Common circuit defect of excitatory-inhibitory balance in mouse models of autism
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One unifying explanation for the complexity of Autism Spectrum Disorders (ASD) may lie in the disruption of excitatory/inhibitory (E/I) circuit balance during critical periods of development. We examined whether Parvalbumin (PV)-positive inhibitory neurons, which normally drive experience-dependent circuit refinement (Hensch Nat Rev Neurosci 6:877–888, 1), are disrupted across heterogeneous ASD mouse models. We performed a meta-analysis of PV expression in previously published ASD mouse models and analyzed two additional models, reflecting an embryonic chemical insult (prenatal valproate, VPA) or single-gene mutation identified in human patients (Neuroligin-3, NL-3 R451C). PV-cells were reduced in the neocortex across multiple ASD mouse models. In striking contrast to controls, both VPA and NL-3 mouse models exhibited an asymmetric PV-cell reduction across hemispheres in parietal and occipital cortices (but not the underlying area CA1). ASD mouse models may share a PV-circuit disruption, providing new insight into circuit development and potential prevention by treatment of autism.
KeywordsParvalbumin VPA Neuroligin GABA
A severe neurobehavioral syndrome with a heterogeneous phenotype, autism spectrum disorders (ASD) are among the most heritable neurodevelopmental disorders of early childhood with an incidence as high as one child in 166. ASD is primarily a genetic disorder of prenatal and early postnatal brain development involving multiple risk genes, disrupted epigenetic pathways and possible environmental insults . At present, there is no wholly effective treatment nor is it understood which biological pathways are specifically disrupted in individuals with autism. The diagnostic indicators of autism are core behavioral symptoms, rather than definitive neuropathological markers. Autistic children exhibit impaired language, abnormal social interactions and repetitive behaviors. One third of ASD patients have seizures and evidence of altered inhibition in the brain .
One unifying explanation for the complexity of ASD may lie in the disruption of excitatory/ inhibitory (E/I) circuit balance during critical periods of development [4, 5, 6]. Neuronal circuits are refined by extraordinary levels of plasticity during sensitive periods in early development, which are diminished in adulthood. It is during these “critical periods” that single neurons acquire multiple functional properties through an experience-dependent maturation. Recent findings have revealed that E/I circuit dynamics can dictate the normal timing of critical periods in brain development . Gene-targeted deletion of a GABA-synthetic enzyme (GAD65 KO) in mice delays plasticity onset within the visual cortex indefinitely , which can be rescued at any age with benzodiazepines .
In neocortex, the two primary types of PV-cell are axon initial segment-targeting chandelier cells and soma-targeting large basket cells. The latter have a large, round soma with several prominent radial dendrites. PV expression begins near postnatal day P12 in layer 5 and matures in an inside-out laminar progression by around P21 in visual cortex and somewhat earlier in somatosensory and other cortical regions [12, 14]. With age, PV-cells become preferentially enwrapped in chondroitin sulphate proteoglycans that form a perineuronal net, which may buffer the ionic environment surrounding these cells  or act to limit growth and sprouting of impinging thalamic axon terminals . Interestingly, removal of these nets can reactivate critical period plasticity in adult animals .
The mouse model system provides a method to experimentally test whether postnatal neurodevelopmental disorders of human cognition involve dysfunction of such critically timed activity-dependent processes. Mouse models of ASD have been developed that reflect genetic alterations associated with autism . Some are based on monogenic aberrations (Neuroligin-3, Neuroligin-4, MeCP2, TSC1/2, FMR1, ubiquitin protein ligase 3A (Ube3A)) that underlie syndromes associated with autistic-like behavior. Other mutant lines are relevant to loci for autism susceptibility, identified by association or linkage in human populations. Advances have included the evaluation of mouse models with behavioral assays designed to reflect disease symptoms, including impaired social interaction, communication deficits and repetitive behaviors, and symptom onset during the neonatal period.
PV-cell deficits across ASD mouse models
anterior cingulate cortex parietal cortex
prenatal immune challenge
medial prefrontal cortex
hippocampus area CA3/CA1
hippocampus area CA3
Global genetic perturbations are produced by prenatal or neonatal environmental challenges, including early exposure to the HDAC inhibitor valproic acid (VPA) or inflammatory agents that have been suggested as autism risk factors by clinical surveys. A robust model has previously been developed in rats based on the fact that treatment of epilepsy or bipolar disorder in pregnant women around 20–24 days post-conception with the drug VPA is linked to an increased incidence of ASD in their children [19, 20, 21]. The physically healthy offspring of pregnant rats treated with a single dose of VPA at an equivalent gestational time point recapitulate the human ASD phenotype [22, 23]. Related VPA mouse models have been attempted, but most involve postnatal VPA treatment not consistent with human studies [24, 25]. One group used a slightly higher dose at a later time point, but they did not evaluate behaviors implicated in autism .
