Sensory Reflexes in SCN2A-associated ASD: Impaired Cerebellar Plasticity Hypersensitizes

Neuron
Impaired cerebellar plasticity hypersensitizes

sensory reflexes in SCN2A-associated ASD
Graphical abstract Authors
Chenyu Wang, Kimberly D. Derderian,
Elizabeth Hamada, ..., Nadav Ahituv,
SCN2A/Nat.2 Sen2a wild type Scn2aSo's heterozygote
_Serzewidupe Nore Guy Bouvier, Kevin J. Bender
functionin DNDN TXDAN
cerebellum ‘7NDN Correspondence
Vestibulo-ocular reflex measurement guy.bouvier@cnrs.fr (G.B.),
kevin.bender@ucsf.edu (K.J.B.)
Normal VOR Elevated VOR
10° UKK)K) In brief
oscillation head angle eyeangle head angle eye angle Impaired function in the sodium channel
fin protocol to reduce VOR gain gene SCN2A is a major risk factor for
CLT Ih Plastic Inflexible autism spectrum disorder. Here, Wang
ae
~ =tee
and colleagues show that heterozygous
loss of SCN2A in the cerebellum alters
reflexive eye movements in both mouse
CRISPR-activation rescue of Scn2a corrects VOR plasticity
anal apa
normal transcription CRISPRa-boosted
and man and that a translatable gene
Normal & plastic therapy rescues such movements
GAWKWY in mouse.
1.2
Highlights
e Vestibulo-ocular reflex (VOR) is hypersensitized by SCN2A
loss of function (LoF)
e Hypersensitivity is present in children with SCN2A LoF and

Scn2a*"” +/— mouse models
e VOR gain cannot be reduced in Scn2a*’~ mice due to

cerebellar plasticity deficits
e VOR gain plasticity can be rescued with a CRISPR-activator-

based therapeutic
Wang et al., 2024, Neuron 112, 1-12

May 1, 2024 © 2024 Elsevier Inc.
https://doi.org/10.1016/j.zneuron.2024.01.029 @ CelPress
Please cite this article in press as: Wang et al., Impaired cerebellar plasticity hypersensitizes sensory reflexes in SCN2A-associated ASD, Neuron
(2024), https://doi.org/10.1016/j.neuron.2024,01.029
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Impaired cerebellar plasticity hypersensitizes

sensory reflexes in SCN2A-associated ASD
Chenyu Wang,’ Kimberly D. Derderian,” Elizabeth Hamada,’ Xujia Zhou,* Andrew D. Nelson,”? Henry Kyoung,”
Nadav Ahituv,°° Guy Bouvier,°”* and Kevin J. Bender?:*.8.*
‘Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA, USA
2Department of Neurology, University of California, San Francisco, San Francisco, CA, USA
Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA
Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA.
SInstitute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA
®Department of Physiology, University of California, San Francisco, San Francisco, CA, USA
Université Paris-Saclay, CNRS, Institut des Neurosciences Paris-Saclay, 91400 Saclay, France
®Lead contact
“Correspondence: guy.bouvier@cnrs.fr (@.B.), kevin.bender@uesf.edu (K.J.B.)
https://doi.org/10.1016/j.neuron.2024.01.029
SUMMARY
Children diagnosed with autism spectrum disorder (ASD) commonly present with sensory hypersensitivity or
abnormally strong reactions to sensory stimuli. Such hypersensitivity can be overwhelming, causing high
levels of distress that contribute markedly to the negative aspects of the disorder. Here, we identify a mech-
anism that underlies hypersensitivity in a sensorimotor reflex found to be altered in humans and in mice with
loss of function in the ASD risk-factor gene SCN2A. The cerebellum-dependent vestibulo-ocular reflex (VOR),
which helps maintain one’s gaze during movement, was hypersensitized due to deficits in cerebellar synaptic
plasticity. Heterozygous loss of SCN2A-encoded Nay1.2 sodium channels in granule cells impaired high-fre-
quency transmission to Purkinje cells and long-term potentiation, a form of synaptic plasticity important for
modulating VOR gain. VOR plasticity could be rescued in mice via a CRISPR-activator approach that in-
creases Scn2a expression, demonstrating that evaluation of a simple reflex can be used to assess and quan-
tify successful therapeutic intervention.
INTRODUCTION ASD appears to be sensitive to both fast and slow movements,

reflecting a hypersensitivity not seen in neurotypical chil-
Altered sensitivity to sensory input is a hallmark of autism dren.'°"? The mechanisms for this heightened sensitivity are
spectrum disorder (ASD); over 90% of children with ASD unknown.
experience heightened sensitivity to sensory stimuli, spanning Here, we show that VOR gain is hypersensitized in children
sensory modalities.“ Although the neuronal mechanisms and mouse models of SCN2A loss of function (LoF), a condition
that contribute to this hypersensitivity have been examined that carries the highest risk for ASD of any gene identified via
extensively in forebrain circuits,>° emerging evidence sug- clinical exome sequencing.'*"* SCN2A encodes the neuronal
gests that changes in sensory sensitivity can occur even sodium channel Nay1.2."° This channel supports action potential
at much earlier stages of sensory processing, including (AP) initiation and propagation in multiple cell classes,'°"°
those that support sensory reflexes.”'* Thus, changes in re- including cerebellar granule cells whose activity is key for modu-
flexive behavior can provide a window into the cellular and cir- lating VOR gain.°° SCN2A LoF impaired synaptic plasticity
cuit dysfunctions that underlie altered sensory experience between granule cells and Purkinje cells, in turn hypersensitizing
in ASD. VOR by preventing the synaptic plasticity that typically readjusts
The vestibulo-ocular reflex (VOR) is one such reflex that is VOR amplitude.’’** Remarkably, VOR plasticity could be
affected in ASD. This reflex transforms vestibular sensory infor- restored in adolescent mice by upregulating Scn2a expression
mation generated by head movements in one direction into eye levels via a CRISPR-activator (CRISPRa)-based approach.
movements in the opposite direction, thereby helping stabilize Overall, these data demonstrate how innate reflexes provide
images on the retina. In neurotypical subjects, this reflex is a window into cerebellar dysfunction in ASD that is both
engaged well by fast head movements, but not by slower well conserved across species and sensitive to therapeutic
head movements. By contrast, VOR assessed in children with intervention.
Neuron 712, 1-12, May 1, 2024 © 2024 Elsevier Inc. 1

(2024), https://doi.org/10.1016/j.neuron.2024.01.029
| @ CelPress Neuron
A Human VOR B_ Neurotypical SCN2A LoF
° .— Inertial head angle

IR Camera : ween wo (scaled)
“eo pe eee
eye eye
Sitting in angle angle
swivel chair
Mouse VOR
IR Camera
+/+
+H
&
o
VOR Gain
VOR Gain
Oo
a
%
ie)
>
+/- 0.05 0.1 02 04 0.6 08 1.0

Rotation Frequency (Hz)
Figure 1. VOR gain is elevated in Scn2a haploinsufficiency conditions

(A) Schematic of purpose-built eye-tracking apparatus for VOR assessment in children. Head movements are measured from a device located at the center of the
head, while the right eye is imaged under infrared illumination (840 nm LED). Participants are seated ina swivel chair and oscillated +5° at ~0.4 Hz to assess VOR gain.
(8) Head angle (purple) and contraversive eye angle for neurotypical (Nt, black) and SCN2A LoF conditions (cyan). Lines represent the average of a single cycle
from all participants; shaded area is SEM. Dashed red lines indicate +5° range.
(C) VOR was assessed in mice head-fixed at the center of a rotating table, imaged under IR illumination.
(0) VOR at 0.4 Hz rotation frequency, displayed as in (B), for Scn2a”’* (left, black) and Sen2a“’~ mice (right, cyan).
(©) Baseline VOR gain in human and mouse. Circles are individuals; box plots are medians, quartiles, and 90% tails. n: 1 Nt, 5 LOF humans; 13 +/+, 12 +/— mice.
Mann-Whitney test p values shown.
(F) VOR gain across rotation frequencies in mouse. Lines connect repeated tests in single mice. Asterisks: p < 0.0001, Friedman teston overall distribution; Mann-
Whitney test on individual frequencies, Holm Sidak correction.
RESULTS VOR gain was assessed with +5° sinusoidal head oscillations
in the dark (~0.4 Hz, peak angular velocity: 12.5°/s). Consistent
SCN2A LoF hypersensitizes VOR gain in humans with previous work, VOR gain was well below unity in neurotyp-
and mice ical children."° By contrast, VOR gain was near unity in children
Children with SCN2A LoF variants typically have limited to no with SCN2A LoF variants (Figures 1B and 1E).
verbal repertoire and have difficulty following instructions but Protein truncation and resultant nonsense-mediated decay
often are very comfortable with caregivers.'° With these behav- accounts for the vast majority of SCN2A LoF cases."*'° As
ioral aspects in mind, we developed a lightweight, helmet- such, mice heterozygous for Scn2a are an ideal model system
mounted infrared eye-tracking system paired with an inertial for studying LoF-related physiology. We therefore assessed
measurement unit (IMU) to assess both eye and head move- VOR in Scn2a”~ mice head-fixed on a rotational platform using
ment, respectively (Figures 1A and Si). Children aged 3-10 similar test conditions as above (5° rotation at 0.4 Hz in the
years were seated either alone or on a caregiver's lap, and dark). Like children with SCN2A LoF, Scn2a"’~ mice showed a
2 Neuron 112, 1-12, May 1, 2024

