Brinp1−/− mice exhibit autism-like behaviour, altered memory, hyperactivity and increased parvalbumin-positive cortical interneuron density

Background BMP/RA-inducible neural-specific protein 1 (Brinp1) is highly conserved in vertebrates, and continuously expressed in the neocortex, hippocampus, olfactory bulb and cerebellum from mid-embryonic development through to adulthood. Methods Brinp1 knock-out (Brinp1−/−) mice were generated by Cre-recombinase-mediated removal of the third exon of Brinp1. Knock-out mice were characterised by behavioural phenotyping, immunohistochemistry and expression analysis of the developing and adult brain. Results Absence of Brinp1 during development results in a behavioural phenotype resembling autism spectrum disorder (ASD), in which knock-out mice show reduced sociability and changes in vocalisation capacity. In addition, Brinp1−/− mice exhibit hyper-locomotor activity, have impaired short-term memory, and exhibit poor reproductive success. Brinp1−/− mice show increased density of parvalbumin-expressing interneurons in the adult mouse brain. Brinp1−/− mice do not show signs of altered neural precursor proliferation or increased apoptosis during late embryonic brain development. The expression of the related neuronal migration genes Astn1 and Astn2 is increased in the brains of Brinp1−/− mice, suggesting that they may ameliorate the effects of Brinp1 loss. Conclusions Brinp1 plays an important role in normal brain development and function by influencing neuronal distribution within the cortex. The increased cortical PV-positive interneuron density and altered behaviour of Brinp1−/− mice resemble features of a subset of human neurological disorders; namely autism spectrum disorder (ASD) and the hyperactivity aspect of attention deficit hyperactivity disorder (ADHD). Electronic supplementary material The online version of this article (doi:10.1186/s13229-016-0079-7) contains supplementary material, which is available to authorized users.


Background
Many neurodevelopmental disorders (NDDs) result from rare genetic alterations including copy number variations (CNVs), single nucleotide polymorphisms (SNPs) and de novo mutations in genes affecting a variety of cellular processes [1][2][3]. Autism spectrum disorder (ASD) is a NDD that affects an estimated 0.6 % of children [4,5], with similar prevalence in adults [6]. Clinical diagnosis is based on atypical social behaviour, impairments in verbal and nonverbal communication, and patterns of restricted interests and repetitive behaviours [7]. Autistic patients commonly have moderate to severe impairments in expressive language [8,9]. ASD also frequently presents with at least one other NDD, for example attention deficit hyperactivity disorder (ADHD), anxiety disorder, or intellectual disability [10,11].
Notably, approximately 28 % of patients diagnosed with ASD also present with ADHD, diagnosed in childhood by inattention, hyperactivity and impulsivity [7,12]. Whist the underlying pathology of such NDDs is not fully understood, interneuron abnormalities have shown to be a key contributor to ASD, and other NDD pathologies [13][14][15][16][17].
Members of the Membrane Attack Complex/Perforin (MACPF) protein superfamily share a domain that facilitates oligomerisation and membrane association [18,19]. BMP/RA-inducible neural-specific protein 1 (BRINP1) is one of five MACPF members expressed predominantly in the nervous system. The 85-kDa BRINP1 protein is over 50 % identical to two other MACPF proteins, BRINP2 and BRINP3 [20], and all three are expressed in an overlapping pattern in neurons in the developing and mature brain.
The physiological role of BRINP1 is not understood. It has been suggested to function in cell cycle regulation [20,21]. Kobayashi et al. recently produced mice carrying a homozygous exon-8 deletion in Brinp1. Adult mutant mice display increased neuronal proliferation in the hippocampus and more parvalbuminpositive interneurons in the CA1 region of the hippocampus. The mice also show behavioural alterations suggested to be relevant to schizophrenia or ADHD [22]. Brinp1 has also been implicated with neural plasticity of song learning in birds [23,24] and is reported to be lost in multiple cancers [25][26][27]. The subcellular localisation of Brinp1 is unknown, but EGFP-tagged Brinp1 has been observed in the endoplasmic reticulum of the PC12 (rat) cell line, suggesting it functions in the secretory/endolysosomal system or is released from cells [20].
To further investigate the role of BRINP1 in brain development and cognitive function, we generated conditional exon 3-deleted Brinp1 −/− mice, via the LoxP/Cre-recombinase system. We demonstrate that these mice exhibit reduced reproductive fecundity, altered parvalbumin interneuron density in both the neocortex and hippocampus, with no change in cell proliferation in the developing embryonic brain (E18.5). In addition, the mice exhibit a social

Gene targeting
A targeting vector was constructed to alter the Brinp1 locus in mouse embryonic stem (ES) cells by homologous recombination following the general strategy outlined by Teoh et al., 2014 [38]. The vector was built using bacterial artificial chromosome (BAC) clone RP23-85B13 as a source of Brinp1 DNA. This vector comprised a neomycin transcriptional unit flanked by flippase (Flp) recognition target (FRT) elements placed in intron 3. A loxP element was placed in the same intron immediately downstream of the neomycin cassette, whilst an upstream loxP element was placed in intron 2. Crerecombinase-mediated deletion of exon 3 was designed to generate a frame shift, resulting in a stop codon in the fourth exon of Brinp1. The targeting construct was electroporated into Bruce 4 C57BL/6derived embryonic stem (ES) cells, and the targeted clone carrying the targeted allele (Brinp1 tm1Pib (MGI: 5604540)) was identified by Southern analysis. A correctly targeted clone was injected into BALB/c blastocysts to generate chimeric mice, which were crossed to C57BL/6 Cre deleter transgenic mice Tg(CMV-cre)1Cgn to remove exon 3 and the neomycin cassette from the targeted allele. This produced animals carrying the Brinp1 tm1.1Pib mutation (MGI: 5604542). In parallel, chimeric mice were crossed to C57BL/6 Flp deleter transgenic mice to remove the neomycin cassette only (Brinp1 tm1.2Pib (MGI: 5604543)). 'Floxed' mice heterozygous for the Brinp1 tm1.1Pib mutation were inter-crossed to generate mice of all three genotypes: Brinp1 +/+ (wild type, WT); Brinp1 +/tm1.1Pib (het); and Brinp1 tm1.1Pib/tm1.1Pib (Brinp1 −/− ).

