Neuroanatomical phenotypes in Sema6A mutant mice
As neurodevelopmental mutations typically affect multiple brain regions, any of which might contribute to behavioural or physiological phenotypes in mice or to the broad array of symptoms in humans, we set out to comprehensively characterize the anatomical defects across the brain due to Sema6A mutation. We were particularly interested, however, in areas most strongly implicated in the psychopathology of psychiatric disorders, including the prefrontal cortex and limbic system, which had not been previously investigated in these mutants.
The prefrontal cortex in rodents encompasses the agranular insular cortex, the orbitofrontal cortices and some of the cingulate cortices 
. Some neurons in these areas normally send axons to the opposite hemisphere across the posterior limb of the anterior commissure (pAC). In embryonic (E16–17.5, n
3, not shown) and newborn (postnatal days (P)0–2, n
9) Sema6A homozygous mutants, all axons of the pAC project ventromedially rather than medially and fail to cross the midline (). Many misrouted pAC axons can be found in adult Sema6A−/− mice (n
5, ), where they extend into the hypothalamus and septum (). Retrograde tracing of pAC axons in homozygous mutant animals also reveals that the cell bodies of these neurons are ventrally misplaced and located within piriform, rather than insular cortex, where they are observed in control animals (Supporting Information S1
Prefrontal cortex dysconnectivity in Sema6A mutants.
Piriform cortex and olfactory structures
Staining for PLAP, Nissl, or with anti-NeuN antibodies at various ages highlighted a dramatic change in morphology of the piriform cortex in Sema6A homozygous mutants. This three-layered structure receives direct input from the olfactory bulb and connects reciprocally with prefrontal cortex and many other structures 
. In Sema6A mutants, the folding of the piriform cortex, most readily visualized in the densely packed cells of layer 2, is greatly exaggerated and is evident far further caudally than normal (; see also and ). In addition, axons from the lateral olfactory tract (LOT), which normally extend very superficially along the surface of the piriform cortex () are displaced internally in Sema6A mutants at E16.5 () and in adulthood () and also extend considerably further caudally ().
Defects in piriform cortex and olfactory projections.
Staining with Nissl, anti-NeuN antibodies or DAPI also revealed a defect in the cellular organisation of the neocortex (n
14 in total; adult: Nissl, n
4; DAPI, n
5; NeuN, n
3; P10: NeuN, n
2). In Sema6A−/−
animals, the normally distinct border between layers 1 and 2 is difficult to distinguish, as many neurons from layers 2/3 invade into the normally cell-sparse neuropil of layer 1 (). This defect is not evident at late embryonic stages (E16.5–E17.5) but can be observed from P3 onwards (data not shown).
Aberrant lamination of the neocortex.
We performed antibody staining for NeuN, a marker of mature neurons, and Prox1, a marker for postmitotic granule cells in the hippocampal dentate gyrus, on brain sections at P10 (). In Sema6A−/− mice, we found a subtle but consistent malformation of the infrapyramidal (lower) blade of the dentate gyrus. At the tip of this blade, many misplaced Prox1-positive granule cells were observed, producing a broadened or forked appearance of the tip. In addition, isolated ectopic granule cells were frequently observed within the molecular layer on the infrapyramidal side, some close to the ventricular wall (, arrows). These misplaced cells persist in the adult ().
Alterations in hippocampal lamination and projections.
We also observed defects in long-range connectivity of the hippocampus. In Sema6A mutants, the post-commissural fornix is often defasciculated and reduced in size, sometimes containing far fewer axons than normal. This phenotype is quite variable, even from one side of the brain to the other (). On average, the cross-sectional area of the fornix was significantly reduced (p<0.05, T-Test, ) to 83.7% (±9 vs. 100% ±17.0 in Sema6A+/−), as was the degree of compactness (measured by the ratio of actual vs. circular perimeter: 2.53±0.66 in Sema6A−/− vs. 1.82±0.08 in Sema6A+/−; p<0.01, T-Test, ).
Altered cortical activity patterns.
With the exception of the defect in the fornix and some aspects of the previously reported corticospinal tract defect 
, all the anatomical phenotypes we observe are fully penetrant (i.e., observable qualitatively across all mutant animals analysed).
We set out to characterise, in an unbiased fashion, the behavioural consequences of mutation of Sema6A, using a broad panel of behavioural tests, along with ethological observation in the home cage environment.
Analysis of gait in Sema6A−/−
mutants revealed a significantly shorter hind stride length in Sema6A−/−
mice relative to wild-type (WT or Sema6A+/+
) controls (; genotype: F2, 39
<0.05, ). A marginally significant increase in hind/front overlap was also observed in Sema6a−/− mice (; genotype: F2, 33
0.07), as well as a mean decrease in front stride length (WT vs. Sema6A−/−
: 3.3 cm±0.15 vs.