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A__ Baseline (dark) Gain-down plasticity induction Post-induction (dark) Figure 2. Impaired VOR gain-down behav-
Virtual ioral plasticity in Scn2a*”~ mice
64 channel ae (A) Experimental design for VOR gain-down induc-
silicon probe y tion and simultaneous in vivo silicon probe
recording in the cerebellar floccular complex. Inset,
recording location in left hemisphere. Left: baseline
VOR recording in the dark. Middle: VOR gain-down
induction with virtual drum as visual stimulus. During
Scn2a”
gain-down induction, visual stimulus was moved in
Post Gain-down Induction phase with table. Right: post-induction VOR
recording in the dark.
(B) Head and eye angles in Scn2a*’* (black) and
Scn2a*’~ (cyan) during baseline (dark colors) and
post-induction (light colors) with table rotation
(purple). Dashed red lines indicate =5° range.
(C) Head angle (purple) and contraversive eye angle
‘ before (darker shade) and after (lighter shade) gain-
ns. down induction. Data presented as in Figure 1D.
VOR Gain (0,4) ° (0) VOR gain before and after gain-down induction.
Data color-coded as in (B). Bars connect data from
Baseline LS individual mice. *p < 0.001, Wilcoxon signed-rank
test, “p < 0.0001, Mann-Whitney test.
*. (&) Putative Purkinje cell unit recordings before and
after gain-down induction. Traces are aligned from
Base Post Base Post table rotation onset.
Scn2a°"— Scn2a"" (F) Average Purkinje cell simple spike firing fre-
Sen2a*” quency during sinusoidal head rotation, before and
after gain-down induction, displayed as in (0D).
Scn2a“"* (black, closed circles, n = 22 units, 6 mice),
Scn2a*~ mice (cyan, n = 13 units, 5 mice). *p < 0.05,
all conditions, mixed-effects modeling, “p < 0.001,
Wilcoxon signed-rank test.
(G) Normalized Purkinje cell simple spike firing fre-
quency during induction protocol, normalized to
firing rate in first minute per unit. Circles and bars are
mean + SEM, binned per minute.
>g (H) Normalized change in VOR gain vs. normalized
2 ae
6s, 7 change in simple spike rate (normalized per unit and
a & x g oer averaged across units per animal). Circles are color-
s 2 Sa vt coded as in (D) and represent individual animals.
2i o l
>
HE ivd
&2ee
Eg
0
01 Base Post Base Post Time (min)
i
Norm. Spike Rate . . .
Sen2a’ Sen2a’ (Post / Baseline) concert with the animal (Figure 2A). During
induction, both WT and Sen2a"’~ mice
tracked the visual stimulus, completely
VOR gain near unity, significantly higher than wild-type (WT) lit- suppressing VOR despite the animal experiencing head move-
termates. This elevated gain persisted over a wide range of rota- ment (Figures S2A and S2C). Typically, this strong VOR cancel-
tion frequencies (Figure 1F). Together, these results suggest that lation during the induction protocol results in long-lasting reduc-
aberrantly high VOR gain may serve as a readout for SCN2A LoF tions in VOR gain, assessed again in the dark right after
in both mouse and human. induction. This persistent reduction in gain was indeed observed
in WT mice. By contrast, VOR gain, when assessed in the dark
VOR hypersensitivity is associated with an inability to after induction, immediately returned to unity in Scn2a”~ mice
modulate VOR gain (Figures 2B-2D). Efforts to increase gain using a “gain-up” pro-
VOR gain is plastic and is adaptively increased or decreased tocol, where the visual stimulus moved in the opposite direction
throughout life to maintain image stability on the retina. The to the animal, were successful in WT animals. By contrast, gain
high gain in humans and mice suggests that VOR plasticity has. could not be increased in Scn2a"”~ mice using the same proto-
been compromised and that SCN2A LoF impairs one’s ability col, possibly because gain was near 1.0 at baseline (Figures
to reduce VOR gain. To test this, we implemented a standard SS5A-S5F). Thus, in Scn2a*”- mice, VOR gain is elevated and
protocol that decreases VOR gain, termed “VOR gain-down.” cannot be altered by protocols designed to decrease or increase
Here, instead of rotating the animal in the dark, the animal was gain.
presented with a visual stimulus of high-contrast vertical bars, In the cerebellum, the floccular complex is critical for modu-
displayed on surrounding computer monitors, that moved in lating VOR gain.*°°° VOR plasticity has been proposed to be
Neuron 112, 1-12, May 1, 2024 3

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Figure 3. Impaired cerebellar plasticity in

Scn2a*’- conditions
(A) Sagittal section of mouse cerebellum in an ani-
mal with eGFP knocked into the Scn2a locus. Note
GFP expression in granule cell layer and molecular
layer and lack of expressing in Purkinje cell layer.
(8) High-magnification single-optical confocal sec-
tion of region highlighted in yellow square in (A).
‘Area co-stained for eGFP, VGlut1, and DAPI. Top
panels are single color. Bottom panels are overlays
of 2 oF 3 colors to show coexpression of eGFP and
valutt.
(C) Left: two EPSCs (20 Hz) before and after 166-Hz-
burst LTP induction in Sen2a‘* (black, n = 6 cells
from 4 mice) and Scn2a*’~ (cyan, n = 6 cells from
4 mice) in the floccular region of the cerebellum.
Inset: paired-pulse ratio before and after LTP in-
duction. “p< 0.01, Wilcoxon signed-rank test. Right:
c Flocculus first EPSC amplitude, normalized to pre-induction
Scn2a"* Scn2a"* a 1p average. Data are binned each minute. Averaged
5 _ &E | induction
‘uct Senza" baseline first EPSC amplitude (in pA) in flocculus:
ih Baseline Fd
atttteHeetetta
'
ad Scn2a*'*: 451.54 + 66.30; Scn2a~: 417.41 + 37.28.
Baseline Circles and bars are mean « SEM.
| Post * a4 =
a {D) Same as (C), but for cells in vermis. Scn2a the
14d E ae
black, n = 6 cells from 6 mice; Scn2a*’, cyan,
100 pA =& a a”a 2 to 10 30 n= 10 cells from 4 mice. Averaged baseline first
ems oad we on Time (min) EPSC amplitude (in pA): Scn2a*’*: 359.50 + 67.89,
Scn2a"’: 383.42 + 94.33,
D ‘Scn2a"* ‘Sen2a* Vermis
5 - 1 ‘Sen2a""
6 induction
Baseline Ee< =|
Baseline 9 in their parallel fiber axons that make
Post 2 excitatory synapses onto Purkinje cells.
af j Gy
e& Consistent with this, we find that a cyto-
100 pa 2 . 52E solic eGFP expression in an animal with
lasms Wak nr) Time 40(min) eGFP knocked into the Scn2a locus is
found in both the granule cell layer and mo-
lecular layer and that eGFP expression in
mediated in part by the modulation of Purkinje cell activ- the molecular layer overlaps with VGlutt, a marker of granule
ity.°?°"5> To test whether the firing rate of Purkinje cells cell parallel fibers (Figures 3A and 3B).°"
changes following VOR gain-down induction in Scn2a"’ VOR gain-down plasticity is correlated with long-term poten-
mice, we used silicon probes to monitor extracellular activity tiation (LTP) of parallel fiber-to-Purkinje cell synapses.°*°7°*
from the left floccular complex before, during, and after gain- Thus, deficits in granule cell function in Scn2a*/~ mice may
down induction, contralateral to the imaged eye (Figures 2 affect the synaptic plasticity that mediates VOR plasticity. To
and S8). Putative Purkinje cell units were modulated during test this, we made acute slices of cerebellum and stimulated
head rotation in both WT and Scn2a‘’~ mice, on average parallel fibers with a high-frequency, burst-based LTP protocol
preferring clockwise head rotations over counterclockwise ro- and monitored excitatory postsynaptic current (EPSC) ampli-
tations (Figure S4). In addition, Purkinje cell firing rates were tude and paired-pulse ratio (PPR) in Purkinje cells. Experiments
increased following gain-down induction in WT mice (Figures were performed both in vermis, as this region is commonly
2E and 2F). Firing rate changes occurred within 10 min of in- studied in ex vivo cerebellar studies, and in flocculus, as this re-
duction onset (Figure 2G) and were consistent across all ani- gion is important for VOR plasticity. This protocol evoked
mals studied (Figure 2H). But in Scn2a"’~ mice, Purkinje cell ac- robust potentiation of EPSCs in WT mice (Figures 3C and 3D)
tivity did not increase following gain-down induction. Instead, and was correlated with a reduction in PPR, suggesting that
firing rate decreased modestly, indicating that normal plasticity this induction protocol acts, at least in part, to increase release
mechanisms were impaired in Scn2a’”~ mice (Figures 2E-2H). probability.°*"° By contrast, LTP was not observed in Scn2a*’
Thus, both Purkinje cell activity in vivo and VOR plasticity are mice, and PPR was unchanged (Figures 3C and 3D). Instead,
affected in Scn2a"”~ mice. a small decrease in EPSC amplitude was apparent, and
this decrease could be blocked by the mGluR1 antagonist
Scn2a heterozygosity alters granule-to-Purkinje cell CPCCOEt (Figure S5H). This suggests that mGluR1-dependent
synaptic transmission and plasticity long-term depression (LTD) mechanisms were co-activated by
Within the cerebellum, Nay1.2 is expressed predominantly in this LTP induction protocol and that LTD was intact in Scn2a*’
granule cells (Figures 3A-3C),"®°° with the highest density conditions. To test this, we induced LTD by pairing EPSCs with
4 Neuron 112, 1-12, May 1, 2024

