Decision letter: Oxytocin improves behavioral and electrophysiological deficits in a novel Shank3-deficient rat

Abstract

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Mutations in the synaptic gene SHANK3 lead to a neurodevelopmental disorder known as Phelan-McDermid syndrome (PMS). PMS is a relatively common monogenic and highly penetrant cause of autism spectrum disorder (ASD) and intellectual disability (ID), and frequently presents with attention deficits. The underlying neurobiology of PMS is not fully known and pharmacological treatments for core symptoms do not exist. Here, we report the production and characterization of a Shank3-deficient rat model of PMS, with a genetic alteration similar to a human SHANK3 mutation. We show that Shank3-deficient rats exhibit impaired long-term social recognition memory and attention, and reduced synaptic plasticity in the hippocampal-medial prefrontal cortex pathway. These deficits were attenuated with oxytocin treatment. The effect of oxytocin on reversing non-social attention deficits is a particularly novel finding, and the results implicate an oxytocinergic contribution in this genetically defined subtype of ASD and ID, suggesting an individualized therapeutic approach for PMS. https://doi.org/10.7554/eLife.18904.001 eLife digest Phelan-McDermid syndrome is a genetic disorder on the autism spectrum that affects how children develop in several ways, with additional symptoms including attention deficits, delays in learning to speak and motor problems. This syndrome is known to be caused by changes in a single gene known as SHANK3 that disrupt communication between brain cells involved in memory and learning. However, we do not know how these changes relate to the symptoms of Phelan-McDermid syndrome. To understand how genetic changes affect the human brain, researchers often carry out experiments in rats or other small rodents because they have brains that are similar to ours. Harony-Nicolas et al. genetically modified rats to carry changes in the SHANK3 gene that reflect those found in people with Phelan-McDermid syndrome. The rats had disabilities related to those seen in Phelan-McDermid syndrome, including limits in long-term social memory and reduced attention span. They also showed changes in the connections between important parts of the brain. Therefore, studying these rats could help us to understand the link between molecular and cellular changes in the brain and how they affect people with Phelan-McDermid syndrome, and associated symptoms. Previous studies have shown that a chemical called oxytocin, which is naturally produced by the brain, helps to form bonds between individuals and can cause positive feelings in relation to certain memories. Harony-Nicolas et al. found treating the rats with oxytocin boosted social memory and led to improvements in other symptoms of Phelan-McDermid syndrome. In particular, oxytocin treatment helped to increase the attention span of the rats. Rats with changes in the SHANK3 gene will be a useful tool for future research into Phelan-McDermid syndrome, particularly in understanding how it affects the connections between brain cells, leading to the symptoms of Phelan-McDermid syndrome. A future challenge will be to find out whether oxytocin has the potential to be developed into a therapy to treat Phelan-McDermid syndrome in humans. Since there is evidence that SHANK3 is involved in other forms of autism, these rats will also be useful in understanding the other ways in which autism can develop. https://doi.org/10.7554/eLife.18904.002 Introduction Phelan McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by intellectual disability (ID), absent or delayed speech, neonatal hypotonia, attention deficits and autism spectrum disorder (ASD). The neurobehavioral manifestations of PMS are caused by heterozygous mutations/deletions in the SHANK3 gene leading to a reduced expression of the SHANK3 protein. Shank3 is a key structural component of the glutamatergic postsynaptic density (PSD), and interacts with glutamate receptors and cytoskeletal elements to regulate glutamate signaling and synaptic plasticity (Kreienkamp, 2008). It has been estimated that more than 80% of individuals with PMS meet ASD diagnostic criteria and that ~0.5–1% of ASD cases, 1–2% of ID cases, and up to 2% of cases with both ASD and ID harbor a SHANK3 mutation, which makes it one of the more common single locus causes of ASD and ID (Gong et al., 2012; Soorya et al., 2013; Leblond et al., 2014). Despite its prevalence, PMS is less well studied than other single locus genetic disorders such as Fragile X or Rett syndromes. To date, no pharmaceutical compounds targeting core symptoms of PMS are available. To address this lack of effective therapeutics, several mouse lines with distinct Shank3 gene mutations have been developed to help understand the