Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate Biosynthetic Pathway in the Brain

Article

Moderate UV Exposure Enhances Learning and

Memory by Promoting a Novel Glutamate

Biosynthetic Pathway in the Brain

Graphical Abstract

Authors

Hongying Zhu, Ning Wang, Lei Yao, ...,

Xiaoyuan Song, Guangming Huang,

Wei Xiong

UCA Presynaptic

neuron

GLU

Correspondence

gmhuang@ustc.edu.cn (G.H.),

wxiong@ustc.edu.cn (W.X.)

CA1

GLU

In Brief

AMPAR

Hippocampus

NMDAR

Na Ca

+

2+

+

Na

UV-induced upregulation of a small

molecule feeds into an intraneuronal

metabolic pathway for glutamate

biosynthesis that can enhance learning

and memory.

CA3

Action potential Postsynaptic

neuron

UVB

Blood

Vessel

UCA

Motor

Object

Learning Recognition Memory

Highlights

d

Single-cell mass spectrometry reveals a novel glutamate

biosynthetic pathway in brain

UV exposure leads to elevated blood and brain levels of

urocanic acid in mice

Urocanic acid promotes glutamate biosynthesis and release

in various brain regions

Moderate UV exposure improves motor learning and object

recognition memory in mice

Zhu et al., 2018, Cell 173, 1–12

June 14, 2018 ª 2018 Elsevier Inc.

https://doi.org/10.1016/j.cell.2018.04.014

Please cite this article in press as: Zhu et al., Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate

Biosynthetic Pathway in the Brain, Cell (2018), https://doi.org/10.1016/j.cell.2018.04.014

Article

Moderate UV Exposure Enhances Learning

and Memory by Promoting a Novel Glutamate

Biosynthetic Pathway in the Brain

1

,3,6

1,6

1

4

1

Hongying Zhu,

Ning Wang, Lei Yao, Qi Chen, Ran Zhang, Junchao Qian, Yiwen Hou, Weiwei Guo, Sijia Fan,

Siling Liu, Qiaoyun Zhao, Feng Du, Xin Zuo, Yujun Guo, Yan Xu, Jiali Li, Tian Xue, Kai Zhong, Xiaoyuan Song,¹

Guangming Huang, * and Wei Xiong *

Hefei National Laboratory for Physical Sciences at the Microscale, Neurodegenerative Disorder Research Center, School of Life Sciences,

5

1

5

1,2

2,4

3,

1,2,7,

1

University of Science and Technology of China, 230026 Hefei, China

²Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, 200031 Shanghai, China

³Hefei National Laboratory for Physical Sciences at the Microscale, School of Chemistry and Materials Science, University of Science and

Technology of China, 230026 Hefei, China

⁴High Magnetic Field Laboratory, Chinese Academy of Sciences, 230031 Hefei, China

⁵Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences & Yunnan Province,

Kunming Institute of Zoology, Chinese Academy of Sciences, 650223 Kunming, China

⁶These authors contributed equally

⁷Lead Contact

*Correspondence: gmhuang@ustc.edu.cn (G.H.), wxiong@ustc.edu.cn (W.X.)

https://doi.org/10.1016/j.cell.2018.04.014

SUMMARY

T cell lymphoma, vitiligo, and uremic pruritus (Ada et al., 2005;

Archier et al., 2012; Johnson-Huang et al., 2010; Radack et al.,

Sunlight exposure is known to affect mood, learning, 2015). Interestingly, UV light not only causes peripheral effects,

and cognition. However, the molecular and cellular but also is associated with various neurological behaviors

linked to the CNS. Speciﬁcally, moderate UV exposure has

been shown to affect mood, addiction, cognition, and memory

mechanisms remain elusive. Here, we show that

moderate UV exposure elevated blood urocanic

(

Beecher et al., 2016; Benedetti et al., 2001; Dominiak et al.,

015; Fell et al., 2014; Keller et al., 2005; Kent et al., 2009; Parrott

acid (UCA), which then crossed the blood-brain

barrier. Single-cell mass spectrometry and isotopic

labeling revealed a novel intra-neuronal metabolic

pathway converting UCA to glutamate (GLU) after

UV exposure. This UV-triggered GLU synthesis pro-

moted its packaging into synaptic vesicles and its

2

and Sabini, 1990). However, relatively little is known about the

molecular and cellular mechanisms that mediate UV-induced

neurobehavioral changes.

Chronic UV exposure results in alterations in levels of proteins,

peptides, and small molecules in the skin or blood, such as

release at glutamatergic terminals in the motor cor- melanin, b-endorphin, vitamin D, and nitric oxide (Bouillon,

tex and hippocampus. Related behaviors, like ro- 2017; D’Orazio et al., 2006; Fell et al., 2014; Hart et al., 2011; Hol-

tarod learning and object recognition memory, were liman et al., 2017). Mounting evidence suggests that these pe-

ripheral changes in chemical substances in the blood induced

enhanced after UV exposure. All UV-induced meta-

bolic, electrophysiological, and behavioral effects

could be reproduced by the intravenous injection of

UCA and diminished by the application of inhibitor

or short hairpin RNA (shRNA) against urocanase,

an enzyme critical for the conversion of UCA to

by UV exposure may affect the brain. For example, UV exposure

elevates skin b-endorphin, which may cross the blood-brain bar-

rier (BBB), thus resulting in opioid-related antinociception and

inducing addiction to UV light in mice (Fell et al., 2014). This

ﬁnding suggests a connection between UV light-induced periph-

eral changes in circulating substances in the blood stream and

GLU. These ﬁndings reveal a new GLU biosynthetic

pathway, which could contribute to some of the

sunlight-induced neurobehavioral changes.

brain function. In addition, levels of a small molecule called uro-

canic acid (UCA) in both epidermis and plasma have been found

to increase immediately after UV exposure (Baden and Pathak,

1967; Kammeyer et al., 1997; Ruegemer et al., 2002). This com-

INTRODUCTION

pound absorbs UV light and thus confers UV ray-resistance (Bar-

resi et al., 2011; Gibbs et al., 2008; Kammeyer et al., 1997; Rue-

Although UV exposure is associated with several skin diseases, gemer et al., 2002). The biological effects of elevated UCA in

moderate UV exposure is beneﬁcial for human health. For blood on the human body, particularly on brain function, have

example, UVB light, one form of radiant energy from sunlight, not been reported. Overall, it remains challenging to link UV

promotes vitamin D generation in the skin (Bouillon, 2017; Hart exposure-induced alterations in the small molecules levels in

et al., 2011). UV radiation has been used to treat certain dis- the blood with their changes in the brain, owing to the lack of

eases, such as psoriasis, atopic dermatitis, eczema, cutaneous appropriate technical support for the sensitive and accurate

Cell 173, 1–12, June 14, 2018 ª 2018 Elsevier Inc.

1

Please cite this article in press as: Zhu et al., Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate

Biosynthetic Pathway in the Brain, Cell (2018), https://doi.org/10.1016/j.cell.2018.04.014

A

B

C

UCA (Neuron)

m/z 139

Patch clamp

100

1

21

HO

N

50

O

[

M+H]⁺

139

6

9 81 95

UCA (Standard)

0

100

m/z 139

1

UCA

1

39.04994

50

m/z

NH

_M₊_H_]+

[

95

8

1

69

139

0

Structural formula of

UCA

5

0

70

90

110

130

150

InESI

MS

m/z

D

E

F

1

500

000

5

4

3

2

1

000

0

1

25

y = 13.44x + 48.22

2

r = 0.9999

100

7

5

2

5

0

5

00

0

100

200

300

400

0

PFC

M₂CeA NAc DS HPC

Concentration of UCA Standard (μM)

CSF

Figure 1. Identiﬁcation of UCA in Neurons by Using Single-Cell MS

(

A) Schematic of single-neuron MS. The expanded view depicts the mass spectra of UCA in single HPC neurons.

B) Chemical structure of UCA.

C) MS/MS to identify UCA in single HPC neurons and the UCA standard.

D) Intensities of intracellular UCA in neurons from the following brain regions: PFC, M

2

, CeA, NAc, DS, and HPC. The digits within the columns represent number

of the neurons with intracellular UCA MS signal and number of total measured neurons from 3 mice.

E) UCA calibration curve constructed by plotting MS intensity against the concentration of the UCA standard. The area marked in gray shows the range of

intracellular UCA concentrations in single neurons.

F) UCA intensity in mouse CSF (N = 6 mice).

(

Data are represented as mean ± SEM.

See also Figure S6.

Evidence has emerged suggesting that the projecting neurons the accelerating rotarod task was dramatically improved after

from MC to DS are also glutamatergic (Hintiryan et al., 2016; UVB exposure (Figure 6B). This effect was markedly inhibited

Kreitzer and Malenka, 2008; Rothwell et al., 2015; Yin et al., by the i.c.v. administration of GG 15 min before UVB exposure

2

009). Therefore, we next measured GLU release by neuronal (Figure 6C). To mimic the UVB-induced increase in circulating

projections from M to DS because intracellular GLU levels in UCA, we intravenously injected UCA and observed that the

also increased after UVB exposure (Figure 4B). Anterograde rotarod skill learning ability of the mice was also signiﬁcantly

tracing with pAAV-mCamKIIa-hChR2(H134R)-EGFP indeed enhanced (Figure S6A). Additionally, this effect was blocked by

veriﬁed the glutamatergic pathway from M to DS (Figure S5F). the i.c.v. injection of GG (Figure S6B).

2

M

2

M2/DS

A similar scenario occurred in the GLU

UVB exposure signiﬁcantly increased the mEPSC amplitude GLU

circuit, where

Additionally, a large body of evidence suggests a role of

CA3/CA1

neurons in long-term recognition memory (Brun

and presynaptic quantal size of GLU (Figures S5F–S5P).

et al., 2002; Clarke et al., 2010). Thus, we next carried out a novel

object recognition test to determine whether UVB exposure af-

fects the recognition memory of mice and whether the intracellular

UCA-GLU metabolic pathway in HPC CA3 neurons is involved.

Mice were exposed to two identical objects placed at an equal

UVB Exposure Improves Motor Learning and

Recognition Memory via the UCA-GLU Metabolic

Pathway in the Brain

Emerging evidence has suggested that the glutamatergic circuit distance (Figure 6D). The time spent exploring each object was

from the MC to the DS is involved in motor learning (Rothwell recorded, and the object preference percentage demonstrated

et al., 2015; Yin et al., 2009). Therefore, we asked whether no distinction between both objectson the left and right sides (Fig-

UVB exposure affects motor learning ability by initiating the ure S6C). The next day, the mice were allowed to explore the open

2

UCA-GLU metabolic pathway in M neurons and subsequently ﬁeld in the presence of a familiar object and a novel object to test

M2/DS

2

circuits. Mice received 50 mJ/cm UVB recognition memory. Long-term (24 hr), but not short-term (2 hr),

activating GLU

exposure before 3 trials with typical accelerating rotarod training recognition memory of mice was signiﬁcantly improved by the

each day for 2 days (Figure 6A). The motor skill learning of mice in UVB exposure (Figure 6E) as well as the i.v. administration of

Cell 173, 1–12, June 14, 2018

9

Please cite this article in press as: Zhu et al., Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate

Biosynthetic Pathway in the Brain, Cell (2018), https://doi.org/10.1016/j.cell.2018.04.014

UCA (Figures S6D and S6E). Interestingly, despite of distinct sen- physical and mental health at multiple levels. Moderate UV-light

sitivities of the skin of different genders to UV light, there was little exposure affects behaviors related to the CNS, such as emotion,

difference between male and female mice in terms of UVB expo- learning, and memory (Beecher et al., 2016; Benedetti et al.,

sure-induced changes in brain UCA-GLU metabolic pathway, ro- 2001; Cunningham, 1979; Dominiak et al., 2015; Fell et al.,

tarod learning, and object recognition memory (Figures S6–S6H). 2014; Keller et al., 2005; Parrott and Sabini, 1990), although it

The UVB exposure-induced improvement of recognition memory is far from clear what mechanism mediates this phenomenon.

was markedly diminished by either i.c.v. injection of a urocanase We demonstrate that UV exposure triggers elevation of blood

inhibitor (Figures 6F, S6I, and S6J) or knockdown of urocanase UCA, which in turn crosses the BBB and enters neurons to

expression in the HPC CA3 regions by using a lentiviral shRNA participate in the biosynthesis of GLU. The increase in neuronal

against urocanase (Figures 6G, S6K, and S6L).

