dissecting the phenotypes of dravet syndrome by gene deletion · dissecting the phenotypes of...

Dissecting the phenotypes of Dravet syndromeby gene deletion

Moran Rubinstein,� Sung Han,� Chao Tai,� Ruth E. Westenbroek, Avery Hunker,Todd Scheuer and William A. Catterall

*These authors contributed equally to this work.

Neurological and psychiatric syndromes often have multiple disease traits, yet it is unknown how such multi-faceted deficits arise

from single mutations. Haploinsufficiency of the voltage-gated sodium channel Nav1.1 causes Dravet syndrome, an intractable

childhood-onset epilepsy with hyperactivity, cognitive deficit, autistic-like behaviours, and premature death. Deletion of Nav1.1

channels selectively impairs excitability of GABAergic interneurons. We studied mice having selective deletion of Nav1.1 in

parvalbumin- or somatostatin-expressing interneurons. In brain slices, these deletions cause increased threshold for action potential

generation, impaired action potential firing in trains, and reduced amplification of postsynaptic potentials in those interneurons.

Selective deletion of Nav1.1 in parvalbumin- or somatostatin-expressing interneurons increases susceptibility to thermally-induced

seizures, which are strikingly prolonged when Nav1.1 is deleted in both interneuron types. Mice with global haploinsufficiency of

Nav1.1 display autistic-like behaviours, hyperactivity and cognitive impairment. Haploinsufficiency of Nav1.1 in parvalbumin-

expressing interneurons causes autistic-like behaviours, but not hyperactivity, whereas haploinsufficiency in somatostatin-express-

ing interneurons causes hyperactivity without autistic-like behaviours. Heterozygous deletion in both interneuron types is required

to impair long-term spatial memory in context-dependent fear conditioning, without affecting short-term spatial learning or

memory. Thus, the multi-faceted phenotypes of Dravet syndrome can be genetically dissected, revealing synergy in causing epilepsy,

premature death and deficits in long-term spatial memory, but interneuron-specific effects on hyperactivity and autistic-like be-

haviours. These results show that multiple disease traits can arise from similar functional deficits in specific interneuron types.

Department of Pharmacology, University of Washington, Seattle, WA 98195-7280

Correspondence to: William A. Catterall,

Department of Pharmacology,

Box 357280 University of Washington Seattle,

WA 98195-7280,

USA

E-mail: [email protected]

Keywords: Dravet syndrome; sodium channel; Nav1.1; interneurons; parvalbumin; somatostatin

Abbreviations: EPSC/P = excitatory postsynaptic current/potential; IPSC = inhibitory postsynaptic currents; PV = parvalbumin;SST = somatostatin

IntroductionVoltage-gated sodium (Nav) channels regulate neuronal ex-

citability by initiation and propagation of action potentials

and amplification of subthreshold synaptic events.

Heterozygous truncation mutations and other severe loss-

of-function mutations in the SCN1A gene, which encodes

the pore-forming subunit of Nav1.1 sodium channels, cause

doi:10.1093/brain/awv142 BRAIN 2015: 138; 2219–2233 | 2219

Received November 4, 2014. Revised March 17, 2015. Accepted April 4, 2015. Advance Access publication May 27, 2015

� The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.

For Permissions, please email: [email protected]

Dravet syndrome (also called severe myoclonic epilepsy of

infancy), a rare genetically dominant, intractable convulsive

disorder. Dravet syndrome symptoms begin during the first

year of life, with seizures often associated with fever, and

progress to prolonged refractory seizures and frequent epi-

sodes of status epilepticus. During and after the second year

of life, patients develop psychomotor delay, ataxia, cogni-

tive impairment, and social interaction deficits (Genton

et al., 2011; Li et al., 2011).

Studies of mouse genetic models demonstrated that

Dravet syndrome is caused by disinhibition due to reduced

excitability of GABAergic inhibitory interneurons without a

corresponding change in the activity of excitatory neurons

(Yu et al., 2006; Ogiwara et al., 2007, 2013; Cheah et al.,

2012; Dutton et al., 2012; Kalume et al., 2013; Rubinstein

et al., 2014; Tai et al., 2014). Moreover, specific heterozy-

gous deletion of Nav1.1 in forebrain GABAergic inter-

neurons leads to seizures, premature death (Cheah et al.,

2012; Kalume et al., 2013; Ogiwara et al., 2013), cognitive

deficits, and autistic features (Han et al., 2012), similar to

those in patients with Dravet syndrome.

Parvalbumin (PV, encoded by PVALB)-expressing and

somatostatin (SST)-expressing interneurons account for a

large fraction of total interneurons. For instance, in layer

V of the cerebral cortex these two classes include 480% of

total interneurons (Rudy et al., 2011). Previous studies sug-

gested that dysfunction of PV interneurons might contrib-

ute to seizures and premature death in Dravet syndrome.

Deletion of both Nav1.1 alleles in PV-expressing neurons, a

more severe genetic ablation than Dravet syndrome, caused

seizures and premature death (Ogiwara et al., 2013). In

addition, heterozygous deletion of Nav1.1 in PPP1R2-

expressing interneurons, which include forebrain PV-

expressing interneurons but also interneurons positive for

reelin (RELN) and neuropeptide Y (NPY) (Belforte et al.,2010), also increased sensitivity to chemically-induced seiz-

ures (Dutton et al., 2012).

Here we investigated the specific contributions of PV- and

SST-expressing interneurons to the physiological and behav-

ioural consequences of Dravet syndrome using mice with

selective heterozygous and homozygous deletion of Nav1.1

in PV-expressing interneurons, SST-expressing interneurons,

or both. Our results demonstrate that haploinsufficiency of

Nav1.1 in either PV-expressing or SST-expressing inter-

neurons results in reduced excitability of these interneuron

types, spontaneous epileptic cortical discharge patterns with-

out convulsions, consistent with absence or partial seizures,

and increased sensitivity to thermally-induced seizures.

Moreover, deletion in both of these interneuron types

increased the susceptibility and severity of thermally-induced

seizures and the frequency of premature death synergistic-

ally. In contrast, contributions to behavioural deficits in

Dravet syndrome segregated by interneuron type. Reduced

excitability of PV-expressing neurons caused autistic-like

social interaction deficits, whereas impaired excitability of

SST-expressing neurons resulted in hyperactivity. Deletion

in both PV and SST interneurons impaired long-term spatial

memory in context-dependent fear conditioning, but was not

sufficient to recapitulate the full extent of the cognitive im-

pairment observed in Dravet syndrome mice. Our results

dissect the differential contributions of interneuron subtypes

to the complex phenotypes of Dravet syndrome. These data

reveal that impairment of different classes of interneurons is

responsible for distinct aspects of the Dravet syndrome

phenotype, and suggest that cellular deficits in different

classes of interneurons may underlie multi-faceted disease

traits in complex genetic syndromes.