Genomic analysis
The WT, Brinp1-targeted, and Brinp1-floxed mouse lines were all verified by Southern analysis. Genomic DNA isolated from the spleen was digested with Pst I and probed with a 500 bp 5′ homology probe. A 3′ homology arm probe was used for blotting of genomic DNA digested with Bgl III. An internal Bgl III probe was used to rule out random integration into the genome. PCR analysis confirmed absence of the neomycin cassette in floxed animals.
Immunoblotting Whole brain lysates were made from Brinp1 −/− and WT mice at postnatal day 12. Samples were run on a 10 % SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The blots were probed with a custom-made rat polyclonal antibody to human BRINP1 (1:200). For BRINP1 antibody production, rats were immunised with gel slices containing purified, denatured recombinant BRINP1 produced in a Baculovirus expression system. Antiserum was validated by indirect immunofluorescence of COS-1 cells transiently expressing human BRINP1 and immunoblotting of corresponding COS-1 cell lysates, following approaches described by Teoh et al. [39]. No signal was detected in mock-transfected (control) COS-1 cell samples. The secondary antibody was an anti-Rat HRP (Rockland, 1:5000); loading control: βIII-tubulin (Covance, 1:1000).
qPCR Primers were designed to produce a single PCR product within a range of 80-190 bp (optimal size of 150 bp). Primers were first validated by RT-PCR, checking for a single PCR product of the predicted size. PCR reactions were set up in a 96-well plate format as 10 μl reactions: 5 μl SYBR Green (Sigma), 4.1 μl of water, 0.2 μl primer 1, 0.2 μl primer 2, and 0.5 μl cDNA. Reactions were run on a Roche Light Cycler 96: 95°C 60 s, (95°C 30 s, 61°C 30 s) x 45, 72°C 120 s. Reference genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin were used for normalisation. Results are represented as the fold change relative to WT and were analysed using unpaired Student's t tests.
Animals C57BL/6 Brinp1 −/− animals and wild-type littermates were generated from heterozygous breeders in all studies, except for monitoring of homozygous matings. Mice were genotyped from tail snips collected at postnatal day 10 (P10). Mice were kept under standard housing conditions with a 12 h light/dark cycle. Mice were housed with mixed genotype littermates; maximum of five adults per box. All breeding and experiments were approved by the Monash University Animal Ethics Committee.

Weighing
Brinp1 −/− , het, and WT littermates were weighed weekly between 3 and 12 weeks (n = 8 males, 8 females per genotype). Repeat measures two-way ANOVAs were performed for comparisons between WT and knockout mice weights.

Reproductive phenotyping
Four breeders per genotype (WT/WT, Het/Het, Brinp1 −/− /Brinp1 −/− and WT ♂/Brinp1 −/− ♀) were set up and monitored over four months. Mice were used as breeders at 7-8 weeks of age. The number of pups was recorded at birth, 48 h post birth, and at weaning (postnatal day 10).

Behavioural testing
Cohort sizes were 9-12 mice per genotype, aged 3-4 months. A 1:1 ratio of females/males was tested. Mice were habituated to the testing facility for 1 week then habituated to the testing room overnight. Mice were tested blind to genotype and in random order. Tests were separated by a minimum of 1 day. WT and knock-out mice were tested in the same testing sessions. Lighting conditions were 30 lux for all behavioural tests. Testing arenas were cleaned with Equinade disinfectant (lavender scent) between trials. In all instances, mice had previously been habituated to the disinfectant whilst housed at the testing facility.

Visual placing test
Mice were lifted by the tail to a height of 15 cm and lowered onto a mesh grid within 1 s, decelerating as the grid approached. One trial was performed per mouse. The distance of the animal's nose from the grid was measured the moment before the mouse extended its forelimbs towards it. A single trial was performed per animal.

Rotarod
Mice were pre-trained on the Rotarod (Ugo Basil) for two initial trials at a constant speed of 4 rpm for 5 min, followed by a third trial accelerating from 4-40 rpm over 5 min. Testing was carried out the following day by 4 × 5 min accelerating trials with an inter-trial interval of 30 min.

