Organization of the human superior olivary complex in 15q duplication syndromes and autism spectrum disorders

Neuroscience 286 (2015) 216–230

ORGANIZATION OF THE HUMAN SUPERIOR OLIVARY COMPLEX IN 15Q DUPLICATION SYNDROMES AND AUTISM SPECTRUM DISORDERS R. LUKOSE, a K. BEEBE b AND R. J. KULESZA Jr. b*

American Psychiatric Association, 2013). The vast majority of individuals with ASD have some degree of auditory dysfunction (Greenspan and Wieder, 1997; Tomchek and Dunn; 2007; Gomes et al., 2008; Bolton et al., 2012). The degree to which hearing is affected in ASD varies by subject but ranges from deafness to hyperacusis and includes difficulty listening in noisy environments (Rosenhall et al., 1999; Roper et al., 2003; Alca´ntara et al., 2004; Khalfa et al., 2004; Szelag et al., 2004; Kellerman et al., 2005; Lepisto¨ et al., 2005; TederSa¨leja¨rvi et al., 2005; Gravel et al., 2006; Tharpe et al., 2006; Russo et al., 2009). Testing of auditory brainstem responses and the stapedial reflex in subjects with ASD provide evidence implicating dysfunction of lower auditory brainstem neurons (Skoff et al., 1980; Gillberg et al., 1983; Rumsey et al., 1984; McClelland et al., 1992; Klin, 1993; Maziade et al., 2000; Rosenhall et al., 2003; Kwon et al., 2007; Roth et al., 2012; Lukose et al., 2013). Accordingly, there is evidence of consistent and severe hypoplasia in the superior olivary complex (SOC), an essential component of the auditory pathway, in subjects with ASD (Rodier et al., 1996; Kulesza and Mangunay, 2008; Kulesza et al., 2011). Presently, the majority of ASD cases are idiopathic, although up to 20% are attributed to genetic disorders (e.g. Fragile X syndrome), genetic mutations, or chromosomal copy number variations, such as chromosome 15q duplication (Gillberg and Coleman, 1996; Sebat et al., 2007; Boddaert et al., 2009; Pinto et al., 2010). The incidence of chromosome 15q duplication syndrome [dup(15q)] is quite low; isodicentric chromosome 15 duplications affect only about 1 in 30,000 (Schinzel and Niedrist, 2001; Battaglia, 2008). This syndrome refers specifically to duplications of the 15q11–13 region where maternally derived duplications are associated with developmental problems (Martinsson et al., 1996; Browne et al., 1997; Cook et al., 1997; Schroer et al., 1998; Repetto et al., 1998; Mao et al., 2000; Bolton et al., 2004; Roberts et al., 2002; Battaglia et al., 2010). Approximately 1–3% of ASD cases are associated with chromosome 15 abnormalities and duplications in the 15q region are of the most common chromosomal duplications associated with ASD (Cook et al. 1998; Schroer et al., 1998; Bolton et al., 2004; Battaglia, 2005; Vorstman et al., 2006; Abrahams and Geschwind, 2008; Depienne et al., 2009; Moreno-De-Luca et al., 2013). In fact, ASD is diagnosed in nearly 70% of subjects with maternal 15q11.2–q13 duplications (Rineer et al., 1998; Kent et al., 1999; Borgatti et al., 2001). Patients with

a

University of Pittsburgh Medical Center – Hamot, Department of Neurology, United States b Lake Erie College of Osteopathic Medicine, Department of Anatomy, United States

Abstract—Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by a number of behavioral and social features. Although the etiology of most cases of ASD is idiopathic, a significant number of cases can be attributed to genetic causes, such as chromosome 15q duplications [dup(15q)]. Recent neuropathological investigations have provided evidence for distinct patterns of heterotopias and dysplasias in ASD and subjects with both ASD and dup(15q). Individuals with ASD characteristically have hearing difficulties and we have previously demonstrated significant and consistent hypoplasia in a number of auditory brainstem nuclei in subjects with ASD. Herein, we compare results from a morphometric investigation of auditory brainstem nuclei in subjects with ASD, dup(15q) and controls. Our observations in subjects with ASD support our previous reports. However, in subjects with dup(15q), we find significantly fewer neurons and in many nuclei, neurons were significantly smaller than in ASD subjects. Finally, we find a notably higher incidence of ectopic neurons in dup(15q). These results suggest that in the brainstem, these neuropathological conditions may evolve from some of the same developmental errors but are distinguished on microscopic features. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: brainstem, ectopic, auditory.

INTRODUCTION Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by difficulties with communication and social interactions, restricted, repetitive behaviors and sensory abnormalities (Allen, 1988; Wing, 1997; *Corresponding author. Address: Auditory Research Center, Lake Erie College of Osteopathic Medicine, 1858 West Grandview Boulevard, Erie, PA 16504, United States. Tel: +1-814-866-8423. E-mail address: [email protected] (R. J. Kulesza Jr.). Abbreviations: ASD, autism spectrum disorder; CN, cochlear nucleus; dup(15q), chromosome 15q duplication syndrome; FN, facial nucleus; LNTB, lateral nucleus of the trapezoid body; LSO, lateral superior olive; MNTB, medial nucleus of the trapezoid body; MSO, medial superior olive; SOC, superior olivary complex; SPON, superior paraolivary nucleus; tz, trapezoid body; VNTB, ventral nucleus of the trapezoid body. http://dx.doi.org/10.1016/j.neuroscience.2014.11.033 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 216

R. Lukose et al. / Neuroscience 286 (2015) 216–230

dup(15q) characteristically display hypotonia, motor and cognitive delays, autistic behaviors and seizures (Battaglia, 2008; Park et al., 2013; Urraca et al., 2013). Furthermore, social, communicative and behavioral features are similarly affected in subjects with ASD and combined dup(15q) and ASD (Wisniewski et al., 1979; Gillberg et al., 1991; Rineer et al., 1998; Wolpert et al., 2000). Finally, deafness and hearing loss have been reported in subjects with dup(15q) syndrome (Bingham et al. 1996; Simic and Turk, 2004). Throughout the brain of subjects with ASD, there is evidence for dysregulated neurogenesis, stunted neuronal maturation, including aberrant neuronal migration (ectopic neurons) and alterations in cell body size, number and dendritic morphology (Bauman and Kemper, 1985; Ritvo et al., 1986; Gaffney et al., 1988; Arin et al., 1991; Piven et al., 1992; Hashimoto et al., 1993; Raymond et al., 1996; Palmen et al., 2004; Schumann and Amaral, 2006; Kulesza and Mangunay, 2008; Whitney et al., 2008; Weigiel et al., 2010; Kulesza et al., 2011; Stoner et al., 2014; Wegiel et al., 2014). Brains from subjects with dup(15q) syndrome reportedly weigh less than brains from both ASD and control subjects and microcephaly is more commonly found in dup(15q) subjects (Wegiel et al., 2012b). Additionally, there are more heterotopias and significantly more dysplasias in the hippocampal region of dup(15q) subjects compared to ASD alone and subjects with both dup(15q) and ASD have a much higher incidence of epilepsy/seizures compared to controls (Battaglia et al., 1997; Schinzel and Niedrist, 2001; Dennis et al., 2006; Wegiel et al., 2012b). Based on these observations, it has been proposed that subjects with dup(15q) syndrome can be distinguished from subjects with ASD based on neuropathological observations (i.e. the number of developmental abnormalities and early onset Ab plaques; Wegiel et al., 2012a,b; Frackowiak et al., 2013). In conjunction with our previous studies of the auditory brainstem in ASD (Kulesza and Mangunay, 2008; Kulesza et al., 2011), we hypothesize that the SOC nuclei are differentially affected in these two neurodevelopmental disorders [ASD and dup(15q)]. Herein, we investigate this hypothesis through a quantitative analysis of neuronal number and cell body morphology in the auditory brainstem of subjects with dup(15q) and compared these observations to ASD and control subjects.