Materials and methodsFor thermal induction of seizures and electrophysiology, weused male and female mice aged 21–24 days (postnatal Day21 age group) or 32–37 days (postnatal Day 35 age group).Behavioural tests were performed on males aged 2–6 months.For EEG recording, fine silver wire EEG electrodes were im-planted under ketamine/xylazine (130/8.8 mg/kg) anaesthesia(Kalume et al., 2013). EEG recordings were collected in con-scious mice using a low impedance head-stage connected toRZ2D bioamp processor (Tucker Davis Technologies) sampledat 6 kHz. EEG signals were processed off-line with a 0–70 Hzlowpass filter. Brain slices were prepared and electrophysiolo-gical studies were carried out as described (Rubinstein et al.,2014; Tai et al., 2014). Thermal induction of seizures wasperformed as described (Oakley et al., 2009, 2013). Theopen field test, the three chamber social interaction test,Barnes circular maze, novel object recognition and Y-mazespontaneous alteration test were performed as described(Han et al., 2012). Our experimental techniques are describedin detail in the Supplementary material.

Results

Impairment of excitability of PV- andSST-expressing cortical interneuronsby selective deletion of Nav1.1channels

Layer V pyramidal cells are the principal output neurons of

the cerebral cortex, and their activity is tightly controlled by

layer V PV-expressing, fast-spiking interneurons and SST-

expressing Martinotti cells (Rudy et al., 2011).

Electrophysiological recordings in cortical slices from mice

with global deletion of Nav1.1 demonstrated reduced excit-

ability of both PV-expressing and SST-expressing inter-

neurons (Tai et al., 2014). Similar recordings were made

here to assess the degree of functional haploinsufficiency of

Nav1.1 upon specific Cre-dependent deletion. Cre expression

in Scn1a+ / + (wild-type) and Scn1afl/ + (Dravet syndrome,

DS) was monitored by the tdTomato reporter, and record-

ings were made from the most brightly labelled cells.

Haploinsufficiency of Nav1.1 in PV-expressing interneur-

ons (PVCre, Scn1afl/ + , termed PV-DS) or SST-expressing

interneurons (SSTCre, Scn1afl/ + , termed SST-DS) resulted

2220 | BRAIN 2015: 138; 2219–2233 M. Rubinstein et al.

http://brain.oxfordjournals.org/lookup/suppl/doi:10.1093/brain/awv142/-/DC1

in increased threshold for action potential generation

(Fig. 1A and B) and higher rheobase (Fig. 1C). In addition,

both the number and frequency of action potentials fired in

response to incremental steps of depolarizing current

injection were reduced by deletion of Nav1.1 in either cell

type (Fig. 1D–G and Supplementary Fig. 1). Layer V inter-

neurons in PV-DS and SST-DS mice also exhibited

increased failure of action potential firing in response to

Figure 1 Deletion of Nav1.1 results in reduced excitability in layer V cortical interneurons. (A) Examples of action potentials

recorded in PV- and SST-expressing layer V cortical interneurons. Scn1a + / + are depicted in black. (B and C) Threshold (B) and rheobase for

action potentials (C) in cortical PV (blue) and SST (yellow) neurons. Action potentials were evoked by 10 ms depolarizing current injection. (D–

G) Firing of cortical neurons with selective deletion of Nav1.1 in response to 1-s long depolarizing current injection at the indicated intensities.

(D) Sample traces of whole-cell current-clamp recordings of PV-expressing neurons in response to injection of 240 pA current. (E) Average

number of action potentials in response to 1 s depolarizing current injection at the indicated intensity (n = 15–19). (F) Sample traces of whole-cell

current-clamp recordings of SST-expressing neurons in response to injection of 160 pA current (n = 15–17). (G) Average number of action

potentials in response to 1 s depolarizing current injection at the indicated intensity. (H–K) Failure rates for action potential generation in cortical

neurons with selective deletion of Nav1.1. Per cent failure of action potentials in PV (H and I) and SST (J and K) interneurons. A train of 100

pulses, each 10 ms long at the minimal current required to trigger action potentials, was given at the indicated frequencies. Sample traces for 100

pulses at 10 Hz are shown (H and J) n = 15–17. Statistical analysis was done using Student’s t-test. *P5 0.05, **P5 0.01, ***P5 0.001.

Dissecting Dravet syndrome phenotypes BRAIN 2015: 138; 2219–2233 | 2221


short repetitive stimulation at frequencies ranging from 5 to

50 Hz (Fig. 1H–K).

These results reveal a common deficit in electrical excit-

ability of these two classes of interneurons, as reflected in

their increased threshold and rheobase for action potential

firing and failures of action potential firing during trains.

The functional impairments observed here are similar or

identical in magnitude to the deficits previously recorded

in mice with Nav1.1 channels deleted globally (Tai et al.,

2014), demonstrating that the Cre-Lox method has been

fully effective in creating functional haploinsufficiency of

Nav1.1 channels in both of these cell types. In addition,

the similar impairment indicates that network changes

caused by loss of Nav1.1 channels in excitatory neurons

and other classes of interneurons in our global knockout

mice do not substantially alter the functional cellular def-

icits beyond those caused by cell-autonomous heterozygous

deletion of Nav1.1 in these PV- and SST-expressing

interneurons.

Spontaneous synaptic activity ofcortical interneurons

As a measure of the impact of deletion of Nav1.1 channels

on the function of neural circuits, we examined the effects of

PV-DS and SST-DS mutations on spontaneous, action poten-

tial-driven, excitatory and inhibitory synaptic activity re-

corded from layer V pyramidal neurons. Deletion of

Nav1.1 in PV-expressing neurons led to reduction in spon-

taneous inhibitory postsynaptic current (IPSC) frequency

Figure 2 Deletion of Nav1.1 in PV neurons, but not in SST neurons, alters the excitation/inhibition balance. Spontaneous IPSC

and spontaneous EPSC were recorded in layer V pyramidal neurons from mice with specific deletion of Nav1.1 in PV or SST mice. (A–D) PV-DS

mice. Example traces of spontaneous IPSC (A) and cumulative histogram of inter-event intervals (B, P = 0.05, Kolmogorov-Smirnov test) with

mean values � SEM (inset) of spontaneous IPSC frequency. Spontaneous IPSC amplitude was 35.9 � 3.1 pA in PVCre Scn1a + / + and 36 � 3.4 pA in

PVCre Scn1afl/ + , P = 0.49. n = 21–24. Example traces of spontaneous EPSC (C) and cumulative histogram of interevent intervals (D, P = 0.038,

Kolmogorov-Smirnov test) with means � SEM (inset) of sEPSC frequency. Spontaneous EPSC amplitude was 25.3 � 1 pA in PVCre Scn1a + / + and