Three-chamber social interaction test
Adapted from methods previously described by Silverman et al, 2010: Identical rectangular wire cages were placed in equivalent positions in the left and right chambers of a 3chamber plexiglass box (600 × 400 × 250 mm). Mice were habituated to the empty cages in the left and right chambers (trial 1) and time interacting with each cage was recorded. In trial 2, an unfamiliar C57BL/6 J WT sexmatched mouse was introduced to one of the cages, and time interacting with each cage was again recorded. In trial 3, a novel mouse was added to the empty cage, and interaction time was compared for each mouse. Each trial lasted 10 min. The mice serving as strangers were habituated to placement under the wire cage for 5 min prior to the test. Mice were tracked using CleverSys Tracking and Topscan software. The interaction zone was defined by the software as an unmarked perimeter zone of 2 cm around the metal cages. Interaction time was defined as nose within the interaction zone. The chambers were cleaned between trials with Equinade disinfectant (lavender scent).
Ultrasonic vocalisation (USV) recording of male mice Based on methods described by Maggio et al. [41]: A cohort of adult male mice was presented with female urine to induce ultrasonic vocalisation in a test setting. Twelve hours prior to testing of males, vaginal smears were taken from an experimentally naïve cohort of female WT littermates of a similar age. After the smear was air-dried, nuclei were visualised by a Diff-Quick stain, and the three stages of the estrus cycle were identified by light microscope. Only urine from females predicted to be in a proestrous or estrus on the day of testing were used. In order to reduce variability and ensure that mice make USVs, prior to testing, males were put in a cage with a proestrous or estrus female for 5 min. For testing, 5 min prior to the urine exposure, male mice were habituated to a cotton-tipped applicator. The applicator was then switched for a new applicator dipped in urine from estrus or proestrous female mice. USVs were recorded for 3 min using an Avisoft Recorder. Parameters were analysed using Avisoft-SASLab Pro software. Call types were designated using call classifications previously assigned by Scattoni et al., 2008 [42].

Locomotor cell
Mice were habituated to the locomotor arena (27.5 cm 2 , TruScan) for 10 min and then allowed to explore freely for 30 min. Parameters measured included floor plane movement, vertical plane movement and stereotypic movement. To investigate the effect of methylphenidate (MPH) on locomotor activity, a cohort of 20 WT/20 Brinp1 −/− mice were divided into four groups: (i) WT MPH, (ii) WT saline, (iii) Brinp1 −/− MPH, (iv) Brinp1 −/− saline. Mice were placed in the locomotor cell for a 15 min habitation phase, then injected by acute intraperitoneal (IP) administration. Locomotor activity was recorded at 5 min intervals for 60 min post injection. Doses of 1.25 and 2.5 mg/kg MPH or saline were trialled. Testing was carried out blind of genotype and drug administration.

Elevated plus maze
Mice were placed on an elevated platform (material: Perspex, colour: beige) at a height of 40 cm above the floor. The platform comprised of two open arms and two closed arms (each 4.5 cm wide, 30 cm in length), connected by a central square (6 cm × 6 cm). The two closed arms were protected by a 15 cm high wall. Mice were placed on the centre square and video recorded whilst exploring the maze for 5 min. Time and frequency of entry into each arm was recorded; tracking software: Noldus Ethovision 3.0.

Y-maze
Mice were tested in two trials of a plexiglass Y-maze (material: Perspex, colour: grey) with each of the three arms having a distinctive visual cue at the end. Dimensions of each arm were 30 cm × 10 cm, with a triangle centre zone of 10 cm equal sides. Mice received a random association between visual cues and arm location. In trial 1, a partition blocked off the left arm of the maze. The mouse was placed at the end of the home arm, facing away from the centre. The time spent in each of the two available arms was recorded over 10 min. Mice were rested for 2 h. In trial 2, testing was repeated in a second trial with the partition removed and all three arms made accessible. Each of the two trials lasted 10 min; tracking software: Noldus Ethovision XT 5.0.

Acoustic startle and pre-pulse inhibition (PPI)
Mice were placed individually inside a Perspex cylinder, closed at both ends. The cylinder was placed upon a platform sensitive to weight displacement, within a sound attenuating box with a background sound level (San Diego Instruments Startle Response System). The background white noise level was set to 70 dB. To measure acoustic startle, a strong 40 ms startle sound was played and startle response was measured by the jumping reflex (<1 s) as weight displacement on the platform. Pre-pulse inhibition was measured as the percentage reduction in startle response when a non-startling 20 ms pre-pulse of (a) 4 dB, (b) 8 dB, or (c) 16 dB above the 70 dB background sound was played 100 ms prior to the startle sound.

Self-directed digging and grooming behaviours
Mice were placed in a plexiglass test cage (40 × 40 × 35 cm) with clean sawdust covering the base. Selfdirected behaviour was video recorded for 20 min from first introduction into the novel cage. Videos were scored manually for duration and frequency of grooming and digging behaviours.

Morris water maze
The test was performed as described by Vorhees and Williams, 2006. Briefly, a 1.9 m diameter pool was filled to a depth of 30 cm with 25°C of water. A 15 cm diameter platform was submerged 1 cm below water level and approximately 500 ml of non-toxic white paint added to hide the platform from the animal. The test mouse was placed in a random quadrant (North, South, East or West) and allowed 2 min to find the platform. Once the platform was found, the mouse was allowed to remain there for 30 s before being removed. If the platform was not found within 2 min, the mice were guided to the platform. Mice received four training trials per day, each with a different start point, for six consecutive days; tracking software: Noldus Ethovision 3.0.

Statistical analysis of behavioural tests
Behavioural data was analysed by the unpaired Student's t test or analysis of variance (ANOVA) and represented as the mean ± standard error. Statistical analysis of reproductive phenotyping was performed by chi-square test.

