autism spectrum disorder ( asd ) is characterized by behavioral , cognitive and language impairment . over the past decade ,
studies on monogenetic syndromes with high prevalence of asd , such as fragile x ( fx ) and tuberous sclerosis ( ts ) , have provided insights into the pathophysiology of diseases that can cause autism . for example , it has been shown that altered signaling downstream of metabotropic glutamate receptor 5 ( mglur5 ) plays a pivotal role in the pathogenesis of fx , and that genetic and pharmacological modulation of mglur5 can ameliorate numerous impairments in fx animal models .
chromosomal copy number variations ( cnvs ) have been associated with 510% of patients with asd .
variation at human chromosome 16p11.2 is the most common of these and accounts for approximately 0.51% of all asd cases .
the affected region harbors ~27 annotated protein - coding genes , many of which are expressed in the brain .
the common clinical presentations in individuals carrying chr16p11.2 microdeletion are language impairment , intellectual disability ( i d ) , asd , anxiety , attention deficit hyperactive disorder ( adhd ) , and epilepsy .
recent studies on animal models of human chr16p11.2 microdeletion have demonstrated morphological , behavioral and electrophysiological deficits , however the synaptic pathophysiology remains largely unexplored . using electrophysiology , biochemistry and behavioral tests we characterized hippocampal function of a mouse model for human chr16p11.2 microdeletion .
we uncovered alterations in mglur5-mediated synaptic plasticity , arc expression , basal protein synthesis , and hippocampus - dependent learning that are reminiscent of previous observations in mouse models of syndromic autism and i d .
importantly , we were able to ameliorate the behavioral abnormalities using an mglur5 negative allosteric modulator ( nam ) , 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1h - imidazol-4-yl)ethynyl)pyridine ( ctep ) , suggesting a pathophysiology shared with fx .
mutant mice ( termed 16p11.2 df/+ ) , engineered to be heterozygous null at the region of chromosome 7qf3 that is syntenic to human chromosome 16p11.2 , were back - crossed for 510 generations to c57bl/6n mice ( charles river ) to allow a comparison of synaptic physiology with previous studies .
as noted previously , loss of the genes in this region can compromise survival , and we found that this effect was amplified in successive generations on the c57bl/6n background ( supplementary fig .
we first characterized basal synaptic transmission at the schaffer collateral - ca1 synapse using hippocampal slices from 45 week old mice and found no difference from wild type ( wt ) in input - output or paired - pulse facilitation ( ppf ) ( supplementary fig .
2 ) . to investigate nmda receptor - dependent synaptic plasticity , we induced long - term potentiation ( ltp ) with theta - burst stimulation ( tbs ) , and long - term depression ( ltd ) with low - frequency ( 1 hz ) stimulation ( lfs ) .
b ) , suggesting basic excitatory synaptic transmission and plasticity mechanisms are intact in the mutant mice .
mglur - ltd was induced either by brief application of the mglur1/5 agonist s-3,5-dihydroxyphenylglycine ( dhpg ) or by applying a series of paired pulses at 50 ms interval ( pp - lfs ) .
we again found no difference between wt and the 16p11.2 df/+ mutant with either induction protocol ( fig .
f ) . a distinctive property of mglur - ltd in wt animals is a requirement for mrna translation at the time of induction . in slices from fmr1 mice , however , mglur - ltd is unaffected by translation inhibitors because basal synaptic synthesis of ltd - regulatory proteins such as arc is elevated downstream of constitutive mglur5 activity due to loss of the translational repressor fmrp .
we were therefore compelled to investigate the protein synthesis - dependence of mglur ltd in the 16p11.2 df/+ mice , and discovered a striking difference from wt . like fx model mice ,
mglur - ltd in the 16p11.2 df/+ mice was unaffected by cycloheximide ( chx ) ( fig .
as reported previously in wt animals , dhpg induces ltd via two mechanisms : a postsynaptic reduction in ampa receptors and a presynaptic reduction in glutamate release probability .
only the postsynaptic mechanism is chx sensitive . to test whether the different sensitivity of ltd to chx in the mutant was due to a qualitatively different expression mechanism , we analyzed ppf at the beginning and end of each dhpg - ltd experiment .
these findings point to a deficiency in postsynaptic regulation of protein synthesis in the 16p11.2 df/+ mice .
we next tested the mutant mice in two hippocampus - dependent behavioral assays , contextual fear conditioning ( cfc ) and inhibitory avoidance ( ia ) , which have been shown in previous studies to reveal cognitive impairments in fmr1 mice .
cfc requires intact mglur5 signaling and new protein synthesis at the time of conditioning . in this assay
, mice are exposed to a distinctive environmental context in which a foot - shock is delivered , and 24 h later the mice are returned to either the same ( familiar ) or a different ( novel ) context ( fig .
wt mice expressed fear memory by freezing in the familiar context , and demonstrated an ability to discriminate different contexts by freezing less in the novel context .
in contrast , the mutant mice showed significantly less freezing in the familiar context , and were unable to distinguish between the familiar and novel context ( fig .
2c ) , suggesting the difference in freezing at 24 h was due to an impairment in memory formation in the mutant mice . in the ia assay , mice received a foot shock upon entry into the dark side of a two - chamber box ( fig .
memory strength and extinction were measured as the latency to enter the dark side when given the opportunity at 6 , 24 , and 48 h intervals .
similar ia deficits in fmr1 mice have been ameliorated by chronic post - adolescent treatment with the mglur5 nam ctep .
therefore we repeated the ia assay on wt and mutant mice that were treated every second day with ctep ( 2 mg / kg p.o . ) or vehicle for 4 weeks .
although treatment had no effect in the wt mice , it corrected the deficits in the mutants both in terms of acquisition and extinction ( fig .
we were motivated to investigate the possibility that fx and 16p11.2 microdeletion have shared pathophysiology for several reasons , including the fact that four deleted genes are targets of fmrp ( maz , sez62l , taok2 , aldoa ) and disruption of several genes in this region are predicted to affect mglur5 signaling ( mvp , cdipt , mapk3 ) or protein turnover ( kctd13 ) .
the most straightforward prediction from our results is that synaptic protein synthesis downstream of mglur5 is exaggerated by the 16p11.2 microdeletion , and this gives rise to cognitive impairment . in the fmr1 mouse ,
however , we found that basal protein synthesis in hippocampal slices in the 16p11.2 df/+ mice was reduced , possibly explained by the decrease in erk pathway activity ( fig .
therefore , constitutive elevation of arc could render mglur - ltd insensitive to acute inhibition of protein synthesis and contribute to cognitive impairment .
first , the data suggest that some cognitive and neuropsychiatric symptoms of 16p11.2 microdeletion disorder arise from altered synaptic signaling that is amenable to targeted drug therapy .
second , the data strengthen the hypothesis that multiple causes of asd and i d converge on common pathophysiological processes , and one of these is the synaptic regulation of protein synthesis .
 human chromosome 16p11.2 microdeletion is the most common gene copy number variation in autism , but the synaptic pathophysiology caused by this mutation is largely unknown . here 
 we show using a mouse with the same genetic deficiency that metabotropic glutamate receptor 5-(mglur5- ) dependent synaptic plasticity and protein synthesis is altered in the hippocampus , and that hippocampus - dependent memory is impaired . 
 remarkably , chronic treatment with a negative allosteric modulator of mglur5 reverses the cognitive deficit . 
Introduction
Results and Discussion
Supplementary Material