26.7 � 2.2 pA in PVCre Scn1afl/ + , P = 0.28, n = 19–20. (E–H) SST-DS mice. Example traces of spontaneous IPSC (E) and cumulative histogram of

interevent intervals (F) with means � SEM (inset) of spontaneous IPSC frequency. Spontaneous IPSC amplitude was 39.5 � 5.3 pA in SSTCre

Scn1a + / + and 39.4 � 3 pA in SSTCre Scn1afl/ + , P = 0.48. n = 25. Example traces of spontaneous EPSC (G) and cumulative histogram of interevent

intervals (H) with means � SEM (inset) of spontaneous EPSC frequency. Spontaneous EPSC amplitude was 28 � 1.4 pA in SSTCre Scn1a + / + and

27.9 � 1.3 pA in and SSTCre Scn1afl/ + , P = 0.37, n = 19–21. Statistical analysis was done using Student’s t-test. *P5 0.05, **P5 0.01.


with no alteration in the spontaneous IPSC amplitudes

(Fig. 2A and B). Associated with this reduction in inhibitory

synaptic neurotransmission, we observed a significant en-

hancement in spontaneous excitatory postsynaptic current

(EPSC) frequency with no change in amplitude (Fig. 2C

and D). These results indicate that the cortical network in

layer V is hyperexcitable and hyperactive in PV-DS mice. In

contrast to the results with PV-DS mice, the frequencies of

spontaneous IPSCs and spontaneous EPSCs were not chan-

ged in neurons from SST-DS mice (Fig. 2E–H), probably

because they make synapses on the distal dendrites far

from our recordings in cell bodies (see ‘Discussion’ section).

Deficits of excitability and synapticamplification in hippocampalinterneurons

Interneurons in the hippocampus also express PV and SST,

and were shown to be involved in seizure generation in

Dravet syndrome (Liautard et al., 2013). As a complement

to our studies of cortical interneurons, we tested the func-

tional consequence of selective deletion of Nav1.1 in CA1

horizontal stratum oriens lacunosum-moleculare (O-LM)

cells. O-LM cells are critical for regulation of action poten-

tial firing of CA1 pyramidal cells and thereby exert import-

ant inhibitory control of circuit function and prevent

epilepsy (Maccaferri and McBain, 1995; Pouille and

Scanziani, 2004; Coulter et al., 2011). These cells represent

a distinct subtype of interneurons that express both SST

and PV (Maccaferri et al., 2000; Ferraguti et al., 2004),

and their excitability was shown to be reduced in Dravet

syndrome mice with global deletion of Nav1.1 (Rubinstein

et al., 2014). Similar recordings were made here in mice

expressing Cre recombinase in both PV-expressing and

SST-expressing interneurons (PV&SST). Recordings were

made from the brightly labelled horizontal tdTomato-ex-

pressing cells, and all of the cells displayed the characteris-

tic sag and firing pattern of O-LM cells (Supplementary

Fig. 2A) (Maccaferri and McBain, 1996; Zemankovics

et al., 2010). Similar to cortical interneurons, haploinsuffi-

ciency of Nav1.1 (PV&SST-DS) resulted in increased

threshold and rheobase for action potential firing

(Fig. 3A–C) and reduced number of action potentials in

prolonged depolarizations (Supplementary Fig. 2B), demon-

strating the importance of Nav1.1 in excitability of OL-M

cells.

To test the effects of selective deletion of Nav1.1 on

evoked synaptic activity, a stimulating electrode was

placed in the alveus to activate the axons of CA1 pyramidal

cells antidromically (Maccaferri and McBain, 1995; Pouille

and Scanziani, 2004), and the stimulation intensity was set

to produce a firing probability of 50% during trains of

stimuli (i.e. five action potentials out of 10 stimuli delivered

to the alveus at 1 Hz). This protocol effectively normalized

for changes in intrinsic excitability (Rubinstein et al.,

2014). The threshold for synaptically evoked action

potentials in PV&SST-DS interneurons was �7 mV more

depolarized (Fig. 3D and E), and accordingly the near-

threshold evoked excitatory postsynaptic potential (EPSP)

amplitude was increased (Fig. 3G). Sodium currents com-

bine to enhance and amplify the response to subthreshold

synaptic inputs (Carter et al., 2012), and deletion of Nav1.1

resulted in a marked shortening of the EPSP half-width

(Fig. 3F and H), which was accompanied by reduction in

the time integral of depolarization during the EPSP (Fig.

3I). Associated with the larger and shorter EPSPs, the la-

tency to spike generation and the spike jitter were signifi-

cantly reduced (Fig. 3D and Supplementary Fig. 2C and D).

Together, these data demonstrate that selective deletion

of Nav1.1 hampers the excitability of multiple types of

interneurons in both the cerebral cortex and the hippocam-

pus. This reduced excitability in both brain regions recap-

itulates the cellular effects observed previously in Dravet

syndrome mice with global deletion of Nav1.1

(Rubinstein et al., 2014; Tai et al., 2014), confirming ef-

fective reduction of Nav1.1 currents in these classes of

interneurons upon Cre expression.

Seizures in mice with selectivedeletion of Nav1.1 channels inPV-expressing interneurons

Loss-of-function mutations in SCN1A are associated with a

range of epilepsies, all involving infantile onset of febrile

seizures that proceed beyond childhood. With global

Dravet syndrome mice, seizures can be induced by mild

elevation of core body temperature late in the third week

of life (Oakley et al., 2009), providing a natural patho-

physiological stimulus for measurement of seizure suscepti-

bility. Therefore, we tested mice with selective deletion of

Nav1.1 for their susceptibility to thermally-induced seizures

at two different ages, postnatal Day 21, the age at which

Dravet syndrome mice with global Nav1.1 deletion first

experience spontaneous seizures (Yu et al., 2006; Oakley

et al., 2009), and postnatal Day 35 following the period of

most intense spontaneous seizures (Kalume et al., 2013).

Body core temperature was increased up to 42�C, and

none of the Scn1a + / + mice tested experienced seizures in

this temperature range. PV-DS mice did not have thermally-

induced seizures at postnatal Day 21 (Fig. 4A); however,

seizures could be evoked in 50% of PV-DS mice at post-

natal Day 35 (Fig. 4B). Previous studies report that ad-

equate Cre expression in this mouse line is restricted to

neurons with the strongest PV expression (Madisen et al.,

2010; Ogiwara et al., 2013). Therefore, we also tested mice

that were heterozygous for deletion of Scn1a but homozy-

gous for expression of PV-Cre (PVCre/Cre, Scn1afl/ + ). In

these mice, the expression of Cre protein is potentially

2-fold higher, and therefore Scn1a deletion should be

achieved in PV neurons with lower activation of the PV

promoter. Indeed, homozygous expression of PV-Cre

increased the number of Cre-expressing cells by �25%







(Supplementary Fig. 3). PV-DS mice with homozygous Cre

expression were already sensitive to thermal induction of

seizures at postnatal Day 21 (Fig. 4D, blue). Moreover, at

postnatal Day 35, all the tested PVCre/Cre, Scn1afl/ + mice

had thermally-induced seizures (Fig. 4E, blue), and their

seizures were longer compared to PVCre, Scn1afl/ + mice

(Fig. 4C, F, blue; 19.3 � 3 s and 135.2 � 18.7 s in PVCre,

Scn1afl/ + and PVCre/Cre, Scn1afl/ + , respectively, P50.01).