Neuron @ CellPress
A. Granule cell excitability B Vermis Flocculus C Vermis — Flocculus

Scn2a"* Son2a . 5bo 30 S407,
2, . 40 Le
=
hau —
so DMM —
=
8 20
bo } coffe]
£
so]
iT im 2 10 E-cof* + 60
< 0 0” oe
7 UN R40 a 20” 40 6" 2 "a <ey te
i mM Current (pA) Current (pA)
SUL ao INN = Zale
>
S200
*
™deH *
an 3 BS FEL 100
20 1 3g S i! a
UL -200 a8 0 Oy ay 0 4) 4/-
10 pA s
D Purkinje cell excitability (vermis) E500 _

e F40p | Soy,
= = = 400 18g
senza” |IMINIININNININM 20 mv
=| Eo gle] syPes
S g % 2004
100 ms & -605 * 4
Scn2a’ a" | ems -500 I
woo OE 4 ie4 Boda Jap. +
V,, (mV)
F Parallel fiber volley (vermis) G Parallel fiber to
Purkinje cell EPSC
50 Hz 166 Hz 10. 50 Hz it
Scn2a‘* gt ”
\ ite S 166 Hz
4 | > a+ | 100 pA
tH i itt Sos ia) eame
= 166 Hz B20
| S10
Son2a* 2 2i”
e Es 10
4 \ aa | | n 5 28
[ Ik 20s, 0SE
100 | 200 S 0°-—____
| [250.5 msmv Time (ms) 0 400
Time (ms)
Figure 4. Impaired cerebellar granule cell excitability in Scn2a*” conditions

(A) APs generated by current injection (10-50 pA, 300 ms) in Scn2a*’* (black) and Scn2a*’~ (cyan).
(8) Top: APs (spikes) per 300-ms stimulation epoch for each current amplitude in the vermis (left) and flocculus (right) (lines and shadow are mean + SEM of
population, *p < 0.05 of slope between 20 and 40 pA). Bottom: near-rheobase APs plotted as dV/dt vs. voltage (phase-plane plot) from Scn2a“’* and Scn2a*’~
(©) AP threshold (top) and peak dV/dt (bottom) in Scn2a‘* (vermis: 13 cells from 3 mice; flocculus: 11 cells from 1 mouse) and Scn2a“’~ (vermis: 16 cells from
3 mice, flocculus: 15 cells from 2 mice). “p < 0.05, Mann-Whitney test. Circles are single cells, while boxes are medians and quartiles with 90% tails.
(0) Spontaneous Purkinje cell AP train in Scn2a*’* and Scn2a‘’~. A single AP is highlighted on the right.
(&) Left to right: single AP, phase-plane plot of all APs in train, and summary AP threshold and peak dV/dt in Scn2a”’* oa and Scn2a
(F) Left: parallel fiber volleys evoked in trains of 10 APs at 50 Hz or 20 APs at 166 Hz. Right: fiber volley amplitude, normalized per volley to first event in train, then
averaged across recordings. Lines and bars are mean + SEM. 50 Hz (top) in Scn2a*’* (black, n= 5 from 2 mice) and Sen2a*~ (cyan, n= 7 from 4 mice). 166 Hz
(bottom) in Scn2a*’* (black, n = 9 from 3 mice) and Scn2a*/-~ (cyan, n = 9 from 4 mice).
(G) Top: parallel-fiber-evoked EPSCs in Purkinje cells from train of 20 stimuli at 166 Hz. Bottom: charge transfer, normalized to transfer from EPSC1 in Scn2a oa
(black, n = 10 cells from 2 mice) and Sen2a‘/~ (cyan, n= 9 cells from 2 mice) conditions.
the depolarization of Purkinje cells to mimic climbing fiber features of the granule cell-Purkinje cell circuit. Consistent with
input.°°°* LTD was no different across WT and Scn2a”~ con- immunostaining localization of Nay1.2,"° intrinsic excitability
ditions (Figure S5G). Thus, a lack of VOR gain-down plasticity was impaired in granule cells in Scn2a"’~ mice in both vermis
may be due to difficulty in potentiating synapses between and flocculus. Granule cell APs were slower to rise, and fewer
granule and Purkinje cells in Scn2a’”~ mice. Furthermore, the ‘APs were evoked per given somatic current stimulus (Figures
decrease in Purkinje cell firing rates observed after gain-down 4A-4C). By contrast, APs in Purkinje cells, which lack Nay1.2
induction in Scn2a*/~ animals may be associated with LTD channels,”°°° were unaffected by Scn2a heterozygosity (Figures
mechanisms that remain intact. 4D and 4E). This suggests that changes to Purkinje cell firing rate
To determine the underlying cellular excitability deficits that plasticity in vivo are due to alterations in presynaptic granule
impair plasticity, we examined intrinsic and synaptic excitability cell input.
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A Senzainal = Bg6-Gre (wT) a6-Cre::Sen2za" CY Figure 5. Scn2a heterozygosity in granule