We translated the well-established rat model into mice by using the same dose at an equivalent injection time point and evaluated behavior of the offspring [26, 27]. Five out of nine VPA-injected pregnant females (500 mg/kg, i.p.) gave birth to viable litters, and of these, two litters included some mice with minor skeletal defects like bent tails and fused fingers. We concluded that this dose was appropriate because it was strong enough to have a teratogenic effect, but not too strong to cause gross impairments. We then compared PV-cell anomalies in the brain by immunostaining of VPA-treated mice and a representative single-gene mutant of Neuroligin-3 (NL-3 R451C) reported to exhibit autistic features  concomitant with enhanced cortical inhibitory synapse function . Note that an independently generated R451C mutant mouse line revealed a different behavioral phenotype, but was not characterized for expression of mutant NL-3 and its electrophysiological consequences , making it unclear how similar the line is to the original R451C mutant mice.
Common disruption of parvalbumin-positive GABA circuits in ASD models
The precise location and extent of PV-empty zones were slightly variable among littermates that shared concomitant prenatal VPA exposure, including a small subset that did not exhibit any noticeable abnormality. However, the majority of VPA-treated mice shared a common PV-empty zone in a region around the caudal part of primary somatosensory cortex. We therefore decided to analyze this region quantitatively, by defining a region of interest between Bregma −1.70 mm and −2.06 mm (rostro-caudal level, Paxinos) in the upper half of the neocortex (see Suppl. Fig. 1 and Methods for details). In order to investigate whether the PV deficiency in the neocortex was generally present in other brain regions, we analyzed PV-cell numbers in the hippocampal CA1 area beneath the affected cortex (same rostro-caudal level; see Suppl. Fig. 1 and Methods for details of the region of interest). We found no significant difference of PV-cell densities in CA1 between VPA- and saline-treated animals (Fig. 2b).
To address the possibility that the lack of PV-cells in neocortex and an interhemispheric imbalance in inhibitory circuits is a more robust hallmark of ASD models, we investigated a single-gene mutation model of ASD, the NL-3 R451C mutant mouse . We found that NL-3 mutant mice exhibited very similar PV-cell deficiency and interhemispheric imbalance as in the VPA model. NL-3 mutant mice were analyzed in the same way as described above for prenatal VPA-treated animals. Once again, a highly significant reduction of PV-cells in one hemisphere was observed specific to the neocortex, as it was absent from underlying CA1 (Fig. 2b). Inter-hemispheric ratios of PV-cell numbers in NL-3 mutant mice were reduced >50% specifically in the neocortex (Fig. 3). Whether hemispheric asymmetries of PV-cells are evident in other ASD mouse models (Table 1) remains to be explored.
We further confirmed an increase of GAD65 puncta in the NL-3 mutant (Suppl. Fig. 2), consistent with the original report by Tabuchi et al . Normally, PV regulates the dynamics of calcium levels and GABA release during action potential bursts in this specific interneuron subset . Interestingly, PV knockout mice themselves have recently been reported to exhibit autistic features (B. Schwaller, personal communication). A loss of PV expression in VPA-treated or NL-3 mutant mice may then exacerbate an E/I imbalance in favor of inhibition , as in other models of ASD (Table 1). Thus, disparate genetic origins may underlie a common circuit defect in ASD.
Impact of PV-cell defects in ASD
Unlike principal excitatory neurons, GABAergic interneurons comprise a diverse group that consists of dozens of different types. Cortical interneuron subtypes can be classified by their morphology, electrophysiological properties, molecular content and specific connectivity patterns. The PV-positive, large basket cells have been implicated in two functions (Fig. 4): 1) initiation of a critical period for cortical plasticity , and 2) generation and synchronization of gamma (γ)-oscillations in the hippocampus and neocortex . Both phenomena have been suggested to be impaired in the etiology of cognitive developmental disorders such as ASD, and can strikingly both be accounted for by the common PV-cell deficit reported here.
Hubel and Wiesel were among the first to find physiological evidence for developmental time windows when specific brain circuits are more susceptible to experience-dependent changes . Monocular occlusion of one eye produces a shift of neuronal response (ocular dominance) in favor of the open eye only when deprivation occurs during a specific ‘critical period’. Since then critical periods have been identified across a variety of brain regions, sensory systems and species . Electrophysiological recording and morphological analyses in vivo in genetically manipulated mice have furthered our understanding of the mechanisms and demonstrated the importance of PV-cells.