Histology
Twenty-five organs per mouse were compared for 7week-old WT and Brinp1 −/− tissue as formalin-fixed, paraffin wax-embedded sections (10 μm), H&E staining, by the Australian Phenomics Network (http://www.australianphenomics.org.au/). Histopathological assessments of two male and two female mice, and clinical haematological analysis of one male and one female mouse, were performed. The following organs were examined for macromorphological abnormalities: testes, epididymis, seminal vesicles, prostate glands, penis, preputial gland, mammary tissue, ovaries, oviducts, uterus, cervix, vagina, clitoral gland, bladder, liver, gall bladder, stomach, duodenum, jejunum, ileum, cecum, colon, mesenteric lymph node, spleen, pancreas, kidney, adrenal glands, salivary glands and regional lymph nodes, thyroids, trachea, lungs, thymus, heart, skin, tail, eyes, harderian glands, brain, spinal cord and hind leg. For haematological analysis, blood samples from 7-week-old mice, collected into EDTA tubes, were run on the Advia 2120 haematology system: giving a red blood cell count (with indices), platelet count, and a white blood cell differential by size, granularity and peroxidase absorption. Brain sections were from the forebrain, midbrain and cerebellum. E18.5 embryos were prepared for H&E staining by the same method.
For immunostaining, WT and Brinp1 −/− mice were anaesthetised, then a transcardial perfusion was performed using phosphate-buffered saline (PBS) followed by 4 % paraformaldehyde (in PBS). Dissected brains were incubated overnight in 4 % paraformaldehyde (PFA) at 4°C, then cryoprotected with 20 % sucrose in PBS for 72 h. Brains were frozen in OCT blocks, using an isopentane bath cooled with liquid nitrogen. Coronal sections were cut to 14 μm on a cryostat and mounted onto Superfrost Plus slides (VWR). A minimum of 4 mice per genotype were analysed, and 3-4 sections per mouse were considered for quantitative analysis. The following antibodies were used: mouse anti-NeuN
Images for all immunostaining were obtained using a Nikon C1 confocal microscope. Cell count analysis were performed blind of genotype using Imaris 7.6.3. A minimum of three representative sections per mouse were analysed by dividing cortical regions into equal bins. Repeat measures two-way ANOVAs were performed for comparisons between WT and knock-out tissue. Statistical tests were performed with GraphPad Prism 5 (GraphPad).