EXPERIMENTAL PROCEDURES Subjects The tissue used in this study was obtained through the Autism Tissue Program (http://www.autismtissueprogram. org). All brain specimens were donated to the program by the subjects’ family and all clinical diagnoses were obtained from the subjects’ medical records and/or family surveys. All identifying information was removed from the records and LECOM’s IRB granted exempt status for all procedures. All brainstems used in this study were preserved in 10% buffered formalin for at least 6 weeks, sectioned into 30-mm blocks, dehydrated in a series of ascending alcohols, embedded in

217

polyethylene glycol (PEG), cut at a thickness of 50 lm, mounted onto glass slides and stained for Nissl substance with Cresyl Violet (for further details see Wegiel et al., 2012a,b). The distribution of amyloid-b was previously described in the forebrain and cerebellum of nine of the dup(15q) subjects (Wegiel et al., 2012a). Nine of the 12 dup(15q) subjects used in this study were characterized by genotyping as previously described (Wegiel et al., 2012a; Frackowiak et al., 2013). Data collection In all subjects studied, the nuclei of the SOC were identified within the tegmentum of the rostral medulla and caudal pons, anterior and medial to the facial nucleus (FN), lateral to axon bundles of the abducens nerve and medial lemniscus and posterior to the pontine nuclei (Kulesza, 2007, 2008; Kulesza et al., 2011). In subjects with ASD or dup(15q) syndrome, neurons of the SOC were arranged in topographically similar patterns as previously described for control subjects (Kulesza, 2007, 2008, 2014; Kulesza et al., 2011). Thus, each nucleus was identified based on location relative to brainstem nuclei, axon bundles (facial nerve, trapezoid body (tz)) and other SOC nuclei. Because the nuclei of the trapezoid body are nearly contiguous and subjects with ASD or dup(15q) syndrome exhibited dysmorphology (Kulesza and Mangunay, 2008; Kulesza et al., 2011), it is possible that a very small percentage (1%) of these neurons were misclassified. Occasionally, large pools of neurons were found within or near the SOC, but outside of observed nuclear boundaries – such neurons were considered ectopic (see later). All tissue sections were examined with an Olympus BX45 microscope and photographed with an Olympus DP12 digital camera. Data on cell body morphology were collected from all specimens used in this study (Table 2). For analysis of cell body morphology, tissue sections were randomly selected throughout the rostrocaudal extent of each nucleus. Cell bodies that were completely within the thickness of the tissue section and had a visible nucleolus were traced with the aid of a camera lucida attachment (Olympus; using a 40 objective with a final on paper magnification of 675). Tracings were digitized into jpeg format using a flatbed scanner and these digitized tracings were imported into ImageJ (calibrated to a standard scale bar; available at http://rsb.info.nih.gov/ij/). Using the ‘‘Analyze’’ feature, measurements were made of cell body area, perimeter, long and short axes, circularity and orientation of the long axis (‘‘angle’’). For each soma, an index of circularity was calculated as follows: Circularity ¼ ½4p  Area=Perimeter2 

The products of this equation range from 0 to 1 and provide an estimate of cell body shape independent of size (Yin et al., 1990). A perfectly circular contour will have a value of ‘‘1’’ while a triangular contour will have a value 0.5. Angle measurements for neurons in the medial superior olive (MSO) were made relative to the anatomical midline (i.e. the raphe). Neurons with a long axis parallel to the midline (i.e. vertical) will have an angle

218

R. Lukose et al. / Neuroscience 286 (2015) 216–230

measurement of 90° and neurons with a long axis perpendicular to the anatomical midline (i.e. horizontal) will have an angle measurement of 0°. See the inset in Fig. 4A which shows reference coordinates (0°, 90° and 180°) used for the angle measurements. For the sake of comparison between specimens (right vs. left halves), all angle data were normalized to the right side of the brain. Cell counts from control subject UMB1706 and ASD subjects B6399, HSB4640, B6349 and CAL105 were published previously (Kulesza et al., 2011) and have been excluded from this analysis. In all the remaining specimens, neuronal packing density was calculated by counting nucleoli along the rostro-caudal extent of each nucleus. These counts were corrected using Konigsmark’s (1970) formula (for recent application see

Thompson and Brenowitz, 2005; Kulesza, 2008; Wagoner and Kulesza, 2009; Kulesza et al., 2011) to correct for profile splitting (rearranged): N ¼ nðt=t þ 2aÞ

In this equation, N is the estimated number of neuronal profiles in a given nucleus, n is the actual number of profiles counted, t is section thickness and a is the square root of r2  (k/2)2. In this expression r is the average radius of the nucleoli and k indicates the diameter of observed nucleoli fragments (we observed no nucleoli fragments, but instead used the average minor axis of observed nucleoli, thereby providing a more conservative estimate). These corrected counts were then divided by the tissue volume from which they were counted, yielding neuronal density. The number of

Table 1. Subjects included in study

sz h a b

Group

Sex

Brain Bank #

Age

Cause of death

Diagnosis

PMI (h)

Control Control Control Control Control Control Control Control Control Control ASD ASDh

M M M F F M M M F F M F

IBR-M10–10 IBR-M4–06 M7–10 IBR-M9–10 UMB1706 M1–10 UMB-4722 M8–10 UMB-4637 B-7827 B-6399 B-7002

3 4 5 6 8 10 14 15 31 32 2 5

CO poisoning Drowning Hanging Hanging Rejection of cardiac allograft CO poisoning Multiple injuries CO poisoning Trauma [automobile accident] Undetermined Drowning Drowning

– – – – – – 16 – 17 – 4 33

ASD ASD

M M

B-5569 HSB4640

5 8

ASD ASDsz,h ASDsz ASD ASDsz,h ASD ASD ASDsz ASD ASD ASD ASD dup(15q)sz,h dup(15q)sz dup(15q)sz dup(15q) dup(15q)sz,h dup(15q) dup(15q)sz dup(15q)sz,h dup(15q)sz dup(15q)sz dup(15q)sz dup(15q)sz

M M M M M F M M F F M M M M M M F F M M F M F F

B6349 B-5807 CAL105 B-7079 B-5891 B-6115 B-7596 B-6994 B-6469 B-7376 B-7459 B-6276 B-5733 B-7359a,b B-7741a,b B-7041a,b B-6973a,b B-7619b B-8130 B-7014a,b B-7982 B-7436a,b B-6856a B-7723a

9 10 11 15 15 17 23 28 49 52 54 56 5 9 10 11 15 15 16 19 22 24 26 39

No known disorder No known disorder No known disorder No known disorder No known disorder No known disorder No known disorder No known disorder No known disorder No known disorder Autism Autism, developmental delay not otherwise specified Autism Autism [pervasive developmental disorder – not otherwise specified (PDD-NOS)] Autism [PDD-NOS] Autism Autism Autism Autism Asperger Autism Autism Autism Autism Autism Autism Ch. 15q Dup. Syn. + Autism Ch. 15q Dup. Syn. Ch. 15q Dup. Syn. + Autism Ch. 15q Dup. Syn. Ch. 15q Dup. Syn. + Autism Ch. 15q Dup. Syn. + Autism Ch. 15q Dup. Syn. + Autism Ch. 15q Dup. Syn. + Autism Ch. 15q Dup. Syn. + Autism Ch. 15q Dup. Syn. + Autism Ch. 15q Dup. Syn. + Autism Ch. 15q Dup. Syn.

Medical history of seizures. Medical history of hearing difficulties. Characterized in Wegiel et al. (2012). Characterized in Wegiel et al. (2014).