neurobiology of the syndrome and as a means of ultimately developing and assessing potential therapeutics (Bozdagi et al., 2010; Peça et al., 2011; Wang et al., 2011; Yang et al., 2012; Kouser et al., 2013; Kloth et al., 2015; Bidinosti et al., 2016; Mei et al., 2016; Wang et al., 2016). The various Shank3-deficient mouse models have displayed PMS and ASD-related behavioral phenotypes including impaired social behavior, increased repetitive behaviors, and motor deficits, as well as altered synaptic transmission and neuronal morphology in the brain (Bozdagi et al., 2010; Peça et al., 2011; Wang et al., 2011; Yang et al., 2012; Kouser et al., 2013; Drapeau et al., 2014; Wang et al., 2016). Recently, the translational relevance of these mouse models has been highlighted by our observation that the hormone IGF-1 improves motor and synaptic deficits observed in a Shank3-deficient mouse line (Bozdagi et al., 2013), a result which directly led to a safety and preliminary efficacy clinical trial of IGF-1 in children with PMS (Kolevzon, 2014a). Here, we report the generation and characterization of the Shank3-deficient rat, representing a novel genetic model of PMS, which demonstrates clear PMS-related behavioral and electrophysiological phenotypes that can be ameliorated by intracerebroventricular (ICV) oxytocin administration. Results Production and developmental phenotyping of the Shank3-deficient rat model Founder Shank3-deficient rats were generated using zinc-finger nucleases (ZFN) technology, targeting exon 6 of the ankyrin repeat domain. This domain was targeted because five patients had been described with mutations in it (Figure 1A) (Durand et al., 2007; Moessner et al., 2007; Hamdan et al., 2011; Boccuto et al., 2013; Yuen et al., 2015). Interestingly, the predicted truncated protein generated upon ZFN targeting of the rat Shank3 gene is quite similar to one of the human mutations that have been described (Hamdan et al., 2011) (Figure 1A, middle sequence). In rat, this mutation leads to a significant reduction in the number of Shank3 transcripts (Figure 1—figure supplement 1A) and reduces expression levels of the full-length Shank3a protein (Figure 1B and Figure 1—figure supplement 1B). We also observed that the levels of the PSD scaffolding protein Homer1 are decreased in the Shank3-deficient rats (Figure 1—figure supplement 1C), consistent with changes in the PSD and with well-replicated findings from Shank3-deficient mice. We noted that, at weaning, there was a modest reduction in the number of homozygous knockout (KO) animals from Heterozygous (Het) x Het matings, compared to expectation (292:555:200, corresponding to ratios of 0.53:1:0.36). Figure 1 with 1 supplement see all Download asset Open asset Gene-targeting of the rat Shank3 gene. (A) The top schematic shows the Shank3 protein domains [ankyrin repeats domain (ANK), a Src homology 3 (SH3) domain, a PDZ domain, and a sterile α-motif (SAM) domain] and indicates the published de novo mutations observed within the ANK domain in PMS patients (light blue text, top schematic) (Durand et al., 2007; Moessner et al., 2007; Hamdan et al., 2011; Boccuto et al., 2013; Yuen et al., 2015). The 68 bp deletion that we introduced in exon 6 of the Shank3 rat gene (middle schematic) produces a stop codon in exon six that truncates the Shank3 protein. In a PMS patient, the c.601–1G>A mutation in intron 5 of the SHANK3 gene abolishes the normal acceptor site and leads to utilization of a cryptic acceptor site introducing a premature stop codon in exon 6 (red lines, bottom schematic), which also results in a similar truncated Shank3 protein (Hamdan et al., 2011). Lower-case letters, genomic sequence; upper-case letters, amino acids; *, premature stop codons. (B) Western blot showing anti-Shank3 staining, using antibodies targeted against the SH3 domain. https://doi.org/10.7554/eLife.18904.003 To evaluate the impact of Shank3-deficiency in rats, basic developmental processes as well as early motor and sensory function were assessed. These assessments were carried out as previously described (Brunner et al., 2015) and included weight, stomach milk content, body temperature, locomotion, grooming, rearing, pup ultrasonic vocalization, geotaxis, and the righting reflex. We found no genotype-related deficits in these basic developmental or functional processes. In addition, when tested on the elevated plus maze, Shank3-Het and KO rats did not exhibit increased anxiety-like behaviors (See Materials and methods for details and Supplementary file 1 for results). These results enabled us to examine more complex PMS and ASD-relevant behaviors. Social memory deficits in Shank3-deficient rats We first measured preference for a social stimulus versus an object (Figure 2—figure supplement 1A), as well as juvenile social play (Figure 2—figure supplement 1B) and adult dyadic social interactions (Supplementary file 1) in freely interacting animals. We found no differences between Shank3-deficient and wildtype (WT) rats in any of these measures of social behavior or social preference. Next, we used social habituation-dishabituation and social discrimination (SD) tests to examine social recognition memory (SRM). In the SD experiments, the subject rat was ultimately tested in its ability to discriminate between familiar and unfamiliar juvenile rats simultaneously introduced to it for 5 min. We used two versions of the SD test, in order to examine both short- and long-term SRM (with short and long referring to the interval between social memory acquisition and recall). To assess short-term SRM, the SD test was performed 30 min after the subject encountered the familiar rat for 5 min (Figure 2A), while, in the more challenging long-term SRM, which requires longer exposure to the social stimulus (Gur et al., 2014), the same test was performed 24 hr after a 1 hr encounter with the familiar rats (Figure 2B). We found that Shank3-deficiency does not impair short-term SRM as shown by the comparable findings between WT and Shank3-deficient rats on the social habituation-dishabituation (Figure 2—figure supplement 1C) and short-term SD tests (Figure 2A). In contrast, in two independent cohorts, we found that Shank3-Het and KO rats were unable to discriminate between novel and familiar social stimuli in the long-term SD test (Figure 2B) as they spent equal time investigating both the familiar and novel social stimuli. Notably, total investigation time (toward both familiar and unfamiliar rats) did not differ across genotypes in any of the SD tests (Figure 2—figure supplement 1D), indicating there is not a decreased interest in social exploration. Moreover, the fact that the Shank3-Het and KO rats performed well on the short-term SD test and were able to perceive and remember their conspecifics, even when only given 5 min for the first interaction, also ruled out perceptual deficits. Figure 2 with 1 supplement see all Download asset Open asset Shank3-deficient rats exhibit deficits in social memory. (A–B) Above each figure are schematics of the short- and long-term social discrimination (SD) paradigms. The examined adult subject is shown in white, and the juveniles are shown in black and grey. Bar plots show behavior in short-term (WT, n = 12; Het, n = 13; KO, n = 9) and long-term (WT, n = 31; Het, n = 37: KO, n = 25) SD tests. For long-term SD, similar results were observed in two independent cohorts, therefore the results were pooled and presented here as a single cohort. Bars ± SEM at left show test subjects average investigation time of a familiar and unfamiliar juvenile rat. The light overlaid gray lines in A and B show the corresponding individual subject data that comprise each bar. Right scatter plots, presented with mean ± SEM, show the ratio of the investigation time (RDI= (Unfamiliar-Familiar)/(Unfamiliar+Familiar) for individual subjects. (C) Above the figure is a schematic of the long-term object location memory paradigm. The same plotting conventions as bar plots in A and B are used, but here they quantify investigation times of an object (WT, n = 12; Het, n = 12; KO, n = 12) in a novel or familiar location. (D) Above, Contextual fear conditioning paradigm schematic. Scatter plots (mean ± SEM) represent percent time freezing during retrieval of a 1-day-old conditioned fear memory (WT, n = 12; Het, n = 12; KO, n = 6). *, p<0.05, **, p<0.01, ***, p<0.001; see Supplementary file 1 for detailed statistical results. https://doi.org/10.7554/eLife.18904.005 To determine whether this observed impairment is selective to social memory or if it also involves more general memory processes, we tested the rats on two long-term non-social memory paradigms that, similarly to the SD test, are known to be hippocampal-dependent, specifically, the object location memory test and the contextual fear conditioning memory test. In contrast to the impaired behavior in the long-term SD test, Shank3-Het and KO rats performed similarly to their WT littermates in the object location memory and contextual fear conditioning memory tests (Figure 2C and D). These results indicate that Shank3 deficiency selectively impairs long-term social memory, but leaves intact both short-term social memory and non-social long-term memory. Attention deficits in Shank3-deficient rats Attention deficits are often associated with PMS. Thus, we assessed performance in the attentionally demanding 5-choice serial reaction time (5-CSRT) task in which rats must respond quickly to briefly presented light cues (Figure 3) (Mar et al., 2013). This task requires training the rats in stages where the duration of the light stimulus is slowly decreased from 32 to 1 s by halving the stimulus duration across sessions once performance criteria are met (i.e. accuracy rates