Generation of a mouse genetic model for Infantile Spasms Syndrome
The human ARX
(GCG)10+7 mutation most closely associated with West syndrome/X-linked infantile spasms syndrome (ISSX) expands the first polyalanine tract of amino acids 100–115 from 16 to 23 residues through the insertion of seven GCG alanine codons within ten consecutive GCG alanine codons () (reviewed in Guerrini et al., 2007
; Poirier et al., 2008
). Alanine is encoded by GCX, where X is any nucleotide. The mouse Arx gene (Locus NP_031518, UniGene Mm.275547) has 15 alanine codons in the first polyalanine tract, but the longest GCG repeat is four, not ten. In keeping with the mixed alanine codon usage, the mouse Arx
expansion knockin mutation was generated with GCT repeats, resulting in a total of 23 alanine codons in tract 1. This mouse Arx knockin reproduces the 23 alanine codons in human ISSX-ARX (GCG)10+7, so we have designated the resultant mouse line Arx (GCG)10+7
The original construct has homologous arms of 4.2 and 2.2 kb, a TK cassette and an frt-flanked neomycin resistance cassette (, Knock-in plus Neo). Exon 2 with the knock-in is flanked by loxP sites. Recombination into embryonic stem cells was confirmed by Southern blotting of Ase-digested DNA and screening with a 3′ probe (). Eight of 210 clones had a 3.5 kb Ase fragment resulting from recombination. Confirmatory Southern blot screening used a 5′ probe for an Eco R1 fragment increased by 2 kb due to the neomycin-resistance cassette. To ensure the mutant transcript would properly splice, the frt-flanked neomycin-resistance cassette was recombined out of intron 2 by Flp recombinase. PCR for a product that was 2 kb smaller confirmed this event (). PCR with the same primers distinguished cells with wildtype genomic DNA or knock-in DNA and was also used for genotyping (). The larger knock-in PCR product is due to the presence of the loxP and frt sites 3′ of exon 2. Their presence in the knock-in stem cell clones was confirmed by cloning and sequencing the PCR product. Mating heterozygous Arx (GCG)10+7 females to wildtype males yielded Arx (GCG)10+7 males at the expected 25% Mendelian frequency. The presence of the 8-alanine expansion in 3 mutant males used for experimental analysis was verified by sequencing the PCR product of exon 2.
Normal development of non-neuronal organ systems, pancreatic metabolism, and fertility of the Arx (GCG)10+7 mutant
is expressed in the developing nervous system, pancreas and testes, as well as in adult brain, skeletal muscle, heart and liver (Kitamura et al., 2002
; Ohira et al., 2002; Poirier et al., 2004
; Colombo et al., 2004
knock-out mice developed by two strategies have severe defects in organs where the gene is expressed during development, and die within days of birth (Kitamura et al., 2002
; Collombat et al., 2003
). In contrast, Arx (GCG)10+7
males and heterozygous as well as homozygous Arx (GCG)10+7
females are viable, attain normal adult body weight, and are fertile. In adult male and female Arx (GCG)10+7
mutants, muscle and bone and all organs are normal in gross appearance. The heart, lungs, kidney, liver, stomach, small and large intestine, pancreas, spleen, thymus lymph nodes have normal histological appearance (n=2 each genotype). Arx (GCG)10+7
testicles, epididymis, ductus deferens, prostate, coagulating gland and seminal vesicle are also structurally normal. The adrenal glands appear normal in both cortical and medullary layers (data not shown). Since Arx
null mutants die perinatally of apparent dehydration and hypoglycemia due to skewed differentiation of pancreatic exocrine cells (Kitamura et al., 2002
; Collombat et al., 2005
), feeding and fasting glucose levels of Arx (GCG)10+7
mutants and wildtype littermates were measured (n = 4 each). Consistent with their viability and the normal histological appearance of pancreatic islets, blood glucose levels were within the normal range in fed, 4-hr fasted, and 24-hr fasted mutant mice (fed: wt 176 ± 4 and mutant 162 ± 9; 4 hr fasted: 166 ± 14 and 146 ± 5; 24-hr fasted: 126 ± 2 and 140 ± 9: p
= 0.16 to 0.23). The finding that the mutant mice show normal development, fertility, and pancreatic function suggests that the mutant ARX protein retains at least partial function in some non-CNS pathways.
