contribute to the observed

643 learning and memory deficits.

644 Collectively, this work supports the importance of DAGL-α in modulating hippocampal

645 learning and memory to the extent that reductions in DAGL-α expression or function may

646 contribute to cognitive pathologies. Age-related decreases in DAGL-α expression (Piyanova et

647 al., 2015) suggest DAGL-α dysregulation may be a useful target for the study of age-related

648 cognitive decline (Bilkei-Gorzo et al., 2017). In sum, the present findings that DAGL-α disruption

649 lead to cellular spatial memory impairments and in vivo deficits selective for the integration of

650 new spatial information, implicate this enzyme as playing a key role in spatial learning and

651 memory.

652 653 654 655 656 657 658 659 660 661 662

DAGL-α regulates spatial memory

663 664 References

665 Allen A, Gammon C, Ousley A, McCarthy K, Morell P (1992) Bradykinin stimulates arachidonic

666 acid release through the sequential actions of an sn-1 diacylglycerol lipase and a

667 monoacylglycerol lipase. J Neurochem 58:1130–1139.

668 Assini FL, Duzzioni M, Takahashi RN (2009) Object location memory in mice: pharmacological

669 validation and further evidence of hippocampal CA1 participation. Behav Brain Res

670 204:206–211.

671 Baggelaar MP, van Esbroeck ACM, van Rooden EJ, Florea BI, Overkleeft HS, Marsicano G,

672 Chaouloff F, van der Stelt M (2017) Chemical proteomics maps brain region specific

673 activity of endocannabinoid hydrolases. ACS Chem Biol 12:852–861.

674 Balsinde J, Diez E, Mollinedo F (1991) Arachidonic acid release from diacylglycerol in human

675 neutrophils. Translocation of diacylglycerol-deacylating enzyme activities from an

676 intracellular pool to plasma membrane upon cell activation. J Biol Chem 266:15638–15643.

677 Basavarajappa BS, Nagre NN, Subbanna S (2014) Elevation of endogenous anandamide

678 impairs LTP, learning, and memory through CB1 receptor signaling in mice. Hippocampus

679 24:808–818.

680 Bilkei-Gorzo A, Albayram O, Draffehn A, Michel K, Piyanova A, Oppenheimer H, Dvir-Ginzberg

681 M, Racz I, Ulas T, Imbeault S, Bab I, Schultze JL, Zimmer A (2017) A chronic low dose of .

682 ∆9-tetrahydrocannabinol (THC) restores cognitive function in old mice. Nat Med 23:782–

683 787.

684 Bisogno T, Howell F, Williams G, Minassi A, Cascio M, Ligresti A, Matias I, Schiano-Moriello A,

685 Paul P, Williams E, Gangadharan U, Hobbs C, Di Marzo V, Doherty P (2003a) Cloning of

686 the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid

687 signaling in the brain. J Cell Biol 163:463–468.

688 Bisogno T, Howell F, Williams G, Minassi A, Cascio MG, Ligresti A, Matias I, Schiano-Moriello

DAGL-α regulates spatial memory

689 A, Paul P, Williams EJ, Gangadbaran U, Hobbs C, Di Marzo V, Doherty P (2003b) Cloning

690 of the first sn1-DAG lipases points to the spatial and temporal regulation of

691 endocannabinoid signaling in the brain. J Cell Biol 163:463–468.

692 Bonini JS, Da Silva WC, Bevilaqua LR, Medina JH, Izquierdo I (2007) On the participation of

693 hippocampal PKC in acquisition, consolidation and reconsolidation of spatial memory.

694 Neuroscience 147:37–45.

695 Brittis P, Silver J, Walsh F, Doherty P (1996) Fibroblast growth factor receptor function is

696 required for the orderly projection of ganglion cell axons in the developing mamalian retina.

697 Mol Cell Neurosci:120–128.

698 Carlson G, Wang Y, Alger BE (2002) Endocannabinoids facilitate the induction of LTP in the

699 hippocampus. Nat Neurosci 5:723–724.