Figure 3 Deletion of Nav1.1 results in reduced excitability of CA1 stratum oriens hippocampal interneurons. (A–C) Action

potentials evoked by direct depolarization of cell body via the patch pipette. (A) Examples of action potentials. (B and C) Threshold (B) and

rheobase (C) of action potentials, n = 7. (D–G) Properties of synaptically evoked action potentials and near-threshold EPSPs in stratum oriens

neurons. Stimulating electrode was placed in the alveus and the stimulation strength was set to produce firing probability of 50% during trains of

10 stimuli at 1 Hz. Representative action potentials (D) and mean values � SEM for threshold for action potential generation (E). (F–I)

Parameters for near-threshold EPSPs. Examples of EPSPs (F). Mean values � SEM for EPSP amplitude (G), duration (H) and time integral (I),

n = 7–9. To control for changes in properties of voltage-dependent currents activated by EPSPs of different amplitude, we examined the duration

of EPSPs with similar amplitude. These were also of shorter duration in PV&SST-DS (Supplementary Fig. 2E and F), indicating that loss of Nav1.1

currents, and not voltage-dependent changes in the kinetics of the EPSP due to its higher amplitude, cause reduced amplification. Statistical

analysis was done using Student’s t-test. **P5 0.01, ***P5 0.001.





Figure 4 Epileptic phenotype of mice with deletion of Nav1.1 in PV- and SST-expressing interneurons. (A–C) Epileptic phenotypes

of mice with heterozygous expression of Cre. (A–B) Percentage of mice remaining free of behavioural seizures (SZ) at the indicated body core

temperatures at postnatal Day 21 (P21, A) and postnatal Day 35 (P35, B). Selective deletion of Nav1.1 in PV-expressing neuron (blue, n = 10), SST-

expressing neurons (yellow, n = 8), or combined deletion of Nav1.1 in both PV and SST expressing neurons (green, n = 17). (C) Mean

values � SEM for seizure duration at postnatal Day 35. (D–F) Epileptic phenotypes of mice with homozygous expression of PV Cre. Mean

values � SEM for the percentage of mice remaining free of behavioural seizures (SZ) at the indicated body core temperatures at postnatal Day 21

(D) and postnatal Day 35 (E). (F) Mean values � SEM for seizure duration at postnatal Day 35. Statistical analysis was done using one-way

ANOVA, **P5 0.01. PVCre/Cre-DS, n = 9, SSTCre/Cre-DS, n = 5, PVCre/Cre&SST-DS, n = 8. The epileptic phenotype SSTCre, Scn1afl/ + was indistin-

guishable from that of SSTCre/Cre, Scn1afl/ + (P4 0.05). Similarly the phenotype of PVCre/Cre, SSTCre, Scn1afl/ + was indistinguishable from that of

PVCre/Cre, SSTCre/Cre, Scn1afl/ + (n = 4 for each) and these data were pooled. Scn1a + / + mice of either genotype do not have seizures at the tested

temperatures (n = 3–7 for each genotype). (G–I) Examples of EEG recordings from cortical lead depicting a spontaneous electrographic seizure in

PVCre/Cre-DS (G), SSTCre-DS (H) and PVCre/Cre&SST-DS (I).


Thus, we observed a gene-dosage effect in the epileptic

phenotypes of PV-DS mice. Heterozygous deletion of

Nav1.1 in some PV interneurons is sufficient to cause ther-

mally-induced seizures, and increasing the number of

affected PV interneurons by homozygous expression of

PV-Cre leads to earlier onset of temperature induced seiz-

ures and longer duration of seizures. We did not observe

any spontaneous convulsive seizures in PVCre/Cre-DS mice

during routine handling or continuous home cage video

recordings between postnatal Days 22–26 (n = 5), the

period of most intense and frequent spontaneous seizures

in mice with global deletion of Nav1.1 (Kalume et al.,

2013). In contrast, video-EEG recordings of PVCre/Cre,

Scn1afl/ + revealed brief (5 s) bilateral abnormal cortical dis-

charge pattern that was associated with a short pause of

behaviour without convulsions, consistent with absence or

partial seizures (Fig. 4G). Together, these results

demonstrate that deletion of Nav1.1 only in PV neurons

is sufficient to cause mild spontaneous seizures and ther-

mally-induced generalized tonic-clonic seizures, but not

premature death.

Seizures in mice with selectivedeletion of Nav1.1 channels inSST-expressing interneurons

Loss of function of Nav1.1 in SST neurons was also suffi-

cient to induce thermal seizures at postnatal Day 35 but

not at postnatal Day 21 (Fig. 4A–C). Cre expression under

the SST promoter is highly efficient (Taniguchi et al.,

2011), and mice with homozygous expression of Cre

(SSTCre/Cre, Scn1afl/ + ) were indistinguishable from mice ex-

pressing only one allele of SST-Cre (SSTCre, Scn1afl/ + )

(Fig. 4A–F). Video-EEG recordings revealed brief episodes

of abnormal cortical discharge patterns that were not asso-

ciated with convulsions (Fig. 4H). No premature death or

convulsive seizures were observed during routine handling

or continuous home cage video recordings from postnatal

Days 22–26 (n = 3).

Together, these results demonstrate that deletion of Nav1.1

only in SST neurons is sufficient to cause susceptibility to

thermally-induced generalized tonic-clonic seizures as well as

mild spontaneous seizures, but not premature death.

Synergistic seizure susceptibility andseverity with combined deletion ofNav1.1 in PV and SST interneurons

As PV-expressing and SST-expressing neurons both con-

tributed to epilepsy in Dravet syndrome, we wondered

whether deletion of Nav1.1 in both types of interneurons

would have synergistic effects. Although PV-DS and SST-

DS mice were not susceptible to thermally-induced seizures

at postnatal Day 21, 88% of PV&SST-DS mice (15/17) had

thermally-induced seizures (Fig. 4A). Moreover, at postna-

tal Day 35, seizure duration in PV&SST-DS mice was

�6-fold longer than in PV-DS mice and �3-fold longer

than in SST-DS mice (Fig. 4C). In addition, while deletion

of Nav1.1 in PV or SST alone did not cause increased mor-

tality, we did observe occasional premature deaths in

PV&SST-DS mice (93% survival at 3 months). As observed

in PV-DS mice, homozygous expression of PV-Cre in PVCre/

Cre&SST-DS mice increased the sensitivity to thermally-

induced seizures (Fig. 4D and E) as well as seizure duration

(Fig. 4F). Thus, heterozygous deletion of Nav1.1 simultan-

eously in both types of interneurons results in earlier onset

of seizure susceptibility and longer duration of thermally-

induced seizures, and those effects are greater than additive

in several respects. Nevertheless, similar to PV-DS and

SST-DS, no spontaneous convulsive seizures were observed

in PVCre/Cre&SST-DS mice during routine handling or con-

tinuous home cage video recordings between postnatal

Days 22 and 26 (n = 4); however, EEG recordings of

PVCre/Cre&SST-DS revealed abnormal cortical discharge

patterns that were not associated with observable convul-

sions (Fig. 4I).