GLU synthesis may result in more GLU packaging into synaptic

vesicles and more GLU release from nerve terminals, thus

M2/DS

strengthening glutamatergic neural circuits such as GLU

DISCUSSION

CA3/CA1

or GLU

projections and related behaviors such as motor

This study provides the ﬁrst evidence of a novel GLU biosynthetic learning and recognition memory. Our results may suggest a uni-

pathway in neurons, as detected with our recently developed sin- versal mechanism for UV exposure-induced neurobehavioral

gle-cell InESI/MS technology (Zhu et al., 2017). Although the HIS- changes related to the CNS: certain chemical substances gener-

UCA-GLU pathway in the liver has been previously described ated in the blood by UV exposure enter the brain through the

(

Brown and Kies, 1959; Cook, 2001; Merritt et al., 1962), it is un- BBB and then alter neuronal excitability. Additional strong evi-

clear whether this pathway exists in the CNS. All metabolites and dence supporting this hypothesis consists of a recent ﬁnding

enzymes related to the liver HIS-UCA-GLU pathway were also that UV exposure elevates circulating b-endorphin, which acts

present in single neurons in various brain regions. A detailed on the CNS and results in opioid receptor-mediated addiction

pathway was further conﬁrmed by direct evidence showing that to UV light (Fell et al., 2014). Because GLU plays multiple roles

1

3

13

incubation with C-HIS and i.v. injection of C-UCA both re- in the CNS, including protein biosynthesis at the amino acid level

1

3

13

sulted in a signiﬁcant increase in C-UCA and C-GLU in single (Nedergaard et al., 2002), disposal of excess or waste nitrogen

neurons. These discoveries expand knowledge of other biosyn- (Melo-Oliveira et al., 1996), and signal transduction between

thetic GLU pathway in neurons beyond the quintessential path- neurons as the most abundant excitatory neurotransmitter (Mel-

ways, such asthe tricarboxylic acid cycleoriginating with glucose drum, 2000; van den Pol et al., 1990), the UV-activated intracel-

and the GLU-glutamine cycle between neurons and astrocytes lular UCA-GLU metabolic pathway in neurons is likely to be

(

B e´ langer et al., 2011). The presence of HIS-UCA-GLU pathway involved in sunbathing-related neurological conditions in addi-

in the CNS is supported by the clinical ﬁndings showing that his- tion to learning and memory, such as mood improvement, addic-

tidinemia and urocanic aciduria, which are autosomal recessive tion, cognition, and brain development.

metabolic disorders due to histidase and urocanase deﬁciency

respectively, are both associated with multiple central develop-

mental symptoms, including learning difﬁculties, speech or lan-

guage disturbances, subnormal intelligence, and mental retarda-

STAR+METHODS

Detailed methods are provided in the online version of this paper

tion (Davies and Robinson, 1963; Espin o´ s et al., 2009; Kalafatic

and include the following:

et al., 1980; Lott et al., 1970; Raisov a´ and Hy a´ nek, 1986), presum-

ably because of lower GLU synthesis in neurons during brain

development, based upon our ﬁndings.

d KEY RESOURCES TABLE

d CONTACT FOR REAGENT AND RESOURCE SHARING

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Mice

This study indicates that the single-cell MS technology is a

valuable tool to study novel metabolic pathways in the brain at

the single-cell level. The use of single-cell MS to study cell meta-

bolism is more advantageous than conventional methods using

tissue homogenates or cell populations, which are limited to

the measurement of average values. For example, UCA has pre-

viously been identiﬁed in the peripheral system, including the

blood, skin, liver, and urine (Baden and Pathak, 1967; Cook,

B Cell Culture

d METHOD DETAILS

B UVB Irradiation

B Mouse Serum and Cerebrospinal Fluid (CSF)

Preparation

B Brain Slice Preparation

2

001; Kammeyer et al., 1997; Ruegemer et al., 2002). However,

B Single-Neuron MS

13

it has not previously been detected in the CNS. Conventional

chemical analysis using brain tissue homogenates may result

in several drawbacks, such as the over-dilution of constituents

at low concentrations, cellular diversity and complexity, and

possible decomposition during tedious sample pretreatment

and chromatographic separation (Altschuler and Wu, 2010; Zen-

obi, 2013), all of which greatly affect the precise measurement of

target compounds.

B

C-Isotope Tracing Experiments

B Calibration Curves of UCA

B Lentivirus Production and Transduction

B RNA Interference

B Synaptosomal Preparation

B Western Blotting

B Brain Tissue Preparation and Immunohistochemistry

B Fluorescent in Situ Hybridization (FISH) Experiment

B Magnetic Resonance Imaging Experiments

B Electrophysiological Recording

Although overexposure to UV radiation may cause several

adverse health effects, moderate UV exposure greatly beneﬁts

10 Cell 173, 1–12, June 14, 2018

Please cite this article in press as: Zhu et al., Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate

Biosynthetic Pathway in the Brain, Cell (2018), https://doi.org/10.1016/j.cell.2018.04.014

B AMPA/NMDA Ratio Measurement

Archier, E., Devaux, S., Castela, E., Gallini, A., Aubin, F., Le Ma ıˆ tre, M., Ara-

ctingi, S., Bachelez, H., Cribier, B., Joly, P., et al. (2012). Carcinogenic risks

of psoralen UV-A therapy and narrowband UV-B therapy in chronic plaque

psoriasis: a systematic literature review. J. Eur. Acad. Dermatol. Venereol.

B Stereotaxic Surgery, Cannula Placement and

Microinjection

B Immunoﬂuorescence after Intracerebroventricular

2

6 (Suppl 3 ), 22–31.

(ICV) administration of GG

Baden, H.P., and Pathak, M.A. (1967). The metabolism and function of uroca-

B Motor Learning

nic acid in skin. J. Invest. Dermatol. 48 , 11–17.

B Novel Object Recognition

d QUANTIFICATION AND STATISTICAL ANALYSIS

B MS Data Analysis

Barresi, C., Stremnitzer, C., Mlitz, V., Kezic, S., Kammeyer, A., Ghannadan, M.,

Posa-Markaryan, K., Selden, C., Tschachler, E., and Eckhart, L. (2011).

Increased sensitivity of histidinemic mice to UVB radiation suggests a crucial

role of endogenous urocanic acid in photoprotection. J. Invest. Dermatol. 131 ,

188–194.

B MR Spectra Analysis

B Electrophysiological Data Analysis

B Western Blot Analysis

Beecher, M.E., Eggett, D., Erekson, D., Rees, L.B., Bingham, J., Klundt, J.,

Bailey, R.J., Ripplinger, C., Kirchhoefer, J., Gibson, R., et al. (2016). Sunshine

on my shoulders: weather, pollution, and emotional distress. J. Affect. Disord.

B Immunoﬂuorescence Analysis

2

05 , 234–238.

SUPPLEMENTAL INFORMATION

B e´ langer, M., Allaman, I., and Magistretti, P.J. (2011). Brain energy meta-

bolism: focus on astrocyte-neuron metabolic cooperation. Cell Metab. 14 ,

Supplemental Information includes six ﬁgures and three tables and can be

found with this article online at https://doi.org/10.1016/j.cell.2018.04.014 .

7

24–738.

Benedetti, F., Colombo, C., Barbini, B., Campori, E., and Smeraldi, E. (2001).

Morning sunlight reduces length of hospitalization in bipolar depression.

J. Affect. Disord. 62 , 221–223.

ACKNOWLEDGMENTS

The authors thank Dr. Yang Pan (National Synchrotron Radiation Laboratory,

University of Science and Technology of China) for instrumentation supports.

The authors thank Dr. Li Zhang (NIH/NIAAA), Dr. Yong Shen (University of Sci-

ence and Technology of China), and the anonymous reviewers for valuable

comments and suggestions to improve the quality of the paper. We acknowl-

edge support from National Key R&D Program of China (2016YFC1300500-2

to W.X. and 2016YFA0201300-2 to G.H.), National Natural Science Foundation

of China (31471014, 91432103, and 91649121 to W.X., 21775143 and

Bouillon, R. (2017). Comparative analysis of nutritional guidelines for vitamin D.

Nat. Rev. Endocrinol. 13 , 466–479.

Brown, D.D., and Kies, M.W. (1959). The mammalian metabolism of L-histi-

dine. II. The enzymatic formation, stabilization, puriﬁcation, and properties

of 4(5)-imidazolone-5(4)-propionic acid, the product of urocanase activity.

J. Biol. Chem. 234 , 3188–3191.

Brun, V.H., Otnass, M.K., Molden, S., Steffenach, H.A., Witter, M.P., Moser,

M.B., and Moser, E.I. (2002). Place cells and place recognition maintained

by direct entorhinal-hippocampal circuitry. Science 296 , 2243–2246.

21475121 to G.H., and 81701068 to H.Z.), the Strategic Priority Research Pro-

gram of the Chinese Academy of Sciences (XDB02010000 to W.X.), the Funda-

mental Research Funds for the Central Universities (W.X. and G.H.), the Major/

Innovative Program of Development Foundation of Hefei Center for Physical

Science and Technology (2017FXZY006 to W.X. and 2017FXCX003 to G.H.),

and Recruitment Program of Global Experts (W.X. and G.H.).

Clarke, J.R., Cammarota, M., Gruart, A., Izquierdo, I., and Delgado-Garc ı´ a,

J.M. (2010). Plastic modiﬁcations induced by object recognition memory pro-

cessing. Proc. Natl. Acad. Sci. USA 107 , 2652–2657.

Cook, R.J. (2001). Disruption of histidine catabolism in NEUT2 mice. Arch.

Biochem. Biophys. 392 , 226–232.

AUTHOR CONTRIBUTIONS

Cunningham, M.R. (1979). Weather, mood, and helping behavior: quasi exper-

iments with the Sunshine Samaritan. J. Pers. Soc. Psychol. 37 , 1947–1956.

W.X. and G.H. designed research and supervised the project. H.Z., N.W., and

Q.C. conducted electrophysiological recordings and single-cell MS. L.Y. con-

ducted western blot experiments. W.G. and S.F. conducted immunohisto-

chemistry. S.L. and J.L. conducted in situ hybridization. Y.H., F.D., Q.Z.,

Y.G., X.Z., and Y.X. conducted animal behavioral tests. R.Z. and X.S. pro-

duced the lentiviral shRNA. T.X. generated the triple knockout blind mice.

J.Q. and K.Z. conducted magnetic resonance imaging experiments. H.Z.,

N.W., W.X., and G.H. analyzed data. W.X., H.Z., and G.H. wrote the

manuscript.

D’Orazio, J.A., Nobuhisa, T., Cui, R., Arya, M., Spry, M., Wakamatsu, K., Igras,

V., Kunisada, T., Granter, S.R., Nishimura, E.K., et al. (2006). Topical drug

rescue strategy and skin protection based on the role of Mc1r in UV-induced

tanning. Nature 443 , 340–344.

Davies, H.E., and Robinson, M.J. (1963). A case of histidinaemia. Arch. Dis.

Child. 38 , 80–82.

Dominiak, M., Swiecicki, L., and Rybakowski, J. (2015). Psychiatric hospitali-

zations for affective disorders in Warsaw, Poland: Effect of season and inten-

sity of sunlight. Psychiatry Res. 229 , 287–294.

DECLARATION OF INTERESTS

Dull, T., Zufferey, R., Kelly, M., Mandel, R.J., Nguyen, M., Trono, D., and Nal-

dini, L. (1998). A third-generation lentivirus vector with a conditional packaging

system. J. Virol. 72 , 8463–8471.

The authors declare no competing interests.

Espin o´ s, C., Pineda, M., Mart ı´ nez-Rubio, D., Lupo, V., Ormazabal, A., Vilaseca,

M.A., Spaapen, L.J., Palau, F., and Artuch, R. (2009). Mutations in the uroca-

nase gene UROC1 are associated with urocanic aciduria. J. Med. Genet. 46 ,

Received: August 17, 2017

Revised: February 21, 2018

Accepted: April 12, 2018

Published: May 17, 2018

4

07–411.

Fell, G.L., Robinson, K.C., Mao, J., Woolf, C.J., and Fisher, D.E. (2014). Skin

b -endorphin mediates addiction to UV light. Cell 157 , 1527–1534.

REFERENCES

Garwood, M., and DelaBarre, L. (2001). The return of the frequency sweep:

designing adiabatic pulses for contemporary NMR. J. Magn. Reson. 153 ,

155–177.

Ada, S., Se c¸ kin, D., Budako g ꢀ lu, I., and Ozdemir, F.N. (2005). Treatment of ure-

mic pruritus with narrowband ultraviolet B phototherapy: an open pilot study.

J. Am. Acad. Dermatol. 53 , 149–151.

Gibbs, N.K., Tye, J., and Norval, M. (2008). Recent advances in urocanic acid

photochemistry, photobiology and photoimmunology. Photochem. Photobiol.

Sci. 7 , 655–667.

Altschuler, S.J., and Wu, L.F. (2010). Cellular heterogeneity: do differences

make a difference? Cell 141 , 559–563.

Cell 173, 1–12, June 14, 2018 11

Please cite this article in press as: Zhu et al., Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate

Biosynthetic Pathway in the Brain, Cell (2018), https://doi.org/10.1016/j.cell.2018.04.014

Hart, P.H., Gorman, S., and Finlay-Jones, J.J. (2011). Modulation of the im-

mune system by UV radiation: more than just the effects of vitamin D? Nat.