700 Cavener VS, Gaulden A, Pennipede D, Jagasia P, Uddin J, Marnett LJ, Patel S (2018) Inhibition

701 of diacylglycerol lipase impairs fear extinction in mice. Front Neurosci 12:eCollection.

702 Chau L, Tai H (1981) Release of arachidonate from diglyceride in human platelets requires the

703 sequential action of a diglyceride lipase and a monoglyceride lipase. Biochem Biophys Res

704 Commun 100:1688–1695.

705 Chevaleyre V, Castillo PE (2004) Endocannabinoid-mediated metaplasticity in the

706 hippocampus. Neuron 43:871–881.

707 Deacon E, Pettitt T, Webb P, Cross T, Chahal H, Wakelam M, Lord J (2001) Generation of

708 diacylglycerol molecular species through the cell cycle: a role for 1-stearoyl, 2-arachidonyl

709 glycerol in the activation of nuclear protein kinase C-βII at G2/M. J Cell Sci 115:983–989.

710 Deng W, Aimone J, Gage F (2010) New neurons and new memories: how does adult

711 hippocampal neurogenesis affect learning and memory? Nat Rev Neurosci 11:339–350.

712 Elphick M, Egertova M (2005) The phylogenetic distribution and evolutionary origins of

713 endocannabinoid signalling. Handb Exp Pharmacol 168:283–297.

714 Faul F, Erdfelder E, Lang AG, Buchner A (2007) G*Power 3: A flexible statistical power analysis

DAGL-α regulates spatial memory

715 program for the social, behavioral, and biomedical sciences. Behav Res Methods 39:175–

716 191.

717 Gao Y et al. (2010) Loss of retrograde endocannabinoid signaling and reduced neurogenesis in

718 diacylglycerol lipase knock-out mice. J Neurosci 30:2017–2024.

719 Gomez-Gonzalo M, Navarrete M, Perea G, Covelo A, Martin-Fernandez M, Shigemoto R, Lujan

720 R, Araque A (2015) Endocannabinoids induce lateral long-term potentiation of transmitter

721 release by stimulation of gliotransmission. Cereb Cortex 25:3699–3712.

722 Goncalves M, Suetterlin P, Yip P, Molina-Holgado F, Walker D, Oudin M, Zentar M, Pollard S,

723 Yanez-Munoz R, Willams G, Walsh F, Pangalos M, Doherty P (2008) A diacylglycerol

724 lipase-CB2 cannabinoid pathway regulates adult subventricular zone neurogenesis in an

725 age-dependent manner. Mol Cell Neurosci 38:526–536.

726 Hama H, Hara C, Yamaquchi K, Miyawaki A (2004) PKC signaling mediates global

727 enhancement of excitatory synaptogenesis in neurons triggered by local contact with

728 astrocytes. Neuron 41:405–415.

729 Hill MN, Patel S, Carrier EJ, Rademacher DJ, Ormerod BK, Hillard CJ, Gorzalka BB (2005)

730 Downregulation of endocannabinoid signaling in the hippocampus following chronic

731 unpredictable stress. Neuropsychopharmacology 30:508–515.

732 Hindenes JO, Nerdal W, Guo W, Di L, Small DM, Holmsen H (2000) Physical Properties of the

733 Transmembrane Signal Molecule, sn-1-Stearoyl 2-Arachidonoylglycerol. J Biol Chem

734 275:6857–6867.

735 Hsu K-L, Tsuboi K, Adibekian A, Pugh H, Masuda K, Cravatt BF (2012) DAGLβ inhibition

736 perturbs a lipid network involved in macrophage inflammatory responses. Nat Chem Biol

737 8:999–1007 Available at: http://dx.doi.org/10.1038/nchembio.1105.