Generation of Brinp1 knock-out mice
A conditional Brinp1-targeted allele (Brinp1 tm1/Pib ) was designed to allow Cre-recombinase-mediated, tissuespecific deletion of Brinp1 (Fig. 2a). In the first Fig. 2 Brinp1 targeting. a The Brinp1 targeting vector was designed with a neomycin resistance cassette after exon 3, and FRT sites positioned before and after the neo r cassette. The 190 bp third exon of Brinp1 contains the start of the MACPF domain. LoxP sites flank exon 3 and the neo r cassette. When crossed with a mouse line expressing Cre-recombinase, the recombination of LoxP sites resulted in the deletion of exon 3 and the neo r cassette. b Genomic DNA isolated from the spleen was cleaved with Pst I and Bgl III and hybridised to 500 bp genomic DNA probes from the 5′ region (Pst I) and 3′ region (Bgl III) of the targeting construct. In wild-type DNA, species of 7.1 kb (Pst I) and 18.4 kb (Bgl III) were detected. These products were not present in DNA from Brinp1 −/− mutants, replaced with shorter species of 5.4 kb (Pst I) and 11.4 kb (Bgl III). c cDNA from the brain tissue of WT or Brinp1 −/− mice was tested for exon 3 deletion by RT-PCR. Primers designed to regions of exon 2 and exon 6 resulted in a PCR product size corresponding to the removal of the 190 bp exon 3 for Brinp1 −/− cDNA. d Sequencing of the Brinp1 −/− allele RT-PCR product showed the expected absence of exon 3, and that splicing fuses exons 2 and 4, resulting in a frame shift that introduces a stop codon after 24 residues. e By immunoblotting, full-length 85 kDa BRINP1 was present in lysates of mouse brains at postnatal day 12 and absent in Brinp1 −/− mice instance, mice lacking Brinp1 in all tissues were generated by breeding animals carrying the targeted allele with animals expressing Cre-recombinase from the two-cell embryonic stage onwards (global Cre deleter). Progeny exhibiting deletion of the selection cassette and third exon of Brinp1 (Brinp1 tm1.1/Pib ) were inter-bred to generate homozygous Brinp1 tm1.1Pib/tm1.1Pib (Brinp1 −/− ) animals and WT littermates. Correct targeting and deletion of exon 3 was confirmed by Southern analysis (Fig. 2b), RT-PCR (Fig. 2c) and DNA sequencing of RT-PCR products (Fig. 2d). The resultant messenger RNA (mRNA) lacking exon 3 (Brinp1Δe3) reflects forced splicing between the intron 2 donor and intron 4 acceptor, fusing exons 2 and 4, and changing the reading frame to introduce a truncating stop codon (Fig. 2d). The predicted mutant protein would comprise the cleavable signal peptide (19 aa) and 54 aa of the 741 aa mature BRINP1 protein and contain no recognisable functional domains. Hence, this 54 aa form is missing over 93 % of the BRINP1 amino acid sequence and would be highly unlikely to fold correctly. It is therefore likely degraded shortly after synthesis, which is the generally accepted fate of truncated or misfolded proteins [43]. Importantly, the absence of BRINP1 protein in the Brinp1 −/− mouse brain was demonstrated by immunoblotting (Fig. 2e).
Brinp1 knock-out mice exhibit decreased postnatal survival and a reduced body weight Brinp1 −/− mice born from heterozygous breeders were not observed at the expected Mendelian frequency. Only 10 out of 75 (13 %) of surviving pups were Brinp1 −/− , indicating that half of the Brinp1 −/− mice died in utero or did not survive to weaning. Analysis of 18 full-term embryos showed that Brinp1 −/− foetuses were present at expected frequencies (6 out of 18, 33 %). We therefore conclude that Brinp1 −/− mice have impaired postnatal viability.
To investigate the reduced survival of Brinp1 −/− mice, the fecundity of WT × WT, het × het, Brinp1 −/− × Brinp1 −/− and WT × Brinp1 −/− breeding pairs was compared (Table 1). Litter numbers at birth were normal for all breeder genotypes, with no delay in first breeding or time between litters. However, post-partum survival of progeny of Brinp1 −/− × Brinp1 −/− breeders was significantly reduced (Fig. 3a) with an average of only one mouse surviving per litter after 3 days. Litter survival from het × het breeders was also reduced (average of four mice surviving per litter). Specifically, only 19/98 (22 %) of all pups born to Brinp1 −/− × Brinp1 −/− parents, and 75 % born to het × het parents, survived longer than 3 days. This was in contrast to a 91 % survival rate of progeny from WT × WT breeders.
The offspring of Brinp1 −/− × Brinp1 −/− parents were observed post-partum. Although pups survived birth and were of normal appearance, most were found dead 12-48 h later. Little to no milk was present in the stomach of the dead animals, indicating lack of adequate nutrition as a likely cause of death. Taken together, these results suggest that mothers lacking Brinp1 are deficient in postnatal care of their offspring, resulting in neonatal death. The higher pup survival rate when a Brinp1 −/− female was mated with a WT male indicates that paternal care also contributes to postnatal survival.
Weekly weighing of pups from heterozygous parents revealed that Brinp1 −/− mice have impaired postnatal growth (Fig. 3b). These mice exhibited a significant delay in weight gain from age of first weighing (week 3) until adulthood in both male and females. Heterozygous females showed delayed weight gain close to that of Brinp1 −/− mice, whereas heterozygous male mice were smaller as juveniles, but their weight recovered close to WT. Despite a 10 % reduction in adult body weight, Brinp1 −/− mice had normal body length and normal brain size (data not shown).
A full pathological examination of mice at embryonic day 18.5 (E18.5) and 7-week-old (P49) Brinp1 −/− mice  Brinp1 knock-out mice behaviour To evaluate the effect of Brinp1 loss on neurological function, the behaviour of Brinp1 −/− mice was assessed. In an initial screen, Brinp1 −/− mice showed normal auditory, visual and olfactory capabilities, and normal motor co-ordination on the Rotarod (Additional file 1: Figure S1a-c). No depressive-like behaviours were indicated via the tail-suspension test.
Brinp1 knock-out mice exhibit reduced sociability and altered ultrasonic vocalisation (USV) A pronounced decrease in sociability was observed by the Brinp1 −/− mice tested for social interaction with an unfamiliar mouse. In the first trial of the threechamber social interaction test, Brinp1 −/− and WT mice showed no preference between the left and right empty cages. Male Brinp1 −/− mice spent slightly less time investigating the empty cages, which may indicate altered exploratory behaviour (Fig. 4a). In the second trial, when presented with a stranger mouse in one of the cages, Brinp1 −/− mice spent significantly less time interacting with the intruder, whilst showing no difference in interaction time with the empty cage (Fig. 4b). Brinp1 −/− mice also exhibited hyperactivity during both trials (Fig. 4c). Results for this test were reproduced with a second cohort (n = 12 mice per genotype, p < 0.05) which also showed reduced time interacting with a stranger mouse. Communication by Brinp1 −/− mice was investigated by analysing the ultrasonic vocalisation (USV) calls of male mice presented with pheromones from estrus or proestrous female mice [44]. Compared to WT, Brinp1 −/− mice showed a trend towards reduced number of calls, a longer latency to call and longer latency to investigate the cotton bud, but these parameters did not reach the 95 % confidence level (Fig. 4d, e). No change in call amplitude was evident, although a trend of increased peak frequency of calls was observed (p = 0.089) (Fig. 4f ). Notably, Brinp1 −/− mice showed significantly shorter call durations (p < 0.05) (Fig. 4g). This reduced call length reflected changes to the distribution of call types. Brinp1 −/− mice emitted a higher percentage of 'short' calls and lower percentage of 'complex' or 'composite' longer calls (Fig. 4h, i). This reduction in complex and A Tukey HSD multiple comparison test found female Brinp1 −/− mice to weight significantly less than WT littermates of the same sex (p < 0.001). Brinp1 het female mice were also found to weigh significantly less than WT (p < 0.001). No significant effect of genotype was found between female Brinp1 het and Brinp1 −/− mice (p = 0.362). ii) Male Brinp1 −/− mice also show a significant reduction in body weight by repeat measures two-way ANOVA: F(2,23) = 8.312, p = 0.002. A Tukey HSD multiple comparison test found a significant difference between male WT and Brinp1 −/− mice (p = 0.002) and male Brinp1 het and Brinp1 −/− mice (p = 0.013). No significant effect of genotype found between male WT and Brinp1 het mice (p = 0.704). Results represented as the mean ± SD *p < 0.05, **p < 0.0.1, ***p < 0.001, ****p < 0.0001 composite calls indicates that Brinp1 influences vocalisation.