Beginning with the discovery that GABA-deficient GAD65 knockout mice do not initiate a critical period and that this deficit could be rescued at any age with diazepam [7, 8], the theory emerged that a requisite E/I balance within the cortex enables plasticity at the appropriate time (Fig. 4a). Diazepam is a positive allosteric modulator of GABAA channels that binds specific subunits to increase channel open probability and increase inhibition. Diazepam administration can prematurely open the critical period of wild-type mice, but targeted point mutation of the α1 receptor subunit prevents diazepam binding as well as its precocious initiation of plasticity . GABAA receptors containing the α1 subunit are located on the soma-proximal dendrite region of pyramidal cells. PV-large basket cells preferentially target the same area (Fig. 1), which undergoes an experience-dependent regulation of receptor number during the critical period . The non-cell autonomous homeoprotein Otx2 is crucial for the maturation of PV-cells and, consistently is an essential transcription factor for critical period initiation .
Taken together, these findings underscore the crucial role PV-cells play in timing neocortical plasticity (Fig. 4a), suggesting that PV-cell deficits may predictably delay critical periods in specific ASD brain regions (Table 1). Rescue of ASD models, such as the FMR1 knockout mouse by mGluR5 manipulation , can then be reconsidered in terms of rebalancing PV-cell recruitment, as mGluR5 is essential for long-term potentiation of excitatory synapses onto these fast-spiking GABAergic neurons . Other direct strategies to enhance PV-cell function, such as benzodiazepine agonists or the delivery of Otx2 , may also prove fruitful. Conversely, to recalibrate PV-cell discrepancies across hemispheres in adult mice (Fig. 3), the administration of chondrotinases to remove peri-neuronal nets that preferentially enwrap mature PV-basket cells may be effective .
EEG gamma (γ)-oscillations (30–80 Hz fluctuations of neuronal activity) have received special attention because of their suspected roles in “higher” cognitive functions such as sensory binding , short-term memory storage  and attention [37, 38]. γ-oscillations are prevalent in the hippocampus and sensory cortex and have been studied both in vivo and in vitro. They are generated or reset in vivo by sensory stimulation in mouse visual, auditory and somatosensory cortex [39, 40, 41], exploratory behavior, sniffing in rodents, visual attention in primates and during REM sleep [38, 42]. Persistent γ-oscillations can be stimulated in vitro with carbachol to activate muscarinic cholinergic receptors or by application of kainate to increase excitability of glutamatergic receptors [43, 44]. They are also reliably generated in slices of auditory and somatosensory cortex by thalamic stimulation, but are short in duration (200–500 ms) .
Regardless of stimulus protocol, γ-oscillations can be abolished by application of the GABAAR antagonist bicuculline . Current-source density analysis combined with voltage-dye imaging reveal an alternating current sink and source near pyramidal cell somata, consistent with peri-somatic inhibition during the oscillation . When excitatory drive is selectively reduced onto PV-cells either by GluR-D knockout or conditional ablation of GluR-A subunit of their AMPA receptors, γ-power is reduced , but not when synaptic inhibition is ablated in PV-cells . Most recently, direct optical activation of PV-cells is shown to be sufficient for inducing γ-rhythms and controlling sensory responses to enhance cortical circuit performance [48, 49].
Both chandelier and large basket PV-cells are fast-spiking (FS) cells that are interconnected via gap junctions and inhibitory chemical synapses to form distributed networks. FS-cells have short duration action potentials and respond to a depolarizing current with high frequency, non-adapting action potential trains [51, 52]. PV-cells express Kv3.1 channels, a class of inward rectifying K+ channels that contributes to their fast-spiking characteristics. PV-cells normally undergo maturation of a number of intrinsic physiological properties that contribute to their precise and rapid firing including an experience-dependent increase in Kv3.1 channels , a decrease in input resistance and action potential width and an increase in action potential amplitude and frequency [53, 54]. Kv3.1 knockout mice exhibit an increase in γ-power, most prominently during waking in the 40–55-Hz range .
Gap junctions strongly aid the synchrony and strength of γ-oscillations, since the blockers octanol and carbexenolone eliminate oscillations [56, 57], but deletion of the major neuronal gap junction subunit, connexin 36, still allows weak and less synchronous γ-oscillations both in vitro  and in vivo . Careful study of the spike timing of different classes of interneurons has shown that while both excitatory and different classes of inhibitory neuron are capable of firing phase-locked with the γ-oscillation, inhibitory neurons are more likely to be synchronized with FS interneurons capable of firing on every cycle (Fig. 4b) [60, 61].
Impaired oscillations in cognitive disorders
A dysregulation of oscillations could prevent discrimination of intrinsic versus extrinsic signals . In schizophrenic subjects, abnormal γ-oscillations are thought to promote hallucinations. Decreased GABA signaling is now well-established in schizophrenia , which is seen not only as a reduction of GABA concentration and GAD67 in postmortem brain, but also as a restricted deficit of PV staining [67, 68]. A specific association between disrupted γ-oscillations, PV-cell reduction, and schizophrenic traits has recently been demonstrated in the methyl azoxy-methanol acetate (MAM) model of schizophrenia. These rats exhibit a specific loss of PV-cells in the prefrontal cortex and a correlated lack of sound induced γ-oscillations in the prefrontal cortex during a fear-conditioning task .