3.16±0.07) and hind base width (WT vs. Sema6A−/−
: 2.61 cm±0.10 vs.
2.38±0.07), although neither of these differences were statistically significant (; p>0.05). Examination of grip strength during the wire hang suspension task failed to reveal any genotypic differences (p>0.05, not shown).
Gait abnormalities and hyperlocomotion.
Examination of open-field exploratory locomotor activity in Sema6A−/−
mice revealed a hyperactive phenotype in both sexes, as indexed by significant increases in activity counts (; genotype: F2, 46
4.78, p<0.05) and distance travelled (; genotype: F2, 46
5.65, p<0.05) over a 6-hour observation period. Relative habituation of exploration to the novel environment did not differ between genotypes (genotype×hours interaction: F10, 230
0.429). Ethologically-based assessment of exploratory behaviours in a novel environment (the ethogram 
) revealed significant increases in locomotion and rearing in mutants relative to controls (data not shown and ). Prior treatment with the antipsychotic clozapine (0.25 mg/kg) reversed the hyper-exploratory phenotype in Sema6A−/−
mice (; genotype×drug interaction: F 4, 67
3.37, p<0.05) to a level indistinguishable from wild-type littermate control mice treated with vehicle or clozapine. Treatment with haloperidol (0.5 mg/kg) reduced control activity count values somewhat, though not significantly (from 2526+/−284 (SEM) in 8 vehicle-treated animals, to 1637+/−545 in 7 haloperidol-treated animals, p
0.16), but completely reversed the hyperlocomotion in Sema6A
mutants (from 5116+/−837 to 1556+/−192, n
8 and 7, respectively, p<0.01). The NMDA-receptor antagonist MK-801 (0.2 mg/kg) stimulated locomotor activity in both Sema6A−/−
mice and controls, with no treatment-genotype interaction; additionally, Sema6A−/−
mice did not show any differential responsiveness to a subthreshold dose of MK-801 (0.05 mg/kg) relative to controls (; genotype×drug treatment interaction; F4, 56
0.67, p>0.05). Heterozygous animals were not significantly different from wild-type in gait or locomotion tests.
Memory defects, decreased anxiety, and increased social interaction.
Recognition memory was examined in the Novel Object Recognition (NOR) paradigm. Sema6A−/−
mice demonstrated increased exploration of both objects relative to wild-type controls during the sample phase (WT vs. Sema6A−/−
0.05) and during test 1 (; WT vs. Sema6A−/−
2.19, p<0.05). Sema6A−/−
mice evidenced impairment in the NOR task, as indexed by decreased exploration of the novel object compared to the familiar object at the 1-hr retention interval (; WT vs. Sema6A−/−
2.18, p<0.05). Sema6A−/−
mice were also examined for spatial working memory in the spontaneous alternation task. Impaired performance, as indicated by decreased alternation across the three arms of the Y maze, was observed in male Sema6A−/−
mice relative to wild-type controls (; t16
2.98; p<0.01). This was accompanied by an increase in total number of arm entries, a measure of activity levels in this task (WT vs. Sema6A−/−
2.85, p<0.05). These effects were not observed in females.
Emotionality and anxiety-related behaviour were examined in Sema6A−/−
mice in the elevated plus maze. Male Sema6A−/−
mice demonstrated decreased anxiety, as evidenced by increased percent time spent in the open arms relative to the enclosed arms of the maze (; WT vs. Sema6A−/−
2.36, p<0.05). The hyperactive phenotype of Sema6A−/−
mice was also evident in this measure, in terms of an increased number of overall arm entries vs.
wild-type controls for both sexes (; genotype: F2, 57
10.304, p<0.0001). No falls from the open arms of the maze were noted for WT or Sema6A−/−
animals, indicating no effect of genotype on overall sensorimotor function in this task. When tested in an alternative assay of anxiety, marble-burying behaviour, both male and female Sema6A−/−
mice demonstrated an anxiolytic phenotype, as measured by fewer marbles buried, relative to wild-type controls (; t39
Social interaction, including various affiliative and agonistic behaviours, was assessed in Sema6A−/−
and wild-type controls by placing them with an unfamiliar age-, sex-, and weight-matched C57/BL6 mouse in a novel environment. Sema6A−/−
mice displayed increased social sniffing episodes towards an unfamiliar mouse relative to controls (Figure I; genotype: F2, 29
7.403, p<0.05). This increase in social investigative behaviour was accompanied by a decrease in walkover episodes (genotype: F2, 29
7.403, p<0.01) and walkover time (genotype: F2, 29
4.33, p<0.01) compared to control mice, indicating a reduction in social dominance-related behaviour ().