Rev. Immunol. 11 , 584–596.

Parrott, W.G., and Sabini, J. (1990). Mood and memory under natural

conditions: evidence for mood incongruent recall. J. Pers. Soc. Psychol. 59 ,

321–336.

Hattar, S., Lucas, R.J., Mrosovsky, N., Thompson, S., Douglas, R.H., Hankins,

M.W., Lem, J., Biel, M., Hofmann, F., Foster, R.G., and Yau, K.W. (2003).

Melanopsin and rod-cone photoreceptive systems account for all major

accessory visual functions in mice. Nature 424 , 76–81.

Radack, K.P., Farhangian, M.E., Anderson, K.L., and Feldman, S.R. (2015).

A review of the use of tanning beds as a dermatological treatment. Dermatol.

Ther. (Heidelb.) 5 , 37–51.

Raisov a´ , V., and Hy a´ nek, J. (1986). Speech disorders associated with histidi-

nemia and other hereditary disorders of amino acid metabolism. Folia Pho-

niatr. (Basel) 38 , 43–48.

Hintiryan, H., Foster, N.N., Bowman, I., Bay, M., Song, M.Y., Gou, L., Yama-

shita, S., Bienkowski, M.S., Zingg, B., Zhu, M., et al. (2016). The mouse cor-

tico-striatal projectome. Nat. Neurosci. 19 , 1100–1114.

Rothwell, P.E., Hayton, S.J., Sun, G.L., Fuccillo, M.V., Lim, B.K., and Malenka,

R.C. (2015). Input- and output-speciﬁc regulation of serial order performance

by corticostriatal circuits. Neuron 88 , 345–356.

Hirata, H., Takahashi, A., Shimoda, Y., and Koide, T. (2016). Caspr3-deﬁcient

mice exhibit low motor learning during the early phase of the accelerated

rotarod task. PLoS ONE 11 , e0147887.

Ruegemer, J., Schuetz, B., Hermann, K., Hein, R., Ring, J., and Abeck, D.

(2002). UV-induced skin changes due to regular use of commercial sunbeds.

Photodermatol. Photoimmunol. Photomed. 18 , 223–227.

Holliman, G., Lowe, D., Cohen, H., Felton, S., and Raj, K. (2017). Ultraviolet

radiation-induced production of nitric oxide:a multi-cell and multi-donor anal-

ysis. Sci. Rep. 7 , 11105.

Scheuss, V., and Neher, E. (2001). Estimating synaptic parameters from mean,

variance, and covariance in trains of synaptic responses. Biophys. J. 81 ,

Hunter, J.K., and Hug, D.H. (1989). Speciﬁc inhibition of bacterial and bovine

1

970–1989.

urocanases by glycylglycine. Pept. Res. 2 , 240–245.

Silver, R.A. (2003). Estimation of nonuniform quantal parameters with multiple-

probability ﬂuctuation analysis: theory, application and limitations. J. Neurosci.

Methods 130 , 127–141.

Johnson-Huang, L.M., Su a´ rez-Fari n˜ as, M., Sullivan-Whalen, M., Gilleaudeau,

P., Krueger, J.G., and Lowes, M.A. (2010). Effective narrow-band UVB radia-

tion therapy suppresses the IL-23/IL-17 axis in normalized psoriasis plaques.

J. Invest. Dermatol. 130 , 2654–2663.

Sun, Y., Nguyen, A.Q., Nguyen, J.P., Le, L., Saur, D., Choi, J., Callaway, E.M.,

and Xu, X. (2014). Cell-type-speciﬁc circuit connectivity of hippocampal CA1

revealed through Cre-dependent rabies tracing. Cell Rep. 7 , 269–280.

Kalafatic, Z., Lipovac, K., Jezerinac, Z., Juretic, D., Dumic, M., Zurga, B., and

Res, L. (1980). A liver urocanase deﬁciency. Metabolism 29 , 1013–1019.

Suska, A., Lee, B.R., Huang, Y.H.H., Dong, Y., and Schl u¨ ter, O.M. (2013).

Selective presynaptic enhancement of the prefrontal cortex to nucleus accum-

bens pathway by cocaine. Proc. Natl. Acad. Sci. USA 110 , 713–718.

Kamat, P.K., Kalani, A., and Tyagi, N. (2014). Method and validation of synap-

tosomal preparation for isolation of synaptic membrane proteins from rat

brain. MethodsX 1 , 102–107.

Tak a´ cs, V.T., Klausberger, T., Somogyi, P., Freund, T.F., and Guly a´ s, A.I.

Kammeyer, A., Pavel, S., Asghar, S.S., Bos, J.D., and Teunissen, M.B.M.

(

2012). Extrinsic and local glutamatergic inputs of the rat hippocampal CA1

area differentially innervate pyramidal cells and interneurons. Hippocampus

2 , 1379–1391.

(

1997). Prolonged increase of cis-urocanic acid levels in human skin and

urine after single total-body ultraviolet exposures. Photochem. Photobiol.

5 , 593–598.

2

6

van den Pol, A.N., Wuarin, J.P., and Dudek, F.E. (1990). Glutamate, the

Keller, M.C., Fredrickson, B.L., Ybarra, O., C oˆ t e´ , S., Johnson, K., Mikels, J.,

Conway, A., and Wager, T. (2005). A warm heart and a clear head. The contin-

gent effects of weather on mood and cognition. Psychol. Sci. 16 , 724–731.

dominant excitatory transmitter in neuroendocrine regulation. Science 250 ,

1

276–1278.

Vanhamme, L., van den Boogaart, A., and Van Huffel, S. (1997). Improved

method for accurate and efﬁcient quantiﬁcation of MRS data with use of prior

knowledge. J. Magn. Reson. 129 , 35–43.

Kent, S.T., McClure, L.A., Crosson, W.L., Arnett, D.K., Wadley, V.G., and

Sathiakumar, N. (2009). Effect of sunlight exposure on cognitive function

among depressed and non-depressed participants: a REGARDS cross-

sectional study. Environ. Health 8 , 34.

Wikgren, J., Mertikas, G.G., Raussi, P., Tirkkonen, R., A¨ yr a¨ v a¨ inen, L., Pelto-

Huikko, M., Koch, L.G., Britton, S.L., and Kainulainen, H. (2012). Selective

breeding for endurance running capacity affects cognitive but not motor

learning in rats. Physiol. Behav. 106 , 95–100.

Kreitzer, A.C., and Malenka, R.C. (2008). Striatal plasticity and basal ganglia

circuit function. Neuron 60 , 543–554.

Leger, M., Quiedeville, A., Bouet, V., Haelewyn, B., Boulouard, M., Schumann-

Xue, T., Do, M.T., Riccio, A., Jiang, Z., Hsieh, J., Wang, H.C., Merbs, S.L.,

Welsbie, D.S., Yoshioka, T., Weissgerber, P., et al. (2011). Melanopsin signal-

ling in mammalian iris and retina. Nature 479 , 67–73.

Bard, P., and Freret, T. (2013). Object recognition test in mice. Nat. Protoc. 8 ,

2531–2537.

Lott, I.T., Wheelden, J.A., and Levy, H.L. (1970). Speech and histidinemia:

Yin, H.H., Mulcare, S.P., Hil a´ rio, M.R., Clouse, E., Holloway, T., Davis, M.I.,

Hansson, A.C., Lovinger, D.M., and Costa, R.M. (2009). Dynamic reorganiza-

tion of striatal circuits during the acquisition and consolidation of a skill. Nat.

Neurosci. 12 , 333–341.

methodology and evaluation of four cases. Dev. Med. Child Neurol. 12 ,

596–603.

Meldrum, B.S. (2000). Glutamate as a neurotransmitter in the brain: review of

physiology and pathology. J. Nutr. 130 (4S, Suppl), 1007S–1015S.

Zamboni, N., Fendt, S.M., R u¨ hl, M., and Sauer, U. (2009). (13)C-based meta-

Melo-Oliveira, R., Oliveira, I.C., and Coruzzi, G.M. (1996). Arabidopsis mutant

analysis and gene regulation deﬁne a nonredundant role for glutamate dehy-

drogenase in nitrogen assimilation. Proc. Natl. Acad. Sci. USA 93 , 4718–4723.

bolic ﬂux analysis. Nat. Protoc. 4 , 878–892.

Zenobi, R. (2013). Single-cell metabolomics: analytical and biological perspec-

tives. Science 342 , 1243259.

Merritt, A.D., Rucknagel, D.L., Silvermanm, and Gardiner, R.C. (1962). Urinary

urocanic acid in man: the identiﬁcation of urocanic acid and the comparative

excretions of urocanic acid and N-formiminoglutamic acid after oral histidine

in patients with liver disease. J. Clin. Invest. 41 , 1472–1483.

Zhu, H., Zou, G., Wang, N., Zhuang, M., Xiong, W., and Huang, G. (2017).

Single-neuron identiﬁcation of chemical constituents, physiological changes,

and metabolism using mass spectrometry. Proc. Natl. Acad. Sci. USA 114 ,

2

586–2591.

Mitchell, S.J., and Silver, R.A. (2000). Glutamate spillover suppresses inhibition

Zielman, R., Wijnen, J.P., Webb, A., Onderwater, G.L.J., Ronen, I., Ferrari,

M.D., Kan, H.E., Terwindt, G.M., and Kruit, M.C. (2017). Cortical glutamate

in migraine. Brain 140 , 1859–1871.

by activating presynaptic mGluRs. Nature 404 , 498–502.

Mon, A., Ab e´ , C., Durazzo, T.C., and Meyerhoff, D.J. (2013). Effects of fat

on MR-measured metabolite signal strengths: implications for in vivo MRS

studies of the human brain. NMR Biomed. 26 , 1768–1774.

Zurkovsky, L., Bychkov, E., Tsakem, E.L., Siedlecki, C., Blakely, R.D., and

Gurevich, E.V. (2013). Cognitive effects of dopamine depletion in the context

of diminished acetylcholine signaling capacity in mice. Dis. Model. Mech. 6 ,

171–183.

Nedergaard, M., Takano, T., and Hansen, A.J. (2002). Beyond the role of gluta-

mate as a neurotransmitter. Nat. Rev. Neurosci. 3 , 748–755.

12 Cell 173, 1–12, June 14, 2018

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Biosynthetic Pathway in the Brain, Cell (2018), https://doi.org/10.1016/j.cell.2018.04.014

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

Antibodies

SOURCE

IDENTIFIER

Mouse monoclonal anti-HAL

Rabbit anti-AMDHD1

Abnova

Sigma

Cat# H00003034-M04,RRID:AB_566289

Cat# HPA039720, RRID:AB_10672967

Cat# NBP1-93883, RRID:AB_11028801

Cat# MAB374, RRID:AB_2107445

Cat# G2404, RRID:AB_477002

Rabbit anti-UROC1

Novus

Mouse monoclonal anti-GAPDH

Mouse monoclonal anti-FTCD

mouse monoclonal anti-NeuN

Goat anti-Mouse IgG (H+L), Alexa Fluor 568

Millipore

Sigma

Millipore

invitrogen

Cat# MAB377, RRID:AB_2298772

Cat# A-11004, RRID:AB_141371

Bacterial and Virus Strains

pAAV-mCamKIIa-hChR2(H134R)-EGFP

Shanghai Sunbio Medical

Biotechnology

N/A

siHAL

Genepharma

Hanbio

N/A

pHBAAV-U6-shUROC1-CMV-EGFP

pHBAAV-U6- shAMDHD1-CMV-EGFP

pHBAAV-U6- shFTCD -CMV-EGFP

pHBAAV-U6- shControl -CMV-EGFP

pAAV-CAG-EGFP

Hanbio

Obio Technology

Chemicals, Peptides, and Recombinant Proteins

Picrotoxin

Sigma

P1675; CAS: 124-87-8

v900090; CAS: 67-68-5

ab120054; CAS: 4368-28-9

859796; CAS: 104-98-3

709638; CAS: 1173097-34-1

V900459; CAS:71-00-1

588644

dimethyl sulfoxide

Tetrodotoxin

Sigma

Abcam

4

-Imidazoleacrylic acid (Urocanic acid)

3

Sigma-Aldrich

Sigma

1

1,2,3-

3

C -Urocanic acid

Histidine

1

3

1-

C-Histidine

Sigma-Aldrich

Sigma

Glycyl-Glycine

piracetam

v900403; CAS: 556-50-3

A506285;CAS:7491-74-9

11668-019

Sangon Biotech (Shanghai)

Invitrogen

Lipofectamine 2000

avidin-biotin complex

Vector Laboratories

Cat# PK-6101, RRID:AB_2336820

Cat# SK-4100, RRID:AB_2336382

0

3

,3 -diaminobenzidine

Experimental Models: Cell Lines

HEK293T cell line

Gift from Dr. Xue T lab (USTC)

N/A

Experimental Models: Organisms/Strains

Mouse:Triple knockout (TKO)

Mouse:C57BL/6J

Hattar et al., 2003

N/A

213

Beijing Vital River Laboratory

Animal Technology

Oligonucleotides

urocanase cDNA: Forward: GAACTGGACACAACGGGG

urocanase cDNA: Reverse: CCATTGGAGACATCCCAG

Primers for genotyping of TKO mice, see Table S1

Primers for shRNA plasmid construction, see Table S2

This paper

N/A

Recombinant DNA

Urocanase plasmids

IPPAnase plasmids

OriGene

N/A

(Continued on next page)

Cell 173, 1–12.e1–e7, June 14, 2018 e1

Please cite this article in press as: Zhu et al., Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate

Biosynthetic Pathway in the Brain, Cell (2018), https://doi.org/10.1016/j.cell.2018.04.014

Continued

REAGENT or RESOURCE

FTCD plasmids

pRSV-Rev

SOURCE

IDENTIFIER

OriGene

N/A

Dull et al., 1998

Gift from Didier Trono

Addgene Cat# 12253

Addgene Cat# 12251

Addgene Cat# 12259

pMDLg/pRRE

pMD2.G

(unpublished data)

Software and Algorithms

ImageJ

National Institutes of Health

GraphPad Software

jMRUI

https://imagej.nih.gov/ij/

GraphPad Prism 6

https://www.graphpad.com

jMRUI time domain analysis software package

http://www.jmrui.eu/welcome-to-the-

new-mrui-website//

Clampﬁt

Molecular Devices

Synaptosoft

https://www.moleculardevices.com

http://www.synaptosoft.com

Mini Analysis software

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact, Wei Xiong

wxiong@ustc.edu.cn ).