Asphyxia from drowning Asthma attack Cardiopulmonary arrest Cerebral edema, seizures Cardiac arrest, drowning Asphyxiation due to hanging Aspiration, pneumonia Cardiac arrest, dil. cardiomyopathy Cardiopulmonary arrest Sudden unexpected death in epilepsy Pulmonary arrest Undetermined Cancer Arteriosclerotic cardiovascular disease Acute respiratory distress Cardiac arrest, seizure Epilepsy Seizure disorder and heart failure Seizure suspected Aspiration pneumonitis Seizure disorder Cardiopulmonary arrest Sudden unexpected death in epilepsy Sudden unexpected death in epilepsy Asphyxia/seizure Epilepsy

– 13.8 3.75 74.5 – 23.23 2.5 25.01 33.25 43.25 16.33 39.15 28.25 3.35 15.58 13.63 17.7 10.5 24 – 39.91 28.08 11.83 36.36 28.67 32.83

219

R. Lukose et al. / Neuroscience 286 (2015) 216–230

neurons in each nucleus was finally estimated by multiplying neuronal density by the total estimated volume of each nucleus (Thompson and Brenowitz, 2005). This method of estimating neuronal number has produced results statistically similar to the optical dissector (Thompson and Brenowitz, 2005; Kulesza, 2007). Ectopic neurons were not included in the estimates of neuronal number as the classification of these neurons (auditory or non-auditory) was not apparent in this material. Data analysis All data sets were compared against a normal distribution using the D’Agostino & Pearson omnibus test. If a normal distribution was achieved (p > .05), comparisons were made using parametric tests (t-tests, ANOVA with Tukey’s multiple comparison test) and results are presented as mean ± standard deviation. The only normally distributed data sets were estimations of neuronal number for the MSO, superior paraolivary nucleus (SPON) and ventral nucleus of the trapezoid body (VNTB). Data sets that failed to meet a normal distribution (p < .05) were compared using nonparametric tests (i.e. Mann–Whitney or Kruskal– Wallis with Dunn’s multiple comparison test) and only the data median is provided. The distribution of neuronal subtypes within each nucleus was compared using the v2 test. Correlations between the number of neurons in the MSO and ADI-R scores were made using Pearson’s correlation. All statistical analyses were made in Prism 6 (GraphPad Software Inc., La Jolla, CA, United States) with a 0.05 significance level.

RESULTS

with dup(15q) + ASD and those with dup(15q) alone (t-test, p < .05). Correlations of neuronal number and ADI-R scores were limited as only two of the dup(15q) subjects had complete ADI-R records. Regardless, our data show no correlation between the number of neurons in the MSO and social (n = 8) or verbal (n = 6) ADI-R scores in ASD subjects (Pearson, r = 0.27, p = .50 and .60, respectively). Of all the subjects with ASD, 25% had a medical history of seizures and 19% had a history of auditory dysfunction (e.g. hearing loss, hyperacusis); of the subjects with dup(15q) syndrome, 83% had a history of seizures and 25% had a history of auditory dysfunction. Finally, there was no difference in the post mortem interval (PMI) between the ASD and dup(15q) subjects (Table 1; 24.5 h compared to 23.5 h respectively; p = .69). Hypoplasia The nuclei of the SOC exhibited obvious hypoplasia in both ASD and dup(15q) subjects compared to controls (Figs. 1 and 2). However, as we have described previously (Kulesza et al., 2011), the degree of hypoplasia varied considerably among subjects. In most ASD and dup(15q) subjects, there was an obvious reduction in the number of SOC neurons and this was indicated by smaller nuclei, diminished rostrocaudal dimension of the complex and reduced neuronal packing density (Fig. 2). In control subjects, the SOC contained approximately 36,000 neurons. In ASD subjects, the SOC included only about 20,000 neurons while in dup(15q) subjects there were only about 17,000 neurons. This decrease represents a 45% loss of neurons in ASD and a 53% loss of neurons in dup(15q).

Subjects

MSO

The results described below are based on study of 10 normally developing control subjects (average age = 12.8 ± 3.4 [mean ± SD]; 6 male/4 female), 16 subjects diagnosed with ASD (average age = 22.4 ± 4.8; 12 male/4 female) and 12 subjects diagnosed with dup(15q) (average age = 17.6 ± 2.7; 7 male/5 female; Table 1). Of the 12 dup(15q) subjects, nine were also clinically diagnosed with ASD (75%). Brain weights between the ASD and dup(15q) groups differed significantly. Subjects with ASD had an average brain weight of 1483 ± 203 g (mean ± SD) and subjects with dup(15q) syndrome had an average brain weight of 1242 ± 221 g (t-test; p = .008). There were no differences in brain weights between males and females in any of the groups (t-test, p > .05) and there was no difference in brain weights between subjects

In both ASD and dup(15q) subjects, the MSO was significantly smaller in both rostro-caudal and anteriorposterior dimensions and there was a significant reduction in the number of MSO neurons (compared to controls; Figs. 2–5). In control subjects, there was an average of 12,687 ± 623 (mean ± SD) neurons. However, ASD and dup(15q) subjects had significantly fewer MSO neurons (5432 ± 610 and 5457 ± 540, respectively; ANOVA, p < .0001, both Tukey’s p < .0001; Figs. 4, 5A), representing a decrease of about 57%. There were no differences in the number of MSO neurons between males and females in the ASD or dup(15q) groups (t-test, p = .75 and .76, respectively). Additionally, there was no difference in the number of MSO neurons between subjects with dup(15q) + ASD and dup(15q) alone (t-test, p = .56). In control subjects,

Table 2. Number of individual brains studied and the total number of neurons analyzed for each condition Brains examined

Control ASD dup(15q)

No. of neurons analyzed

Cell Counts

Morphology

MSO

LSO

SPON

MNTB

VNTB

LNTB

9 12 12

10 16 12

591 1032 904

456 678 519

301 642 466

373 732 400

257 462 278

475 882 644

220

R. Lukose et al. / Neuroscience 286 (2015) 216–230

Fig. 1. Organization of the human SOC. Shown in this figure is a transverse section through the human caudal pons containing the SOC from a control subject. The SOC nuclei are situated between the facial nucleus (FN), the facial nerve (fn), the pontine nuclei (PN), the central tegmental tract (ctt) and the medial lemniscus (ml). The principal nuclei of the SOC are outlined with heavy dashed lines (MSO and LSO); on either side of the main cell column of the MSO is a zone rich in MSO dendritic arbors but few neurons (spaced-dashed line). The nuclei of the trapezoid body (MNTB, VNTB and LNTB) are situated among axons of the tz. Scale bar = 1000 lm.

MSO somata had a median cross-sectional area of 260 lm2. MSO somata were significantly smaller in ASD and dup(15q) subjects (173 lm2 and 171 lm2, respectively; Kruskal–Wallis, p < .0001, both Dunn’s p < .0001; Figs. 3, 5B). There were no differences in the cross-sectional area of MSO neurons between male and female subjects in any of the groups (Mann–Whitney, p > .05). In control subjects, MSO somata had a median circularity measure of .496 while in affected subjects MSO somata were significantly more round (.630 in ASD and .617 in dup(15q) subjects; Kruskal–Wallis, p < .0001, both Dunn’s p < .0001; Fig. 5C). In control subjects, MSO somata had a median long axis oriented at 54.9°, while the angle measurements were 87.6° in ASD subjects and 71.6° in dup(15q) subjects (Kruskal–Wallis, p < .0001, both Dunn’s p < .0001; Figs. 4, 5D). In control subjects, the distribution of MSO angles was heavily biased toward values between 49° and 60° (95% CI of median; skewness 1.073; kurtosis.29). However in ASD and dup(15q) subjects, the angle measurements follow a more uniform distribution (kurtosis = 1.083 and 1.001, respectively). These changes in the orientation of MSO neurons in ASD and dup(15q) indicate a loss of the normal laminar organization of the MSO cell column. In the current series of control subjects, the MSO was composed of 45% fusiform, 43% stellate and 12% round neurons (Fig. 5E). There was a significant shift in the neuronal composition of the MSO in ASD and dup(15q) subjects (Fig. 5E). In ASD, the MSO was composed of 21% fusiform, 38% stellate and 41% round neurons while in dup(15q) subjects, the MSO was composed of 23% fusiform, 37% stellate and 41% round neurons. The

