with a concentration of 0.2 mg/ml for in vivo
experiment with a dosage of 2 mg/kg (0.1 ml/
10 g, intraperitoneally). 5 mM flupirtine stock
solution (0.9% saline containing 0.3% dimethyl
sulfoxide/DMSO, v/v) was added to artificial
cerebrospinal fluid (ACSF) to reach a concen-
tration of 50mM for in vitro electrophysiology.
Flupirtine was prepared at a concentration of
2 mg/ml in 0.9% saline containing 0.3% DMSO
(v/v, vehicle) for in vivo experiments with
adosageof20mg/kg(0.1ml/10g,intra-
peritoneally). Flupirtine solution was ultrasoni-
cated prior to application. Counterbalanced
crossover design was used for in vivo pharma-
cology experiments to reveal the drug’s effect.
Two rounds of drug administrations were
separated by at least one week for a complete
wash-out of drug’s effect. 4-Aminopyridine
(4-AP) was purchased from Sigma-Aldrich (Cat.
no. 275875). 100 mM 4-AP stock solution (0.9%
sodium chloride/saline as vehicle) was added
to ACSF to reach a concentration of 50mM for
in vitro electrophysiology ofIM experiment.
MK6096 (Merck) was prepared at a concen-
tration of 2 mg/ml in a mixture (v/v, vehicle) of
50% 0.9% saline and 50% Poly ethylene glycol
(average Mn 400, PEG400, Sigma-Aldrich Cat.
no. 202398) for in vivo pharmacology experi-
ment as previously described ( 13 ).
In vitro electrophysiology
All the in vitro electrophysiology experiments
were performed during the light phase (ZT3-
ZT9). 3-9 mice were used each group. Slices
were randomly assigned to groups examining
effects of XE991 or flupirtine on M current in
the in vitro pharmacology experiments.
Slice preparation
Mice from both groups were decapitated after
anesthesia with sevoflurane or perfused with
ice-cold slicing solution under anesthesia. To
increase the chances of acquiring a healthy
slice, we used a sucrose-based or choline-based
ACSF for brain slice preparation to reduce the
cell excitotoxicity and loss during slice prepa-
ration ( 53 ). After decapitation, the brain was
rapidly dissected and immersed in ice-cold
sucrose/choline-based ACSF slicing solution
(pH 7.4, 95% O 2 /5%CO 2 ). 300mm-thick coronal
brain slices containing Hcrt neurons with eYFP
fluorescence were sectioned using a VT1200s
vibratome (Leica Microsystems). After ~20 min
incubation at ~35°C, the slices were stored
at room temperature. Slices were used for
maximally 5 hours after dissection. Experi-
ments were performed at room temperature
21° to 24°C.
Recording and data analysis
During experiments, slices were superfused
with a physiological extracellular solution con-
taining: 125 mM NaCl, 2.5 mM KCl, 25 mM
NaHCO3, 1.25 mM NaH2PO4, 25 mM D-glucose,
2mMCaCl2,and1mMMgCl2(pH7.4in95%
O 2 /5% CO 2 , ~325 mOsm). Neurons were cho-
sen based on eYFP expression and visualized
with an Olympus BX51WI with Nomarski optics
connected to a camera (Q-imaging). Thick
wall borosilicate pipettes (1B150F-4, World
Precision Instruments Inc.) were pulled using
a P-97 puller (Sutter Instruments) and electro-
des with a resistance of 3-6 megohms were
used for recording. Intracellular solution used
for whole-cell recording contained: 120 mM
K-methyl-sulfonate, 10 mM NaCl, 10 mM EGTA,
1 mM CaCl2, 10 mM HEPES, 0.5 mM NaGTP,
5 mM MgATP, pH adjusted to 7.2 with KOH, os-
molarity adjusted to 305 mOsm with sucrose;
0.2% biocytin was added for post-hoc staining.
Neurons were recorded under current-clamp
to examine excitability, or under voltage-clamp
to examine PSCs. 1 s step currents from–50 pA to
300 pA were used to evoke AP firing. For
optogenetic stimuli, a 15-ms blue light pulse
(3.4 mW, calibrated with Thorlab light meter)
was given at 1 Hz, 5 Hz, 10 Hz, 15 Hz, and
20 Hz in a randomized manner for 10 s to
compare light-induced activity between the
young and aged groups, and the interval be-
tween sweeps was 20 s. Data were acquired
with a Multiclamp 700B amplifier (Axon In-
struments, USA), and sampled at 10 kHz.