granule cells 3 28, cells alone impairs VOR gain-down plasticity
COD ONY Ne 2" =<] (A) Experimental design for VOR gain-down induc-
/foos\ = ey tion as in Figure 2, but for Sen2a™ mice crossed to
——s wy, \ & the alpha6-Cre driver line (6-Cre), which restricts
06-CresSen2a"" & 4 Jease post Base Post Cre expression largely to cerebellar granule cells.
es Ul, AS 7 0" "a6-Cre a6-Cre _(B) Head angle (purple) and contraversive eye angle
Fi Ae Re (WT) Scn2a*" before (darker shade) and after (lighter shade) gain-
down induction for #6-Cre not crossed to Scn2a"””
D a6-Cre (WT) 6-Gre::Sen2a°" animals (black, WT-equivalent), or 6-Cre::Scn2a"”™
Baseline mice (green).
(©) VOR gain before and after gain-down induction.
[some Data color-coded as in (B). Bars connect data from
‘\ vn individual mice. “p < 0.001, Wilcoxon signed-rank
Post. att wil ett hr 400 pv |wii r i test, *p < 0.0001, Mann-Whitney test.
Induction (O) Putative Purkinje cell unit recordings before and
after gain-down induction. Traces aligned to table
E ~~ F is G rotation onset.
160) 5 Fal s (©) Average Purkinje cell simple spike firing frequency
a 2 . ° 214 so . during sinusoidal head rotation, before and after
= 100 . ‘ 3 or O51 gain-down induction, displayed as in (C). 6-Cre WT
2 s * = S8 * (gray, open circles, n = 24 units, 5 mice) and 6-
e : E210 as e, Cre::Scn2a*" mice (green, n = 28 units, 5 mice).
2 50 ‘ we, 53 *p < 0.005, all conditions, mixed-effects modeling,
a : 3 aon *p=0.01, Mann-Whitney test. Note that baseline firing
7 ~ 5 D 5 7 rates are not different than those observed in consti-
Base Post Base Fost Time (min) Norm. Spike Rate tutive Sen2a~ mice: Scn2a“’~ baseline firing: 45.5 +
(Post / Baseline) 7.5 Hz, n= 13 units; a6-Cre::Sen2a"”: 52.5 + 5.2 Hz,
n= 28 units; p = 0.34, Wilcoxon signed-rank test.
(F) Average Purkinje cell simple spike firing frequency during induction protocol, normalized to firing rate in first minute per cell. Circles and bars are mean + SEM,
binned per minute.
(@) Normalized change in VOR gain vs. normalized change in simple spike rate (normalized per unit and averaged across units per animal). Circles are color-
coded as in (C) and represent individual animals. Data from Son2a‘“* (gray) and Scn2a‘’~ (light cyan) are in background for comparison.
Granule cells often fire in bursts in vivo, with average instanta- mediated entirely by Scn2a heterozygosity in granule cells alone.
neous frequencies of 160-170 Hz in mice.*°" These high-fre- To test this, we crossed animals heterozygous for a conditional
quency bursts are critical for gain-down plasticity, as they are a Scn2a knockout allele (Scn2a") to the alpha6-Cre driver line,
major signal that evokes LTP,’?~“° which we show is impaired which expresses Cre largely in cerebellar granule cells.'°“® As in
in Scn2a*’~ mice. Ahigh axonal Nay densityis required to sustain constitutive Scn2a heterozygotes, VOR gain was elevated at
a burst of APs at high frequency,*° suggesting that lower Nay1.2 baseline and could not be reduced by gain-down induction proto-
density in parallel fibers may impair transmission. To test this, we cols (Figures SA-5C). Furthermore, Purkinje cell firing rate—which
assayed AP conduction fidelity in parallel fibers. At a lower fre- was not different from that in constitutive Scn2a"”” mice at base-
quency (50 Hz), parallel fiber volleys were sustained at normal line—was not increased during or after VOR gain-down induction
levels in Scn2a‘’~ mice; however, fiber volley amplitude was (Figures SE-5G), confirming that heterozygous loss of Nay1.2 in
attenuated markedly at the higher frequency used for LTP induc- granule cells alone can impair VOR gain-down plasticity.
tion (166 Hz) (Figure 4F). Because the amplitude of axonal APs af- While the alpha6-Cre line allows for selective expression of
fects transmitter release probability,’ these smaller fiber volleys Cre in granule cells within the cerebellum, there is sparse expres-
would be expected to evoke less transmitter at granule-Purkinje sion of Cre in some other brain regions.*° To control for this, and
synapses. To test this, we again stimulated parallel fibers at high to further confirm that floccular complex granule cell function is
frequency and recorded resultant EPSCs in Purkinje cells. Similar critical for VOR gain-down plasticity, we injected Cre bilaterally
to fiber volley measurements, we found that EPSC charge trans- into the floccular complex of Scn2a* mice. Injections were per-
fer was impaired in Scn2a*’~ mice, with more pronounced defi- formed at post-natal day 30 (P30) to further determine which
cits occurring later in trains when volley waveform was more components of VOR gain and its plasticity require normal
markedly reduced (Figure 4G). Together, these data suggest Nay1.2 levels at this later state of development. Because granule
that deficits in VOR plasticity in Scn2a"’~ mice are strongly asso- cells are the only cell class at the injection site that express
ciated with deficits in the synaptic transmission required for LTP Scn2a, this approach will result in selective Scn2a heterozygos-
at parallel fiber-to-Purkinje cell synapses. ity in floccular granule cells alone. As observed in alpha6-
Cre::Scn2a*" mice, VOR gain-down plasticity was impaired.
Scn2a heterozygosity in granule cells alone is sufficient Interestingly, it did not alter baseline VOR gain from WT-like
to alter VOR gain plasticity levels (Figure S6), even 3 months after injection (Figure S7).
SCN2A is expressed across the brain. However, our results sug- This suggests that baseline gain is maintained despite loss of
gest that alterations in VOR behavior in Scn2a”~ mice are likely gain-down plasticity, perhaps because there is no pressure to
6 Neuron 112, 1-12, May 1, 2024

Neuron @ CellPress
change baseline values once established in early development, VOR can shed light on dysfunction in cellular, circuit, and behav-
as there is likely limited error in visual scene stabilization that ioral plasticity in neurodevelopmental disorders as well. VOR
would drive such changes. Consistent with this, VOR measured function is conserved across Mammalia. We observed that its
while presenting a static visual stimulus was near unity for both dysfunction is also conserved in mouse and human SCN2A hap-
WT and Scn2a*’~ mice, confirming that Scn2a"’~ mice can loinsufficiency. This parallels observations in non-syndromic
respond to visual stimuli (Figure S2). Combined, these data indi- ASD, where heightened VOR gain or dysfunction in other as-
cate that Scn2a heterozygosity in floccular granule cells drives pects of vestibular sensation can also be observed, '”"'* though
impairments in VOR plasticity. such observations may depend critically on the cohort studied or
on the parameters under investigation. '*"°*°° VOR is detectable
VOR gain alterations can be rescued by upregulating within the first months of life in humans,°”-"° well before ASD is
Scn2a expression typically diagnosed.°° Given the importance of early diagnosis,°°
These observations add to a growing body of literature showing examination of VOR and related innate behaviors may help
that Scn2a is a key gene for synaptic and behavioral plas- improve patient and family outcomes.
ticity.'°“°°" In neocortical circuits, we recently found that Vestibular deficits could be rescued by upregulating Scn2a
synaptic plasticity could be reinvigorated if Scn2a expression expression. Upon retro-orbital injection of CRISPRa, we
was restored in Scn2a"”- mice.°* This was achieved using a observed an upregulation of Scn2a gene expression at both P3
CRISPRa-based therapeutic approach that targets a transcrip- and P30. Notably, while injections at either age rescued VOR
tional activator to the residual functional allele present in Scn2a gain-down plasticity, only those at P3 rescued baseline gain to
heterozygotes. Because neurons are non-dividing cells, a single normal levels. Consistent with this result, bilateral injection of
CRISPRa administration can effectively upregulate gene activity Cre into the floccular complex of Scn2a"” animals at P30 sup-
throughout life. Furthermore, previous research has shown that pressed VOR gain-down plasticity but did not alter baseline
this approach only upregulates genes in tissues where the tar- VOR gain from WT-like levels (Figure S6). This lack of rescue ap-
geted regulatory element is active.°° Therefore, Scn2a upregula- pears to not correlate with the degree of Scn2a rescue, as we
tion would only occur in cells that express Scn2a endogenously, observed comparable rescue of VOR gain-down plasticity inde-
such as cerebellar granule cells. To test whether cerebellar plas- pendent of the degree of Scn2a expression upregulation.
ticity could be similarly rescued with this approach, we injected Instead, these results parallel earlier studies demonstrating
a pair of blood-brain-barrier-penetrant adeno-associated viruses that baseline VOR is dependent on visual experience early in
containing all necessary CRISPRa components and the fluores- life. In these works, animals dark-reared during an early critical
cent protein mCherry via the retro-orbital sinus to upregulate period never exhibited normal baseline VOR gain.°’°° This sug-
Scn2a across the brain. CRISPRa was administered via this route gests that baseline VOR gain crystalizes between P3 and P30,
to best mimic the broad, but incomplete infection predicted to but it remains unclear whether enhanced experience or behav-
occur with similar gene therapy approaches in future clinical trials. ioral therapy later in life may help modify baseline VOR gain
Inducing Scn2a heterozygosity at P30 impairs VOR gain-down even with later-in-life Scn2a rescue.
plasticity, but not baseline gain (Figure S6), suggesting that these Here, deficits in VOR were due specifically to deficits in
two processes may be differentially regulated across develop- granule cell transmission. This, in turn, altered mechanisms for
ment. Therefore, we tested for rescue at two ages, with regulation of Purkinje-cell-mediated inhibition of the VOR circuit
CRIPSRa administration at P3 and P30. Quantitative PCR through the vestibular nuclei. The net result, for both human and
(qPCR) for Scn2a mRNA in cerebellar lysates from all mouse models of SCN2A LoF, was a VOR gain saturated near
CRISPRa-injected animals revealed increases in expression in unity, even at slow head angular velocities. These effects appear
6 out of 6 animals injected at P3 and 5 out of 6 animals injected due, at least in part, to the alterations at parallel fiber-to-Purkinje-
at P30 (Figures 6A-6C). Remarkably, VOR gain-down plasticity cell synapses. Our understanding of interactions between paral-
was restored in all animals but the lone animal injected at P30 lel fiber firing rates and plasticity at synapses onto Purkinje cells
that did not show Scn2a upregulation (Figures 6D and 6E). There is consistent with impaired LTP, but not LTD, in Scn2a"”~ condi-
was no correlation between mRNA upregulation levels and the tions. At parallel fiber-to-Purkinje cell synapses, high-frequency
degree of plasticity rescued in the 11 animals with successful in- bursts of APs are required for LTP related to VOR gain-down
jections (Figure 6C). Plasticity restoration coincided with in- plasticity.°° By contrast, LTD can be supported by shorter bursts
creases in Purkinje cell firing rate during and after VOR gain- of granule cell activity paired with Purkinje cell depolarization
down induction (Figures 6F-6H), indicating that both cellular and likely remains intact even in Scn2a””~ conditions (Fig-
and behavioral plasticity could be rescued even when restored ure 3F).°7°4°% Thus, loss of parallel fiber-to-Purkinje cell
in later stages of development. By contrast, baseline VOR gain LTP, but not LTD, may bias VOR gain toward unity and render
was restored only in animals injected at P3, suggesting that there VOR inflexible to gain-down protocols. However, synaptic plas-
is an early developmental window between P3 and P30 in which ticity between granule cells and molecular layer interneurons
baseline gain is crystallized. (MLIs), which provide feedforward inhibition to Purkinje cells,
can also be influenced during extended high-frequency bursts
DISCUSSION of parallel fibers.°°°° MLI activity is necessary for VOR gain-
up, but not for gain-down, plasticity.°° While it is likely that gain
Oculo-motor reflexes are commonly assessed for diagnosis of cannot be further increased in Scn2a*’~ animals, as their gain
neurological disorders. Here, we highlight how examination of is already near 1.0 at baseline, we also cannot rule out additional
Neuron 112, 1-12, May 1, 2024 7