distribution of cell body shapes was significantly different between these populations (Chi-square, p < .0001). Lateral superior olive (LSO) In control subjects, the caudal aspect of the LSO has a characteristic bi-lobed or ‘‘bowtie’’ appearance (Moore and Linthicum, 2004; Kulesza, 2007 – Figs. 1, 2A and B). Such lobulation was not observed in any of the subjects with ASD or dup(15q) and like the MSO, the LSO was markedly reduced in the rostral-caudal dimension (Fig. 2C–F). In control subjects, the LSO contained approximately 3790 neurons (median; Fig. 6A). In the affected brains, there were significantly fewer LSO neurons. In ASD subjects, the LSO contained only 1928 (Kruskal–Wallis, p < .005, Dunn’s p < .01) neurons and in dup(15q) subjects the LSO contained 1969 neurons (Dunn’s, p < .05). ASD subject M13–10 had no identifiable LSO. The difference in LSO neuronal number between ASD and dup(15q) subjects was not significant (Dunn’s, p > .05). In control subjects, LSO somata had a median cross-sectional area of 169 lm2. LSO somata were significantly smaller in ASD (154 lm2, Kruskal–Wallis, p < .0001, Dunn’s p < .05) and in dup(15q) subjects (121 lm2, Dunn’s p < .0001; Fig. 6B). In addition, LSO somata in dup(15q) subjects were significantly smaller than in ASD subjects (Dunn’s, p < .0001). In control subjects, LSO somata had a median circularity measure of .71. LSO somata in ASD subjects (.67) were significantly less round than controls and dup(15q) subjects (.73; Kruskal–Wallis, p < .0001, Dunn’s, p < .001). In control and ASD subjects, the LSO was composed of mainly

R. Lukose et al. / Neuroscience 286 (2015) 216–230

221

Fig. 2. Topography of the SOC in control, ASD and dup(15q) subjects. Schematics of the SOC nuclei (transverse plane) are shown for two control (A and B), two ASD (C and D) and two dup(15q) subjects (E and F). All schematics are based on tracings made from transverse sections at approximately the same rostro-caudal level and magnification. In all subjects with neurodevelopmental disorders (C–F), there is a significant reduction of nuclear volume which was most obvious in the MSO. Scale bar = 1000 lm.

round (68%; 56%) and stellate neurons (26%; 33%) with few fusiform neurons (6%; 11%). However, in dup(15q) subjects, the LSO contained significantly more round neurons (83%) and fewer stellate neurons (12%; Chi-square, p = .03). SPON In control subjects, the SPON contained 3800 ± 342 neurons (mean ± SD). There were significantly fewer SPON neurons in ASD (2110 ± 172; ANOVA, p < .001,

Tukey’s p < .0001) and dup(15q) subjects (2170 ± 176; Tukey’s, p < .001; Fig. 6C). In control subjects, SPON neurons had a median cross-sectional area of 226 lm2. SPON somata were significantly smaller in ASD (180 lm2, Kruskal–Wallis, p < .0001, Dunn’s, p < .0001) and dup(15q) subjects (151 lm2, Dunn’s, p < .0001). Furthermore, SPON somata in dup(15q) subjects were significantly smaller than in ASD (Dunn’s, p < .0001; Fig. 6D). In control subjects, SPON somata had a median circularity measure of .61. There was no difference in circularity between control and ASD

222

R. Lukose et al. / Neuroscience 286 (2015) 216–230

Fig. 3. Morphology of MSO neurons. Transverse sections of the MSO are shown from two control (A and B), two ASD (C and D) and two subjects with dup(15q) (E And F). In control subjects, MSO neurons are typically fusiform or stellate in morphology, stacked from anterior to posterior and issue prominent dendrites to both sides of the cell column (arrows in A and B). In ASD subjects, MSO neurons are smaller and abnormally oriented (arrows in C) or round (D). In subjects with dup(15q), MSO neurons are smaller, and abnormally oriented (arrows in E). In F, there is a patch of MSO neurons that as a group are abnormally oriented (arrowheads). Scale bar = 100 lm.

subjects (.64). However, SPON somata in dup(15q) subjects were significantly more round than controls (.67; Kruskal–Wallis, p < .01, Dunn’s p < .01). In control subjects, the SPON was composed of mostly round neurons (53%) with nearly equal populations of fusiform (23%) and stellate neurons (24%). There was no difference in the distribution of cell types in ASD (Chi-square, p = .6) or dup(15q) subjects (p = .23). Medial nucleus of the trapezoid body (MNTB) In control subjects, the MNTB contained approximately 4296 neurons (median). There were significantly fewer MNTB neurons in ASD (2618, Kruskal–Wallis, p < .0001, Dunn’s p < .05) and dup(15q) subjects (1434; Dunn’s p < .0001; Fig. 6E). In control subjects,

MNTB somata had a median cross-sectional area of 249lm2. In ASD and dup(15q) subjects, MNTB somata were significantly smaller (181 lm2 and 154 lm2, Kruskal–Wallis, p < .0001, both Dunn’s p < .0001; Fig. 6F). Furthermore, MNTB somata in dup(15q) subjects were significantly smaller than in ASD (Dunn’s, p < .0001; Fig. 6F). Control MNTB somata had a median circularity measure of .77. MNTB somata in ASD and dup(15q) were significantly less round (.70 and .71, respectively; Kruskal–Wallis, p < .0001, both Dunn’s, p < .0001). In control subjects, the MNTB was composed mainly of neurons with round/oval somata (93%) with few stellate (5%) or fusiform neurons (2%). In ASD and dup(15q) subjects, the MNTB contained significantly fewer round/oval neurons (76% and 75%, respectively; Chi-square, p < .004 and .001).

R. Lukose et al. / Neuroscience 286 (2015) 216–230

223

Fig. 4. Schematics of the MSO. Schematic reconstructions of the MSO cell column from two control (A and B), two ASD (C and D) and two subjects with dup(15q) (E And F) are provided. In subjects with ASD and dup(15q) the packing density of MSO neurons is reduced. In ASD, there are many fewer neurons and in dup(15q) there are often highly disorganized aberrant lobules in the MSO (E and F). Note the abnormal orientation of MSO neurons in E. The orientation arrows (0°, 90°, 180°) are reference for the angle measurements. Scale bar = 100 lm.

Fig. 5. Quantitative assessment of the MSO. Shown in A is a box plot of neuronal number in the MSO. There were significantly more neurons in control subjects. Shown in B is a box plot of cell body area of MSO neurons and shown in C is a box plot of circularity measures for MSO neurons. In control subjects, MSO neurons are larger and less round. In A–C, the whiskers represent the 1–99th percentile. Figure D demonstrates a histogram of MSO neuron angle measurements. In control subjects, there is a more asymmetric distribution of angles of orientation. The scale shown at the top left of D is in degrees. Shown in E is a bar graph showing the distribution of different cell body morphologies in control, ASD and dup(15q) subjects. There are many more round neurons in the neurodevelopmental conditions. Key to symbols: ++++ = Tukey’s p < .0001; ⁄⁄⁄⁄ = Dunn’s p < .0001; #### = Chi-square p < .0001.