Stimulus generation and data acquisition were
performed using pClamp 10. Data were ana-
lyzed using Stimfit 0.14.9 (www.stimfit.org)
and R 3.5.1 (the R project for statistical com-
puting). RMP values were measured and
averaged from temporal windows (at least
50 ms prior to the peak of a given AP for
spontaneously firing neurons) with minimal
membrane potential variance ( 54 ). The RMPs
were determined without predicted/measured
junction potential correction. All the ampli-
tudes of APs and spikelets were calculated
from RMPs. Depolarization events with a peak
value above–20 mV, and with a half width
shorter than 6 ms were qualified for spikelet
analyses. PSC recording from non-fluorescent
neuron innervated by fluorescent Hcrt neuron
expressing ChR2-eYFP was performed as
illustratedinfig.S4A.ForthePSCfailure
analysis, a success PSC was considered when a
current deflection bigger than 10 pA occurred
time-locked to the light pulse. The investiga-
tors were blind to the group information while
conducting the data analyses.
LC neurons were recorded in slices prepared
from WT young (2 to 3 months) and aged (18
to 21 months) mice, infused with biocytin,
followed by antibody staining against tyrosine
hydroxylase (TH). Only the neurons positive
for both biocytin and TH were included for
data analyses.IMrecording
For recording of the slowly deactivating
M-current (IM) mediated by KCNQ2/3, perfo-rated patch recordings were used to maintain
the integrity of second messenger signaling
cascades and minimize current rundown. The
pore-forming antibiotic nystatin was dissolved
in DMSO at 50 mg/ml. This stock solution was
diluted in an internal pipette solution and
vortexed and ultrasonicated for a final con-
centration of 100 to 200mg/ml. Pipette tips were
prefilled by brief immersion into antibiotic-
free solution and then pipettes were back
filled with nystatin. After the cell-attached con-
figuration was attained, the access resistance
was periodically monitored with hyperpolariz-
ing voltage steps (10 mV, 20 ms) and capaci-
tative transients were cancelled. After 10 to 20 min,
recording was started once the access resistance
stabilized. The recording was terminated if a
sudden change in access resistance occurred.
Extracellular solution contained 4-AP (5 mM)
to minimize contamination by other potas-
sium currents, and AMPARs, glycine receptors
and GABAA receptors were blocked by 6,7-
dinitroquinoxaline-2,3-dione (DNQX, Tocris
Cat. no. 0189, 10mM), strychnine (Sigma-
Aldrich Cat. no. S0532, 1mM), (2R)-amino-5-
phosphonopentanoate (APV, Tocris Cat. no.
0106, 100mM) and bicuculline (Sigma-Aldrich
Cat. no. 285269, 10mM).IMwas recorded using
a standard deactivation protocol (1000 ms hy-
perpolarizing steps to -30 mV from a holding
potential of–20 mV every 10 s, intersweep
holding potential–20 mV).IMdid not inacti-
vate with this protocol, while contamination
by other voltage-gated currents was minimized.
IMwas measured as the inward relaxation
current caused by deactivation ofIMduring
this voltage step (Fig. 4, G and H). After ob-
tainingatleastastable5minbaseline,XE991
(50mM) or flupirtine (50mM) was applied. The
effect of XE991 or flupirtine was determined
by comparing averagedIMamplitudes over a
5 min period just before XE991 or flupirtine
application with averagedIMamplitudes dur-
ing the 5 to 10 min period after XE991 or
flupirtine application.Array tomography (AT)
Tissue preparation
Array creation and immunohistochemistry
were described in detail in a previous publi-
cation ( 55 ). In short, a small piece of tissue
(~2mmhighby1mmwideby1mmdeep),
covering the LH containing eYFP-labeled Hcrt
neurons, was microwave-fixed in 4% Para-
formaldehyde (PFA). The fixed tissue was
then dehydrated in graded steps of ethanol,
and then embedded in LR White resin over-
nightat50°C.Theembeddedtissuewassec-
tioned on an ultramicrotome at a thickness of
70 nm and placed as a ribbon array directly on
gelatinorcarboncoatedglasscoverslips.The
ultrathin physical sectioning allows AT to
achieve true isotropic voxels of ~100 nm. To
assure that the brain tissue from animals wereLiet al.,Science 375 , eabh3021 (2022) 25 February 2022 10 of 14
RESEARCH | RESEARCH ARTICLE