Infant Arx (GCG)10+7 mutants show spontaneous spasm-like myoclonic events
Rodent pups display spontaneous limb and tail movements during normal sleep or upon awakening, including brief focal myoclonic twitches, kicks, and generalized startles (Blumberg et al., 2007
). Startles are sudden, spontaneous, and simultaneous contractions of skeletal muscles throughout the body and have been defined behaviorally as “abrupt, high-amplitude, synchronous movements of at least three limbs” (Karlsson et al., 2006
). We designed a ‘littermate array’ for our behavioral videomonitoring studies to reproducibly sample and analyze aberrant motor movements; pups were placed unrestrained in separate wells of a transparent plate that allowed full range of motion and simultaneous observation of all littermates under the same temperature-controlled conditions (). In addition to motor startles as defined above, we observed another distinct category of spontaneous high-amplitude movements: sustained spasm-like movements so strong as to cause the pup to flip or fall over, axial contractions that bowed or twisted the body, and abrupt lateral displacements of the pup across the floor of the compartment.
Figure 2 Infant Arx (GCG)10+7 pups display twice as many spontaneous, severe spasm-like movements as do wildtype littermates. A, Still image from a video-recording of a wildtype (#1) and three mutant 9-day old male mouse littermates. Mutant pup #9 in lower right (more ...)
The most severe spasm-like movements, those causing a pup to flip or fall over (see video 1 in Supplemental data
), were increased nearly four-fold in mutants at all ages compared to wildtype siblings. Mutant pups at 7 days had between 0–11 such episodes in a 30 min period while non-mutant pups had a maximum of two (means of 2.8 ± 0.8 vs. 0.6 ± 0.02, p
< 0.002). At 9-days old, 31% of mutant pups had 1–2 severe spasms, while only one non-mutant pup had a single episode in a 30 min period. At 11 days old, 38% of mutant pups displayed 1–3 severe spasms per 30 min while none were observed in their non-mutant counterparts. Overall, when compared to their non-mutant littermates, the Arx (GCG)10+7
mutant pups showed twice as many total spontaneous, high-amplitude movements (including the severe spasms described above as well as startles, body displacement and rapid trunk flexion), at all three of the ages monitored (). The frequency of these monitored events declined between days 9 and 11, to about half. Hemizygous males and homozygous females showed similar rates at the three ages (p
= 0.49, 0.29, 0.67). Similarly, and consistent with the normal development of females heterozygous for the Arx
null mutation, the spectrum and frequency of spontaneous myoclonic movements in wildtype males and female heterozygotes did not significantly differ (p
= 0.42, 0.12, 0.52 for the three ages).
The final category of movements scored was low-amplitude, phasic movements, including myoclonic twitches and short-distance kicks that did not disturb body posture. These are exhibited by every pup at all ages. There was no significant difference between the rate of low amplitude movements in mutant and non-mutant pups at the three tested ages () (p = 0.29–0.36). The incidence of movements in this category declined steadily and at a similar rate from postnatal day 7 to day 11 in both mutant and non-mutant pups. Unlike the high-amplitude spasm movements, the Arx mutation had no significant effect on the frequency of low-amplitude motor activities at infantile stages.
Spontaneous epileptic EEG activity in Arx (GCG)10+7 mutants
To determine whether the Arx (GCG)10+7
mutation alters cortical excitability, we performed prolonged video-electroencephalographic recordings from chronically implanted mutant pups 11–21 days of age (n = 5), mutant juveniles and adults between 3.5 and 10 weeks of age (n = 12), and age-matched wildtype littermates (n = 7). Due to their small size, it was not possible to reliably record from mice as young as those monitored for spasm-like movements (7–11 days old). Recordings for periods exceeding 4 hr revealed multiple 4–5 sec episodes that began with a high-voltage (up to 3.9 mV) slow wave transient followed by attenuation of the background EEG amplitude and a transient increase of higher frequency activity (). The pups exhibited a very brief myoclonic jerk involving the head and body at the onset of the attenuation event (arrow in ). These stereotyped ‘electrodecremental’ episodes associated with myoclonic jerks are similar to those ictal events seen in infantile spasm patients (Hrachovy and Frost, 2003
). High amplitude cortical spikes and sharp waves also occurred frequently, up to 20 times/hr. These discharges were multifocal in origin (arising independently in both hemispheres), and were more frequent during nonREM sleep. In addition, high-voltage slow waves associated with a spike or sharp component occurred independently over both hemispheres, usually infrequently but sometimes 10–20 times per hr. These high voltage patterns resemble those in except that the activity before and after the high voltage event are similar; there is no attenuation or increase in frequency and no obvious motor behavior linked to the discharge.