VNTB In control subjects, the VNTB contained an average of 2219 ± 224 neurons and in ASD the VNTB contained 1830 ± 224 neurons (this difference was not significant;

Fig. 6G). However, in dup(15q) subjects there were significantly fewer VNTB neurons compared to controls (1272 ± 206, ANOVA, p < .05, Tukey’s, p < .05; Fig. 6G). In control subjects, VNTB somata had a

224

R. Lukose et al. / Neuroscience 286 (2015) 216–230

Fig. 6. Quantitative assessment of SOC nuclei. The top row shows the total number of neurons in the remaining SOC nuclei. The whiskers represent the 10–90th percentile. There are significantly fewer neurons in all nuclei in ASD and dup(15q) except for the VNTB in ASD (G). The bottom row of figures show box plots of cell body cross sectional area. The whiskers represent the 1–99th percentile. Neurons in subjects with ASD and dup(15q) were significantly smaller than in controls. Additionally, in all nuclei neurons in dup(15q) subjects were smaller than in ASD. Key to symbols (compared to control): Dunn’s, ⁄ = p < .05, ⁄⁄ = p < .01, ⁄⁄⁄⁄ = p < .001; Tukey’s, + = p < .05, +++ = p < .001, ++++ = p < .0001; comparing dup(15q) to ASD (Dunn’s): ### = p < .001, #### = p < .0001.

median cross-sectional area of 221 lm2 and in ASD subjects VNTB somata measured 241 lm2 (this difference was not significant; Fig. 6H). However in dup(15q) subjects, VNTB somata were significantly smaller than in controls and ASD (126 lm2; Kruskal– Wallis, p < .0001, both Dunn’s p < .0001; Fig. 6H). In control subjects, VNTB somata had a median circularity measure of .70. VNTB somata had a median circularity measure of .66 (Kruskal–Wallis, p < .01, Dunn’s p < .01) in ASD and .67 in dup(15q) subjects (not significant). In all subjects, the VNTB was composed mainly of round (67–77%) and stellate neurons (22–27%) with few fusiform neurons (5–8%). There was no statistical difference in the distribution of the cell body types between controls and ASD or dup(15q) subjects (Chi-square, p > .05). Finally, the human VNTB is known to include a population of giant neurons (somata area > 500 lm2; Kulesza, 2008; Fig. 6H). In control subjects, this giant cell population made up only 3% of the total. In ASD subjects, 5% of VNTB neurons were classified as giant cells, but in dup(15q) subjects there were only 1% giant cells.

Lateral nucleus of the trapezoid body (LNTB) In control subjects, the LNTB contained 7885 neurons (median). In affected brains, there were significantly fewer LNTB neurons (ASD – 5275, Kruskal–Wallis, p < .0005, Dunn’s p < .01; dup(15q) – 4856, Dunn’s,

p < .01; Fig. 6I). In control subjects, LNTB somata had a median cross-sectional area of 234 lm2. However, LNTB somata were significantly smaller in both ASD (189 lm2, Kruskal–Wallis, p < .0001, Dunn’s, p < .0001) and dup(15q) subjects (170 lm2, Dunn’s, p < .0001; Fig. 6J). Additionally, LNTB somata in dup(15q) subjects were significantly smaller than in ASD (p < .001; Fig. 6J). In control subjects, LNTB somata had a median circularity measure of .72. LNTB somata in ASD subjects were significantly less round (.68, Kruskal–Wallis, p < .0001, Dunn’s p < .0001) than controls and dup(15q) subjects (.72, Dunn’s, p < .005). In control, ASD and dup(15q) subjects the LNTB was composed of mainly round somata (74–79%), a minor population of stellate neurons (16–19%) and few fusiform neurons (4–6%).

Ectopic neurons In a number of ASD and dup(15q) subjects, we identified clusters of ectopic neurons in or around the SOC (Fig. 7). Because of the spacing of the tissue sections in our sample and the apparently small rostro-caudal dimension of these clusters, we were unable to estimate the total number of ectopic neurons in these subjects. Of the 16 ASD subjects examined, we identified ectopic neurons near the SOC in 2 of them (13%; Fig. 7A). However, of the 12 dup(15q) subjects examined, 5 (42%) had ectopic neurons in the vicinity of the SOC

R. Lukose et al. / Neuroscience 286 (2015) 216–230

(Fig. 7B, C). These ectopic clusters ranged from 15 to over 200 neurons (per tissue section) and were more commonly found along the rostral third of the SOC, posterior to the MSO (Fig. 7C) or lateral to the LNTB (within the tz; Fig. 7B, C). In two of the dup(15q) subjects there were more subtle ectopic populations. In subject M2–11 there were ectopic neurons medial and within the MSO (Fig. 4F) and in subject M1–12 there were neurons within the boundaries of the FN (intermingled with motor neurons) and within the LNTB which contained neuromelanin.

DISCUSSION Herein, we have provided the first quantitative morphometric analysis of the auditory brainstem in subjects with dup(15q) syndrome. We have also provided data supporting our previous findings of significant hypoplasia and dysmorphology of the SOC in ASD (Kulesza and Mangunay, 2008; Kulesza et al., 2011). In each subject we examined [with ASD or dup(15q)], we found significantly fewer neurons in the SOC but constituent neurons were significantly smaller than in control subjects. Specifically, within the SOC we found a 44% decrease in neuronal number in ASD while in dup(15q) subjects there was a 53% decrease. In this group of subjects with ASD, we find no correlation between the number of MSO neurons and ADI-R scores – this supports our previous observations (Kulesza et al., 2011). Additionally, we identified a high incidence of ectopic neurons in the brainstem of dup(15q) subjects – nearly one-half of these individuals had abnormal neuronal clusters within or around the SOC. We interpret the significant dysmorphology in the SOC of ASD and dup(15q) subjects to contribute significantly to the auditory processing difficulties in these neurodevelopmental conditions. Finally, we have identified a number of morphological features that appear to differentiate ASD and dup(15q) brains, suggesting somewhat different etiologies (discussed below). In the MSO of subjects with ASD and/or dup(15q), we found significantly fewer neurons but the remaining MSO neurons were smaller and more round (in contrast to the normal fusiform or stellate morphology). In control brains (sectioned in the transverse plane), human MSO neurons are elongated and arranged in a highly ordered, laminar column, indicative of the presumed tonotopic axis. However, in ASD and dup(15q) subjects we found MSO neurons to be more round and for the nucleus to lack a clear laminar arrangement. We propose that this disordered arrangement of MSO neurons in ASD and dup(15q) is associated with a disrupted tonotopic axis in the MSO and/or disorganized inputs from the cochlear nucleus (CN). We also found that the normal neuronal population of the MSO was shifted toward more round, immature neurons – from 12% in controls to 41% in ASD and dup(15q). Together, we interpret these findings to indicate that the normal function of the MSO is significantly impacted in these neurodevelopmental conditions. Here and in previous reports, we provided evidence that the MSO