Epileptic phenotype of mice withhomozygous deletion of Scn1a

Mice with homozygous deletion of Scn1a have severe

ataxia, frequent seizures and all die by postnatal Day 14

(Yu et al., 2006; Kalume et al., 2007; Ogiwara et al.,

2007). Homozygous deletion of Scn1a in PV-expressing

neurons (PVCre, Scn1afl/fl) resulted in severe ataxia, spon-

taneous convulsive seizures, high sensitivity to thermally-

induced seizures, and frequent premature death, with only

40% survival at 3 months of age (Fig. 5A–C). Thus, the

degree of loss of Nav1.1 function in PV-expressing neurons

has dramatic effects on the epileptic phenotype. In contrast,

the epileptic phenotype of mice with complete deletion of

Scn1a in SST-expressing neurons (SSTCre, Scn1afl/fl) was

similar to SST-DS mice. These mice had seizures at high

temperature (Fig. 5A and B) and no premature death was

observed (Fig. 5C). Mice with complete deletion of Nav1.1

in both PV and SST interneurons (PVCre, SSTCre, Scn1afl/fl)

exhibited ataxia, frequent spontaneous convulsive

seizures, and thermally induced seizures at low

temperatures (Fig. 5A and B). Moreover, spontaneous

death was frequent (Fig. 5C). These results with homozy-

gous deletion of Nav1.1 in PV&SST-DS mice support the

conclusion that deletion in both PV- and SST-expressing

neurons has synergistic effects on epilepsy and premature

death.

Specific effect of deletion of Nav1.1 inSST-expressing interneurons onhyperactivity

Our previous results show that Dravet syndrome mice with

global deletion of Scn1a display behavioural traits that are

found in patients with Dravet syndrome, including


hyperactivity, autistic-like social interaction deficits, and

cognitive impairments (Han et al., 2012). Moreover,

while floxed Scn1a + / + mice were indistinguishable from

wild-type mice, Scn1afl/ + mice with specific heterozygous

deletion of Nav1.1 in forebrain interneurons displayed all

Dravet syndrome-associated behavioural deficits (Han

et al., 2012). However, which types of interneurons are

responsible for these behavioural phenotypes is not

known. To answer this question, we performed behavioural

tests with PV-DS, SST-DS, and PV&SST-DS mice, focusing

on behaviours that we have previously shown to be altered

in Dravet syndrome mice. As a first step, we measured the

total distance moved during 10 min in the open field test to

examine general locomotor activity in a novel environment.

PV-DS mice displayed normal locomotor activity (Fig. 6A).

However, SST-DS mice (Fig. 6B) and PV&SST-DS mice

(Fig. 6C) travelled significantly longer distances. These

data indicate that deficit of excitability in SST-expressing

interneurons contributes to hyperactivity in Dravet syn-

drome mice, whereas impairment of excitability in PV-

expressing interneurons does not.

Specific effect of deletion of Nav1.1in PV-expressing interneurons onautistic-like behaviours

We tested the social behaviour of these mice with the three-

chamber test of social interaction. In this test, mice are

placed in the central compartment of a three-chambered

plexiglass box and allowed to interact with an object (a

small empty cage) in one side chamber or with a stranger

mouse (in an identical small cage) in the other side chamber.

Scn1a+ / + mice spent �2-fold more time interacting with a

stranger mouse than with the empty cage (Fig. 7A and B). In

contrast, PV-DS mice displayed a social interaction deficit

and exhibited no preference for interaction with a stranger

mouse compared to the empty cage (Fig. 7A and B). No

social deficit was apparent for SST-DS mice, which displayed

a strong preference for interaction with the stranger mouse

(Fig. 7C and D). The double Cre-expressing PV&SST-DS

mice recapitulated the autistic-like social deficits of the PV-

DS mice (Fig. 7E and F). These data provide evidence that

reduced electrical excitability in PV-expressing interneurons

contributes specifically to social interaction deficits in Dravet

syndrome, but impairment of excitability of SST-expressing

interneurons does not.

Effect of deletion of Nav1.1 in PV- andSST-expressing interneurons onspatial learning and memory

Cognitive impairment is characteristic in patients with

Dravet syndrome. Similarly, Dravet syndrome mice with

global or specific heterozygous deletion of Scn1a in fore-

brain interneurons, have deficits in short-term and long-

term context-dependent fear conditioning and in spa-

tial memory (Han et al., 2012). In contrast, PV-DS,

Figure 5 Epileptic phenotype of mice with selective dele-

tion of both alleles encoding Nav1.1. (A–C) Epileptic pheno-

types of Scn1afl/fl mice. (A) Percentage of mice remaining free of

behavioural seizures (SZ) at the indicated body core temperatures

at postnatal Day 21. (B) Means values � SEM for the temperature of

seizure induction. (C) Survival plots for PVCre, Scn1afl/fl mice

(n = 22, blue), SSTCre, Scn1afl/fl mice (n = 15, yellow) and PVCre,

SSTCre, Scn1afl/fl mice (n = 8, green). Statistical analysis was done

using one-way ANOVA. *P5 0.05.

Figure 6 Selective deletion of Nav1.1 in SST-expressing

neurons causes hyperactivity. Distance moved in the open field

test for PV (A, n = 8), SST (B, n = 6), and PV&SST mice (C, n = 10).

The behaviour of SSTCre and SSTCre/Cre mice was indistinguishable in

all the tests performed and the data were pooled together.

Statistical analysis was done using Student’s t-test. *P5 0.05.


PVCre/Cre-DS, SST-DS and PV&SST-DS mice did not dem-

onstrate deficits in context-dependent fear-conditioning. All

of these mice displayed robust freezing behaviour when they

returned to the fearful spatial context of a foot shock 30 min

and 24 h later (Fig. 8A–C). In contrast, PVCre/Cre&SST-DS

mice demonstrated normal freezing behaviour immediately

following a foot shock and 30 min later, but their freezing

behaviour was significantly reduced after 24 h. These results

indicate that simultaneous deletion of Nav1.1 in both PV-

and SST-expressing neurons causes impairment of long-term

context-dependent fear memory (Fig. 8D).