higher than 80% for two consecutive days with omission rates lower than 20%). Shank3-Het and KO rats learned the task and were able to reach baseline, similar to WT controls. However, both the Shank3 Het and KO rats had lower accuracy and lower omission rates, when compared to WT rats, even after extensive training. Moreover, after reaching baseline criterion Shank3-deficient rats did not maintain even this level of performance across the 10-day test period, during which they performed significantly fewer correct trials (Figure 3A), made more errors (Figure 3B), and exhibited higher omission rates than WT rats (Figure 3C). Even on trials with a correct response, Shank3-deficient rats responded more slowly and with more variable latencies than WT rats (Figure 3D). Figure 3 with 1 supplement see all Download asset Open asset Shank3-deficient rats exhibit deficits in attention. (A) Traces and clouds indicate mean percentage of trials with a correct response ± SEM (WT, n = 10; Het, n = 13; KO, n = 12) across 10 5-CSRT sessions. The right side in all panels is the cross-rat median (dot) and middle quartiles (vertical lines). (B) Traces represent mean percentage of trials where an incorrect response was made. (C) Mean percentage of trials with no cued response. (D) Average reaction times on trials with a correct response. Results were observed in two independent cohorts; therefore, the results were pooled. https://doi.org/10.7554/eLife.18904.007 Slow, inaccurate and omitted responses to very brief visual stimuli are commonly interpreted as reflecting an attention deficit (Robbins, 2002). While changes in accuracy might also be attributed to deficits in sensory perception, we excluded this possibility by carrying out studies with less bright visual cues and Shank3-deficient rats performed at WT levels (not shown). It was only when the duration of the light cues was shortened that the deficits were manifested, which indicates impaired vigilance and/or spatial attention. Furthermore, these deficits were not due to decreased motivation for food, because the latency of Shank3-deficient rats to collect reward after a correct response was similar to WT rats (Figure 3—figure supplement 1A), as was task performance when light cues were of a longer duration (Figure 3—figure supplement 1B). In summary, these results indicate that Shank3-deficient rats are impaired in an attentionally demanding task, which, when considered together with the deficits in long-term social memory, indicates this model demonstrates face validity for some features of PMS. Synaptic plasticity deficits in Shank3-deficient rats Memory deficits, while not considered core symptoms of autism, have been associated with ASD (Boucher et al., 2012). Both impaired working (Barendse et al., 2013) and episodic memory (Maister et al., 2013) have been observed in human subjects with ASD, which has been attributed to aberrant connectivity of the hippocampus and medial prefrontal cortex (mPFC) (Ben Shalom, 2003). The behavioral deficits we observe in Shank3-deficient rats are consistent with dysfunction in these circuits. The hippocampus and mPFC are both important for SRM (Watson et al., 2012; Harvey and Lepage, 2014; Jacobs and Tsien, 2014). Moreover, performance in the 5-CSRT task depends on mPFC function (Rogers et al., 2001) and attention, working memory, and decision-making require intact hippocampal-prefrontal functional connectivity (Jones and Wilson, 2005). We therefore evaluated the effect of Shank3 deficiency on synaptic function and plasticity in hippocampal-PFC circuitry. Extracellular field excitatory postsynaptic potential (fESPs) recordings at Schaffer collateral-CA1 synapses were similar between genotypes, with no differences in paired-pulse facilitation (Supplementary file 1) or the input-output relationship (Figure 4—figure supplement 1). These results suggest that basal synaptic transmission is generally intact in Shank3-deficient rats. We found, however, in independent cohorts, that plasticity at these synapses was not intact. Long-term potentiation (LTP) induced by high-frequency stimulation (HFS) was reduced in both Shank3-Het and KO rats (Figures 4A and 6A, and Figure 6—figure supplement 1A), while mGluR-dependent long-term depression (LTD) was reduced only in KO rats (Figure 4B). Figure 4 with 1 supplement see all Download asset Open asset Synaptic plasticity is impaired in Shank3-deficient rats. (A) High-frequency stimulation (HFS, arrow)-induced long-term potentiation (LTP) at hippocampal Schaffer collateral-CA1 synapses (n = 6 rats/genotype, 1–2 slices per rat). (B) Long-term depression induced by the mGluR agonist DHPG (50 µM, 5 min) is indicated by the horizontal line (n = 6 rats/genotype, six slices per rat). (C) HFS-induced LTP in the prelimbic PFC after stimulation of ipsilateral CA1 in ventral hippocampus of intact anesthetized WT (n = 