@ CelPress Neuron
A Systemic c .
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Figure 6. Rescue of VOR gain-down behavioral plasticity with CRISPR activation

(A) Two AAV-PHP vectors are injected together for systemic infection via the retro-orbital sinus. AAV1 contains a guide RNA and mCherry. AAV2 contains dCas9
and the transcriptional regulator VP64.
(8) Cerebellar coronal section from animals injected at P30 and P3. Note broad, but incomplete, infection across cerebellum, as signaled by mCherry fluores-
cence. A region of cerebellum containing the left floccular complex was dissected immediately after harvesting brain for quantitative PCR and is missing from
section.
(C) Quantitative PCR of Scn2a mRNA mouse cerebellum from CRISPRa-injected mice. In 5/6 animals injected at P30, marked upregulation was noted (closed
circles). In 1 animal, no upregulation was noted (open circle). At P3, all animals exhibited robust upregulation. Data are plotted vs. the normalized change in VOR
gain (e.g., slope per animal in E). Dashed line is a linear regression.
(0) Example VOR before and after gain-down induction in CRISPRa-treated animals (P30 and P3).
(©) VOR gain before and after gain-down induction in Sen2a"’~ mice with Scn2a CRISPRa at P30 (red, 6 mice), P3 (purple, 6 mice), and empty vector (blue, 4 mice),
depicted as in Figure 2D. WT and Scn2a*’” data replotted from Figure 2D for comparison. *p < 0.05 Wilcoxon signed-rank test; all data included
(F) Average Purkinje cell simple spike firing frequency during sinusoidal head rotation, before and after gain-down induction in all retro-orbital sinus-injected mice.
Circles are color-coded as in (D) and represent individual units. *p < 0.005, mixed-effects modeling,
(@) Normalized Purkinje cell simple spike firing frequency during gain-down induction for all retro-orbital sinus-injected mice. Circles and bars are mean + SEM,
binned per minute.
(H) Normalized change in VOR gain vs. normalized change in simple spike firing frequency (normalized per unit and averaged across units per animal). Circles are
color-coded as in (D) and represent individual units. Data from Sen2a*’* (gray) and Scn2a*’~ (light cyan) are in the background for comparison. Note the overlap of
CRISPRa population with Scn2a"* le population (except open circle) and empty vector with Scn2a’~ population.
changes to MLI activity due to impairments in their parallel fiber that established a role for parallel fiber-Purkinje cell synaptic
inputs. Future work examining these circuit elements, as well as plasticity and VOR adaptation to understand links between
changes in these elements over development, will be critical for cellular dysfunction and alterations in reflexive behavior in
understanding the full extent of dysfunction in cerebellar circuits SCNZA haploinsufficiency. While this behavior requires the floc-
in Scn2a haploinsutficiency conditions. cular complex alone, Nay1.2 is expressed ubiquitously in granule
The simplicity of cerebellar anatomy—composed of relatively cells across the cerebellum, as is high-frequency, burst-based
few cell classes with stereotyped synapses—belies its functional parallel fiber LTP to Purkinje cells.“?"°°”° Thus, the vestibular
complexity. Indeed, the cerebellum contributes to a range of plasticity deficits observed here may hint toward broader cere-
processes, including the social and cognitive processing that bellar plasticity deficits, affecting a range of phenotypes in chil-
are affected in ASD.°”-”? Here, we built upon decades of work dren with SCN2A LoF."° Furthermore, this work parallels studies
8 Neuron 112, 1-12, May 1, 2024

Neuron @ CellPress
of cerebellar plasticity in mouse models of other ASD-associated © Histology

genes.”*”® Thus, cerebellum-dependent reflexes may prove to © Data analysis
be generalizable and translatable biomarkers for ASD and poten- © Ex vivo electrophysiology
tial therapeutics, even though these reflexes may have no causal @ QUANTIFICATION AND STATISTICAL ANALYSIS,
role in core ASD phenotypes themselves.
SUPPLEMENTAL INFORMATION
Limitations of the study
This work represents the first study, to our knowledge, in which Supplemental information can be found online at https://doi.org/10.1016/j
VOR gain was assessed in children with profound developmental neuron.2024.01.029.
delay. Children with SCN2A LoF, including those in this study,
are often non-verbal, unlikely to follow complex instruction, ACKNOWLEDGMENTS
and, from consultations with caregivers, very unwilling to un- We are grateful to children, parents, and caregivers associated with the
dergo head restraint in an apparatus traditionally used to assess FamilieSCN2A Foundation for participating in this study. We thank Drs. B. Bar-
VOR gain in the clinic. Given these limitations, we designed a hel- bour, M. Casado, J. Christie, F. Dunn, A. Nelson, P. Reeson, M. Scanziani, and
met-mounted VOR apparatus that allows a child to move more M. Schonewille for feedback on this work and Dr. J. Christie for discussions
freely and restricted our analysis to baseline VOR gain. In that motivated this study. This work was supported by an Action Potential
contrast to our experiments in Scn2a"’~ mice, we did not Award from the FamilieSCN2A Foundation (C.W.), a research grant from Bio-
Marin Pharmaceutical Inc. (K.J.B.), the Weill Neurohub Investigator Program
attempt to assess VOR gain plasticity with any induction proto- (KJ.B. and N.A), SFARI 629287 (K.J.B. and N.A.), Howard Hughes Medical
cols in children. In future studies, this may be possible, perhaps Institute funds awarded to M. Scanziani (G.B.), and National Institutes of
by using virtual reality headsets. In addition, VOR measurements Health grants F32 MH125536, K99 MH135209 (A.D.N.), and R01
in much younger children may be useful as an early biomarker of MH125978 (K.J.B.).
cerebellar dysfunction that may be evident long before other
ASD-associated symptoms emerge. AUTHOR CONTRIBUTIONS
This study was limited to children that presented with SCN2A Conceptualization, C.W., G.B., and K.J.B.; methodology, C.W., K.D.D., E-H.,
LoF without seizure or anti-seizure medication within the previ- XZ., AD.N., H.K., NA, G.B., and K.J.B.; software, C.W., K.D.D., H.K., and
ous 6 months. An estimated 20%-30% of children with LoF G.B.; formal analysis, C.W.; investigation, C.W., K.D.D., E.H., X.Z., A.D.N.,
variants present with seizure, usually with an onset after the first H.K.,.N.A., G.B., and K.J.B.; writing - original draft, C.W.; writing - review & ed-
3-6 months of life.’° Seizures are treated with a range of medica- iting, all authors; visualization, C.W., G.B., and K.J.B.; supervision, N.A., G.B.,
tions, but those that interfere with sodium channel function are and K.J.B.; funding acquisition, C.W., A.D.N., N.A., and K.J.B.
typically avoided.’° How such medications interact with the
physiology that underlies VOR is unknown. Furthermore, mouse DECLARATION OF INTERESTS
models with constitutive or conditional Scn2a deletion were N.A. is the cofounder and on the scientific advisory board of Regel Tx. K.J.B. is
used. While this models the majority of LoF cases in the human on the scientific advisory board of Regel Tx. N.A. and K.J.B. receive funding
population, it does not model missense variants that either from BioMarin Pharmaceutical Incorporated.
dampen or eliminate channel function but do not lead to
nonsense-mediated decay of Nay1.2. How VOR is encoded in Received: May 22, 2023
such conditions will require the creation of new missense Revised: January 3, 2024
Accepted: January 29, 2024
models. Published: February 26, 2024
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STAR*METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit anti-RFP Rockland Immunochemicals Cat#600-401-379S; RRID: AB_11182807
Chicken anti-GFP Aves Labs Cat#GFP-1010; RRID: AB_2307313
Guinea pig anti-VGlut Millipore Cat#AB5905; RRID: AB_2301751
Alexa Fluor 568 goat anti-rabbit Invitrogen Cat#A-11011
Alexa Fluor 488 goat anti-chicken Invitrogen Cat#A-11039
Alexa Fluor 568 goat anti-guinea pig Invitrogen Cat#A-11075
Bacterial and virus strains
‘AAV-EF1a-Cre-mCherry UNC Viral Core N/A
AAV-PhP.eb-CMV-sadCas9-VP64 Tamura et al.°? N/A
AAV-PhP.eb-U6-sasgRNA-CMV-mCherry Tamura et al.*° N/A
Chemicals, peptides, and recombinant proteins
DNQXx disodium salt Tocris Cat#2312
Picrotoxin Tocris Cat#1128
CPCCOEt Tocris Cat#1028
Pilocarpine hydrochloride Sigma-Aldrich Cat#P6503-5G
Phosphate buffered saline Sigma-Aldrich Cat#SIAL-P4417-100TAB
Paraformaldehyde WR International Cat#100504-858
Sodium azide Sigma-Aldrich Cat#S2002-100G
Triton X-100 Sigma-Aldrich Cat#X100-100mI
Normal goat serum Fisher Scientific Cat#NC9660079
ProLong Gold with DAPI Life Technologies Cat#P36931
Vybrant Dil Cell-Labeling Solution Invitrogen Cat#V22885
Experimental models: Organisms/strains
Mouse: C57BL/6J The Jackson Laboratory JAX Strain #:000664;
RRID: IMSR_JAX:000664
Mouse: Scn2a*” Planelis-Cases et al."° N/A
Mouse: Alpha6 - Cre Mutant Mouse Recourse & RRID: MMRRC_015966-UCD
Research Centers
Mouse: Sen2a*" N/A
Software and algorithms
IGOR Pro Wavemetrics RRID: SCR_000325; v6.3
Prism Graphpad RRID: SCR_002798; v9
LabView NI RRID: SCR_014325; v2020
MATLAB MathWorks RRID: SCR_001622; vR2020a
DeepLabCut Mathis et al.°" RRID: SCR_021391; v2.2.0.5
KiloSort2 Pachitariu et al.°>°° RRID: SCR_016422; https://github.com/
MouseLand/Kilosort
Phy Cyrille Rossant https://github.com/cortex-lab/phy
Mouse eye tracking acquisition code in LabView Liu et al.°"; Bouvier et al.°° N/A
Visual stimuli code in MATLAB Liu et al.°* N/A
Human eye tracking acquisition code Dave Jones https://github.com/waveform80/picamera
Human motion tracking (MU) acquisition code This paper https://doi.org/10.5281/zenodo.10573756
Neuron 112, 1-12.e1-e5, May 1, 2024 e1