(

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice

All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Science and Tech-

nology of China (USTC) and the Chinese Academy of Sciences (CAS). Mice were group-housed in groups of 2-5 with a 12-h light/

dark cycle (lights off at 7 p.m.) and free access to food and water ad libitum under speciﬁc pathogen-free conditions. Male

C57BL/6J mice were used for all experiments unless otherwise speciﬁed. Male triple knockout (TKO) mice (rod a transducin

ꢁ/ꢁ

knockout, Gnat ; cyclic nucleotide gated channel alpha 3 knockout, Cnga3 ; melanopsin knockout, Opn4 ) were used for

sampling. The C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology. Genotyping of the TKO

mice was performed using the primers provided in Table S1. Experimenters were blinded to all experimental conditions until all

data were obtained. Unless otherwise speciﬁed, the mice were randomly assigned to control or treatment groups in all experiments.

Cell Culture

HEK293T cell line (female) was a kind gift from Dr. Xue T (USTC) and accompanied by authentication documents verifying the identity

according to their short tandem repeat proﬁles. HEK293T cell line was cultured in Dulbecco’s Modiﬁed Eagle’s medium (DMEM)

ꢂ

(

HyClone) supplemented with 10% fetal bovine serum (GIBCO) at 37 C and 5% CO

2

.

METHOD DETAILS

UVB Irradiation

Mice were dorsally shaved 2 days before the start of radiation exposure. During the UVB irradiation experiment, mice were kept in an

acrylic chamber (15 3 30 3 50 cm) with a power-adjustable UVB lamp (G15T8E, Sankyo Denki, Japan) on top. The shaved mice were

2

exposed to 50 mJ/cm of UVB (7 mW/cm , 120 min) and then placed back to their home cages before testing. The irradiation intensity

of UV was detected using a UV irradiometer (Photoelectric instrument factory of Beijing normal university, Beijing, China) placed on

the bottom of the acrylic chamber. The suberythematic dose was determined on the basis of previous studies (D’Orazio et al., 2006;

Fell et al., 2014).

Mouse Serum and Cerebrospinal Fluid (CSF) Preparation

For serum collection, the mouse blood was collected into regular 1.5 mL Eppendorf tubes and stored at room temperature for 30 min,

ꢂ

and then centrifuged for 10 min at 4500 rpm (PICO 17, Thermo Scientiﬁc, USA) at 4 C. The supernatant was transferred to a vial and

ꢂ

stored at ꢁ20 C until analyzed by mass spectrometry (MS).

To collect mouse CSF samples, the Isoﬂurane-anesthetized wild-type male C57BL/6J mice were mounted on a stereotaxic system

68015, RWD Life Science, China). After drilling through the skull, a 5 mL syringe (Hamilton) was lowered into the lateral ventricle

(

1.0 mm lateral, 0.3–0.5 mm posterior, 2.3–2.5 mm depth from bregma). CSF was withdrawn at a rate of 400 nL/min. Approximately

mL of CSF was collected per mouse.

1

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2

All mouse serum (5 ml) and CSF samples were diluted 30 folds using methanol-H O solution (30:70, v/v). Atenolol (38-188 pM) was

added as an internal standard for MS analysis.

Brain Slice Preparation

2

The secondary motor cortex (M ), hippocampus (HPC), central nucleus of the amygdala (CeA), dorsal striatum (DS), nucleus accum-

bens (NAc) and prefrontal cortex (PFC) slices were prepared from C57BL/6J mice aged P28-P30. The mice were sacriﬁced and then

the brains were rapidly removed from the skull and immersed in ice-cold pre-oxygenated cutting solution containing sucrose

1

94 mM, NaCl 30 mM, NaHCO

300 mm) were cut using a Vibratome (VT 1200S, Leica, Germany). The tissue slices were incubated in a holding chamber containing

oxygenated (95% O and 5% CO ) artiﬁcial cerebrospinal ﬂuid (aCSF), which consisted of NaCl 124 mM, NaHCO 26 mM, KCl

.5 mM, NaH PO 1.2 mM, MgCl 1 mM, CaCl 2 mM, and glucose 10 mM (pH 7.4; osmolality, 315 mOsm/kg). For the mouse brain

3 2 2 4

26 mM, MgCl 1 mM, glucose 10 mM, KCl 4.5 mM, and NaH PO 1.2 mM, pH 7.4. Coronal sections

(

2

3

4

2

4

2

ꢂ

slices (coronal or sagittal planes) recovered from mechanical injury, they were incubated at 32 C oxygenated (95% O

ꢂ

2

and 5% CO

2

)

aCSF for approximately 30 min and then cooled to room temperature (21-23 C) at least 30 min before recording.

Single-Neuron MS

After extraction of the cell cytoplasmic chemical constituents, the capillary was coupled to the single-neuron MS, which had been

fabricated as previously described (Zhu et al., 2017). After the brain slices recovered from mechanical injury, they were transferred

to the recording chambers. The neurons were randomly chosen for the subsequent electrophysiological recording and MS analysis.

We approached the neuron using a micromanipulator (MP-285, Sutter) and patched the neuron with borosilicate glass pipettes ﬁlled

4 3 4

with pipette solution (NH HCO 185 mM and NH Cl 80 mM) by applying negative pressure. The neurons were clamped at ‒70 mV after

the patched cell membrane was broken by rapidly applied negative pressure. After the electrophysiological data were recorded, the

cytoplasmic chemical constituents were obtained from the assayed neuron by applying negative pressure. Only neurons with tightly

held seals (> 1 GU) and nonruptured membranes were selected for analysis, avoiding the dilution of intracellular ﬂuid by aCSF. Once a

sufﬁcient amount of ﬂuid was withdrawn from the cell, the patch pipette was quickly removed from the slice and then assayed via MS.

An AC voltage with an amplitude of 4 kV at ꢀ500 Hz was applied outside of the spray capillary micropipette. The tip of the spray

micropipette was kept ꢀ5 mm away from the oriﬁce of the MS instrument. High resolution mass measurements were analyzed using

an Exactive plus MS instrument (Thermo Fisher Scientiﬁc, San Jose, CA, USA). Its main experiment parameters were set as follows:

ꢂ

Capillary temperature, 275 C; S-lens radio frequency (RF) level, 50%; mass resolution, 70,000; maximum inject time, 10 ms; and

microscan, 1. Other MS experiments were conducted with a LTQ Velos Pro. MS instrument (Thermo Fisher Scientiﬁc, San

ꢂ

Jose, CA, USA). MS parameters were capillary temperature 275 C, S-lens RF level 42%, maximum injection time 300 ms, and micro-

scan 1. Chemical structure were conﬁrmed by tandem MS using LTQ Velos Pro. MS instrument based on collision-induced disso-

ciation (CID) with helium (He) as background gas at 28% energy. Commercial electrospray ionization (ESI) source was removed

before our experiments. Positive ion mode was used in our experiments.

1

3

C-Isotope Tracing Experiments

1

To trace HIS metabolism, 1- C-HIS was added into aCSF at a ﬁnal concentration of 3 mg/ml. After the mouse brain slices (coronal

3

1

3

or sagittal planes) recovered from mechanical injury, they were transferred to oxygenated aCSF with or without 1- C-HIS and

13

incubated for 1 h at room temperature. After the mouse slices incubated with aCSF containing 1- C-HIS, they were washed three

times with oxygenated aCSF at room temperature and then transferred to the recording chambers. Other experimental procedures

were the same as those for single-neuron MS.

13

To trace UCA metabolism in vivo, we intravenously (i.v.) injected 1,2,3-

3

C -UCA (20 mg/kg, 2.5 mg/ml) diluted in saline into the

mice 30 min before serum/CSF collection and brain slices preparation. Saline is the corresponding vehicle solution. All the collected

samples were analyzed using MS. All drug treatment or control groups were blinded from experimenters.

Calibration Curves of UCA

Estimations of intracellular UCA levels in neurons were calculated from calibration curves with external UCA standards at concen-

trations ranged from 1 mM, 10 mM, 100 mM and 300 mM with three replicates per concentration point. All calibrations were obtained

0

with artiﬁcial intracellular solution: potassium gluconate 130 mM, NaCl 6 mM, ethylene glycol-bis(b-aminoethyl ether)-N,N,N ,N -tet-

raacetic acid (EGTA) 11 mM, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) 10 mM, CaCl 1 mM, NaOH 4 mM, MgCl

mM, Mg-ATP 2 mM and Na-GTP 0.2 mM (pH was adjusted to 7.3).

2

1

Lentivirus Production and Transduction

HEK293T cells in a 10-cm plate were seeded at 70% conﬂuence 1 day before transfection. The cells were transfected the next day at

5% conﬂuence with 6 mg of plasmid containing Uroc1 shRNA or control shRNA, 6 mg of pRSV-Rev, 6 mg of pMDLg/pRRE and 3 mg of

8

pMD2.G (Addgene) together with 21 mL of Lipofectamine 2000 (Invitrogen). The medium was replaced 10 h after transfection. Viral

ꢂ

supernatant was harvested 70 h post-transfection, ﬁltered with a 0.45-mm ﬁlter (Millipore), and centrifuged for 2.5 h at 30,000 g 4 C.

After centrifugation, the supernatant was removed and the pellet was resuspended with 20 mL of PBS containing 10% FBS and

ꢂ

8

mg/ml polybrene and stored at ꢁ80 C.

Cell 173, 1–12.e1–e7, June 14, 2018 e3

Please cite this article in press as: Zhu et al., Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate

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RNA Interference

Small interfering RNA (siRNA) were used for histidase knockdown. HEK293T cells were co-transfected with either targeting siRNA or

control siRNA (Genepharma) and histidase overexpressing plasmid (OriGene) using lipofectamine 2000 reagent for 48 h. For knock-

down of urocanase, IPPAnase or FTCD, short hairpin RNA (shRNA) plasmid constructs (Hanbio) were co-transfected with overex-

pressing plasmids of urocanase, IPPAnase or FTCD (OriGene) into HEK293T cells for 48 h. The subsequent western blotting analysis

were performed to validate the knockdown efﬁcacy of siRNA or shRNA. The most efﬁcient shRNA plasmid construct was used to

package AAV virus (Hanbio) for in vivo knockdown of urocanase, IPPAnase or FTCD. Several independent siRNA duplexes or shRNA

plasmid constructs were used and the sequence were listed in Table S2.

Synaptosomal Preparation

Mouse brain synaptosomes were prepared as previously described (Kamat et al., 2014). Brieﬂy, the mice were anaesthetized and

perfused with ice-cold saline. The brain was removed and kept on ice-cold 0.32 M sucrose HEPES buffer (NaCl 145 mM, KCl

5

mM, CaCl

2

2 mM, MgCl

2

1 mM, glucose 5 mM, HEPES 5 mM, pH 7.4) supplemented with protease inhibitor cocktail. Tissue

ꢂ

was homogenized using a Dounce homogenizer and the homogenate was centrifuged at 800 g, 4 C, 10 min to collect the supernatant

ꢂ

(

S1). The supernatant (S2) containing cytosol was collected for electrophoresis after the S1 was centrifuged at 10,000 g, 4 C, 20 min.