Brinp1 knock-out mice are hyperactive and exhibit changes in exploratory behaviour
Hyperactivity of Brinp1 −/− mice was revealed by testing animals in the locomotor cell where, over a period of 30 min, the Brinp1 −/− mice travelled 50 % further, spent less time resting and showed a consistent increase in velocity compared to WT (Fig. 5a,  b). Brinp1 −/− mice also spent proportionally more time in the centre of the locomotor cell (Fig. 5c). Notably, Brinp1 −/− mice showed an increase in stereotypic episodes (repeat motion within a small area) and increased rearing (vertical plane activity) (Fig. 5d, e).
To examine whether the absence of Brinp1 affects anxiety-like behaviour, mice were allowed to freely explore an elevated plus maze for 5 min. Testing of the Brinp1 −/− mice on the maze showed male Brinp1 −/− mice spent significantly less time in a closed arm. (Fig. f ). The number of entries into each arm of the EPM was unaffected (Additional file 1: Figure S1d).
Brinp1 −/− mice showed a normal preference for investigating an unfamiliar object in a novel object recognition test (Additional file 1: Figure S1f ). To assess mice in an environment similar to their home cage, mice were placed in a sawdust-lined, plexiglass box. Mice in this environment showed increased rearing behaviour at a similar frequency to the locomotor cell. In addition, there was a decrease in the amount of time the Brinp1 −/− mice spent digging compared to wild type (Fig. 5g). Male mice showed no significant differences in grooming behaviour, whilst female mice showed significantly decreased grooming (Fig. 5g).

Methylphenidate does not decrease hyperactivity of Brinp1 knock-out mice
Methylphenidate (MPH) is a psychostimulant drug commonly used to treat hyperactivity and inattention in children diagnosed with ADHD [45]. It has previously been shown to reduce hyperactivity in mouse models of ADHD [46,47]. To investigate whether MPH reduces hyperactivity of Brinp1 −/− mice, animals were tested using a published regime that previously measured activity in the locomotor cell of spontaneous SNAP25 mutant Colombomo mice [48]. Activity was determined as the distance travelled, measured at 5 min intervals. In the habituation phase prior to injection, Brinp1 −/− mice travelled significantly further than WT mice (Fig. 5h). The effect of MPH (2.5 mg/kg) as a stimulant was indicated by the expected increase in activity of the WT animals , peaking at 20 min post injection. This pattern of activity was comparable to that of the saline-treated Brinp1 −/− group. The Brinp1 −/− MPH group also showed an increase in locomotor activity above the level of both the Brinp1 −/− saline and WT MPH groups. Results at 1.25 mg/kg MPH showed a milder effect of increased activity for both the WT and Brinp1 −/− groups (Additional file 1: Figure S1f ). Methylphenidate did not significantly alter rearing  [42]. Unidentified calls (11.7 % WT, 11.3 % Brinp1 −/− ) were excluded. i Representative spectrogram of first calls from a WT and a male Brinp1 −/− mouse. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001. N = 6 females, 6 males per genotype for sociability experiment (A-C). N = 10 WT, 9 Brinp1-/-male mice for USV experiment (D-I). All data represented as the mean ± SD activity at doses of 2.5 mg/kg (Fig. 5i) or 1.25 mg/ kg. Overall these results demonstrate that at two doses, MPH stimulates activity in both controls and Brinp1 −/− animals, and does not ameliorate hyperactivity of the knock-out animals. This is a similar response to the drug tested with SNAP25 mutant mice [48].

Brinp1 knock-out mice exhibit normal sensory gating
The pre-pulse inhibition (PPI) test is used to measure sensory gating in mice, which models deficits in human subjects diagnosed with schizophrenia. No abnormalities in startle response or sensory gating for Brinp1 −/− mice were detected by startle/prepulse inhibition testing, indicating that these mice do not model this aspect of human schizophrenia (Fig. 6a).
Brinp1 knock-out mice have impaired short-term memory Brinp1 −/− mice were tested for spatial learning and memory in the Y-maze test (Fig. 6b). With a 2 h interval between trials, Brinp1 −/− mice did not show the typical increase in time spent exploring a novel arm, indicating impaired short-term memory. To determine whether long-term memory impairment was also evident, Brinp1 −/− mice were tested using the Morris water maze. Brinp1 −/− mice showed no impairment in this test (Fig. 6c). Possibly due to their hyperactivity, male knock-out mice showed faster time, as well as a shorter swim distances (p = 0.048), required to find the platform compared to WT on day 1 of the experiment.