@ CelPress Neuron
RESOURCE AVAILABILITY
Lead contact
Requests for reagents and resources should be directed to Kevin Bender (kevin.bender@ucsf.edu).
Materials availabi
This study did not generate new unique reagents.
Data and code availability
Data reported in this paper are available from the lead contact upon reasonable request. This paper does not report original code. Any
additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
Humans
Experiments were performed on children of either sex, aged 3-10 years. Due to the low sample size, we did not test whether sex had
any influence on the data. All procedures were in accordance with UCSF IRB guidelines and parental consent was obtained for all
participants. Inclusion criteria for SCN2A loss-of-function (LoF) children included those with a genetic diagnosis of SCN2A dysfunc-
tion and a diagnosis of autism spectrum disorder and neurodevelopmental delay. Furthermore, children were limited to those without
seizure or any seizure-related medication within the last 6 months. Neurotypical (Nt) children were limited to those that did not present
with neurodevelopmental delay, autism spectrum disorder, or seizure.
Mice
Allexperimental procedures were performed in accordance with UCSF IACUC guidelines. All experiments were performed on mice of
either sex housed under standard conditions with adlibitumaccess to food and water, with colonies maintained in-house, with health
monitored by UCSF veterinarians. No sex differences were noted in behavior or electrophysiology, and data were pooled across sex.
Wild-type C57B6J were from JAX (000664); Alpha6-Cre mice were initially described in Bahn et al."°; Scn2a*”” mice were initially
described in Planells-Cases et al.°°; conditional knockout Scn2a allele mice were initially described in Spratt et al.’°; conditional
knockin Scn2a mice with GFP in-frame were described in Tamura et al.°? Animals were sampled from once for VOR, unless explicitly
noted otherwise (e.g., repeat VOR baseline assays at different times after viral manipulations).
METHOD DETAILS
Human vestibulo-ocular reflex