The pellet (P2) containing synaptosomes were suspended in 0.8 M sucrose HEPES buffer solution and further centrifuged at 20,000

ꢂ

g, 4 C, 30 min. The pellet was washed in HEPES buffer twice and were suspended in lysis buffer solution (Tris 50 mM, NaCl 150 mM,

1% NP-40, 0.5% Sodium dexyocholate supplemented with protease inhibitor cocktail as the synaptosome). The synaptosomal

preparation was used for western blotting analysis.

Western Blotting

HEK293T cells co-transfected with overexpressing plasmids and shRNA plasmids were collected and lysed in lysis buffer solution.

ꢂ

Mouse brain tissues were homogenized and sonicated followed by centrifugation at 10,000 g, 4 C for 30 min. The supernatants were

ꢂ

diluted 1:1 with laemmli 2X sample buffer and denatured at 99 C for 10 min. The samples were separated on 12% SDS/PAGE gels.

After electro-transferring to a 0.45 mm PVDF membrane, the membrane were blocked in 5% BSA and blotted with following anti-

bodies: anti-HAL (Abcam, 1:500), anti-UROC1 (Novus, 1:250), anti-AMDHD1 (Sigma, 1:250) and anti-GAPDH (Millipore, 1:40000).

HRP-conjugated secondary antibodies and chemiluminescent substrate (Thermo Fisher) were used to detect signals.

Brain Tissue Preparation and Immunohistochemistry

Mice (N = 3) were deeply anesthetized by intraperitoneal (i.p.) injection of pentobarbital (40 mg/kg), and then perfused with chilled

PBS, followed by 4% paraformaldehyde (PFA) in PBS. Brains were removed and immersed in the same ﬁxative solution (4% PFA

ꢂ

in PBS) overnight, then were subsequently incubated in 30% (wt/vol) sucrose in PBS for 24 h at 4 C. Sagittal brain sections

ꢂ

4

0 mm thick were prepared using a cryostat (CM 1860, LEICA Microsystems) at ꢁ20 C and collected into a 12-well plate.

After a PBS wash (three times within 5 min), sections were incubated for 20 min in 3% peroxide mixed in methanol in order to

inactivate the endogenous peroxidase. Sections were incubated with 0.3% Triton X-100 and 5% normal goat serum in PBS. After

4

0 min of blocking, the sections were incubated with rabbit anti-Histidase antibody (1:200, ab154063, Abcam), anti-urocanase

ꢂ

antibody (1:200, NBP1-93883, Novus), or anti-IPPAnase antibody (1:200, HPA039720, Sigma) in PBS for 24 h at 4 C. On the following

day, the sections were washed in PBS and incubated with biotinylated goat anti-rabbit (1:1000, PK-6101, Vector Laboratories) for 2 h.

After a PBS wash, sections were reacted with avidin-biotin complex (ABC, PK6101, Vector Laboratories) for 30 min. Brain sections

0

were washed with PBS and collected on gelatin-coated slides, air-dried. Slides were incubated with 3,3 -diaminobenzidine

(

DAB, SK-4100, Vector Laboratories) for 2-4 min before a distilled-water wash. Slides were dehydrated in alcohol gradient

90, 95, 100%), immersed in xylene, mounted with Neutral Balsam (E675007, Sangon Biotech). All the incubations were done at

room temperature, unless mentioned otherwise.

Fluorescent in Situ Hybridization (FISH) Experiment

For FISH, the detected fragment of mouse urocanase cDNA was cloned into pEASY-T3 with the pair of primers provided in the Key

Resources Table. Probes were prepared with DIG RNA Labeling Mix (Roche) by in vitro transcription with T7 or SP6 polymerases.

Frozen sections with 15 mm thick were collected on PLL-coated slides, then further ﬁxed in 4% PFA/ PBS for 10 min, subsequently

2 2

washed with DEPC-PBS for 10 min, and then treated with 3% H O to block the endogenous peroxidase activity. After that the slides

were treated with 0.5% Triton X-100/DEPC-PBS for 20 min, at last acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine

ꢂ

(

probe for 16 h at 60 C. After washing twice in 2 3 SSC at 60 C for 15 min each, the samples were treated with 5 mg/ml RNase A at

pH 8.0). After acetylation, the sections were prehybridized for 2 h at 60 C, and then hybridized for with either scrambled or positive

ꢂ

37 C. They were sequentially washed twice in 0.2 3 SSC at 60 C for 30 min each. Probes were detected by standard method using

anti-Digoxigenin-POD Fab fragments (Roche) and TSA-Plus Cyanine 3 system (PerkinElmer).

e4 Cell 173, 1–12.e1–e7, June 14, 2018

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Detected sequence:

gaactggacacaacgggggaactcttggtggacctcgggtcagaccagacatcctgtcacaacccgttcaatggcggctactaccctgtgcagctcagcttctcagaggcc

cagagcctcatgtcctccaaccctgctgccttcaagcacctggtgcaggaaagcctgaggaggcatgtcgcagccatcaacaggttggcccaagagaagttcttcttctggg

actatggtaatgccttcctcttggaggctcagagagcaggagcagatgtagagaagaaaggagccaacaagatggagttccgctacccctcctatgtccagcacattatggg

ggatatattctcccagggctttgggcccttccgctgggtatgcacatcaggggacccccaggacctggctgtcactgatcatctggccacatctgtactggagaaagccattg

ctgatggagtgaaggcatctgtgaagctacagtatatggataatatacgctggatccgagaggctgccaagcatcagctggtggtgggctcccaggcaaggatcctgtactc

agaccagaaaggccgtgtggccattgctgtggccattaaccaggccatcgccagtgggaagatcaaggcaccagtggtcctgagccgtgaccaccatgatgtgagtggca

ctgacagccccttcagggagacttcgaatatttatgacggttctgccttctgcgcagacatggccgtgcagaacttcgtgggagatgcctgtcgtggtgccacctgggttgcac

ttcacaatggagggggtgtaggctggggtgaagtcatcaatgggggatttggccttgttctagatggaactgcagaggctgagcagaaagccaggatgatgctcagctggga

tgtctccaatgg.

Magnetic Resonance Imaging Experiments

Male mice aged 8-10 weeks were used. The integrated animal/MRI facility at high magnetic ﬁeld laborary allowed for the measure-

ments to be taken without requiring the mice to be euthanized at the end of experiment, and all animals were returned to the animal

facility after they had fully recovered from the experimental procedures.

All magnetic resonance imaging (MRI) experiments were performed on a 9.4 Tesla 40-cm horizontal bore MR spectrometer (Agilent

Technologies, Santa Clara, CA, USA). The animals were placed in a prone position on a specially designed cradle and inserted into

the magnet. A home-built radiofrequency (RF) coil were used for both RF transmission and signal detection. Mice were anaesthetized

2

with isoﬂurane (3.5% induction, 1.5%–2.0% maintenance in air/O 2:1) for the duration of the scan (approximately 2.5 h). Respiratory

rate and rectal temperature were monitored throughout the experiment with a physiologic monitoring unit (model 1030; SA

ꢂ

Instruments, Stony Brook, NY), and the body temperature was maintained at 36.5 C with a homemade heating pad. Reference

2 2

T -weighted images were acquired, from which the spectroscopic region of interest were determined in the cortical M area as

well as the HPC CA3 area. Subsequently, the i.v. injection of UCA (20 mg/kg, 2.5 mg/ml diluted in saline) began at a constant

rate of 100 ml/min through the tail vein with a syringe micro-pump, and the procedure was completed within 2 min. MR spectra

were acquired at 0 min, 20 min, 40 min, 60 min, 80 min and 100 min post-injection of UCA solution.

T

2

-weighted MR images were acquired using a fast spin echo sequence with a repetition time (TR) of 5000 ms, echo spacing =

1

0 ms, echo train length (ETL) = 4; additionally, localized proton spectra from the M and HPC regions outlined on the T -weighted

2

images were acquired using the following parameters: effective echo time (TE) = 30 ms, ﬁeld of view (FOV) = 16 3 16 mm , matrix

size = 192 3 192, slice thickness = 1 mm (10 slices, gap = 0), and bandwidth (BW) = 25 kHz. A LASER (Localization by Adiabatic

Selective Refocusing) sequence (Garwood and DelaBarre, 2001) was used in this study. The acquisition parameters were: TR/

3

TE = 2000/36 ms, voxel size = 1 3 1 3 2 mm , spectral width = 4006 Hz, and Free induction decay size = 4096 with 512 averages.

Typical spectra line widths for water resonance after 3D shimming were approximately 20 Hz.

Electrophysiological Recording

Slices were transferred into the recording chamber continuously perfused at 3-4 ml/min with aCSF at 28 C. Neurons were visualized

ꢂ

using a ﬁxed-stage microscope (BX50WI, Olympus, Tokyo, Japan) with differential interference contrast and infrared illumination. We

obtained all whole-cell voltage clamp mode recordings using borosilicate glass pipettes (5-7 MU) ﬁlled with internal solutions. The

internal solution (pH 7.2) for excitatory postsynaptic currents (EPSC) recording contained K-gluconate 145 mM, HEPES 5 mM,

0

Mg-adenosine triphosphate (ATP) 5 mM, Na-guanosine 5 -triphosphate (GTP) 0.2 mM, and EGTA 10 mM. The miniature excitatory

postsynaptic currents (mEPSCs) were recorded with a holding potential of ꢁ70 mV and PTX (100 mM), which was used to block the

A

inhibitory neurotransmitter receptors (GABA and glycine receptors), diluted with aCSF. For mEPSC experiments, brain slices from

C57BL/6J mice expressing pAAV-mCamKIIa-hChR2(H134R)-EGFP in M

2

or HPC CA3 were cut for ChR2 stimulation test. Dorsal

M2/DS

CA3/CA1

striatum neurons or HPC CA1 neurons were recorded with single optical stimulation of GLU or GLU

2

respectively. Only neurons respond to a 473-nm laser (AniLab-Opto, 10 mW/mm ) were selected for the following tests. The mEPSCs

projecting axons,

were collected after TTX (ﬁnal concentration: 0.5 mM) was added to the bath perfusion. To measure presynaptic glutamate release of

M

2

-DS or HPC CA3-CA1 circuit, the multiple probability ﬂuctuation analysis was used as previously described (Scheuss and Neher,

2001; Silver, 2003; Suska et al., 2013). Brieﬂy, a train of ﬁve light pulses at 20 Hz frequency and 5-ms duration was used to stimulate

the ChR2-expressing axons every 10 s. 50-100 evoked EPSCs were recorded at ﬁve release probability conditions. Experimental

results were discarded if the series resistance varied by more than 15% or exceeded 25 MU.

AMPA/NMDA Ratio Measurement

To evoke EPSC currents in hippocampal CA1 pyramidal neurons, Schaffer collateral was stimulated using a concentric circular

A

electrode every 20 s. PTX (100 mM) was diluted with aCSF to block the inhibitory neurotransmitter receptors (GABA and glycine

receptors). CA1 Pyramidal neurons were voltage-clamped at ꢁ70 and +40 mV to record AMPAR-mediated and NMDAR-mediated

currents respectively. For each neuron, the total 10 sweeps of AMPAR and NMDAR-mediated currents were recorded and the

average value of currents were then calculated. The NMDAR-mediated current was measured at 50 ms after the stimulation. The

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AMPAR-mediated current was measured as the peak value. Then, the AMPA/NMDA ratio was calculated by dividing average AMPA

EPSC by NMDA EPSC.