Figure 3 EEG abnormalities in Arx (GCG)10+7 mice. A, Mutants under 21 days of age display sharp spike-slow wave transients followed by attenuation of background activity and an increase in high frequency background rhythmic activity. The arrow indicates the beginning (more ...)
Arx (GCG)10+7 mutants between the ages of 3.5 and 10 weeks show spontaneous electrographic seizures characterized by generalized attenuation of the background activity and the appearance of low voltage fast activity, followed by generalized high frequency and high amplitude spike and polyspike activity dissipating at different times in different brain regions (). The episodes of high frequency spiking lasted 18–29 seconds, during which the mice generally made 4–10 slow versive movements of the head and or trunk, and clonic movements followed by vigorous grooming suggestive of limbic seizures with hippocampal involvement. Following generalized seizure discharges, there was prolonged attenuation of the EEG voltage. One juvenile mutant male was observed to have spontaneous tonic/clonic seizures (two in a 7-hr period). A second type of seizure, also seen in pups, was distinguished by 6-Hz spike wave bursts with amplitudes of 150–400 μV (), and accompanied by behavioral arrest. These episodes last up to four seconds, with an average of 1.2/second, and occur between 3–28 times per hr (average rate of 10.6/hr). These spike wave bursts occurred in half (2/4) of the mice that had other seizure patterns during the monitoring period, and in 75% (8/12) of the mutant adults monitored. The third type of abnormality observed in the majority of the mutant mice, whether awake or asleep, were frequent (up to 118/hr) multifocal single spike and sharp wave patterns of complex morphology with amplitudes ranging between 600–1200 μV. Rare (1–3/hour) low voltage spike or sharp waveforms were seen in wildtype mice and were typically less than 600 μV in amplitude.
Cognitive impairment and abnormal behavior in Arx (GCG)10+7 mutant mice
Large groups of mutant and wildtype control two-month old male littermates were screened with a battery of standardized behavioral tests (n = 21 of each genotype). In a prepulse inhibition test to assess sensorimotor gating, Arx (GCG)10+7
and wildtype mice responded to the loudest sound, 120 dB, with a startle movement of similar maximum amplitude (669 ± 150 vs. 703 ± 94) The acoustic startle response in both genotypes was inhibited by approximately 10%, 25% or 44% when warning prepulse sounds of 74, 78 or 82 dB, respectively, preceded the 120 dB startling sound. (). These results demonstrate intact hearing and unaltered brain stem reflexes to a startle stimulus, distinguishing the spontaneous startle events from the hypekplexia due to dysinhibition with the brainstem and spinal cord as seen in mice with glycine receptor α-subunit mutations (Ryan et al, 1994
). Mutant mice were significantly more sensitive than wildtype mice to heat-induced pain, with a 30% shorter latency to exhibiting a hindlimb response (7.6 vs. 9.95 sec; p
= 0.003. Coordination and the ability to learn motor skills were assayed on the accelerating rotarod, in four trials on two successive days. Mutant mice performed significantly better than wildtype mice in most trials (p
= 0.006–0.036), and slightly better but insignificantly in the other trials (). Mutant mice seemed less fearful, often turning around on the rod while it rotated slowly.
Figure 4 Arx (GCG)10+7 mutants behave with abnormally low anxiety, are cognitively impaired and display an autism-like characteristic. A, The startle response of mice of both genotypes habituates similarly as louder prepulse sounds repeatedly precede the 120 dB (more ...)