We further assessed spatial learning in the absence of a

fear stimulus using the Barnes circular maze. PVCre/

Cre&SST-DS and littermate controls were trained three

times per day for four consecutive days to find a specific

target hole. In contrast to mice with global deletion Scn1a

(Han et al., 2012), PVCre/Cre&SST-DS mice were able to

improve their performance, and their latency to reach the

target hole decreased with training similar to littermate

controls (Fig. 8E). During the probe trial on the fifth day,

both groups of mice remembered the spatial location of the

target hole (Fig. 8F). Thus, deletion of Nav1.1 in PV and

SST neurons does not recapitulate the spatial learning def-

icit observed in Dravet syndrome mice (Han et al., 2012).

To further examine short-term memory and working

memory, we tested PVCre/Cre&SST-DS in novel object rec-

ognition and spontaneous alteration in the Y-maze. PVCre/

Cre&SST-DS performance was similar to that of littermate

controls in both tests (Fig. 8G–I), indicating that loss func-

tion of Nav1.1 in PV and SST neurons does not impair

short-term working memory. Altogether, while simultan-

eous deletion of Nav1.1 in PV and SST expressing neurons

can affect long-term fear memory, other class(es) of inter-

neurons are involved in causing more severe short-term

memory impairment and spatial learning deficits observed

in mice with global deletion of Nav1.1.

Dissecting behavioural phenotypes inDravet syndrome

Taken together, our behavioural data demonstrate that im-

pairment of excitability in different subtypes of inter-

neurons is differentially involved in the distinct

behavioural deficits that we have observed in Dravet syn-

drome mice. These results reveal how a single gene muta-

tion can result in multi-faceted neurophysiological and

behavioural traits in a complex neuropsychiatric syndrome.

Hyperactivity, impaired social interactions, and cognitive

impairment are all characteristics of children with Dravet

syndrome (Genton et al., 2011; Li et al., 2011). Therefore,

it is conceivable that these traits in patients with Dravet

syndrome may also involve impaired excitability of differ-

ent classes of interneurons. Dissection of these phenotypes

of Dravet syndrome by specific gene deletion in mice is

considered further below.

DiscussionLoss-of-function mutations in Nav1.1 cause Dravet syn-

drome in mice by selective impairment of excitability in

GABAergic interneurons without detectable effects on exci-

tatory neurons (Yu et al., 2006; Ogiwara et al., 2007;

Cheah et al., 2012; Dutton et al., 2012; Tai et al., 2014).

PV interneurons preferentially form synapses at the periso-

matic region of their target cells, thereby controlling neur-

onal activity and spike output, whereas SST-expressing

interneurons preferentially contact the distal dendrites,

which allow them to efficiently control the impact of syn-

aptic inputs near these sites (Klausberger and Somogyi,

2008; Harris and Mrsic-Flogel, 2013). The complementary

and coordinated functions of these two types of inter-

neurons provide versatile and powerful control of the ac-

tivity in neuronal circuits. However, it is not known how

Figure 7 Selective deletion of Nav1.1 in PV-expressing

neurons causes social deficit. Three-chamber test of social

interaction for PV (A and B, n = 8–9), SST (C and D, n = 11–14) and

PV&SST (E and F, n = 6–7) mice. Time in each chamber (A, C and

E) and total interaction time (B, D and F): O = object (an empty

small mouse cage); C = centre; M = mouse (a stranger mouse in an

identical small cage). Statistical analysis was done using Student’s t-

test., *P5 0.05, **P5 0.01, ***P5 0.001.


impaired electrical excitability in these two classes of inter-

neurons would contribute to the multi-faceted neurological

and behavioural phenotypes in Dravet syndrome. Here we

show that Nav1.1 is important for normal synaptic boost-

ing and action potential firing of both PV- and SST-

expressing interneurons and that reduced excitability of

these major classes of interneurons contributes synergistic-

ally to the epilepsy and differentially to the behavioural

deficits associated with Dravet syndrome.

Deletion of Nav1.1 in pyramidalneurons does not cause Dravetsyndrome phenotypes

Previous studies have shown that global haploinsufficiency

of Nav1.1 channels has no detectable effect on sodium

currents or action potential firing in pyramidal neurons in

hippocampus or cerebral cortex (Yu et al., 2006; Ogiwara

et al., 2007; Rubinstein et al., 2014; Tai et al., 2014).

Selective deletion of Nav1.1 channels in forebrain

GABAergic interneurons fully reproduces the complex epi-

lepsy and behavioural phenotypes of Dravet syndrome in

these mice (Cheah et al., 2012; Ogiwara et al., 2013).

Moreover, selective deletion of Nav1.1 channels in excita-

tory neurons does not cause the disease traits of Dravet

syndrome and can actually oppose the lethal effects of

the disease by reducing the frequency of premature death

(Dutton et al., 2012; Ogiwara et al., 2013). Thus, in mouse

genetic models of Scn1a deletion, which reproduce all of

the complex facets of Dravet syndrome, no evidence has

emerged that alteration of excitability of pyramidal neurons

contributes to causing disease phenotypes.

Figure 8 Cognitive phenotype of mice with selective deletion of Nav1.1. Context-dependent fear conditioning test in PVCre (A, n = 16-

19 for Scn1a + / + and Scn1afl/ + , respectively), SST (B, n = 10), PV&SST mice (C, n = 10) and PVCre/Cre&SST mice (D, n = 7–9). Freezing behaviour

of PVCre (n = 6–9) were indistinguishable from that of PVCre/Cre (n = 10) and these data were pooled in A. Basal = freezing time (%) during

exploration of a cage containing an electrical grid as a floor and markings for identification; training = freezing time (%) during presentation of a

0.6 mA, 2 s long foot shock; 30 min = freezing time (%) upon return to the same cage after 30 min but without shock; 24 h = freezing time (%) upon

return to the same cage after 24 h but without shock. (E) In the Barnes circular maze, both PVCre/Cre&SST-DS mice and littermate controls

decreased their latency to the target hole similarly over the 4-day repeated training trials. (F) Latency to the target hole during the probe trial on

the fifth day (n = 6 for each genotype). (G) In the novel object recognition test PVCre/Cre&SST-DS mice and littermate controls had normal

recognition memory for a preconditioned object (F = familiar), which was presented 2 min before the test, and spent more time with the novel

object (N = novel). (H) The normalized ratio of time spent with the familiar object divided by time spent with the novel object (n = 10–11 for

each genotype). (I) Y-maze task: spontaneous alternation behaviour during a 10-min session (n = 5 for each genotype). Statistical analysis was done

using Student’s t-test. *P5 0.05, **P5 0.01.