Stereotaxic Surgery, Cannula Placement and Microinjection

Mice were anaesthetized with pentobarbital sodium (i.p., 0.5%, 0.01 ml/g) and placed on a stereotaxic apparatus (68015, RWD Life

Science, China). Ophthalmic ointment was applied to prevent the eyes from drying. A midline scalp incision was made, and a crani-

otomy was drilled above each cannula implantation or injection site. For local drug infusion, guide cannula (62001, RWD) were

implanted into lateral ventricles (anteroposterior, A/P: ꢁ0.3 mm; medio-lateral, M/L: ꢁ1.0 mm; dorsoventral, D/V: ꢁ1.8 mm). The

glycyl-glycine (GG) was dissolved in saline (13.2 mg/ml, 0.9% NaCl). Thirty min before the experiment, infusion needles attached to

1

0 mL syringes were inserted into the guide cannula, and 1 mL of GG solution was delivered within 2 min. For lentiviral shRNA or

pAAV-mCamKIIa-hChR2(H134R)-EGFP injection, glass micropipette attached to 10 mL syringes were placed bilaterally into the

cortex (A/P: +1.3 mm; M/L: 0.7 mm; D/V: ꢁ0.8 mm) or the CA3 region of the hippocampus (A/P: ꢁ1.7 mm; M/L: 2.2 mm; D/V:

.25 mm), and 0.5 mL of virus was infused per side at a rate of 50 nl/min using a pump (Legato130, KD Scientiﬁc). Mice were allowed

M

2

to recover for 2 weeks before behavioral tests or electrophysiological recordings. For ﬂuorescence imaging, we injected 0.5 mL of

8

AAV-CAG-EGFP (1.6 3 10 vg/ml, Obio Technology) into the CA3 and waited 3 weeks

Immunoﬂuorescence after Intracerebroventricular (ICV) administration of GG

For NeuN immunoﬂuorescence, after 2-h ICV injection of GG or saline, male C57BL/6J mice from both groups (N = 3 mice each

group) were deeply anesthetized by pentobarbital (i.p., 40 mg/kg), and then perfused with chilled PBS, followed by 4% PFA in

PBS. Brains were collected and immersed in 4% PFA in PBS overnight, then were subsequently incubated in 30% (wt/vol) sucrose

ꢂ

in PBS for 24 h at 4 C as above described. Coronal brain sections (40 mm) were prepared using a cryostat (CM 1860, LEICA

ꢂ

Microsystems) at ꢁ20 C and collected into a 12-well plate. After a PBS wash (three times within 5 min), sections were incubated

for 20 min in PBS containing 10% goat serum and 0.3% Triton X-100.Then slices were incubated with mouse anti-NeuN antibody

ꢂ

(

1:200, Millipore) at 4 C overnight. The slices were then incubated with goat anti-mouse secondary antibody conjugated with

alexa 568 (1:1000, invitrogen) for 1 h at room temperature. After washing with PBS for three times, slices were sealed for imaging.

Immunoﬂuorescence-labeled images were examined by confocal microscopy (LSM 880, Olympus).

Motor Learning

We used the rotarod training system (XR1514, Xinruan, Shanghai, China) to test the motor skill learning of mice as previously

described (Hirata et al., 2016; Wikgren et al., 2012). Before the ﬁrst training sessions, the mice were habituated to stay on a stationary

rod for 2 min. To assess motor learning, a total of six trials for the rotarod test were carried out using an accelerating protocol from 4 to

6

0 rpm in 300 s with 20-min inter-trial intervals. Three training sessions every day were performed for 2 days. After falling, the mice

were immediately placed back to their home cages, and the time to fall was automatically recorded by the rotarod software. The

mice were taken from the rod and the latency was recorded as 300 s, if stayed on the rotarod more than the full 300 s of a trial.

The apparatus and testing area were cleaned with 75% ethanol, when the trials were ﬁnished.

Novel Object Recognition

Novel object recognition methods have been described previously (Leger et al., 2013; Zurkovsky et al., 2013). Brieﬂy, the open-ﬁeld

apparatus consisted of an acrylic chamber (40 3 40 3 30 cm). Two different objects were prepared in duplicate: towers of Lego

bricks (12.5 cm high, 2.5 cm deep and 7.5 cm wide, built from blue, yellow, red and green bricks) and Falcon tissue culture ﬂasks

(

15.5 cm high, 3.5 cm deep and 9 cm wide, transparent plastic with an orange bottle cap). The objects were placed 5 cm away

from the walls and attached to the ﬂoor. Mice were tested during the dark phase (active phase between 7:00 p.m. and 7:00 a.m.).

Before training, mice were placed in the training room for 30 min. During the familiarization session, mice were allowed to freely

explore two identical objects (Lego bricks) placed into the arena at ﬁxed locations for 3 min. The ANY-maze video-tracking system

(

Stoelting, Wood Dale, USA), which is based on nose-point detection, was used to record the time spent exploring objects. Active

exploration was deﬁned as mice snifﬁng or touching the object when the gap between the nose and the object was less than 2 cm.

Climbing over the object or gnawing the object was not considered as exploratory activity. At the end of the test, each mouse was

returned to its home cage, and the chamber and objects were cleaned using 75% ethanol, then air-dried for 3 min. After an interses-

sion interval (ISI) of 2 or 24 h, one of the familiar objects was replaced by a novel object (Falcon tissue culture ﬂasks). The location of

the novel object (left or right) was randomized among the mice and the groups tested. Object preference was calculated by using the

following formula: preference % = (time to explore the individual object/total exploration time for both objects) 3 100%). Data were

excluded if the total of exploration time was less than 10 s.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software). Paired and unpaired two-tailed Student’s t tests

were used to compare difference between treatment and control groups and P values were plotted (p < 0.05 was considered as sta-

tistically signiﬁcant difference). Results are expressed as mean ± SEM (standard error of the mean). The number (N) of mice or cells,

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the statistical approaches used and statistical signiﬁcance are indicated in the Figures and Figure legends. For single-neuron MS and

electrophysiological recording experiments, N = number of neurons collected from at least 3 mice. For mouse serum or CSF exper-

iments, western-blot, immunohistochemistry and behavioral experiments, n = number of mice. Sample sizes were determined based

on previous experience. No statistical methods were used to predetermine sample size. The main authors of this manuscript

re-calculated independently all the results of every experiment. All the data collected from control and experimental groups in our

experiments were analyzed blindly. More information concerning data quantiﬁcation and analysis for different experiments can be

found below.

MS Data Analysis

Metabolites identiﬁcation was based on high resolution mass measurements, isotopic proﬁles and matching tandem mass spectral

data from endogenous substances with chemical standards when available. For high resolution mass measurements, to exclude

13

13 12

data with faulty assignments, mass deviation was set lower than 15 ppm. For analysis of C-labeling results, the D( C/ C)(%) is

deﬁned and calculated by the following formula:

1

3

13

Intensity of C ꢁ M

1

3

12

Dð C= CÞð%Þ =

ꢁ

Intensity of M

ð Clabeled groupÞ

Intensity of M

13

ðnon Clabeled groupÞ

1

3

Where M represents HIS, UCA, IPPA, FMGA or GLU.

MR Spectra Analysis

All MR spectra were processed using the jMRUI time domain analysis software package (http://www.jmrui.eu/

welcome-to-the-new-mrui-website/). Preprocessing steps consisted of exponential apodization at 5 Hz, Fouriers transformation

and phase correction of the transformed spectrum. The chemical shifts of metabolite peaks were referenced to the N-acetylaspartate

3

CH - group at 2.02 ppm. AMARES (advanced method for accurate, robust and efﬁcient spectral ﬁtting) (Vanhamme et al., 1997), a

widely used quantitation tool for MRS data, was employed to ﬁt the spectra and quantify the GLU signal, using the none-suppressed

water peak in spectra as intensity reference to quantify metabolite concentration (Mon et al., 2013).

Electrophysiological Data Analysis

The electrophysiological data were acquired using an Axon MultiClamp 700B ampliﬁer (Molecular Devices, AXON), ﬁltered at 3 kHz

and digitized at 10 kHz using an Axon Digidata 1500A digitizer combined with pCLAMP 10.4 software. Data detection was accom-

plished using Mini Analysis software. For variance-mean analysis (multiple probability ﬂuctuation analysis), 50-100 light-evoked

EPSCs were averaged and variance of peak EPSCs was calculated. Variance plotted against the mean amplitude of EPSCs for

each eliciting condition. We used a binomial model to calculate the following parameter as described previously (Suska et al., 2013).

I = NPrQ

[S1]

2

s = NQ Prð1 ꢁ PrÞ

[S2]

2

Fitting curve indicated that the variance (s ) of EPSC amplitudes has a parabolic relationship with I. Quantal size (Q) is equal to the

slope of the best ﬁt line in a synapse with low release probability (Pr). The number of synapses (N) and Pr were calculated with [S1]

and [S2]. Neurons not ﬁtted were discarded for data analysis.

Western Blot Analysis

The intensity of the bands revealed in western blot photographic ﬁlms was quantiﬁed using the gel quantiﬁcation plugin on ImageJ

software. Absolute values were normalized to the intensity of loading control (GAPDH) bands from the same samples. Results are

expressed as the normalized values of band intensity relative to GAPDH.

Immunoﬂuorescence Analysis

For all Immunoﬂuorescence quantiﬁcations, at least 3 to 6 coronal brain slices per mouse from at least 3 independent mice were

analyzed. Serial sections of several brain regions were immunostained and the number of NeuN-positive neurons in the ﬁeld of

view (FOV) was counted by ImageJ software.

Cell 173, 1–12.e1–e7, June 14, 2018 e7

Supplemental Figures

A

m/z 69

N

+

H

+

NH

-

C H O

3

2

O

CH

2

CH

C

OH

C

+

CH

-

CO₂

- H O

2

+

H⁺

+ H⁺

+

H

+

N

NH

m/z 95

NH

m/z 121

+

m/z 139

[UCA+H]

-

C H O

2

CH

+

H

+

N

NH

m/z 81

B

1

800

[

UCA+H]⁺

m/z 139

PFC

2

CeA

M

1

200

NAc

DS

HPC

6

00

0

9

4

95

m/z

96

Figure S1. Detection of Urocanic Acid in the Neurons from Several Brain Regions, Related to Figure 1

(

A) The possible fragment pathway of protonated UCA in collision induced dissociation.

B) Representative tandem mass (MS/MS) spectra of intracellular UCA in single neurons from brain regions of prefrontal cortex (PFC), secondary motor cortex

2

M ), central nucleus of the amygdala (CeA), nucleus accumbens (NAc), dorsal striatum (DS) and hippocampus (HPC) of mice.

(

legend on next page)

Figure S2. Effects of UVB Exposure and i.v. UCA Administration on UCA Levels in the Serum, CSF, and Single Neurons, Related to Figure 2

A and B) Representative MS/MS spectra of UCA in mouse serum at 0, 30, 60, 90, 120 and 240 min after mock-UV (A) or UVB exposure (B). All spectra were

normalized to their respective controls (0 min, mock-UV).

(

C) Representative MS/MS spectra of UCA in mouse cerebrospinal ﬂuid (CSF) before and after UVB exposure. All spectra were normalized to control group

before UVB).

D and E) The normalized intensities of intracellular UCA, IPPA, FMGA and GLU in neurons of HPC from mice 30 min (D) or 24 h (E) after mock-UV or UVB exposure.

F) The fold changes in MS intensity of serum UCA at 0.5 h after i.v. injection of UCA at 4, 10, 20, 40 and 100 mg/kg (N = 3 mice per dose). The dotted line indicates

2

the fold changes in mouse blood UCA level after 2-h UVB exposure (50 mJ/cm ).

(G) Representative MS/MS spectra of UCA in mouse CSF before (black line) and after (red line) i.v. injection of UCA (20 mg/kg, 30 min). All spectra were normalized

to the control group (before i.v. UCA).

13

(

3 3

H) Representative MS spectra of UCA and 1,2,3- C -UCA in mouse serum after i.v. injection of saline (blue line) or 1,2,3- C -UCA (red line, 20 mg/kg, 30 min).

13

The spectra of

C

3

-UCA were normalized to the spectra of UCA. The 1,2,3-

C

3

-UCA /UCA ratios in the mouse serum after i.v. injection of saline or

3

C -UCA

(

20 mg/kg, 30 min).

I) Representative MS spectra of UCA and 1,2,3-

13

3 3

C -UCA in mouse CSF after i.v. injection of saline or 1,2,3- C -UCA (20 mg/kg, 30 min). The spectra of

1

3

C

3

-UCA were normalized to the spectra of UCA.

1

J) Identiﬁcation of the in vivo UCA-GLU metabolic pathway in single HPC neurons via C-labeling assay. The C-UCA/UCA, C-IPPA/IPPA, C-FMGA/FMGA

3

13

(

13

and C-GLU/GLU ratios in single neurons from the HPC of mice with or without i.v. injection of C-UCA (20 mg/kg, 1 h).

K-O) Representative MS/MS spectra of intracellular UCA in single neurons from brain regions of PFC (K), M (L), HPC (M), NAc (N) and DS (O) with i.v. injection of

UCA (20 mg/kg, 30 min) or saline. All spectra were normalized to their respective controls (i.v. saline group).

P and Q) Effects of UCA-incubation on intracellular level of UCA and related metabolites in NAc neurons. (P) Normalized intensities of intracellular UCA, IPPA,

(

2

(

FMGA and GLU in single HPC neurons with or without UCA preincubation (0.7 mg/ml). Data were normalized to their respective controls (neurons without UCA-

incubation). (Q) Fold changes in intracellular intensities of UCA and GLU in neurons from HPC, M2 and NAc brain slices after UCA preincubation (0.7 mg/ml).

1

R–U) Comparison of the permeability of BBB for exogenous small molecules in NAc. (R) The normalized C-HIS/HIS ratios in single neurons from the NAc and

3

(

13

HPC after i.v. injection of C-HIS (10 mg/kg, 30 min). (S) The normalized C-HIS/HIS ratios in single neurons from the NAc and M

10 mg/kg, 30 min). (T) The normalized intensities of intracellular piracetam in single neurons from the NAc and HPC after i.v. injection of piracetam (2 mg/kg,

0 min). (U) The normalized intensities of intracellular piracetam in single neurons from the NAc and M after i.v. injection of piracetam (2 mg/kg, 30 min). All data

were normalized to NAc group.