In a more direct measure of anxiety, Arx (GCG)10+7 mice behaved abnormally in the light/dark exploration test, spending more than twice as long in the light and 20% less in the dark as did wildtype mice (light time: 38% vs. 17%, dark time 62 vs 83%; p <0.0001) () with an average of 35% longer in the light at each interval (p = 0.007). The mutants made 67% more light/dark transitions (46.2 vs. 27.6; p <0.0001). The latency until the first entry into the darkened chamber was similar in both genotypes. These results indicate that mutant mice initially behave like wildtype mice, but then explore with less anxiety. In the open-field test, Arx mutant mice showed similar speeds and amounts of locomotor activity during a 30 min period of exploration of a novel arena. There was no difference in horizontal, vertical or circling activity, and a slight but insignificant increase in the total number of movements by the mutants. The striking difference was that mutant mice spent 40% more time in the center than did wildtype littermates (0.316 vs. 0.226 Center/Total distance ratio; p = 0.004). The abnormal behavioral profiles in the light/dark and open field exploration tests reveal subnormal anxiety levels in the Arx mutants.
Reduced anxiety may also contribute to cognitive impairments in the Arx (GCG)10+7
mutant mice, as demonstrated by Pavlovian conditioned fear training involving a brief foot shock applied after a conditioning sound (CS) cue (). Trained mutant mice placed in the training context/environment the next day adopted a fear-induced “frozen” posture 48% less often than did wildtype mice (Context, 21% vs. 44%, p
<0.0001). After many variables of the training context were changed, including the chamber floor, shape, and scent, the freezing responses by mutant mice were only 27% as frequent as those of wildtype mice (PreC-cue, 4.6% vs. 16.9 %, p
= 0.018). When the conditioning sound cue was delivered in this altered environment, mutants froze only 47% as frequently as control littermates (CS, 32.9 vs. 69.7%, p
<0.00001). The relative lack of learned fear in mutants was also revealed by a major reduction in freezing responses in mutants post-sound cue versus pre-sound cue (CS-PreCS) compared with control mice (28.4 vs. 52.8%, p
<0.0003). These results indicate an impairment of associative learning and memory in the mutants, with deficits in both context- and sound cue-dependent aspects, despite their normal acoustic response and hypersensitivity to painful stimuli. Finally, we performed the tube test for social dominance, often used as an indicator of autism-like characteristics (Shahbazian et al., 2002
; Spencer et al., 2005
). In three separate trials, each mouse was confronted with a different non-cagemate mouse of the opposite genotype. In 79% of trials Arx (GCG)10+7
mice demonstrated the autistic-like behavior of retreating, while wildtype mice retreated in only 21% of the trials ((49/60 matches; p
Reduction of mature Arx-positive interneuron populations in the Arx (GCG)10+7 mutant
ARX+ GABAergic interneurons are expressed in the mature mouse brain in multiple forebrain regions, with the highest populations present within the neocortex, hippocampus, and adult neural progenitor zones. Using a commercial antibody specific for ARX in wild type mice, ARX+ interneurons were labeled throughout the neocortex, especially in the deeper layers (). As compared to adult male wildtype siblings, only about 68% of ARX+ cells remained in layers I–IV of somatosensory and motor cortex in Arx (GCG)10+7 mutants, and only 53% as many ARX+ cells were present in the deeper layers V–VI (, ). The total reduction throughout the cortex was to approximately 58% of wildtype values. At the cellular level, ARX protein was localized in the nuclei of 72 ± 5 % of the wildtype and 45 ± 11 % of the mutant interneurons (n = 5 each genotype; p < 0.05), therefore remaining in the cytoplasm of a larger percentage of mutant interneurons (, large arrows). There was no difference in ARX+ cell distribution in the parietal cortex (n = 3 each genotype, not shown). In the hippocampal formation, about half as many ARX+ cells were present in the hilar region of the mutant dentate gyrus (). Likewise, ARX+ cell populations in the mutant striatum were reduced to roughly one half of the wildtype level (). Nuclear ARX localization in hippocampal and striatal interneurons appeared similar in both genotypes.
Figure 5 ARX-positive interneurons are significantly reduced from normal in the neocortex, hippocampus and striatum of the Arx GCG(10+7) mutant. A, B, Low magnification views of sections of cortex spanning from the pia to the white matter, immunostained with anti-ARX (more ...)
Relative presence of neuron types that are affected in Arx (GCG)10+7 mutants (WT = 100%)
Selective reductions of interneuron subtypes
We used antibodies specific for the interneuron markers parvalbumin, calretinin, calbindin, and NPY to examine specific subsets of interneurons in the adult mutant, and found that not all were equally affected by the Arx(GCG)10+7 mutation. The density and location of parvalbumin- and calretinin-expressing GABAergic interneurons in mutant brains was similar to that in wildtype littermates in all regions. In contrast, other subtype populations were reduced in specific regions.