In auditory cortex of autistic adolescents, magnetoencephalographic (MEG) recordings show a deficit of evoked γ-oscillations phase-locked to the stimulus — strikingly just in one hemisphere . A similar finding that the onset of γ-oscillations during a visual task is disrupted in autism, suggests that there are signal-to-noise processing problems [71, 72]. Of particular relevance is the recent finding that visual attention preferentially modulates FS interneurons in parietal areas , which we find are prone to PV-cell deficits in ASD mouse models (Table 1).
Clinical studies suggest that decreased inhibitory input and connectivity lead to altered synchronicity between different brain regions, which may be a key contributing factor to the features of ASD [35, 64, 74, 75, 76]. Consistent anatomical evidence reveals narrower cortical minicolumns in autistic patients [77, 78, 79] due to the loss of local inhibitory projections . This may result in further altered long-range connectivity [80, 81], vital for communication between neocortical modules and hemispheres [74, 82, 83, 84, 85].
Interestingly, among several inbred strains , it is the acallosal BTBR T+tf/J mouse that most closely exhibits behavioral changes specific to autism, including reduced social approach, altered ultrasonic vocalization, and resistance to change in routine . Moreover, network architecture and synchronicity are altered in cultured neuronal networks transfected with the autism-associated R451C NL-3 mutation studied here . The MeCP2-null hippocampal CA3 circuit has diminished basal inhibitory rhythmic activity, which in turn renders the circuitry prone to hyperexcitability . Finally, a recent report demonstrates that γ-oscillations are diminished in the FMR1 knockout mouse model of ASD , consistent with their PV-cell deficit (Table 1).
Thus, studying γ-oscillation development in ASD mouse models can serve as a useful readout of impaired PV-cell network activity, which is now amenable to powerful, direct molecular genetic manipulation [eg. 91]. Future work will explore the hemispheric asymmetry in VPA or NL-3 mutant mice described here, whether this can be rescued by treatments aimed at restoring PV-cell balance [11, 17] and ultimately reversing this heterogeneous neuro-developmental disorder [92, 93].
Materials and methods
C57BL/6 females were mated overnight with C57BL/6 males and pregnancy was determined by the presence of a vaginal plug on embryonic day E0. Valproic Acid (VPA) solution was prepared from sodium salt of VPA (NaVPA; Sigma-Aldrich, St. Louis, MO) and was dissolved in 0.9% saline to a final concentration of 150 mg/mL at pH 7.3. At E10.5, pregnant females were briefly anesthetized with isofluorane and injected intraperitoneally with either a single dose of VPA or 0.9% saline (control). Mice were weighed prior to injection and the volume was adjusted for a final dose of 500 mg/kg. This dose and time point is identical to the conditions used in previous studies with rats . NL-3 R451C mice were bred, genotyped and maintained as described previously 
Adult mice (2–3 months old) were transcardially perfused with ice-chilled 4% paraformaldehyde in PBS (50 ml) and post-fixed overnight in the same solution at 4°C. Sixty μm thick coronal sections were cut on a Vibratome (Leica VT1000S). Free-floating sections were permeabilized and blocked for 4 hours at room temperature (RT) in 20% Bovine Serum Albumin (BSA) / 0.5% TritonX100 in PBS. Sections were then incubated with primary, rabbit anti-Parvalbumin (PV) (Swant) or GAD6 (DSHB, Iowa), and subsequently secondary, Alexa Fluor 546 (Invitrogen), antibodies (3–4 h each at RT, 1:500 (PV), 1:1000 (Alexa Fluor) in 5% BSA in PBS). Between and after antibody incubations, sections were washed 3x, 10 min each, in 5% BSA in PBS on a shaker. Sections were then mounted in Vectashield Hard Set (Vector Laboratories) and imaged.
Image acquisition and analysis
Images were acquired on a Nikon Eclipse 80i epifluorescent microscope equipped with a Coolsnap EZ camera (Photometrics) and AR acquisition software (NIS Elements) using a 4x air objective. Microscope images were processed and analyzed using Photoshop (Adobe). For quantification, images of the upper half of coronal brain sections, collected between Bregma −1.70 mm and −2.06 mm (anterior-posterior level, Paxinos), were taken (Suppl. Fig. 1). PV-positive cells were counted in the neocortex and in the hippocampal area CA1. Regions of interest were defined by perpendicular lines along the upper blade and outer edge of the dentate gyrus curve (Suppl. Fig. 1). PV-positive cells were counted separately for the two hemispheres. Analysis was performed blind to genotype or treatment by two independent investigators.
We thank the Simons Foundation (TKH, NG) and HFSP (NG) for funding, and Michael Marcotrigiano for technical support.
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