2

after i.v. injection of C-HIS

(

3

2

All digits within the columns represent numbers of total neurons measured from 3 mice. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

based on unpaired t test.

(

legend on next page)

Figure S3. Identiﬁcation of the HIS-UCA-GLU Metabolic Pathway in Neurons of Various Brain Regions in Mice, Related to Figure 3

(

A and B) Representative MS/MS spectra for intracellular glutamate (GLU) (A, top) and histidine (HIS) (B, top) from single HPC neurons and standards of GLU

A, bottom) and HIS (B, bottom).

C and D) The possible fragment pathway of protonated 4-Imidazolone-5-propionic acid (IPPA) (C) and formiminoglutamic acid (FMGA) (D) in collision induced

dissociation.

E–H) The heatmap of HIS-incubation-induced increases in the intracellular level of metabolites involved in the HIS-UCA pathway in the brain. Sagittal mouse

(

brain slices were incubated with HIS at 0.2 mg/ml for 1 h or artiﬁcial cerebrospinal ﬂuid (aCSF) as a control. Each dot represents a measured neuron at the

approximate position in the brain slice. The color of each dot represents the range of fold changes in the intracellular level of UCA (E), IPPA (F), FMGA (G) or GLU

(H) in neurons from the brain slice after HIS incubation, when compared to the adjacent brain slice incubated with control aCSF. Please be aware that the brain

regions in this map are artiﬁcially ﬂattened within the same plane for viewing convenience. The bar graphs in the right portion indicate the average values of HIS-

incubation-induced fold changes in intracellular levels of UCA (E), IPPA (F), FMGA (G) or GLU (H) in the neurons from certain brain regions. OB (Olfactory bulb,

M/L:-0.6); PFC (Prefrontal cortex, M/L:-0.48); Prcc (Precerebral cortex, M/L:-0.84); Pocc (Posterior cerebral cortex, M/L:-0.96); DS (Dorsal striatum, M/L:-1.08);

NAc (Nucleus accumbens, M/L:-0.96); HPC (Hippocampus, M/L:-1.08); Th (Thalamus, M/L:-0.84); HT (Hypothalamus, M/L:-0.24); CeA (Central amygdaloid

nucleus, M/L:-2.4), VTA (Ventral tegmental area, M/L:-0.6); SN (Substantia nigra, M/L:-0.6); PAG (Periaqueductal gray, M/L:0.48); BS (Brain stem, M/L:0.48);

CB (Cerebellum, M/L:-0.6). M/L: medio-lateral.

1

3

13

(

I) (Top) Representative MS spectra of IPPA and C-IPPA in single HPC neurons with or without preincubation of C-HIS (3 mg/ml, 1h). The spectra of C-IPPA

1

were normalized to the spectra of IPPA either without (blue line) or with (red line) C-HIS incubation. x10: the signal is magniﬁed ten times. (Bottom) Differential

3

13

ratio of intensity of intracellular C-IPPA/IPPA with or without preincubation of C-HIS (3 mg/ml, 1 h).

13

J–L) Representative MS spectra of HIS and C-HIS (J), UCA and C-UCA (K), GLU and C-GLU (L) in the single HPC neurons without or with preincubation of

13

(

1

3

13

C-HIS (3 mg/ml, 1h). The spectra of C-HIS, C-UCA and C-GLU were normalized to the spectra of HIS, UCA and GLU separately either without (blue line) or

13

with (red line) C-HIS incubation. x10: the signal is magniﬁed ten times.

(M) Full images of Western-blot for Figure 3F.

(N–P) Validation of antibodies by western blot analysis of overexpressed histidase (N), urocanase (O) and IPPAnase (P) 48 h after siRNA or shRNA transfection in

HEK293T cells. UT: untransfected cells; Ctrl-si: random control siRNA; Ctrl-sh: random control shRNA.

Q–S) The immunohistochemistry of histidase (Q), urocanase (R) and IPPAnase (S) in the sagittal mouse brain slice using avidin-biotin-peroxidase complex

(

staining assay. Cb: cerebellum; VMH: ventromedial hypothalamic nucleus; PMV: premammillary nucleus, ventral part; PnC, pontine reticular nucleus; AHA,

anterior hypothalamic, anterior; NAc, nucleus accumbens; Hpc, hippocampus; CC, cingulate cortex.

(

T) In situ hybridization for urocanase on cerebellum, frontal cortex and HPC slices from adult mouse brain.

U) Western blot analysis of urocanase, IPPAnase and FTCD in synaptosomes. PSD-95 as the marker of synaptosomes.

V and W) The normalized intensities of intracellular UCA, IPPA, FMGA and GLU in neurons from HPC of mice after intracerebroventricular (ICV) injection of glycyl-

1

2

glycine (GG) (2.6 mg, 1 ml) or saline (1 ml). These mice received intra-HPC injection of AAV-shRNA against urocanase (0.5 ml/side, 1.3 3 10 vg/ml) (V) or random

12

control shRNA (shRandom, 0.5 ml/side, 1.6 3 10 vg/ml) (W) 3 weeks before single-cell MS tests. vg, vector genomes. All data were normalized to their respective

controls (ICV saline group).

(

X-BB) Effects of ICV injection of GG on the basic electrophysiological properties of HPC neurons. The resting membrane potential (X), resistant input (Y), action

potential amplitude (Z), action potential threshold (AA) and action potential frequency (BB) of neurons in HPC slices from mice after ICV injection of GG (2.6 mg,

ml) and saline (1 ml).

CC–II) Immunocytochemical examination of NeuN-positive neurons in different brain regions after 2-h ICV injection of GG. (CC-HH) Sample photomicrographs of

NeuN staining from saline-treated (left) and GG-treated (right) mice. Five brain regions were exhibited and the NeuN+ neurons were counted in the ﬁeld of view

FOV, 300 mm x 300 mm). Scale bar = 100 mm. (II) Cell counting of NeuN+ neurons displayed no difference between saline-treated and GG-treated mice in the

above regions. Th: thalamus; HT: hypothalamus; Au1: primary auditory cortex; S1: primary sensory cortex.

JJ and KK) Effects of ICV injection of GG on intracellular level of metabolites related to neuronal health (JJ) and other GLU biosynthetic pathway (KK) in HPC

1

(

neurons. The normalized intensities of intracellular ATP, pyruvate, GLN and GABA in single neurons from the HPC of mice after ICV injection of GG (2.6 mg, 1 ml) or

saline (1 ml). All data were normalized to their respective controls (ICV saline group).

(

LL–NN) Representative MS/MS spectra of HIS (LL), UCA (MM) and GLU (NN) in single HPC neurons with or without preincubation of HIS (0.2 mg/ml, 1 h) or GG

0.13 mg/ml, 2 h).

OO and PP) Representative MS/MS spectra of UCA (OO) and GLU (PP) in single HPC neurons with or without preincubation of UCA (0.7 mg/ml, 20 min) or GG

0.13 mg/ml, 80 min).

All digits within the columns represent numbers of total neurons measured from 3 mice. Data are represented as mean ± SEM. **p < 0.01, ***p < 0.001, ns, not

signiﬁcant (p > 0.05) based on unpaired t test.

A

E

B

C

D

HPC

Mock-UV

NAc

Mock-UV

DS

Mock-UV

**

CeA

Mock-UV

UVB

ns

UVB

2

.5

***

**

2.0

1.5

6

5

4

3

2

1

0

3

2

1

0

*

**

*

**

2

.0

ns

*

**

ns

***

*

***

1.5

1.0

0.5

0.0

ns

*

**

1.0

ns

1

7

30

17 30

IPPA

17 30

GLU

0.5 29 32

29 32

IPPA

29 32

GLU

29 28

UCA

29 28

IPPA

29 28

GLU

2

4

28

24 28

IPPA

24 28

GLU

0.0

UCA

FMGA

UCA

FMGA

UCA

FMGA

Mock-UV+ICV Saline

UVB+ICV Saline

UVB+ICV GG

Mock-UV+ICV Saline F

UVB+ICV Saline

UVB+ICV GG

shRandom

#2 #3

shRNA

#1 #2

1.5

#1

#3

**

2

1

0

ns

m/z 139

2.5

2.0

***

PFC

ns

***

Urocanase

1.0

7

2 kD

9

4

95

96

2

1

0

m/z

m/z 139

1

.5

.0

M₂

0

.5

1

9

4

95

96

GAPDH

m/z

36 kD

2

1

0

46 34 32

37 29 32

29 35 28

m/z 139

0.5

0.0

HPC

0.0

shRandom

shRNA

9

4

95

96

PFC

M₂

HPC

m/z

G

H

IPPAnase

I

FTCD

Mock-UV UVB +

UVB +

shRandom shRNA

Mock-UV

2

.5

**

ns

shRandom-UVB

shRNA-UVB

shRandom-UVB

shRNA-UVB

2

4

3

2

1

0

***

+

2.0

ns

***

[

UCA+H]

*

***

**

m/z 139

***

3

2

1

0

1

0

.5

.0

.5

***

*

**

ns

***

1

***

2

9

35

36

39

0

9

33 36 33 33 36 33

33 36 33

FMGA

33 36 33

GLU

29 30 31

UCA

29 30 31

IPPA

29 30 31 29 30 31

4

95 9694 95 9694 95 96

0.0

Mock-UV UVB

UVB +

m/z

UCA

IPPA

shRandom shRNA

FMGA

GLU

J

K

L

M

UCA

IPPA FMGA GLU

ShRandom-Urocanase

shRNA-Urocanase

ShRandom-IPPAnase

shRNA-IPPAnase

ShRandom-FTCD

shRNA-FTCD

2

.0

*

2.0

1.5

1.0

0.5

0.0

4

3

2

1

0

4

3

2

1

0

*

***

1.5

**

*

**

*

** *** ***

1

0

.0

.5

**

***

4

5

44

45 44

32

33

32 33

2

6

30

26 30

IPPA

26 30

GLU

25 26

UCA

25 26

IPPA

25 26

GLU

0.0

ICV GG

_

+

_

+

_

UCA

IPPA

FMGA

GLU

UCA

FMGA

+

N

R

O

P

Q

Mock-UV

UVB

Mock-UV

UVB

Mock-UV

UVB

Mock-UV

UVB

3

2

3

+

[

UCA+H]

m/z 139

[GLU+H]

m/z 148

[UCA+H]

m/z 139

[GLU+H]

m/z 148

2

1

0

2

1

0

2

1

+

/+

+/+

Gnat ;Cnga3

;

Gnat ;Cnga3

;

-/-

Gnat ;Cnga3

;

Gnat ;Cnga3 ;

+/+

Opn4 WT mice

1

0

Opn4-/- blind mice

0

101

9

4

95

m/z

96

101

102

m/z

103

94

95

m/z

96

102

m/z

103

i.v. Saline

i.v. UCA

S

i.v. Saline

i.v. UCA

T

i.v. Saline

i.v. UCA

3

2

1

0

+

3

+

[GLU+H]

m/z 148

+

[

GLU+H]

m/z 148

[GLU+H]

m/z 148

2

1

0

2

1

0

HPC

PFC

M₂

1

01

102

m/z

103

101

102

m/z

103

101

102

m/z

103

H O

U

V

2

i.v. Saline

i.v. UCA

x6000

i.v. Saline

i.v. UCA

*

**

4

3

+

4

[

GLU+H]

m/z 148

NAc

DS

GLU

2

1

0

1

3

2

1

0

01

102

103

m/z

4

3

+

[

GLU+H]

m/z 148

2

1

0

4

3

2

1

38

48

33

27

1

01

102

m/z

NAc

DS

5

4

3

ppm

2

Figure S4. Effects of UVB Exposure and i.v. UCA on UCA-GLU Pathway in the Brain, Related to Figure 4

(

A–D) Effects of UVB exposure on intracellular levels of metabolites involved in UCA-GLU pathway, including UCA, IPPA, FMGA and GLU, in single HPC (A), NAc

B), DS (C) and CeA (D) neurons. All data were normalized to their respective controls (mock-UV groups).

(

(legend continued on next page)

2

(E) Representative MS/MS spectra (Left) and normalized intensities (Right) of intracellular UCA in single neurons of PFC, M and HPC of mice with or without

mock-UV treatment, UVB exposure or ICV injection of GG (2.6 mg, 1 ml). Data were normalized to their respective controls (mock-UV groups without ICV injection

of GG).

(

F) Knockdown efﬁcacy of urocanase shRNA constructs. The western blot (Left) and normalized band intensity (Right) of urocanase in HEK293T cells, which were

9

treated by the lentiviral short hairpin RNAs against urocanase (shRNAs, 0.5 ml/side, 10 TU/ml) or random control shRNA (shRandom, 0.5 ml/side, 10 TU/ml).