225

consistently displays hypoplasia and dysmorphology in ASD. Furthermore, subjects with ASD appear to have consistently smaller neurons in some (i.e. cerebellum, claustrum), but not all brain regions (Bauman and Kemper, 1985; Fatemi et al., 2002a; Wegiel et al., 2014). Thus, we propose that dysmorphology in the MSO, claustrum and cerebellar Purkinje cells be considered hallmark neuropathological features of ASD (Wegiel et al., 2014). The neurons of the human MSO are characteristically fusiform or stellate in morphology, although a small population of round neurons is present in control subjects (Kulesza, 2007). Notably, in young subjects (i.e. younger than 10 years of age) we tend to find more neurons in the MSO with round to oval cell bodies (Kulesza et al., 2011) and we consider an increased population of such neurons in the MSO to represent immature or injured cells (Caldero´n-Garciduen˜as et al., 2011). Notably, we have identified some differences in the MSO of ASD subjects in this study compared to our previous report (Kulesza et al., 2011). Specifically, we previously reported the MSO in ASD to include 77% round neurons (n = 9). In this present report, we find the MSO in ASD to include only 41% round neurons (n = 16). We believe two factors likely contribute to this difference: age and variability. First, in the present study the age of ASD subjects averaged 22 years, while in our previous study the age of ASD subjects averaged 15 years. Second, our data suggest there is significant variability between ASD subjects in the degree to which the MSO (and other SOC nuclei) are disrupted. We believe this difference illustrates the variability (i.e. spectrum) of ASD. Finally, we believe our data provide evidence that there are significantly more round MSO neurons in young subjects who are more severely affected with ASD. In subjects with ASD, we found that the majority of the remaining SOC nuclei (LSO, SPON, MNTB and LNTB) had significantly fewer neurons, but again that the constituent neurons were significantly smaller than in controls. Similar to our previous investigation of the SOC in ASD, we find VNTB neurons to be the same size and number as in control subjects. In subjects with dup(15q), we found significantly fewer neurons in the LSO, SPON and each of the nuclei of the trapezoid body (including the VNTB). In addition, we found that subjects with dup(15q) had significantly smaller neurons than both ASD and control subjects in the LSO, SPON, MNTB, VNTB and LNTB. In the LSO and SPON, we also found more round neurons in dup(15q) subjects than in controls. In the MNTB of both ASD and dup(15q) subjects, there were significantly fewer round neurons. We have previously shown that human MNTB principal neurons are round/oval and are associated with large, calyx terminals arising from the globular bushy cells in the contralateral CN (Kulesza, 2014). This alteration in MNTB neuronal morphology may signify disruption of the pathway from the CN to the MNTB in ASD and dup(15q). In this and our previous report (Kulesza et al., 2011), we have examined the nuclei of the SOC in 25 subjects with ASD and have found ectopic neurons in four

226

R. Lukose et al. / Neuroscience 286 (2015) 216–230

Fig. 7. Ectopic neurons. Ectopic clusters were found in both ASD and dup(15q) subjects and are indicated by dashed lines and are highlighted in yellow. Shown in figure A is a view of the SOC and the surrounding pontine tegmentum from a subject with ASD. In this subject, there are two large ectopic islands: one is situated lateral to the LNTB and posterior to the tz (right) and another posterior to the SPON and LSO. Additionally, there is what appears to be an ectopic facial motor neuron (arrow; based on cell body size and morphology). Scale bar in A = 500 lm. Shown in B and C are examples of ectopic neurons from subjects with dup(15q). In B, the ectopic neurons are lateral to the LNTB, but within the tz. In C, there are three ectopic neuronal clusters which extend from the lateral aspect of the SOC over the posterior edge of the MSO. Scale bar in B (corresponding to B and C) = 200 lm.

individuals (16%). In the brainstem of subjects with dup(15q), we identified ectopic neurons in five individuals (42%). These results correlate with increased frequency of cortical ectopic neurons in dup(15q) versus idiopathic ASD at 89% versus 10% respectively (Wegiel et al., 2012b). We were not able to examine the entire brainstem in these subjects and therefore, we cannot rule out the possibility that the rate of brainstem ectopic neurons in ASD and dup(15q) is higher than we report. Our results suggest that neuronal migration defects within the brainstem are a common pathological finding in dup(15q) and that such heterotopias are much more common in dup(15q) syndromes compared to ASD. The MSO is the most severely affected of the SOC nuclei in ASD and

dup(15q) and in these disorders we observed a loss a loss of MSO neurons ranging from 31% to 97% of the nucleus (a loss of up to 13,000 neurons). Since ectopic brainstem neurons are a common finding in these conditions, we hypothesize that a significant number of these ectopic neurons are aberrant or undifferentiated MSO neurons. The literature provides evidence that dup(15q) brains are more severely affected than in ASD. Specifically, dysplasias are much more common in the forebrain and cerebellum of subjects with dup(15q) (Wegiel et al., 2012b). Also, heterotopias affecting the hippocampus and dentate gyrus were present in the majority of dup(15q) subjects but were rarely found in ASD subjects. Further, there is excessive intracellular and occasionally

227

R. Lukose et al. / Neuroscience 286 (2015) 216–230

extracellular accumulations of amyloid-b in cortical, thalamic and Purkinje neurons in ASD and dup(15q) + ASD subjects (Weigiel et al., 2012a). Additionally, seizures are a common comorbidity in ASD, present in up to as many as 30% subjects (Tuchman and Rapin, 2002; Valvo et al., 2013). However, a higher incidence of seizures/epilepsy in subjects with dup(15q) (compared to ASD) has been observed (Wegiel et al., 2012b). In the present work, 25% of the subjects with ASD had a medical history significant for seizures while 82% of the subjects with dup(15q) had a history of seizures. It has been proposed that these ectopic neuronal clusters could act as seizure foci (Wegiel et al., 2012b). Finally, the brains of subjects with dup(15q) weighed significantly less than the brains from subjects with ASD (Weigiel et al., 2012a; this study). Indeed, our investigation of the auditory brainstem supports these observations. Specifically, in dup(15q) we found neurons were smaller in size than in ASD and we found more ectopic neurons than in ASD subjects. There is evidence for disruption of GABAergic circuits in ASD (Fatemi et al., 2002b; Yip et al., 2007, 2009; Blatt and Fatemi, 2011; Paciorkowski et al., 2011). Specifically, there is a significant reduction in the amount of glutamic acid decarboxylase (GAD) in cortical regions and cerebellum and decreased levels of GABAA and GABAB receptors in ASD (Fatemi et al., 2002a,b). Furthermore, the genes for GABAA receptor subunits a5, b3, c3, have been implicated in ASD and coincidentally are located on chromosome 15q11.2–q13 (Cook et al., 1998; Schroer et al., 1998; Menold et al., 2001; Shao et al., 2003; Hogart et al., 2007; Blatt and Fatemi, 2011). These genes are non-imprinted but can show differential parental expression (Hogart et al., 2007) and abnormal expression may contribute to the ASD phenotypes seen in chromosome 15 duplication syndromes (Hogart et al., 2010). The 15q11.2–q13 region additionally encodes ubiquitin E3 ligase (Ube3a; Schroer et al., 1998) which has been linked to synaptic glutamate concentration (Smith et al., 2011). Maternal imprinting of this gene is associated with ASD (Veenstra-VanderWeele et al., 1999) and overexpression appears to induce the ASD phenotype (Hogart et al., 2010; Urraca et al., 2013). Specifically, elevated Ube3a expression in a mouse model has been shown to result in decreased socialization, impaired communication, increased repetitive behaviors, reduced presynaptic glutamate release and reduced neuronal excitability (Smith et al., 2011). Thus, there is clear evidence for dysfunction of and possibly unbalanced GABA and glutamate signaling in the mature brain in ASD [and most likely dup(15q)]. Additionally, abnormal neuronal migration has been linked to neurodevelopmental disorders (Guerrini and Parrini, 2010; Paciorkowski et al., 2011) and is believed to contribute to the pathophysiology of ASD (Weigiel et al., 2010). We believe that much of the dysmorphology we have identified in the brainstem is attributable to developmental errors. The variability of the size and location of these ectopic pools [in ASD and dup(15q)] suggests that the degree to which neuronal migration is affected in these conditions varies significantly between subjects. Beyond their well-defined roles in synaptic transmission, GABA and glutamate have been

implicated as paracrine signaling molecules which impact arrival, position and total neuronal number at a given target (Manent and Represa, 2007). These observations suggest that GABA and glutamate may play important roles in early development of the central nervous system and that disrupted signaling during brain development may contribute to the behavioral and sensory issues associated with ASD and dup(15q) phenotypes. Finally, we propose that disrupted GABA and/or glutamate signaling, both developmentally and postnatally, in dup(15q) syndromes contributes to the microcephaly and the higher prevalence of neurodevelopmental abnormalities in this condition. Acknowledgments—We are grateful to the families of the tissue donors, who have made this study possible. Tissue and clinical data were obtained from the NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, the Harvard Brain Tissue Resource Center, the Brain Bank for Developmental Disabilities and Aging of the NYS Institute for Research in Developmental Disabilities through the Autism Tissue Program. The authors would like to thank Dr Jerzy Wegiel and Dr Jane Pickett for their advice and support, the LECOM Research Collaborative and Dr Bertalan Dudas for continued support.