738 Jenniches I, Ternes S, Albayram O, Otte DM, Bach K, Bindila L, Michel K, Lutz B, Bilkei-Gorzo

739 A, Zimmer A (2016) Anxiety, Stress, and Fear Response in Mice with Reduced

740 Endocannabinoid Levels. Biol Psychiatry 79:858–868.

DAGL-α regulates spatial memory

741 Katona I, Urban G, Wallace M, Ledent C, Jung K, Piomelli D, Mackie K, Freund T (2006)

742 Molecular composition of the endocannabinoid system at glutamatergic synapses. J

743 Neurosci 26:5628–5637.

744 Kim EJ, Pellman B, Kim JJ (2015) Stress effects on the hippocampus: a critical review. Learn

745 Mem 22:411–416.

746 Kwilasz AJ, Abdullah RA, Poklis JL, Lichtman AH, Negus SS (2015) Effects of the fatty acid

747 amide hydrolase (FAAH) inhibitor URB597 on pain-stimulated and pain-depressed

748 behavior in rats. Behav Pharmacol 25:119–129.

749 Lafourcade M, Elezgarai I, Mato S, Bakiri Y, Grandes P, Manzoni O (2007) Molecular

750 components and functions of the endocannabinoid system in mouse prefrontal cortex.

751 PLoS One 2.

752 Larson J, Wong D, Lynch G (1986) Patterned stimulation at the theta frequency is optimal for

753 the induction of hippocampal long-term potentiation. Brain Res 368:347–350.

754 Lieberwirth C, Pan Y, Liu Y, Zhang Z, Wang Z (2016) Hippocampal adult neurogenesis: its

755 regulation and potential role in spatial learning and memory. Brain Res 1644:127–140.

756 MacDonald PL, Gardner RC (2000) Type I error rate comparisons of post hoc procedures for IxJ

757 Chi-square tables. Eductional Psychol Meas 60:735–754.

758 Ogasawara D et al. (2016) Rapid and profound rewiring of brain lipid signaling networks by

759 acute diacyglycerol lipase inhibition. Proc Natl Acad Sci U S A 113:26–33.

760 Okazaki T, Sagawa N, Okita J, Bleasdale J, MacDonald P, Johnston J (1981) Diacylglycerol

761 metabolism and arachidonic acid release in human fetal membranes and decidua vera. J

762 Biol Chem 256:7316–7321.

763 Pan B, Wang W, Long J, Sun D, Hillard CJ, Cravatt BF, Liu Q (2009) Blockade of 2-

764 arachidonoylglycerol hydrolysis by selective monoacylglycerol lipase inhibitor 4-nitrophenyl

765 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) Enhances

766 retrograde endocannabinoid signaling. J Pharmacol Exp Ther 331:591–597.

DAGL-α regulates spatial memory

767 Pan B, Wang W, Zhong P, Blankman JL, Cravatt BF, Liu QS (2011) Alterations of

768 endocannabinoid signaling, synaptic plasticity, learning, and memory in monoacylglycerol

769 lipase knock-out mice. J Neurosci 31:13420–13430.

770 Piyanova A, Lomazzo E, Bindila L, Lerner R, Albayram O, Ruhl T, Lutz B, Zimmer A, Bilkei-

771 Gorzo A (2015) Age-related changes in the endocannabinoid system in the mouse

772 hippocampus. Mech Ageing Dev 150:55–64.

773 Sahay A, Scobie K, Hill A, O’Carroll C, Kheirbek M, Burghardt N, Fenton A, Dranovsky A, Hen R

774 (2011) Increasing adult hippocampal neurogenesis is sufficient to improve pattern

775 separation. Nature 472:466–470.

776 Shonesy BC, Bluett RJ, Ramikie T, Baldi R, Hermanson DJ, Kingsley PJ, Marnett LJ, Winder

777 DG, Colbran RJ, Patel S (2014) Genetic disruption of 2-arachidonoylglycerol synthesis

778 reveals a key role for endocannbinoid signaling in anxiety modulation. Cell Rep 9:1644–

779 1653.