G) Representative MS/MS spectra (Left) and normalized intensities (Right) of intracellular UCA showing the effects of intra-HPC injection of lentiviral shRNA

(

9

against Uroc1 (shRNA, 0.5 ml/side, 10 TU/ml) or random control shRNA (shRandom, 0.5 ml/side, 10 TU/ml) on UVB-induced elevation of intracellular UCA level in

9

single HPC neurons. Data were normalized to their respective controls (mock-UV groups).

(

H and I) The normalized intensities of intracellular UCA, IPPA, FMGA and GLU in single HPC neurons of mice exposed to UVB or mock-UV. These mice received

12

intra-HPC injection of AAV-shRNA (0.5 ml/side, 1.4 3 10 vg/ml) against IPPAnase (H) and FTCD (I) or random control shRNA (shRandom, 0.5 ml/side, 1.6 3 10

vg/ml) 3 weeks before single-cell MS tests. vg, vector genomes. Data were normalized to their respective controls (mock-UV groups).

(

J–L) The normalized intensities of intracellular UCA, IPPA, FMGA and GLU in single HPC neurons of mice which received intra-HPC injection of AAV-shRNA

1

2

12

0.5 ml/side) against urocanase (1.3 3 10 vg/ml) (J) IPPAnase (1.4 3 10 vg/ml) (K) and FTCD (1.4 3 10 vg/ml) (L) or random control shRNA (shRandom,

12

(

12

1

.6 3 10 vg/ml) 3 weeks before single-cell MS tests. vg, vector genomes. Data were normalized to their respective controls (shRandom groups).

(

M) The normalized intensities of intracellular UCA, IPPA, FMGA and GLU in single neurons from the HPC of mice after ICV injection of GG (2.6 mg, 1 ml) or saline

1 ml). All data were normalized to their respective controls (ICV saline group).

+

N–Q) Representative MS/MS spectra of UCA (N and P) and GLU (O and Q) in single HPC neurons of WT (Gnat ;Cnga3 ;Opn4 ) mice (N and O) and triple

/+

+/+

À/À

knockout (Gnat ;Cnga3 ;Opn4 ) blind mice (P and Q) after mock-UV (black line) or UVB (red line) exposure. All spectra were normalized to their respective

controls (mock-UV group).

(

R–T) Representative MS/MS spectra of intracellular GLU in single neurons from brain regions of PFC (R), M

line) or saline (black line).

U) MS/MS spectra (Left) and normalized intensities (Right) of intracellular GLU in single neurons from NAc and DS of mice with i.v. injection of UCA (20 mg/kg,

0 min) or saline. All data were normalized to i.v. saline group.

2

(S) and HPC (T) after i.v. UCA (20 mg/kg, 30 min, red

(

3

1

(

V) In vivo H MR spectra with (inset) and without water suppression, acquired from the M

All digits within the columns represent numbers of total neurons measured from 3 mice. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01,

**p < 0.001, ns, not signiﬁcant (p > 0.05) based on unpaired t test.

2

region in mice brain. GLU signal is highlighted (red) in blow-up spectra.

*

A

B

0

.8

.6

.4

.2

.0

0.8

0.6

0.4

0.2

0.0

3

2

19

17

_

20

29

20

27

_

20

_

+

_

+

Mock-UV

+

_

Mock-UV

UVB

shRandom

shRNA

+

_

+

_

UVB

+

_

+

_

ICV Saline

ICV GG

+

_

+

_

+

C

F

I

D

E

8

ns

4

0

1.0

0

.8

Mock-UV

UVB

+40 mV

30

6

4

2

0

.6

.4

.2

0

+40 mV

20

10

-

70 mV

-70 mV

0

2

0 ms

Mock-UV UVB

Mock-UV

UVB

G

H

pAAV-mCamKIIα

hChR2(H134R)-EGFP

-

Laser

473nm

^M2

M₂

M₁

Recording

CPu

DS

LV

NAc

100 ms

J

K

*

mEPSC

2

1

0

5

0

5

*

8

6

4

2

0

*

**

*

**

ICV Saline + Mock-UV

ICV Saline + UVB

_

ICV GG + UVB

Mock-UV

UVB

Mock-UV

UVB

ICV GG

+

_

Mock-UV

UVB

ICV GG

+

_

1

s

+

_

+

_

+

L

M

N₄

O ₄₀

P ₁_.₀

*

0

*

1

600

200

0

.8

.6

.4

30

20

0

8

00

0

20

0

4

10

0

0.2

0

80 160 240

0

Amplitude of

EPSC (pA)

Mock-UV UVB

5

0 ms

Figure S5. Effects of UVB Exposure on Glutamatergic Synaptic Transmission from HPC CA3 to CA1 and from M

2

to DS, Related to Figure 5

A) Quantitative frequency of mEPSCs of CA1 neurons from mice with or without mock-UV, UVB exposure and ICV injection of GG (2.6 mg, 1 ml).

B) Quantitative frequency of mEPSCs of CA1 neurons from mice with or without mock-UV, UVB exposure and intra-HPC CA3 injection of AAV-shRNA

(

1

2

12

0.5 ml/side, 1.3 3 10 vg/ml) against urocanase or random control shRNA (shRandom, 0.5 ml/side, 1.6 3 10 vg/ml). vg, vector genomes.

C and D) Effects of UVB exposure on number of synapses (N) (C) and release probability (Pr) (D).

(legend continued on next page)

(E) Effects of UVB exposure on the ratio between AMPA and NMDA receptor contribution to EPSCs. (Left) Sample traces representing EPSCs recorded while

holding cells at negative (À70 mV) and positive (+40 mV) membrane potentials. (Right) The ratio of amplitudes of the NMDA EPSC to those of the AMPA EPSC in

neurons of HPC of mice receiving mock-UV or UVB exposure.

(

F–P) Effects of UVB exposure on glutamatergic synaptic transmission from M

mCamKIIa-hChR2(H134R)-EGFP along the neuronal projection from M to DS. (G) Schematic image showing the electrophysiological recording of DS neurons

projecting ﬁbers. (H) Trace records of blue light (473 nm, 5 ms) evoked EPSC in DS neurons. Neurons responding to optical

2

to DS. (F) Representative image showing the viral expression of pAAV-

2

M2/DS

with optical stimulation of GLU

stimulation were selected for the following recordings (J-P). (J-L) The trace records (I), quantitative amplitudes (J) and frequencies (K) of mEPSCs of DS neurons

from mice with or without mock-UV (black dots, n = 23 neurons from 3 mice), UVB exposure (blue dots, n = 25 neurons from 3 mice) or ICV injection of GG (red

dots, 2.6 mg, 1 ml, n = 21 neurons from 3 mice). (L-P) A combination of optogenetic stimulation and MPFA allows the measurement of circuit-speciﬁc synaptic

M2/DS

properties. (L) EPSCs were triggered by optical stimulation of GLU

neuronal ﬁbers. Example traces of 100 overlap synaptic responses from mice exposed

to mock-UV or UVB. (M) MPFA of the example recordings in (L). (N and O) Quantal parameters quantal size (N) and number of synapses (O) were derived from the

parabolic ﬁt of the variance-mean analysis (mock-UV: n = 10 neurons from 3 mice; UVB: n = 12 neurons from 3 mice). (P) The release probability (Pr) was

calculated according to Equation 1 in star methods.

All digits within the columns represent numbers of total neurons measured from 3 mice. Data are represented as mean ± SEM. *p < 0.05, ***p < 0.001, ns, not

signiﬁcant (p > 0.05) based on unpaired t test.

A

E

I

B

C

D

H

L

2

1

00

50

00

7

60

5

40

3

2

1

0

70

60

50

40

30

20

10

0

**

***

2

50

200

*

ns

*

**

0

1

50

00

0

8

9

8

6

5

0

i.v. UCA

i.v. Saline

50

0

ICV Saline+Mock-UV

ICV GG+Mock-UV

0

F-L F-R

F

N

F-L F-R F

i.v. UCA

N

1

2

3 4

5

6

0

1

2

3 4

5

6

F-L

F-R

F-L

F-R

i.v. Saline

Trial

Mock-UV

UVB

F

G

ns

6

0

**

Mock-UV UVB

3

2

150

100

80

60

40

*

** **

*

***

*

**

*

4

ns

*

1

6

16 16 16

16 16 16 16

2

1

0

50

0

Mock-UV

UVB

6

8

2

0

2

8

27

28 27

IPPA

28 27

GLU

0

F-LF-R F

i.v. Saline

N

F-LF-R

F

N

0

i.v. UCA

UCA

FMGA

0

1

2

34

5

6

F-L F-R

F

N

F-L F-R

F

N

Trial

Mock-UV

UVB

J ₇₀

K

70

7

6

5

0

6

4

2

0

ns

6

5

4

3

0

ns

0

6

5

4

3

2

1

0

40

30

20

10

0

6

8

20

10

0

8

7

8

0

F-L

F-R

F-L

F-R

F-L

F-R

F

N

F-L

F-R

F-L

F-R

F-L F-R

F

N

F-L F-R

F

N

ICV Saline+UVB

ICV GG+UVB

ICV GG + Mock-UV

shRandom+UVB

shRNA+UVB

shRandom+Mock-UV shRNA+Mock-UV

Figure S6. Effects of UVB Exposure on Rotarod Learning and Object Recognition Memory in Male and Female Mice, Related to Figure 6

(

A)Time spent on the rotarod in the mice with i.v. injection of UCA (20 mg/kg, 30 min, red dots, N = 10 mice) or saline as control (blue dots, N = 11 mice).

B)Time spent on the rotarod in the mice with ICV administration of GG (13.2 mg, 1 ml, blue dots, N = 5 mice) or saline as control (black dots, N = 13 mice) followed

by mock-UV exposure.

C) The preference on familiar objects placed on different sites (F-L: familiar objects on the left side; F-R: familiar objects on the right side) during 3-min

familiarization in two groups of mice, which were arranged to receive mock-UV or UVB exposure 24 h after familiarization.

D) The preference of mice on familiar and novel objects during 3-min test at 2 h after familiarization. Mice received i.v. injection of UCA (20 mg/kg, 30 min) or saline

before test. F-L: familiar objects on the left side; F-R: familiar objects on the right side.

E) The preference of mice on familiar and novel objects during 3-min test at 24 h after familiarization. Mice received i.v. injection of UCA (20 mg/kg, 30 min) or

saline before test. F-L: familiar objects on the left side; F-R: familiar objects on the right side.

F–H) Effects of UVB exposure on hippocampal intracellular UCA-GLU metabolic pathway and neurobehaviors of female mice. (F) Normalized MS intensities of

intracellular UCA, IPPA, FMGA and GLU in single neurons of HPC of female mice exposed to mock-UV or UVB. Data were normalized to their respective controls

mock-UV groups). All digits within the columns represent numbers of total neurons measured from 3 mice. (G) Latency to fall on the accelerating rotarod for each

(

trial on days 1 and 2 in mice after mock-UV (black dots, N = 10 mice) or UVB (red dots, N = 14 mice) exposure. (H) The preference of female mice to familiar and

novel objects during a 3-min test at 24 h after familiarization. Mice received mock-UV (N = 6 mice) or UVB exposure (N = 8 mice) for 2 h before the test. F-L: familiar

objects on the left side; F-R: familiar objects on the right side; N: novel objects.

(

I) The preference on familiar objects placed on different sites (F-L: familiar objects on the left side; F-R: familiar objects on the right side) during 3-min famil-

iarization in two groups of mice, which were arranged to receive ICV injection of GG (13.2 mg, 1 ml) or saline followed by UVB exposure 24 h after familiarization.

J) The preference on familiar and novel objects during 3-min test at 24 h after familiarization. Mice received ICV injection of GG (13.2 mg, 1 ml) followed by Mock-

UV exposure. F-L: familiar objects on the left side; F-R: familiar objects on the right side.

K) The preference on familiar objects placed on different sites (F-L: familiar objects on the left side; F-R: familiar objects on the right side) during 3-min

(

9

familiarization in two groups of mice, which were arranged to receive intra-HPC CA3 injection of Uroc1 shRNA (0.5 ml/side, 10 TU/ml) or shRandom

9

0.5 ml/side, 10 TU/ml) followed by UVB exposure before test.

(

L) The preference on familiar and novel objects during 3-min test at 24 h after familiarization. Mice received intra-HPC CA3 injection of Uroc1 shRNA

9

0.5 ml/side, 10 TU/ml) or shRandom (0.5 ml/side, 10 TU/ml) followed by mock-UV exposure. F-L: familiar objects on the left side; F-R: familiar objects on the

9

right side.

All digits within the columns represent the numbers of total neurons measured from 3 mice or the numbers of mice in each group. Data are represented as mean ±

SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not signiﬁcant (p > 0.05) based on unpaired t test.

Article Doi

Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate Biosynthetic Pathway in the Brain

DOI: 10.1016/j.cell.2018.04.014

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.cell.2018.04.014

Full text of DOI:10.1016/j.cell.2018.04.014

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