Deletion of Nav1.1 in PV-expressingneurons results in reducedexcitability, seizures andautistic-like behaviour

In contrast to the lack of effect in pyramidal neurons, de-

letion of Nav1.1 in PV-expressing interneurons in cortical

layer V resulted in reduced excitability due to increased

threshold and rheobase, reduced number and frequency

of action potentials, and inability to sustain firing during

trains of depolarizations (Fig. 1). These deficits are similar

to the loss of excitability observed in mice with global de-

letion of Nav1.1 (Tai et al., 2014). Moreover, deletion of

Nav1.1 selectively in PV neurons was sufficient to increase

the excitation/inhibition ratio (Fig. 2), as observed previ-

ously in Dravet syndrome mice (Han et al., 2012).

Together, our physiological recordings confirmed that Cre

expression in PV interneurons results in reduced function of

Nav1.1, and the reduced excitability of PV interneurons in

PV-DS mice is comparable to the functional deficits

observed in Dravet syndrome mice.

These deficits in excitability at the cellular level were suf-

ficient to cause thermally-induced seizures in PVCre, Scn1afl/+

mice (heterozygous expression of Cre and Scn1a) (Fig. 4).

Homozygous expression of Cre in PVCre/Cre, Scn1afl/ +

mice resulted in brief spontaneous non-convulsive seizures,

and increased the sensitivity to thermally-induced general-

ized tonic-clonic seizures. Generalized tonic-clonic seizures

could be evoked as early as postnatal Day 21, and their

duration was longer at postnatal Day 35 (Fig. 4). These

results demonstrate the contribution of PV interneurons

to epilepsy in Dravet syndrome (Fig. 4). Although hetero-

zygous deletion of Nav1.1 is the relevant genetic ablation in

patients with Dravet syndrome, and mice with homozygous

global deletion of Nav1.1 do not survive beyond postnatal

Day 14 (Yu et al., 2006; Ogiwara et al., 2007), we also

tested the epileptic phenotype in mice with cell-specific

homozygous deletion of Scn1a. Similar to previous findings

(Ogiwara et al., 2013), PVCre, Scn1afl/fl mice had increased

mortality and experienced seizures at or slightly above

normal body temperature (Fig. 5). Thus, selective deletion

of Nav1.1 in PV neurons demonstrated gene dosage effects

that exacerbated the epileptic phenotype in two ways: by

increasing the number of cells in which Scn1a gene is

deleted via homozygous expression of Cre in PVCre/Cre

mice and by increasing the loss of excitability via deletion

of both alleles in Scn1afl/fl mice.

Unlike other generalized epilepsy disorders, patients with

Dravet syndrome have autism-spectrum behaviours (Li

et al., 2011). Reduction in the number of PV neurons

was observed in multiple mouse models of autism spectrum

disorders (Gogolla et al., 2009). Strikingly here, PV-DS

mice showed reduced social interaction in the three-

chamber test (Fig. 7), similar to globally deleted Dravet

syndrome mice (Han et al., 2012). PV-DS mice were not

hyperactive (Fig. 6) and performed similarly to control

animals in context-dependent fear conditioning, indicating

normal spatial learning and memory in this experimental

paradigm (Fig. 8). Altogether, our experiments provide a

clear dissection of the complex phenotypes of Dravet syn-

drome and show that dysfunction of PV-expressing inter-

neurons due to loss of Nav1.1 is sufficient to cause epilepsy

and social interaction deficit, but not hyperactivity or loss

of context-dependent fear memory.

Deletion of Nav1.1 in SST-expressingneurons results in seizures andhyperactivity

We tested two types of SST-positive neurons: cortical layer

V Martinotti cells, which express SST (Silberberg and

Markram, 2007), and hippocampal horizontal O-LM

cells, which express both SST and PV (Maccaferri et al.,

2000; Ferraguti et al., 2004). Deletion of Nav1.1 in either

type of SST-expressing neuron resulted in increased thresh-

old and rheobase for action potential firing (Figs 1 and 3).

Moreover, SST-DS neurons failed to sustain firing during

prolonged depolarizations (Fig. 1F and G, and

Supplementary Fig. 2B) and trains of action potentials

(Fig. 1J and K). In the hippocampus, SST-DS interneurons

had higher threshold for synaptically evoked action poten-

tials and shorter duration of near threshold EPSPs (Fig. 3).

Together, these data demonstrate the importance of Nav1.1

in setting the threshold and initiation of action potentials in

multiple types of interneurons (Rubinstein et al., 2014; Tai

et al., 2014), and confirm loss of function of Nav1.1 on

SST-Cre expression. Like PV-DS mice, SST-DS mice had

mild spontaneous seizures and were also susceptible to

thermal induction of generalized tonic-clonic seizures (Fig.

4). However, thermally-induced seizures occurred at high

temperatures with both heterozygous and homozygous de-

letion of Nav1.1, and no premature death was observed

(Fig. 5). Thus, heterozygous deletion of Nav1.1 channels

only in SST-expressing neurons produced a mild epileptic

phenotype.

In layer V of the cerebral cortex, SST-expressing

Martinotti cells are increasingly recruited by sustained

high-frequency stimulation and provide frequency-depend-

ent disynaptic inhibition to pyramidal cells (Silberberg and

Markram, 2007). This local inhibitory circuit is disrupted

in Dravet syndrome mice (Tai et al., 2014). However, this

feedback circuit would not be expected to be active under

basal conditions of electrical activity because an intense

burst of action potentials is required to produce inhibition

(Silberberg and Markram, 2007). Moreover, SST-express-

ing neurons also target PV interneurons (Letzkus et al.,

2011; Pfeffer et al., 2013), and reduction in their excitabil-

ity might have opposing effects in small circuits. In agree-

ment with these characteristics, we found that the

excitation/inhibition balance of spontaneous excitatory

and inhibitory synaptic activity recorded at the cell body

of pyramidal neurons was unchanged in SST-DS cortical



slices (Fig. 2E–H). It may be that stronger stimuli, such as

higher temperatures, are needed to reveal the effects of

reduced synaptic inhibition in SST-DS mice.

Hyperactivity is a well-documented behavioural charac-

teristic in children with Dravet syndrome (Li et al., 2011)

and in Dravet syndrome mice (Han et al., 2012; Ito et al.,

2012). We found that SST-DS mice were hyperactive in an

open field test (Fig. 6). However, SST-DS mice did not

show any deficit in social interaction in the three-chamber

test (Fig. 7) or in the context-dependent fear-conditioning

test (Fig. 8). Taken together, our results with SST-DS mice

further dissect the phenotypes of Dravet syndrome by

showing that Nav1.1 is important for normal function of

SST neurons and that specific loss of excitability of these

neurons contributes to epilepsy and hyperactivity but not to

autistic-like behaviours or deficit in spatial learning and

memory as assessed in the context-dependent fear condi-

tioning test.