780 Silva-Cruz A, Carlstrom M, Ribeiro JA, Sebastiao AM (2017) Dual influence of

781 endocannabinoids on long-term potentiation of synaptic transmission. Front Pharmacol 8.

782 Snyder J, Hong N, McDonald R, Wojtowicz J (2005) A role for adult neurogenesis in spatial

783 long-term memory. Neuroscience 130:843–852.

784 Sugaya Y, Cagniard B, Yamazaki M, Sakimura K, Kano M (2013) The endocannabinoid 2-

785 arachidonoylglycerol negatively regulates habituation by suppressing excitatory recurrent

786 network activity and reducing long-term potentiation in the dentate gyrus. J Neurosci

787 33:3588–3601.

788 Tanimura A, Yamazaki M, Hashimotodani Y, Uchigashima M, Kawata S, Abe M, Kita Y,

789 Hashimoto K, Shimizu T, Watanabe M, Sakimura K, Kano M (2010) The endocannabinoid

790 2-arachidonoylglycerol produced by diacylglycerol lipase alpha mediates retrograde

791 suppression of synaptic transmission. Neuron 65:320–327.

792 Varvel SA, Anum EA, Lichtman AH (2005) Disruption of CB(1) receptor signaling impairs

DAGL-α regulates spatial memory

793 extinction of spatial memory in mice. Psychopharmacology (Berl) 179:863–872.

794 Varvel SA, Lichtman AH (2002) Evaluation of CB1 receptor knockout mice in the Morris water

795 maze. J Pharmacol Exp Ther 301:915–924.

796 Varvel SA, Wise LE, Niyuhire F, Cravatt BF, Lichtman AH (2007) Inhibition of fatty-acid amide

797 hydrolase accelerates acquisition and extinction rates in a spatial memory task.

798 Neuropsychopharmacology 23:1032–1041.

799 Viader A, Ogasawara D, Joslyn C, Sanchez-Alavez M, Mori S, Nguyen W, Conti B, Cravatt B

800 (2015) A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific

801 pathways regulating neuroinflammation. Elife 5:1–24.

802 Wagner AK, Brayer SW, Hurwitz M, Niyonkuru C, Zou H, Failla M, Arenth P, Manole MD,

803 Skidmore E, Thiels E (2013) Non-spatial pre-training in the water maze as a clinically

804 relevant model for evaluating learning and memory in experimental TBI. Neurobiol Learn

805 Mem 106.

806 Wilkerson JL, Donvito G, Grim TW, Abdullah RA, Ogasawara D, Cravatt BF, Lichtman AH

807 (2017) Investigation of Diacylglycerol Lipase Alpha Inhibition in the Mouse

808 Lipopolysaccharide Inflammatory Pain Model. J Pharmacol Exp Ther 363:394–401.

809 Williams EJ, Walsh F, Doherty P (1994) The production of arachidonic acid can account for

810 calcium channel activation in the second messenger pathway underlying neurite outgrowth

811 stimulated by NCAM, N-cadherin, and L1. J Neurochem 62:1231–1234.

812 Williams EJ, Walsh FS, Doherty P (2003) The FGF receptor uses the endocannabinoid

813 signaling system to couple to an axonal growth response. J Cell Biol 160:481–486.

814 Wirz L, Reuter M, Felten A, Schwabe L (2017) A haplotype associated with enhanced

815 mineralocorticoid receptor expression facilitates the stress-induced shift from “cognitive” to

816 “habit” learning. eNeuro 4:1–16.

817 Xu J-Y, Zhang J, Chen C (2012) Long-lasting potentiation of hippocampal synaptic transmission

818 by direct cortical input is mediated via endocannabinoids. J Physiol 590:2305–2315.

DAGL-α regulates spatial memory

819 Yoshida T, Fukaya M, Uchigashima M, Miura E, Kamiya H, Kano M, Watanabe M (2006)

820 Localization of diacyglycerol lipase-alpha around postsynaptic spine suggests close

821 proximity between production site of an endocannabinoid, 2-arachidonoyl-glycerol, and

822 presynaptic cannabinoid CB1 receptor. J Neurosci 26:4740–4751.