For VOR experiments, children were seated in a standard office executive chair with the center of their head situated near the azimuth
swivel. Children were either seated alone or in a caregiver's lap for both Nt and LoF cohorts, with no discernable differences in data
quality due to caregiver presence. Children were then fitted with a helmet (67i skateboard helmet, child's small or medium depending
on participant, Amazon) that was kitted with an inertial movement unit centered at the top of the helmet and an infrared camera (Rasp-
berry Pi NoIR) and 940 nm LED (Luxeon star) with a 10° diffuser lens focused on the right eye. The camera was aligned to ensure that a
corneal reflection from the LED could be visualized. Eye movement was first calibrated in light conditions by having children attend to
a visual stimulus (a chirping yellow sparrow stuffed animal or a pen light clicked on and off as conditions permitted) moved back and
forth from a position either directly ahead or 10 degrees offset. Calibration was repeated >4x for each child and eye position calibra-
tion tracking was averaged over all trials.
Following calibration, the chair was rocked back and forth ~+5° at ~0.4 Hz in the dark by an experimenter wearing night-vision
optics, with oscillations paced by a metronome. Videography and IMU data were acquired at 60 and 300 Hz, respectively. IMU
data were downsampled to 60 Hz for subsequent analysis.
Data acquisition could not be performed blind to LoF/Nt condition given the phenotypes being analyzed. All data was therefore
coded and blinded for subsequent analysis. Post-hoc processing of eye position was performed using DeepLabCut 2.0 Toolbox
(deeplabcut.org).*' DeepLabCut was trained by an experimenter to detect 8 points at regions of maximal contrast at the edge of
the pupil (every 45 degrees) and 4 points at the edge of the corneal reflection (every 90 degrees). Horizontal eye angle was calculated
as the average horizontal pupil value (all 8 points) relative to the average horizontal corneal reflection value (all 4 points). Data acquired
during VOR tests were then normalized to calibration sets to obtain the exact eye movement angle.
Allchildren naturally made saccades and blinked during data acquisition. These events were removed from subsequent analysis by
eliminating all data when eye movement speed exceeded 20 deg/sec and where the DeepLabCut position “confidence” value, aver-
aged over all 12 imaged points, was <0.99 (range: 0 to 1.0). Eye angle and head angular velocity (IMU) data were used to calculate
instantaneous velocity, smoothed with a 2 data-point-wide Gaussian filter. VOR gain was then calculated as the ratio of the integral of
absolute values of instantaneous eye velocity and corresponding values of head velocity over the entire recording epoch.
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Neuron @ CellPress
Surgical procedures
Head plate implantation for head-fixed recordings
Mice were implanted with a head bar composed of three threaded screw inserts arranged in a triangle (McMaster-Carr, 923954109)
at least 14 days before recording. Mice were anesthetized with 2% isoflurane. The scalp was shaved and disinfected with povidone
iodine. The scalp was resected, and the skull was scored with a sterilized bone scraper. The edge of the skin was glued to the skull
and the metal threaded screw inserts were sterilized and mounted using Metabond and dental cement (Ortho-Jet powder; Lang
Dental) mixed with black paint (iron oxide). The inserts were mounted via a stereotax with the triangular center aligned to bregma.
Following the implantation, black dental cement was used to build a recording well surrounding the skull above the targeted floccular
complex region at (from the bregma, in mm): anterior-posterior, -5.40, mediolateral, -2.75. The surface of the skull above the left
hemisphere of the cerebellum was not covered with dental cement but was coated with a thin layer of transparent cyanoacrylate
glue. Mice were injected intraperitoneally with 0.1 mg/kg buprenorphine before and after surgery and were checked daily for
3 days after the head-bar surgery.
Craniotomy for electrophysiological recordings
Following the last day of habituation to head fixation, each mouse was anesthetized with 2% isoflurane and the skull above the
recording site was drilled off. The dura was not removed, and the craniotomy was kept moist with phosphate buffered saline
(PBS). Following craniotomy, the window was sealed with biocompatible silicone sealant until the recording session (Kwik-Cast,
World Precision Instruments).
Stereotaxic transcranial viral injection
Mice with one conditional knockout Sena (Scn2a*") allele at P30 were anesthetized under isoflurane at 2.0% and mounted onto the
stereotaxic machine (Kopf 1900). 500 nL of AAV-EF 1a-mCherry-IRES-Cre-WPRE (UNC Vector Core) was injected into the floccular com-
plexeson both sides at (from the bregma, in mm): anterior-posterior, -5.40; mediolateral, +2.75; dorsoventral: —4.00 at 100nL per minute.
Viral injection via retro-orbital sinus
AAV-PhP.eb-CMV-sadCas9-VP64 with either AAV-PhP.eb-U6-sasgRNA-CMV-mCherry (CRISPRa) or an empty vector containing
AAV-PhP.eb-U6-CMV-mCherry suspended in 5% Sorbitol (Sigma-Aldrich, Inc.) at a total volume of 150 wL and packaged in a
28-gauge needle with 0.5 mL insulin syringe (U-100; Becton, Dickinson and Company). Mice aged P30 were anesthetized under
inhalant isoflurane and were kept warm on a heating pad. Mice aged P3 were immobilized on a sterile surface cooled with ice.
Ophthalmic ointment (Puralube Vet Ointment; Dechra) was applied to both eyes of the anesthetized mouse. The mouse was then
laid on its left side and the needle was subsequently inserted into the medial canthus underneath the left eyeball until it reached
the base. The mixture of viral constructs was then introduced into the retro-orbital sinus. After a 10-s pause, the needle was slowly
withdrawn and additional ocular ointment was applied. The P3 mice were immediately returned to their home cage with the mother.
The mice were monitored for signs of bleeding (none noted) and provided analgesic and monitored for pain for 3 days post-
injection.°°
Behavior
Habituation and eye calibration
Behavioral experiments were performed during the light cycle and the experimenter was blind for genotype during experiment and
analysis. After allowing for at least 1 week of recovery after surgery, each mouse was habituated to the experimental setup by head-
fixing it in a purpose-built restraining tube with a head holder that was positioned at the center of the turntable for 1-2 h each day for 4
consecutive days.” Each mouse was habituated for 1 h in the darkness on the first day, then with a 20-min increment on each sub-
sequent day for the following three days.
On the third habituation day, the right eye of each mouse was calibrated using a standard method, as reported previously. 83,84
Briefly, to calibrate eye movement, we used a corneal reflection generated by an infrared LED as a reference position for the eye.
Astatic virtual drum was presented during calibration. After identifying the pupil and the center of the eye, the camera and the refer-
ence LED were rotated by 10 degrees in both directions to acquire the distance between the pupil reference and the pupil center. The
pupil size was varied by changing the luminance of the virtual drum to obtain increase the dynamic range of pupil size values.
Vestibulo-ocular reflex recordings
Mice were given 3% topical pilocarpine HCI in both eyes 15 min prior to the beginning of each VOR recording session to temporarily
restrict their pupil size in the dark. Compensatory eye movements were recorded using video-oculography. The velocity of head
movements was controlled by the servo-controlled platform that enabled the rotation of the animal along the horizontal plane.
The platform was attached to a gearbox 15:1 (VTR010-015-RM-71 VTR, Thomson) that increased the torque of a servo motor
(AKM53L-ANC2C-00 KEC0432 AC Servomotor 1.83 kW, Kolmorgen). The motor was tuned using a servo drive (AKDB013206-
NBAN-0000 servo drive, Kolmorgen) and controlled in velocity mode using analog waveforms computed in LabView 2020 (NI).
The head of each mouse was positioned with a 20 degrees angle along the sagittal plane of the head (nose pointing 20 degrees
down), such that the plane of the horizontal vestibular canal was approximately parallel to the plane of the rotating platform. VOR
was evoked by rotating the platform in the dark with 5° amplitude in each direction and a temporal frequency of 0.4 Hz unless other-
wise stated. Gain-down VOR induction protocol was performed by presenting a coherent virtual drum with a spatial frequency of
0.8 Hz and a temporal frequency of 0.4 Hz at 5° amplitude in each direction with the platform rotation that oscillates in the same di-
rection to evoke near complete VOR cancellation (see Figure S2A). Gain-up VOR induction was performed by presenting a coherent
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virtual drum with a spatial frequency of 0.8 Hz and a temporal frequency of 0.4 Hz with 2.5° amplitude in each direction with the plat-
form rotation that oscillates in the opposite direction.
In vivo extracellular recordings
All recordings in this study were performed in the left floccular complex (contralateral to the imaged eye). Extracellular recordings
were performed using a 2-shank silicon probe that is 8 mm in length and contains 32 channels on each shank with 25 im inter-chan-
nel distance and 250 um inter-shank distance (ASSY-77 H2, Cambridge Neurotech). The recording electrode was controlled with a
micromanipulator (MP-285; Sutter Instrument) and stained with Dil lipophilic dye (Thermo Fisher) for post-hoc identification of the
electrode location (see Figure S5C). The signals were acquired at 30 kHz using an INTAN system (RHD2000 USB Interface Board,
INTAN Technologies). Recordings in the floccular complex were performed at (from bregma, in mm): anterior-posterior, -5.40; medio-
lateral, -2.75; dorsoventral: —4.00.
On the day of recording, each mouse was head-fixed on the VOR experiment setup. The silicon sealant was then removed, and the
craniotomy was kept moist with artificial cerebrospinal fluid contained (in mM): 140 NaCl, 5 KCl, 10 D-glucose, 10 HEPES, 2 CaCl2,
2 MgSO4 with a pH of 7.4. The recording electrode was lowered into the left floccular complex via the craniotomy at a speed
of ~250 um per minute. Experiments began at least 50 min after reaching the final electrode depth to allow for electrode stabilization.
Histology
For anatomical analysis, mice were perfused transcardially with phosphate buffered saline (PBS, Sigma-Aldrich) and then with 4%
paraformaldehyde (PFA, VWR International, Inc.) in PBS. Brains were extracted from the skulls, post-fixed in 4% PFA overnight at
4°C, switched to 15% sucrose in PBS for 4 h, and then stored in 30% sucrose in PBS until sectioned. Sequential coronal sections
of the cerebellum (30 um) were collected with a Microm HM 525 Cryostat and were stored in 0.05% sodium azide (Sigma-Aldrich)
in PBS. For in vivo recording electrode location identification, sections were rinsed in 1X PBS 3 times for 15 min and then were
then coverslipped with ProLong Gold with DAPI (Life Technologies). For immunofluorescence, sections were rinsed in 1X PBS 3 times
for 15 min and then incubated overnight at 4°C with 10% normal goat serum (Fisher Scientific) with 0.2% Triton X-100 (Sigma-Aldrich)
and appropriate antibodies [mCherry: Rabbit anti-RFP (1:1000, Rockland Immunochemicals), GFP: chicken anti-GFP (1:500, Aves
Labs), VGlutt: guinea pig anti-VGlut (1:1000, Sigma Aldrich)]. The next morning, sections were kept at room temperature for 1 h before
being rinsed with 1X PBS 3 times for 15 min. The sections were then incubated with respective secondary antibodies (Alexa Fluor 568
goat anti-rabbit (mCherry); Alexa Fluor 488 goat anti-chicken (GFP); Alexa Fluor 568 goat anti-guinea pig (VGlut) (1:500 for all second-
aries, Invitrogen). Sections were coverslipped with ProLong Gold with DAPI (Life Technologies). Fluorescence images were acquired
using an Olympus FV3000 confocal microscope under4x or 40x objectives at Nyquist sampling resolution (1.4 NA).
Data analysis
Monitoring eye movements by video-oculography
The movement of the right eye was monitored using a high-speed infrared (IR) camera (IPX-VGA210LMCN; Imperx, Inc.) by capturing
the reflection of the eye on an R mirror (Edmund Optics #64-471) that is transparent to visible light under the control of custom routines
in LabView 2020 (NI) and a frame grabber (PCle-1427, Nl) at an acquisition rate of 100 Hz. The pupil was identified online by thresh-
olding pixel values and its profile was fitted with an ellipse to determine the center. The eye position was measured by computing the
distance between the pupil center and the corneal reflection of a reference IR LED placed along the optical axis of the camera.
VOR gain calculation
Eye tracking data were processed in customized routines in MATLAB. VOR gain was calculated as the ratio of the average of the
difference of the maximum and minimum of the eye position for each sinusoidal cycle and the amplitude of table rotation (10 degrees,
10 s trial duration for all rotation frequencies). VOR gain was calculated using sinusoidal cycles that did not contain saccades. At least
20 trials were collected from each mouse.
Purkinje cell simple spikes identification
Automated spike sorting was carried out using KiloSort (https://github.com/cortex-lab/Kilosort)® by manual curation of the units us-
ing Phy2 (https://github.com/cortex-lab/phy). Multiple parameters were used to identify putative Purkinje cell units. First, we only
included units that exhibited spikes across at least 3 adjacent channels on the linear probe (25 jim inter-channel distance) to bias
selection towards cells that have large somatodendritic area. This is thought to exclude units arising from molecular layer interneu-
rons.®” Second, we excluded units with refractory period violations greater than 1%-2%. Simple spikes from putative Purkinje cell
units were then validated using previously defined standards based on mean firing rates (>22 Hz), median inter-spike intervals, inter-
spike interval (ISI) distributions, and the coefficient of variation of the natural log of the ISIs (Range: 0.05 to 0.4).°® Purkinje cell units
outside of these ranges were excluded from subsequent data analysis. We also excluded complex spikes from our analysis.
Firing frequency and CV of ISI computation
All analysis was carried out via MATLAB (MathWorks) and Python on identified Purkinje cell units. The timings of each simple spike
were aligned with individual trials of VOR recordings using a digital pulse triggered by a photodiode positioned on the virtual drum.
Only simple spikes that occurred during the 10-s sinusoidal oscillation trials were included in subsequent analyses. To determine
directional bias of the recorded PC units, we used the ratio of simple spikes frequency during clockwise (CW) and counterclockwise
(CCW) head rotation in each trial. Overall simple spike firing frequency was calculated by treating each trial (10 s) as a bin and dividing
e4 Neuron 112, 1-12.e1-e5, May 1, 2024