Brinp1 knock-out mice show an increase in PV-positive interneurons in the somatosensory cortex
Brains from adult WT and Brinp1 −/− mice were analysed for changes in cortical anatomy and hippocampal structure, at Bregma co-ordinate −1.94, encompassing the medial hippocampus and the somatosensory cortex. Analysis with an antibody to PV, a marker of a subpopulation of interneurons, showed an increase in cellularity in layers IV and VI of the neocortex (Fig. 7a-c) in Brinp1 −/− mice, as well as an increase in total PV-positive cells in the hippocampus (Fig. 7d). Increased PV interneuron numbers were also suggested in the CA1 region of the hippocampus (p = 0.087). No significant changes in number or distribution of other interneuron subpopulations, marked by calretinin or somatostatin, were detected (Additional file 2: Figure S2a, b). No significant changes in pyramidal neuron layering were detected with a pan-neuronal marker (NeuN) or layer II-IV marker Cux1 (Additional file 3: Figure S3). The medial hippocampus also showed normal morphology with this antibody. Brains were stained with the global astrocyte marker, GFAP. No changes in cortex or hippocampal astrocyte numbers were observed (Additional file 2: Figure S2c). Brinp1 knock-out embryos show normal cortical cell survival and proliferation Brinp1 −/− embryonic brains were examined at E18.5. No significant differences in cortical Ki67-positive cell numbers were detected, indicating that Brinp1 does not affect cortical cell proliferation in the E18.5 mouse brain (Fig. 8a). No significant changes in the mitotic cell numbers were apparent in the cortex using a phosphohistone-H3 (Phh3) antibody, a marker of mitotic activity (Fig. 8b). There were also no significant changes in apoptosic cell numbers in the E18.5 cortex, using a caspase 3 antibody (Fig. 8c). Layer formation during embryonic development was examined at E12.5 (layer V and VI formations), E14.5 (layer IV formation) and E16.5 (layers II/III formation). Pregnant females, from heterozygous matings, were injected with BrdU at one of each of these time points. Due to the poor postnatal survival rates of Brinp1 −/− mice, embryos were harvested just prior to birth, at E18.5. There were no significant differences in BrdU+ cell numbers detected in the subventricular zone in any of the three injection time points. There were also no significant differences in E12.5-, E14.5-or E16.5-born BrdU+ cell numbers in the dentate gyrus of the E18.5 hippocampus (Fig. 8d). Together, these results indicate that Brinp1 has no detectable Increased expression of Astrotactin 1 and Astrotactin 2 in the developing brain of Brinp1 knock-out mice Quantitative PCR was performed on mRNA from the brains of Brinp1 −/− mice, to assess whether homologous MACPF family members are upregulated. The late embryonic brain (E18.5) showed a 2-fold increase in both Astn1 and Astn2 mRNA (Fig. 9a). Levels of the Brinp1 exon 3-deleted, frameshifted mRNA (Brinp1Δe3) also increased by 1.5-fold compared to levels of Brinp1 mRNA in the WT mice. No significant changes to the levels of Brinp2 or Brinp3 mRNA were evident at this developmental time point. At 6 weeks of age, Brinp1 −/− mice showed a 3-fold increase in Astn1 mRNA and a 2-fold increase in Astn2 mRNA in the hippocampus (Fig. 9b). Down-regulation of both Brinp1Δe3 and Brinp2 mRNA was detected in the adult cortex, with no change in expression of either Astrotactin mRNA (Fig. 9c). Normalisation with GAPDH and βactin gave similar results (β-actin data shown in Additional file 4: Figure S4). Our study of Brinp1 −/− mice has revealed behavioural phenotypes that resemble social and communication impairments found within human ASD. The threechamber social interaction test is an accepted behavioural test to investigate social deficits relevant to autism. Using this test, Brinp1 −/− mice spent significantly less time interacting with a stranger sex-matched mouse: a sign of social withdrawal. Furthermore, testing of ultrasonic vocalisations of adult male mice is a key method for behavioural phenotyping of NDDs [44,49]. Male Brinp1 −/− mice exhibited an altered vocalisation phenotype when presented with pheromones from estrus females. The shorter call duration of Brinp1 −/− mice, with fewer complex calls, indicates altered communication ability.
Brinp1 −/− mice showed poor postnatal survival. The marked reduction in sociability and communication observed in Brinp1 −/− adults may manifest in reduced attentiveness towards litters, thereby hindering neonate survival if mothers fail to provide adequate thermal insulation, nourishment and post-parturition care towards their pups. A series of cross-fostering experiments would be required to demonstrate this. Reduced survival may also be a result of flawed two-way communication between dam and pup. Future investigation of the social communication deficits of Brinp1 −/− mice should determine whether Brinp1 −/− pups emit altered vocalisations. It would also be interesting to investigate ultrasonic vocalisation between two interacting Brinp1 −/− mice.
Repetitive movements such as increased grooming are reported in some but not all mouse models of ASD [50,51]. Brinp1 −/− mice did not exhibit increased grooming. However, increased rearing and increased stereotypic episodes were observed in the locomotor cell. Whilst interesting observations, these behaviours do not necessarily model the repetitive movements of autism as such movements could also the result of hyperactivity.
Taken together, the above observations suggest that Brinp1 −/− mice model social communication aspects of human ASD, since poor sociability, delayed language and impaired communication skills are fundamental facets of the pathology.
Brinp1 knock-out mice are hyperactive, with short-term memory impairment Brinp1 −/− mice are hyperactive, evidenced by a 50 % increase in horizontal plane activity in the locomotor cell. There are also some indications of changes in exploratory behaviour of Brinp1 −/− mice; showing increased rearing, and altered time spent in exposed regions of the locomotor cell, as well as less time in the protected arms of the elevated plus maze. The hyperactivity of Brinp1 −/− mice was present in multiple testing arenas, and it therefore should be noted that this may confound other aspects of behaviour, such as the mouse's response to social approach tasks or attention during memory-based tasks.
Brinp1 −/− mice have a 10 % reduction in body mass. This may be because of increased energy expenditure due to hyperactivity, and is consistent with variants in the Brinp1 locus at 9q33.1 in patients with growth delay [52].
Methylphenidate (Ritalin) is a psychostimulant commonly used in the treatment of ADHD symptoms [45,53], although a considerable proportion of patients do not respond [54]. We examined the predictive validity of Brinp1 −/− mice as a model for ADHD by investigating their response to methylphenidate. Methylphenidate did not reduce the hyperactivity of Brinp1 −/− mice, indicating that Brinp1 deletion is unlikely to directly affect the dopaminergic pathways of the prefrontal cortex, as hypothesised for ADHD aetiology [55]. A future direction would be to examine the effect of other drugs, such as d-amphetamine (a psychostimulant that promotes catecholamine release at pre-synaptic terminals) [48], atomoxetine (a norepinephrine reuptake inhibitor) [56] or clonidine (and α2adrenergic receptor agonist) [57], on hyperactivity as well as sociability of Brinp1 −/− mice.
Brinp1 −/− mice did not show preference in exploring the novel arm of the Y-maze, when testing with a 2 h interval between trials, indicating impaired short-term memory. The Brinp1 −/− mice performed normally in the Morris water maze, demonstrating that long-term memory is unaffected. The short-term memory impairment of Brinp1 −/− mice may model cognitive impairment evident in a subset of autistic patients, or possibly psychiatric disorders that show association with the BRINP1 locus [30,33].