823 Yoshida T, Uchigashima M, Yamasaki M, Katona I, Yamazaki M, Sakimura K, Kano M,

824 Yoshioka M, Watanabe M (2011) Unique inhibitory synapse with particularly rich

825 endocannabinoid signaling machinery on pyramidal neurons in basal amygdaloid nucleus.

826 Proc Natl Acad Sci U S A 108:3059–3064.

827 Yoshino H, Miyamae T, Hansen G, Zambrowicz B, Flynn M, Pedicord D, Blat Y, Westphal R,

828 Zaczek R, Lewis D, Gonzalez-Burgos G (2011) Post-synaptic diacylglycerol lipase

829 mediates retrograde endocannabinoid suppression of inhibition in mouse prefrontal cortex.

830 J Physiol 589:4857–4884.

831 Zhang Z, Wang W, Zhong P, Liu SJ, Long JZ, Zhao L, Gao HQ, Cravatt BF, Liu QS (2015)

832 Blockade of 2-arachidonoylglycerol hydrolysis produces antidepressant-like effects and

833 enhances adult hippocampal neurogenesis and synaptic plasticity. Hippocampus2 25:16–

834 26.

835 Zucker RS, Regehr WG (2002) Short-term synaptic plasticity. Annu Rev Physiol 64:355–405.

836

837

838

839

840

841

842

843

844

DAGL-α regulates spatial memory

845 846 Legends

847 Figure 1. DAGL-α-/- mice and C57BL/6 mice treated with the DAGL inhibitor DO34 show

848 disrupted TBS-induced LTP in CA1 of the hippocampus. The magnitude of LTP was

849 significantly decreased in both A, DAGL-α-/- mice (n = 8) compared to DAGL-α+/+ (n = 9) and

850 DAGL-α+/- mice (n = 11), as well as in slices from B, DO34-treated C57BL/6J mice (n = 7)

851 compared with VEH-treated (n = 9) or DO53-treated mice (n = 8). The magnitude of PTP during

852 the initial induction phase was also significantly decreased in the DO34 group compared with

853 VEH or DO53. Values represent mean ± SEM.

854

855 Figure 2. DAGL-α-/- mice are profoundly impaired in MWM fixed platform task

856 performance. A, Fixed platform task experimental timeline (days). B, During MWM fixed

857 platform acquisition, DAGL-α-/- mice (n = 13) exhibited longer distances to the platform than both

858 DAGL-α+/+ mice (n = 15) and DAGL-α+/- mice (n = 13), as well as showed probe trial

859 performance deficits of C, longer distances to the prior platform position D, fewer platform

860 entries and E, a lower spatial preference for the target quadrant. No significant difference in F,

861 cued task performance or G, swim speed suggest the DAGL-α-/- mice performance deficits were

862 unaffected by sensorimotor or motivational impairments. No significant differences were evident

863 in body weight at either H, baseline or I, during fixed platform acquisition. Values represent

864 mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DAGL-α+/+ mice, and $ p < 0.05, $$ p <

865 0.01, $$$ p < 0.001 vs. DAGL-α+/- mice.

866

867 Figure 3. DAGL-α-/- mice exhibit altered MWM search strategy. DAGL-α-/- mice (n = 13) A,

868 used more non-spatial and thigmotaxic swim paths than DAGL-α+/+ mice (n = 15) on fixed

869 platform day 10, as well as B, spent more time in the MWM outer ring than both DAGL-α+/+ and

870 DAGL-α+/- mice (n = 13) during fixed platform acquisition. C, Fixed platform day 10

DAGL-α regulates spatial memory

871 representative swim traces show spatial (direct and self-orienting) swim paths in DAGL-α+/+

872 mice, spatial (direct) and non-spatial (circling) swim paths in DAGL-α+/- mice, and non-spatial

873 (circling and thigmotaxic) swim paths in DAGL-α-/- mice. Values represent mean ± SEM; ** p <

874 0.01, *** p < 0.001 vs. DAGL-α+/+ mice, and $ p < 0.05 vs. DAGL-α+/- mice.