Synergistic effects of deletion ofNav1.1 in PV- and SST-expressingneurons on epilepsy and prematuredeath

Activity in neuronal circuits is tightly regulated by different

subtypes of inhibitory interneurons (Klausberger and

Somogyi, 2008; Wolff et al., 2014). PV-expressing, fast-

spiking interneurons and SST-expressing Martinotti cells

have complementary roles in providing inhibitory synaptic

input at the cell bodies and basal dendrites versus the distal

dendrites of layer V pyramidal cells, respectively. Our re-

sults show that reduced excitability of both PV and SST

interneurons contributes to Dravet syndrome, and further

that deletion of Nav1.1 in both types of interneurons sim-

ultaneously results in more severe epilepsy. PV&SST-DS

mice were sensitive to thermally-induced seizures at post-

natal Day 21, when PV-DS or SST-DS mice were seizure-

free (Fig. 4A). At postnatal Day 35, when all the genotypes

had thermally-induced seizures, the seizures in PV&SST-DS

mice were much longer in duration (Fig. 4C and F).

Moreover, some PV&SST-DS mice died prematurely,

whereas we did not observe any premature death in PV-

DS mice with heterozygous or homozygous expression of

PV-Cre or in SST-DS mice. Comparison of homozygous

Scn1afl/fl mice also demonstrated synergistic effects.

Thermally-induced seizures of PVCre, SSTCre, Scn1afl/fl

mice occurred at lower temperatures compared to either

PVCre, Scn1afl/fl or SSTCre, Scn1afl/fl (Fig. 5B), and all

PVCre, SSTCre, Scn1afl/fl mice died, whereas 40% of PVCre,

Scn1afl/fl survived at 3 months of age, and no mortality was

observed in SSTCre, Scn1afl/fl mice (Fig. 5C). The synergy

observed for deletion of Nav1.1 channels in PV- and SST-

expressing interneurons in epilepsy and premature death

may reflect the synergistic effects of impairing their two

complementary forms of inhibition in the cell body/

proximal dendritic versus distal dendritic compartments

of cortical pyramidal neurons.

Differential effects of deletion inPV- and SST-expressing interneuronson behaviour

Dysfunction of interneurons, and the resulting excitation/

inhibition imbalance are involved in the aetiology of many

neurological and psychiatric syndromes such as epilepsy,

autism and schizophrenia. However, it is unclear how spe-

cific interneuron deficits contribute to pathophysiology of

the complex behavioural phenotypes (Marin, 2012). Our

experiments provide a clear dissection of the roles of PV-

expressing versus SST-expressing interneurons in causing

hyperexcitability and autistic-like phenotypes in Dravet

syndrome mice. SST-DS mice are hyperactive, whereas

PV-DS mice are not. PV-DS mice show autistic-like behav-

iours, whereas SST-DS mice do not. Behavioural tests with

PV&SST-DS mice showed reduced social interaction that

was similar to PV-DS and hyperactivity that was similar

to SST-DS (Figs 6 and 7). These results suggest that the

effects of deletion of Nav1.1 channels in PV-DS mice are

truly primary in causing autistic-like phenotypes, and fur-

ther deletion in SST-expressing interneurons has no add-

itional effect on that phenotype. Similarly, it seems that

deletion of Nav1.1 channels in SST-DS mice is truly pri-

mary in causing hyperactivity and further deletion in PV-

expressing interneurons has no additional effect on that

phenotype, even in the sensitized background of SST-DS

mice.

In contrast, while PV- and SST-DS mice had normal

freezing behaviour in the context-dependent fear condition-

ing test, PVCre/Cre&SST-DS mice had reduced freezing after

24 h, demonstrating synergistic effects of PV and SST in

impairment of long-term fear-related spatial memory (Fig.

8D). Interestingly, it was recently shown that memory re-

trieval circuits change with time (Do-Monte et al., 2015),

so it is possible that normal firing of other classes of inter-

neurons is sufficient for retrieval of fearful memory early

after the event, while function of PV and SST interneurons

is essential for retrieval at later time points. In this context,

it was surprising that PVCre/Cre&SST-Dravet syndrome mice

did not have a deficit in spatial learning in the Barnes maze,

as indicated by their ability to improve their performance

each day and to respond normally in the probe trial (Fig.

8E and F). It is conceivable that repeated training each day

for 4 days is sufficient to overcome the deficit in long-term

memory.

Contribution of other interneuronsto Dravet syndrome

The data presented here clearly dissect the roles of PV- and

SST-expressing interneurons in Dravet syndrome and its

associated comorbidities. However, mice with deletion of


Nav1.1 in all forebrain interneurons recapitulated all the

epileptic symptoms (Yu et al., 2006; Oakley et al., 2009,

2013; Kalume et al., 2013), hyperactivity, reduced sociabil-

ity and cognitive deficits (Cheah et al., 2012; Han et al.,

2012). In contrast, not all of these symptoms were observed

in comparable severity in PV&SST-DS mice. The epileptic

phenotype of PV&SST-DS mice is mild compared to global

or interneuron-specific deletion of Nav1.1 (Yu et al., 2006;

Ogiwara et al., 2007; Oakley et al., 2009; Cheah et al.,

2012; Kalume et al., 2013) because they did not have spon-

taneous generalized tonic-clonic seizures, their thermally-

induced generalized tonic-clonic seizures occurred at

higher temperatures, and premature death was infrequent.

Moreover, while long-term memory in the context-depend-

ent fear conditioning test was impaired (Fig. 8), short-term

fear memory and spatial learning in the Barnes maze were

preserved, indicating less severe cognitive impairment com-

pared to Dravet syndrome mice.

These results show that the severe deficit in spatial learn-

ing and memory in Dravet syndrome mice requires add-

itional deletion of Nav1.1 channels in interneurons other

than those that express PV and SST. Future studies of add-

itional classes of interneurons by specific deletion of Nav1.1

channels using the Cre-Lox method may give further in-

sights into the complete set of interneurons in which

Nav1.1 channels must be deleted to fully replicate Dravet

syndrome.

Complex behavioural deficits inneuropsychiatric disease

Beyond their direct implications for Dravet syndrome, our

results provide the first example, to our knowledge, of dis-

section of complex epileptic and behavioural phenotypes by

gene deletion in single classes of interneurons in any neuro-

psychiatric disease. Because complex combinations of

phenotypes are common in neuropsychiatric syndromes

(Marin, 2012), this study may provide a precedent for

other, more widespread disorders by showing that a

single genetic mutation, which causes a common deficit at

the cellular level in most or all interneurons, can neverthe-

less cause complex disease outcomes through the differen-

tial physiological and behavioural impacts of deficits in

specific classes of interneurons. Such ‘interneuronopathies’

may be a common source of complexity in both genetic and

idiopathic forms of neuropsychiatric disease.

FundingResearch reported in this publication was supported by the

National Institute of Neurological Disorders and Stroke of

the National Institutes of Health under award number R01

NS25704 and by a research grant from the Simons

Foundation Autism Research Initiative. The content is

solely the responsibility of the authors and does not

necessarily represent the official views of the National

Institutes of Health or the Simons Foundation.

Supplementary materialSupplementary material is available at Brain online.

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