REFERENCES Abrahams BS, Geschwind DH (2008) Advances in autism genetics: on the threshold of a new neurobiology. Nat Rev Genet 9(5):341–355. Alca´ntara JI, Weisblatt EJ, Moore BC, Bolton PF (2004) Speech-innoise perception in high-functioning individuals with autism or Asperger’s syndrome. J Child Psychol Psychiatry 45(6):1107–1114. Allen DA (1988) Autistic spectrum disorders: clinical presentation in preschool children. J Child Neurol 3(Suppl.):48–56. American Psychiatric Association (2013) Diagnostic and statistical manual of mental disorders. 5th ed. Arlington, VA: American Psychiatric Publishing. Arin DM, Bauman ML, Kemper TL (1991) The distribution of Purkinje cell loss in the cerebellum in autism. Neurology 41(Suppl.):307. Battaglia A (2005) The inv dup(15) or idic(15) syndrome: a clinically recognisable neurogenetic disorder. Brain Dev 27(5):365–369. Battaglia A, Gurrieri F, Bertini E, Bellacosa A, Pomponi MG, Paravatou-Petsotas M, Mazza S, Neri G (1997) The inv dup(15) syndrome: a clinically recognizable syndrome with altered behavior, mental retardation, and epilepsy. Neurology 48(4):1081–1086. Battaglia A (2008) The inv dup(15) or idic(15) syndrome (tetrasomy 15q). Orphanet J Rare Dis 3:30. Battaglia A, Parrini B, Tancredi R (2010) The behavioral phenotype of the idic(15) syndrome. Am J Med Genet C Semin Med Genet 154C(4):448–455. Bauman M, Kemper TL (1985) Histoanatomic observations of the brain in early infantile autism. Neurology 35:866–874. Bingham PM, Spinner NB, Sovinsky L, Zackai EH, Chance PF (1996) Infantile spasms associated with proximal duplication of chromosome 15q. Neurology 15(2):163–165. Blatt GJ, Fatemi SH (2011) Alterations in GABAergic biomarkers in the autism brain: research findings and clinical implications. Anatom Rec (Hoboken) 294(10):1646–1652. Boddaert N, Zilbovicius M, Philipe A, Robel L, Bourgeois M, Barthe´lemy C, Seidenwurm D, Meresse I, Laurier L, Desguerre I, Bahi-Buisson N, Brunelle F, Arnold Munnich A, Samson Y, Mouren M, Chabane N (2009) MRI findings in 77 children with non-syndromic autistic disorder. PLoS One 4(2):e4415.

228

R. Lukose et al. / Neuroscience 286 (2015) 216–230

Bolton PF, Golding J, Emond A, Steer CD (2012) Autism spectrum disorder and autistic traits in the Avon Longitudinal Study of Parents and Children: precursors and early signs. J Am Acad Child Adolesc Psychiatry 51(3):249–260. Bolton PF, Veltman MW, Weisblatt E, Holmes JR, Thomas NS, Youings SA, Thompson RJ, Roberts SE, Dennis NR, Browne CE, Goodson S, Moore V, Brown J (2004) Chromosome 15q11-13 abnormalities and other medical conditions in individuals with autism spectrum disorders. Psychiatr Genet 14(3):131–137. Borgatti R, Piccinelli P, Passoni D, Dalpra` L, Miozzo M, Micheli R, Gagliardi C, Balottin U (2001) Relationship between clinical and genetic features in ‘‘inverted duplicated chromosome 15’’ patients. Pediatr Neurol 24:111–116. Browne CE, Dennis NR, Maher E, Long FL, Nicholson JC, Sillibourne J, Barber JC (1997) Inherited interstitial duplications of proximal 15q: genotype-phenotype correlations. Am J Hum Genet 61(6):1342–1352. Caldero´n-Garciduen˜as L, D’Angiulli A, Kulesza RJ, Torres-Jardo´n R, Osnaya N, Romero L, Keefe S, Herritt L, Brooks DM, AvilaRamirez J, Delgado-Cha´vez R, Medina-Cortina H, Gonza´lezGonza´lez LO (2011) Air pollution is associated with brainstem auditory nuclei pathology and delayed brainstem auditory evoked potentials. Int J Dev Neurosci 29(4):365–375. Cook Jr EH, Courchesne RY, Cox NJ, Lord C, Gonen D, Guter SJ, Lincoln A, Nix K, Haas R, Leventhal BL, Courchesne E (1998) Linkage-disequilibrium mapping of autistic disorder, with 15q1113 markers. Am J Hum Genet 62(5):1077–1083. Cook Jr EH, Lindgren V, Leventhal BL, Courchesne R, Lincoln A, Shulman C, Lord C, Courchesne E (1997) Autism or atypical autism in maternally but not paternally derived proximal 15q duplication. Am J Hum Genet 60(4):928–934. Dennis NR, Veltman MW, Thompson R, Craig E, Bolton PF, Thomas NS (2006) Clinical findings in 33 subjects with large supernumerary marker(15) chromosomes and 3 subjects with triplication of 15q11-q13. Am J Med Genet A 140(5):434–441. Depienne C, Moreno-De-Luca D, Heron D, Bouteiller D, Gennetier A (2009) Screening for genomic rearrangements and methylation abnormalities of the 15q11-q13 region in autism spectrum disorders. Biol Psychiatry 66:349–359. Fatemi SH, Halt AR, Realmuto G, Earle J, Kist DA, Thuras P (2002a) Purkinje cell size is reduced in cerebellum of patients with autism. Cell Mol Neurobiol 22:171–175. Fatemi SH, Halt AR, Stary JM, Kanodia R, Schulz SC, Realmuto GR (2002b) Glutamic acid decarboxylase 65 and 67 kDa proteins are reduced in autistic parietal and cerebellar cortices. Biol Psychiatry 52(8):805–810. Frackowiak J, Mazur-Kolecka B, Schanen NC, Brown WT, Wegiel J (2013) The link between intraneuronal N-truncated amyloid-b peptide and oxidatively modified lipids in idiopathic autism and dup(15q11.2-q13)/autism. Acta Neuropathol Commun 1(1):61. Gillberg C, Rosenhall U, Johansson E (1983) Auditory brainstem responses in childhood psychosis. J Autism Dev Dis 13(2):181–195. Gaffney GR, Kuperman S, Tsai LY (1988) Morphological evidence of brainstem involvement in infantile autism. Biol Psychiatry 24:578–586. Gillberg C, Steffenburg S, Wahlstro¨m J, Gillberg IC, Sjo¨stedt A, Martinsson T, Liedgren S, Eeg-Olofsson O (1991) Autism associated with marker chromosome. J Am Acad Child Adolesc Psychiatry 30(3):489–494. Gillberg C, Coleman M (1996) Autism and medical disorders: A review of the literature. Dev Med Child Neurol 38:191–202. Gomes E, Pedroso FS, Wagner MB (2008) Auditory hypersensitivity in the autistic spectrum disorder. Pro-Fono Rev Atualizacao Cientifica 20(4):279–284. Gravel JS, Dunn M, Lee WW, Ellis MA (2006) Peripheral audition of children on the autistic spectrum. Ear Hear 27(3):299–312. Greenspan SI, Wieder S (1997) Developmental patterns and outcomes in infants and children with disorders in relating and communicating: a chart review of 200 cases of children with autistic spectrum diagnoses. J Dev Learn Dis 1:87–141.