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the total number of spikes by the duration of the trial. The firing frequency from each trial was then averaged within each experimental
epoch, including pre- and post-gain down induction. The firing frequency of each trial during gain-down induction was calculated in
the same way but was binned every 4 trials and normalized to the first bin to show percentage change over time. The coefficient of
variation (CV) of the inter-spike intervals (ISI) was calculated on the same set of Purkinje cell simple spikes.
Ex vivo electrophysiology
Ex vivo cerebellar tissue preparation
Mice aged P28 through P59 were anesthetized and 250 m-thick acute coronal slices containing the cerebellum were prepared. Slices
were prepared from Scn2a‘”~ or wild-type littermates (genotyped by PCR). All data were acquired and analyzed blind to Scn2a geno-
type. Data were acquired from both sexes (blind to sex). Cutting solution contained (in mM): 87 NaCl, 25 NaHCO, 25 glucose, 75 su-
crose, 2.5 KCI, 1.25 NaHzPO,, 0.5 CaClp and 7 MgCl; bubbled with 5%C0,/95%O2; 4°C. Following cutting, slices were incubated in
recording solution for 30 min at 33°C, then at room temperature until recording. Recordings solution contained (in mM): 125 NaCl,
2.5 KCl, 1.3 CaCl, 1 MgCle, 25 NaHCOs, 1.25 NaHPO,, 25 glucose; bubbled with 5%C0/95%0p; 32-34°C, ~310 mOsm at ~33°C.
Ex vivo electrophysiological recordings
Cerebellar Purkinje cells and granule cells were visualized with differential interference contrast (DIC) optics for conventional visually
guided whole-cell recording. For current-clamp recordings, patch electrodes (Schott 8250 glass, 7-8 MQ tip resistance for granule
cells, 3-4 M2 tip resistance for Purkinje cells) were filled with a solution containing (in mM): 113 K-Gluconate, 9 HEPES, 4.5 MgCl,
0.1 EGTA, 14 Trisp-phosphocreatine, 4 Nap-ATP, 0.3 tris-GTP; ~290 mOsm, pH: 7.2-7.25. For voltage-clamp recordings and synap-
tic activity in Purkinje cells, patch electrodes (Schott 8250 glass, 3-4 Ma tip resistance) were filled internal solution contained (in mM):
110 CsMeSOs, 40 HEPES, 1 KCI, 4 NaCl, 4 Mg-ATP, 10 Na-phosphocreatine, 0.4 Naz-GTP, 0.1 EGTA; ~290 mOsm, pH: 7.22. All
data were corrected for measured junction potentials of 12 and 11 mV in K- and Cs-based internals, respectively.
Electrophysiological data were acquired using Multiclamp 700A or 700B amplifiers (Molecular Devices) via custom routines in
IgorPro (Wavemetrics). For measurements of action potential waveform and fiber volleys, data were acquired at 50 kHz and filtered
at 20 kHz. For all other measurements, data were acquired at 10-20 kHz and filtered at 3-10 kHz. For current-clamp recordings,
pipette capacitance was compensated by 50% of the fast capacitance measured under gigachm seal conditions in voltage-clamp
priorto establishing a whole-cell configuration, and the bridge was balanced. For voltage-clamp recordings, pipette capacitance was
compensated completely, and series resistance was compensated 50%.
Parallel fiber volley recordings were made from coronal slices of the medial posterior cerebellum. The recording electrode was
placed ~1 mm away from a tungsten bipolar stereotrode (WE3ST30.1A10; MicroProbes for Life Science). The strength of electrical
stimulation was adjusted for each recording to maintain the amplitude of the first parallel fiber volley above 1 mV. Parallel fiber volleys
were evoked by field electrical stimulation in either trains of 10 at 20 ms inter-stimulus-interval or trains of 20 at 6 ms ISI. This exper-
iment was performed in the presence of 10 uM 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 25 uM picrotoxin.
In experiments measuring paired pulse ratio and synaptic plasticity, EPSCs were evoked via a bipolar glass theta electrode placed
~200 jum orthogonal and ~200 um lateral to the recorded neuron in the molecular layer in the presence of 25 iM picrotoxin at -80 mV.
Whole-cell voltage-clamp recordings were made from either the lobule 4/5 of the cerebellum or the floccular regions. In short-term
synaptic facilitation protocols, excitatory postsynaptic currents (EPSCs) were evoked byfield electrical stimulation in a train of 20 at
6 ms ISI.
For high-frequency burst-based long-term potentiation (LTP), excitatory postsynaptic currents (EPSCs) were evoked with a theta
stimulating electrode placed ~200 um into the molecular layer and ~200 um lateral to the recorded Purkinje cell. After establishing a
stable baseline of 5 min (EPSC ISI: 15 s) in voltage clamp, EPSPs were evoked by field electrical stimulation in a train of 10 at 6 ms ISI.
Trains were repeated every 1 s for 5 min in the presence or absence of the mGluR1 antagonist CPCCOEt (50 uM, Tocris Bioscience).
Long-term depression (LTD) was induced by pairing Purkinje cell membrane depolarization (40 mV, 120 ms) with a set of two stimuli
delivered to parallel fibers (6 ms ISI, 55 ms after the onset of Purkinje cell depolarization), mimicking climbing fiber-dependent depolar-
ization of Purkinje cell dendrites.°’ LTD induction was repeated 120x (1 s ISI). Following induction, EPSC stimulation frequency was reset
to 0.05 Hz, and changes in EPSC amplitudes were assessed by comparing data 25 - 30 min following induction to baseline (-5.0 - 0 min).
QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analyses were performed using IgorPro8 unless otherwise noted (Wavemetrics). No statistical tests were used to prede-
termine sample size, but our sample sizes are similar to those generally employed in the field. The exact value of n and what n rep-
resents is reported in figure legends. All data are presented in text as mean + standard error (SEM), unless otherwise noted. The
stated P values are the results of the non-parametric Wilcoxon rank sum test to compare values between different mice or recordings,
and the non-parametric Wilcoxon signed rank test to compare values from the same recording in different experimental conditions,
unless otherwise noted. Mixed-effects modeling was used to compare simple spike firing frequency between baseline and post-in-
duction (units nested within parent animals) using Prism. Data were corrected for multiple comparisons when necessary, using the
Holm-Sidak method.
Neuron 112, 1-12.e1-e5, May 1, 2024 e5

Sensory Reflexes in SCN2A-associated ASD: Impaired Cerebellar Plasticity Hypersensitizes

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Sensory Reflexes in SCN2A-associated ASD: Impaired Cerebellar Plasticity Hypersensitizes

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Neuron

Impaired cerebellar plasticity hypersensitizes

e Hypersensitivity is present in children with SCN2A LoF and

e VOR gain cannot be reduced in Scn2a*’~ mice due to

e VOR gain plasticity can be rescued with a CRISPR-activator-

Wang et al., 2024, Neuron 112, 1-12

Impaired cerebellar plasticity hypersensitizes

INTRODUCTION ASD appears to be sensitive to both fast and slow movements,

Neuron 712, 1-12, May 1, 2024 © 2024 Elsevier Inc. 1

A Human VOR B_ Neurotypical SCN2A LoF

° .— Inertial head angle

+/- 0.05 0.1 02 04 0.6 08 1.0

Figure 1. VOR gain is elevated in Scn2a haploinsufficiency conditions

2 Neuron 112, 1-12, May 1, 2024

Neuron 112, 1-12, May 1, 2024 3

Figure 3. Impaired cerebellar plasticity in

4 Neuron 112, 1-12, May 1, 2024

A. Granule cell excitability B Vermis Flocculus C Vermis — Flocculus

D Purkinje cell excitability (vermis) E500 _

Figure 4. Impaired cerebellar granule cell excitability in Scn2a*” conditions

Neuron 112, 1-12, May 1, 2024 5

A Senzainal = Bg6-Gre (wT) a6-Cre::Sen2za" CY Figure 5. Scn2a heterozygosity in granule

6 Neuron 112, 1-12, May 1, 2024

Neuron 112, 1-12, May 1, 2024 7

Figure 6. Rescue of VOR gain-down behavioral plasticity with CRISPR activation

8 Neuron 112, 1-12, May 1, 2024

of cerebellar plasticity in mouse models of other ASD-associated © Histology

Neuron 112, 1-12, May 1, 2024 9

10 Neuron 112, 1-12, May 1, 2024

Neuron 112, 1-12, May 1, 2024 11

12 Neuron 112, 1-12, May 1, 2024

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Neuron 112, 1-12.e1-e5, May 1, 2024 e1

Human vestibulo-ocular reflex

e2 Neuron 112, 1-12.e1-e5, May 1, 2024

Neuron 112, 1-12.e1-e5, May 1, 2024 e3

e4 Neuron 112, 1-12.e1-e5, May 1, 2024

Neuron 112, 1-12.e1-e5, May 1, 2024 e5

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