5), Shank3 Het (n = 6) and KO (n = 6) rats. Inset shows a schematic of the target location of the stimulating and recording electrodes in vivo. Summary data are presented as mean ± SD. *p<0.05, ***p<0.001; See Supplementary file 1 for detailed statistical results. https://doi.org/10.7554/eLife.18904.009 Innervation of the mPFC by the hippocampus (Marquis et al., 2006) is important for attention and working memory, both of which we have shown here are impaired in Shank3-deficient rats. We therefore assessed hippocampal-PFC synaptic transmission in vivo by stimulating hippocampal CA1/subicular regions and recording fEPSPs in the prelimbic area of the PFC in anesthetized rats. We found that LTP was reduced in both Shank3-Het and KO rats (Figure 4C). We also observed that there were no genotype associated differences in the input-output relationship of evoked local field potentials in coronal PFC slices, which demonstrates that these changes were specific to hippocampal-prefrontal circuitry (Supplementary file 1). In summary, these results suggest that Shank3-deficiency impairs plasticity in both the projections from hippocampus to the PFC and within intrinsic hippocampal circuits. Oxytocin improves behavior and synaptic plasticity deficits in Shank3-deficient rats Oxytocin has a central role in SRM formation (Ferguson et al., 2000; Gur et al., 2014) and hence we reasoned that it may underly some of the altered behaviors we observed; in addition and more broadly, oxytocin modulates mammalian social behavior and may be dysregulated in ASD (Harony and Wagner, 2010). To determine whether the behavioral deficits we observed in Shank3-deficient rats could be improved with a pharmacological intervention strategy, we tested Shank3-deficient rats on long-term SD and 5-CSRT tasks following an injection of either oxytocin or saline into the left lateral ventricle. We observed that oxytocin improved both the long-term social memory and attention deficits in Shank3-Het and KO rats (Figure 5A and B). Notably, in WT animals, oxytocin had no effect on long-term SRM (Figure 5—figure supplement 1A) or 5-CSRT task performance (Figure 5—figure supplement 1B). Figure 5 with 1 supplement see all Download asset Open asset Oxytocin improves social memory and attentional deficits in Shank3-deficient rats. Above each figure is a schematic depicting the sequence of oxytocin administration and behavioral testing on the long-term social discrimination (SD) paradigm (in A) or 5-CSRT task (in B). In A, the test subject is shown in white and the juveniles are shown in black and grey. (A) Left bars (± SEM) show the test subject’s average investigation time of a familiar and unfamiliar juvenile rat on long-term SD test following treatment with oxytocin or vehicle for Shank3 Het (n = 15) and KO (n = 8) rats. The light overlaid gray lines show the corresponding individual subject data that comprise each bar. Bars on right show the ratio of the investigation time (RDI= (Unfamiliar-Familiar)/(Unfamiliar+Familiar) for individual subjects with the light overlaid gray lines representing the corresponding individual subject data. (B) Bars (± SEM) represent the percentage of correct (left), omitted (middle), and incorrect (right) trials in saline (solid bars) and oxytocin (open bars) of Shank3 Het (n = 7) and KO (n = 6) rats. Color conventions are identical to those used in Figure 3A–C. The light overlaid gray lines show the corresponding individual subject data that went into each bar. *p<0.05, **p<0.01, ***p<0.001; See Supplementary file 1 for detailed statistical results. https://doi.org/10.7554/eLife.18904.011 Since oxytocin reversed the behavioral deficits in Shank3-deficient rats and has been previously shown to enhance the induction of synaptic plasticity in several systems (Benelli et al., 1995; Tomizawa et al., 2003; Fang et al., 2008; Ninan, 2011; Lin et al., 2012; Gur et al., 2014), we also examined the effect of oxytocin on LTP both in vitro and in vivo. As previously reported, in acute hippocampal slices derived from WT rats, oxytocin enhanced LTP induction after a weak stimulation of one train of 100 Hz pulses (Tomizawa et al., 2003; Lin et al., 2012) but we did not observe this oxytocin-dependent enhancement in Shank3-deficient rats (Figure 6—figure supplement 1A). In WT slices, oxytocin had no effect on LTP induced by stronger stimulation (4 × 100 Hz stimulation trains), but it greatly enhanced LTP in slices prepared from Shank3-deficient rats (in independent cohorts, Figure 6A and Figure 6—figure supplement 1B). In vivo, oxytocin also reversed the impaired LTP at hippocampal-prefrontal synapses in Shank3-deficient rats (Figure 6B). These results indicate that oxytocin treatment restores LTP induction at hippocampal and hippocampal-prefrontal synapses in Shank3-deficient rats and is consistent with a mechanism whereby deficits in synaptic plasticity