Reduction of calbindin-expressing interneurons in Arx (GCG)10+7 mutant forebrain
The most striking loss was in calbindin+ interneurons in multiple regions, including layers I–IV of the neocortex with a lesser reduction in the deeper layers (), accompanied by an approximately equal reduction in these cells in the granule cell layer of the dentate gyrus (), and striatum (, ). The Arx (GCG)10+7 mutation had little effect on calbindin expression itself, as evidenced by strong immunopositivity of other calbindin-expressing structures, such as the mossy fiber axons of granule cells in the dentate hilar region () (n = 4 each genotype).
Figure 6 Calbindin D28K-positive interneurons are less abundant in the neocortex, hippocampus and striatum of the Arx GCG(10+7) mutant. A, B, Neocortex of wildtype (WT) and mutant motor/somatosensory cortex immunostained with anti-calbindin D28K antibody, showing (more ...) Reduction of NPY-expressing Interneurons in the Arx (GCG)10+7 mutant striatum
In contrast, NPY+ interneuron loss was region-specific. A subset of GABA-ergic interneurons express neuropeptide Y (NPY), and likewise, a subset of NPY+ cells express ARX. NPY+ cells were absent from the Arx
null mouse (Kitamura et al 2002
), but in the Arx (GCG)10+7
there was no difference in the number of NPY+ cells in layers I–V or layers V–VI of the motor/somatosensory cortex or the parietal cortex (), and no discernable difference in NPY+ cell numbers in hippocampal sections (). However the mutant striatum showed a 31% loss of NPY+ cells compared to wildtype siblings, although the cell morphology appeared normal (, ) (n = 3 each genotype).
Figure 7 Severe reduction of striatal interneurons. The NPY+ interneuron population is reduced in the striatum of the Arx (GCG)10+7 mutant, although present at wildtype (WT) levels in the neocortex. A, B, Full-thickness views of somatosensory cortex immunostained (more ...) Reduction of striatal cholinergic interneurons in the Arx (GCG)10+7 mutant striatum
Basal ganglia pathology is found in children with ARX mutations who display marked and prolonged dystonia (Juhasz et al, 2001
, Guerrini et al., 2007
), and Arx-null mice show a severe lack of cholinergic interneurons in the basal ganglia (Colombo et el., 2007
). The latter defect may be due to limited migration of cholinergic precursor cells from the ventral subpallium during early development, and a strong reduction in expression of transcription factors such as Lhx7 that are linked to the expression of ARX. The effect of the Arx (GCG)10+7
mutation on striatal cholinergic interneurons was assessed by quantification of choline acetyl transferase (ChAT)-expressing cells in the caudate/putamen. Mutants contained only 39% as many striatal ChAT+ cells compared to their wildtype litter mates (, n = 3 each genotype).
Presence of Dcx-positive neural progenitors
Finally, we examined cerebral progenitor zones in adult mutants and +/+ mice (n = 4 each genotype) for other Arx
-related defects, since ARX is found in adult neural stem cells (Colombo et al., 2004
. Doublecortin (Dcx) is a microtubule-associated protein marker for neural stem cells in germinal zones, expressed shortly after mitosis during the commitment to a neuronal fate and in the early stage of migration (Overstreet-Wadiche and Westbrook, 2006
; von Bohlen und Halbach, 2007
). Prolonged hippocampal seizure activity induces neurogenesis and the expression of Dcx+ cells within these zones (Parent et al, 2006
). The density and location of Dcx+ cells appeared unchanged in the subgranular layer of the dentate gyrus, the tertiary germinal matrix where adult stem cell proliferative activity coexists with GABAergic interneurons. In addition, there was no obvious decrease of Dcx+ cells in the cortical subventricular germinal zone and rostral migratory stream (supplemental Figure 1
). The lack of a clear expansion in the size of this population is consistent with the phenotype of brief seizures observed in these mutants. Consistent with this interpretation, we found no evidence of cell death using the Fluoro-jade technique (not shown) in either the mutants or wildtype siblings.