875

876 Figure 4. DO34 delays MWM fixed platform acquisition, but does not affect probe trial

877 performance. A, Fixed platform task acquisition experimental timeline (days). B, During MWM

878 fixed platform acquisition, mice treated with 30 mg/kg DO34 (n = 12) exhibited longer distances

879 to the platform than VEH (n = 15), 0.3 mg/kg DO34 (n = 12), and 30 mg/kg DO53 (n = 12). C,

880 Fixed platform task probe trial (expression) experimental timeline (days). Following drug-free

881 MWM fixed platform acquisition training, no change in performance was seen at probe trial

882 between drug treatments (VEH n = 9 or 30 mg/kg DO34 n = 8) for any measure; D, distances to

883 the prior platform position E, platform entries or F, spatial preference for the target quadrant. No

884 difference in G, cued task performance suggest the high dose DO34 did not affect sensorimotor

885 or motivational components of MWM performance. H, Mice administered 30 mg/kg DO34

886 showed increased swim speeds during fixed platform acquisition compared to VEH-treated mice

887 on days 1, 2, 4, 5, and 8, but 0.3 mg/kg DO34 reduced swim speeds on days 7, 9, and 10. No

888 significant differences were evident between groups on fixed platform training day 1 across trials

889 (data not shown), or in body weight at I, baseline, yet a significant interaction between drug and

890 day on body weight throughout J, acquisition training showed reductions compared to VEH at

891 30 mg/kg DO34 (days 2 through 15), 3 mg/kg DO34 (days 4, 8,13), 0.3 mg/kg DO34 (day 4),

892 and 30 mg/kg DO53 (days 2, 3, 4, 5, 7, 13). Values represent mean ± SEM; * p < 0.05, ** p <

893 0.01, *** p < 0.001 vs. VEH; $$ p < 0.01 vs. 0.3 mg/kg DO34; and ### p < 0.01 vs. 30 mg/kg

894 DO53.

895

DAGL-α regulates spatial memory

896 Figure 5. DO34 produces modest and selective changes to MWM search strategy. DO34-

897 treated mice (0.3 mg/kg; n = 12, 3 mg/kg; n = 11, 30 mg/kg; n = 12) A, showed no significant

898 change in swim path strategy than VEH-treated mice (n = 15) on fixed platform day 10 B, but

899 mice administered 30 mg/kg DO34 showed a modest increase in the time spent in the MWM

900 outer ring during fixed platform acquisition, than VEH-treated mice. C, Fixed platform day 10

901 representative swim traces show spatial (self-orienting and direct) and non-spatial (circling and

902 scanning) swim paths in VEH, and DO34-treated (0.3, 3, 30 mg/kg) mice. Values represent

903 mean ± SEM; * p < 0.05 DO34 (30 mg/kg) vs VEH-treated mice.

904

905 Figure 6. DO34 does not affect extinction or forgetting tasks. A, Experimental timeline for

906 the extinction (days/weeks) of mice trained drug-free in fixed platform task (same cohort as per

907 probe trial [expression] Figure 4). B, Experimental timeline for additional mice trained drug-free

908 in fixed platform task prior to a forgetting task (days/weeks). C, No change in either the

909 extinction of the fixed platform task, or forgetting performance, was seen between VEH (n = 9

910 and n = 6 respectively) and 30 mg/kg DO34-treated mice (n = 8 and n = 6 respectively), (MWM

911 fixed platform acquisition day 1 and 10 of VEH and 30 mg/kg DO34 for both extinction and

912 forgetting tasks included for comparison). Values represent mean ± SEM; * p < 0.05, ** p <

913 0.01.