across hippocampal-prefrontal circuitry underly PMS-relevant behavioral deficits we observed in these animals. Figure 6 with 1 supplement see all Download asset Open asset Oxytocin improves synaptic plasticity deficits in Shank3-deficient rats. (A) Traces depict Hippocampal HFS-induced LTP (4 × 100 Hz) in WT and Shank3-deficient rats (n = 4 rats/genotype, six slices per rat). Application of 1 µM of oxytocin is indicated by the horizontal line. (B) LTP in the hippocampal to prefrontal synaptic pathway recorded in vivo in WT (n = 3), Shank3 Het (n = 4) and KO (n = 4) rats. Oxytocin (2 ng) or saline was administered in the lateral ventricle 5 min before LTP induction. ***p<0.001. See Supplementary file 1 for detailed statistical results. https://doi.org/10.7554/eLife.18904.013 Discussion Deficits in attention and the presence of ASD-related behaviors are common in PMS. In our genetically modified rat model of PMS, we found that Shank3 deficiency impairs social memory and attention. These impairments are accompanied by attenuated synaptic plasticity within the hippocampus and in the monosynaptic pathway connecting the hippocampus to the PFC. Hippocampal synaptic plasticity deficits have also been reported in Shank3-mouse models and were associated with impairments in actin cytoskeleton remodeling and with changes in the levels of glutamatergic receptors and other PSD scaffolding components including Homer, which we also found to be decreased in the Shank3-deficient rat (Figure 1—figure supplement 1C) (Bozdagi et al., 2010; Wang et al., 2011; Duffney et al., 2013; Kouser et al., 2013; Wang et al., 2016). The fact that long-term social memory was selectively impaired, in contrast to more general memory processes, may reflect the complexity of the information involved in social memory (Adolphs, 2010; Wiley, 2013), which may require more elaborate synaptic mechanisms than simpler forms of memory. These results may also relate to previous findings that correlate ASD-associated episodic memory deficits with the complexity of the memoranda (Lind, 2010), which suggests that sub-optimal thresholds for synaptic plasticity interfere with the ability of complex stimuli, such as social stimuli, to activate memory formation. Deficits in attentionally demanding tasks, like the 5-CSRT task, may also result from blunted synaptic plasticity, where brief events must be rapidly encoded and reliably stored by neural circuits to promote appropriate and timely behavior. It is interesting, and important, to compare findings in different rodent models of PMS and relate them to symptoms observed in patients. In particular, while there is some overlap between the social communicative deficits observed in PMS and those observed in what is considered more classical autism, there are important differences. In contrast to the more typical conception of ASD (whether accurate or not), PMS-associated ASD is not associated with a social aversion or lack of social approach (Soorya et al., 2013; Kolevzon, 2014b). A recent study of functional neuroimaging showed that differential fMRI changes in response to social versus non-social sounds are preserved in PMS, in contrast to studies of idiopathic autism (iASD) (Wang et al., 2016). These findings, together with the fact that not all individuals with PMS are diagnosed with ASD, indicate that animal models for PMS should not necessarily present with social behavioral deficits and imply that behavioral phenotypic diversity in PMS patients may reflect additional processes. This may explain some of the discrepancies that have been reported across distinct Shank3-mouse models (Harony-Nicolas et al., 2015), and those that we report here in the Shank3-deficient rat. These discrepancies could be attributed to (1) mutations/deletions in existing rodent models were targeted to different parts of the Shank3 gene, (2) varying genetic background, (3) different behavioral paradigms, (4) varying age and sex, (5) a focus on heterozygotes or knockouts, or other factors. Beyond these methodological issues, from an evolutionary perspective, mice and rats are separated by millions of years, which likely explain many of the observed discrepancies in behavioral repertoires between the two species. Just as mice and rats differ, so do mice and rats greatly differ from human, and thus one should be careful not to overly anthropomorphize these model systems or look for perfect overlap across species. Further investigation of the mechanisms underlying synaptic plasticity deficits and the molecular pathways affected by Shank3 mutation and the consequent synaptic changes in rat and mouse Shank3 models will likely provide new targets for therapeutic treatments and allow comparison between two different species, thus providing more reliable molecular targets for future drug development studies. Although all the mutations that have been st