Brinp1 knock-out mice exhibit changes in PV interneurons in the neocortex
More parvalbumin-positive interneurons were detected in the adult Brinp1 −/− mouse neocortex and hippocampus. PV interneurons are a subset of GABA-ergic neurons that play an inhibitory role in the cortex. Importantly, changes in PV interneuron populations have been reported in other ASD mouse models, as well as ASD patients [14,15]. The cell-specific increase of this subtype of interneurons is of particular interest as this implies an unknown effect of Brinp1 deletion on fast-spiking basket and chandelier cells. We postulate that the absence of Brinp1 causes dysregulated proliferation or migration of these specific PV interneuron populations, resulting in a greater density of such cells in defined regions of the neocortex. Further investigation is needed to determine the underlying molecular mechanism, whether the increase in PV interneurons in the somatosensory cortex is matched with increases in other cortical regions, and to determine the age at which this phenotype first appears. As cortical interneurons have a critical role in regulating network excitability generated by pyramidal interneurons [58], the gain of PV interneurons may therefore shift the balance between cortical excitation and inhibition, resulting in the reported behavioural changes. An imbalance between excitation and inhibition has been observed in the brains of humans with ASD [59][60][61].
Interestingly, mRNA of the MACPF family members Astrotactins 1 and 2 increased in the embryonic brain (E18.5) and 6-week-old hippocampus of Brinp1 −/− mice. Both Astrotactins 1 and 2 are reported to facilitate neuronal migration [28,29]. This suggests that Astrotactins and Brinp1 may have similar functions, and perhaps work in a common pathway, such that Astrotactins 1 and 2 functionally compensate for the absence of Brinp1 during neuronal migration. The increase of Brinp1Δe3 mRNA in Brinp −/− animals is possibly due to compensatory pressure in the absence of functional protein.
During the final stages of our study, Kobayashi et al. reported a different line of Brinp1 −/− mice, possessing a deletion in exon 8 (Brinp1Δe8) [22]. Our observations are consistent with some but not all of their findings. Both Brinp1 knock-out lines exhibit increased activity, reduced sociability, reduced anxiety and impaired working memory. Importantly, however, Kobayashi et al. did not investigate communication (or report potentially associated breeding difficulties), whereas our findings reveal a profound communication deficit suggesting an ASD-like endophenotype which is consistent with reports of autism and language delay in patients with de novo human mutations at the 9q33.1 locus. Although they suggest a schizophrenia-like phenotype in the Brinp1Δe8 mice, Kobayashi et al. did not test pre-pulse inhibition (PPI). By contrast, we have demonstrated that PPI, used to measure sensory gating, is normal for Brinp1 −/− mice.
Overall, similar behavioural phenotypes were evident in both female and male Brinp1 −/− animals. However, we note some subtle gender-specific effects in Brinp1 −/− mice. For example, reduced anxiety is evident on the EPM for males only, whereas only female Brinp1 −/− mice exhibited reduced grooming behaviour. There is greater weight variability in male mice during postnatal development, whilst heterozygous mice are affected in adult females only.
Kobayashi et al. report that adult Brinp1Δe8 knock-out animals exhibit increased hippocampal neurogenesis and more PV interneurons in the hippocampus, concluding that the altered behaviours reflect a hippocampal-specific defect. No cortical or other perturbations were reported in the Brinp1Δe8 knock-out mice. By contrast, our Brinp1 knockout animals display no evidence of increased cell proliferation in the embryonic cortex and no evidence for increased embryonic cell proliferation. Like Kobayashi et al., we observed an increase in PV interneurons in the adult hippocampus, but this is not hippocampus-specific, as we also detected additional PV interneurons in the neocortex. Given these observations, we therefore disagree with the Kobayashi et al. conclusion that Brinp1 is a negative regulator of general cell proliferation and that the behavioural alterations in mice lacking BRINP1 arise solely due to disruption of the hippocampus. Our findings demonstrate that changes in density of PV interneurons in the Brinp1 −/− neocortex and hippocampus from an unknown mechanism likely underpin the reported behavioural phenotypes.
An explanation for the differences between the two studies may lie in the differences between the two Brinp1 mutant alleles. The Kobayashi study involved a conventionally targeted Brinp1 −/− line carrying a deletion of exon 8, which removes the terminal 418 residues of the 760 residue protein but retained the promoter and the potential to express a smaller protein containing the MACPF domain (No protein analysis was carried out on the Brinp1Δe8 mice). By contrast, our knock-out allele removes exon 3 and truncates the protein after the first 74 amino acids (preceding the MACPF domain), and immunoblotting established that BRINP1 production was abolished. Our targeting strategy also included removal of the neomycin selection cassette, which is known to have effects on survival and physiology if retained in the targeted locus [62,63]. The Brinp1Δe8 allele does not have the selection cassette removed, which may influence the phenotype.

Conclusions
Brinp1 −/− mice exhibit reduced sociability, vocalisation impairments, hyperactivity and alterations in short-term memory. These behavioural phenotypes appear to show face validity for the core social communication deficits of ASD, along with the hyperactivity aspect of ADHD. The reported increase in PV-positive interneurons in the Brinp1 −/− mouse neocortex and hippocampus may underpin such behavioural changes. These findings demonstrate an important role in normal brain development and suggest Brinp1 as a candidate gene for neurodevelopmental disorders, with closest relevance to ASD.