914

915 Figure 7. DO34 delays MWM reversal learning but does not produce perseverative

916 behavior. A, Experimental timeline for mice trained in a reversal task (days) following drug-free

917 fixed platform training. B, During MWM reversal, mice treated with 30 mg/kg DO34 (n = 15)

918 exhibited longer distances to the platform than VEH (n = 15) on reversal days one, two, and five

919 (MWM fixed platform acquisition day 1 and 10 of VEH and 30 mg/kg DO34 included for

920 comparison). C, During the same MWM reversal trials, mice treated with 30 mg/kg DO34 also

921 exhibited a delayed inhibition of spatial preference for the quadrant containing the previous

DAGL-α regulates spatial memory

922 platform location compared to VEH. D, During the same MWM reversal trials, mice treated with

923 30 mg/kg DO34 also exhibited a delayed acquisition of spatial preference for the quadrant

924 containing the new platform location compared to VEH. Values represent mean ± SEM; * p <

925 0.05, ** p < 0.01.

926

927 Figure 8. DAGL-α-/- mice, but not DO34 treated mice, show Object Location spatial

928 memory deficits. A, Experimental protocol for Object Location testing of DAGL-α+/+ , -α+/-, and -

929 α-/- mice. During sample phase, no difference in B, total exploration time C, and no preference

930 for either object (pre-assigned stationary or moved) was seen between DAGL-α+/+ (n = 13),

931 DAGL-α+/- (n = 15), and DAGL-α-/- (n = 7) mice. During choice phase D, a significant decrease in

932 discrimination ratio was evident in DAGL-α-/- and DAGL-α+/- mice compared to DAGL-α+/+ mice.

933 E, In the evaluation of DO34 on OL spatial memory, during sample phase F, DO34 treatment (n

934 = 15) lowered the total exploration time compared to VEH (n = 14), yet G, no preference for

935 either object (pre-assigned stationary or moved) was seen between DO34 and VEH-treated

936 mice. During choice phase H, DO34 produced no significant change in discrimination ratio

937 compared to VEH-treated mice. Values represent mean ± SEM; * p < 0.05 vs either DAGL-α+/+

938 or VEH-treated control mice.

939

940 Figure 9. DAGL-α-/- mice show profound alterations in brain endocannabinoids. A, B, C, D,

941 The DAGL-α substrate SAG, was elevated in all four brain regions in DAGL-α-/- mice (n = 8)

942 compared to both DAGL-α+/+ (n = 8) and DAGL-α+/- mice (n = 8). DAGL-α-/- mice possessed

943 decreased levels of 2-AG and AA, respectively in, E and M, hippocampus, F and N, PFC, G and

944 O, striatum, and H and P, cerebellum. DAGL-α+/- mice showed modest 2-AG reductions in

945 striatum, hippocampus, and cerebellum compared to DAGL-α+/+ mice. AEA levels of DAGL-α-/-

946 mice were significantly reduced in I, hippocampus, and L, cerebellum, but not in J, PFC, or K,

DAGL-α regulates spatial memory

947 striatum. Values represent mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 vs DAGL-α+/+

948 mice, and $$ p < 0.01, $$$ p < 0.001 vs DAGL-α+/- mice.

949

950 Figure 10. DO34-treated mice show brain region selective alterations in

951 endocannabinoids and related lipids. A, B, C, D, The DAGL-α substrate SAG, was elevated

952 in all four brain regions at 2 h post-DO34 injection (n = 5) compared to VEH (n = 5), and

953 compared to 24 h (n = 8) in PFC and striatum. At 2 h post-DO34 injection, 2-AG and AA were

954 reduced respectively in, E and M, hippocampus, F and N, PFC, G and O, striatum, and H and P,

955 cerebellum, while at 24 h post-DO34 injection, 2-AG was reduced only in E, hippocampus and

956 H, cerebellum, whereas AA showed reductions across M, N, O, P, all four brain areas. No

957 changes in, I, J, K, L, AEA were evident compared to VEH. Values represent mean ± SEM; * p

958 < 0.05, ** p < 0.01, *** p < 0.001 vs VEH, and # p < 0.05 vs 24 h.