Guerrini R, Parrini E (2010) Neuronal migration disorders. Neurobiol Dis 38(2):154–166. Hashimoto T, Tayama M, Miyazaki M, Murakawa K, Kuroda Y (1993) Brainstem and cerebellar vermis involvement in autistic children. J Child Neurol 8(2):149–153. Hogart A, Nagarajan RP, Patzel KA, Yasui DH, Lasalle JM (2007) 15q11-13 GABAA receptor genes are normally biallelically expressed in brain yet are subject to epigenetic dysregulation in autism-spectrum disorders. Hum Mol Genet 16(6):691–703. Hogart A, Wu D, LaSalle JM, Schanen NC (2010) The comorbidity of autism with the genomic disorders of chromosome 15q11.2-q13. Neurobiol Dis 38(2):181–191. Kellerman GR, Fan J, Gorman JM (2005) Auditory abnormalities in autism: toward functional distinctions among findings. CNS Spectr 10(9):748–756. Kent L, Evans J, Paul M, Sharp M (1999) Comorbidity of autistic spectrum disorders in children with Down syndrome. Dev Med Child Neurol 41:153–158. Khalfa S, Bruneau N, Roge´ B, Georgieff N, Veuillet E, Adrien JL, Barthe´le´my C, Collet L (2004) Increased perception of loudness in autism. Hear Res 198(1–2):87–92. Klin A (1993) Auditory brainstem responses in autism: brainstem dysfunction or peripheral hearing loss? J Autism Dev Disord 23(1):15–35. Konigsmark BW (1970) Methods for counting of neurons. In: Nauta WJH, Ebbesson SOE, editors. Contemporary research methods in neuroanatomy. Heidelberg: Springer. p. 315–338. Kulesza RJ (2007) Cytoarchitecture of the human superior olivary complex: medial and lateral superior olive. Hear Res 225(1– 2):80–90. Kulesza RJ (2008) Cytoarchitecture of the human superior olivary complex: nuclei of the trapezoid body and posterior tier. Hear Res 241:52–63. Kulesza RJ (2014) Characterization of human auditory brainstem circuits by calcium-binding protein immunohistochemistry. Neuroscience 258:318–331. Kulesza RJ, Lukose R, Stevens LV (2011) Malformation of the human superior olive in autistic spectrum disorders. Brain Res 1367:360–371. Kulesza RJ, Mangunay K (2008) Morphological features of the medial superior olive in autism. Brain Res 1200:132–137. Kwon S, Kim J, Choe BH, Ko C, Park S (2007) Electrophysiologic assessment of central auditory processing by auditory brainstem responses in children with autism spectrum disorders. J Korean Med Sci 22(4):656–659. Lepisto¨ T, Kujala T, Vanhala R, Alku P, Huotilainen M, Na¨a¨ta¨nen R (2005) The discrimination of and orienting to speech and nonspeech sounds in children with autism. Brain Res 1066(1– 2):147–157. Lukose R, Brown K, Barber CM, Kulesza RJ (2013) Quantification of the stapedial reflex reveals delayed responses in autism. Autism Res 6(5):344–353. Manent JB, Represa A (2007) Neuronal and brain maturation: early paracrine actions of GABA and glutamate in neuronal migration. Neuroscientist 13:268–279. Mao R, Jalal SM, Snow K, Michels VV, Szabo SM, BabovicVuksanovic D (2000) Characteristics of two cases with dup(15)(q11.2–q12): one of maternal and one of paternal origin. Genet Med 2(2):131–135. Martinsson T, Johannesson T, Vujic M, Sjo¨stedt A, Steffenburg S, Gillberg C, Wahlstro¨m J (1996) Maternal origin of inv dup(15) chromosomes in infantile autism. Eur Child Adolesc Psychiatry 5(4):185–192. Maziade M, Me´rette C, Cayer M, Roy MA, Szatmari P, Coˆte´ R, Thivierge J (2000) Prolongation of brainstem auditory-evoked responses in autistic probands and their unaffected relatives. Arch Gen Psychiatry 57(11):1077–1083. Menold MM, Shao Y, Wolpert CM, Donnelly SL, Raiford KL, Martin ER, Ravan SA, Abramson RK, Wright HH, Delong GR, Cuccaro ML, Pericak-Vance MA, Gilbert JR (2001) Association analysis of

R. Lukose et al. / Neuroscience 286 (2015) 216–230 chromosome 15 GABAa receptor subunit genes in autistic disorder. J Neurogenet 15(3–4):245–259. McClelland RJ, Eyre DG, Watson D, Calvert GJ, Sherrard E (1992) Central conduction time in childhood autism. Br J Psychiatry 160:659–663. Moore JK, Linthicum Jr FH (2004). In: Paxinos George, Mai Juergen, editors. Auditory System. The Human Nervous System. Elsevier Academic Press. Moreno-De-Luca D, Sanders SJ, Willsey AJ, Mulle JG, Lowe JK (2013) Using large clinical data sets to infer pathogenicity for rare copy number variants in autism cohorts. Mol Psychiatry 18(10):1090–1095. Paciorkowski AR, Thio LL, Rosenfeld JA, Gajecka M, Gurnett CA, Kulkarni S, Chung WK, Marsh ED, Gentile M, Reggin JD, Wheless JW, Balasubramanian S, Kumar R, Christian SL, Marini C, Guerrini R, Maltsev N, Shaffer LG, Dobyns WB (2011) Copy number variants and infantile spasms: evidence for abnormalities in ventral forebrain development and pathways of synaptic function. Eur J Hum Genet 19(12):1238–1245. Palmen SJ, van Engeland H, Hof PR, Schmitz C (2004) Neuropathological findings in autism. Brain 127(Pt 12):2572–2583. Park DH, Lim S, Park ES, Sim EG (2013) A nine-month-old boy with isodicentric chromosome 15: a case report. Ann Rehab Med 37(2):291–294. Pinto D, Pagnamenta AT, Klei L, Anney R, Merico D, Regan R, Conroy J, Magalhaes TR, Correia C, Abrahams BS, Almeida J, Bacchelli E, Bader GD, Bailey AJ, Baird G, Battaglia A, Berney T, Bolshakova N, Bo¨lte S, Bolton PF, Bourgeron T, Brennan S, Brian J, Bryson SE, Carson AR, Casallo G, Casey J, Chung BH, Cochrane L, Corsello C, Crawford EL, Crossett A, Cytrynbaum C, Dawson G, de Jonge M, Delorme R, Drmic I, Duketis E, Duque F, Estes A, Farrar P, Fernandez BA, Folstein SE, Fombonne E, Freitag CM, Gilbert J, Gillberg C, Glessner JT, Goldberg J, Green A, Green J, Guter SJ, Hakonarson H, Heron EA, Hill M, Holt R, Howe JL, Hughes G, Hus V, Igliozzi R, Kim C, Klauck SM, Kolevzon A, Korvatska O, Kustanovich V, Lajonchere CM, Lamb JA, Laskawiec M, Leboyer M, Le Couteur A, Leventhal BL, Lionel AC, Liu XQ, Lord C, Lotspeich L, Lund SC, Maestrini E, Mahoney W, Mantoulan C, Marshall CR, McConachie H, McDougle CJ, McGrath J, McMahon WM, Merikangas A, Migita O, Minshew NJ, Mirza GK, Munson J, Nelson SF, Noakes C, Noor A, Nygren G, Oliveira G, Papanikolaou K, Parr JR, Parrini B, Paton T, Pickles A, Pilorge M, Piven J, Ponting CP, Posey DJ, Poustka A, Poustka F, Prasad A, Ragoussis J, Renshaw K, Rickaby J, Roberts W, Roeder K, Roge B, Rutter ML, Bierut LJ, Rice JP, Salt J, Sansom K, Sato D, Segurado R, Sequeira AF, Senman L, Shah N, Sheffield VC, Soorya L, Sousa I, Stein O, Sykes N, Stoppioni V, Strawbridge C, Tancredi R, Tansey K, Thiruvahindrapduram B, Thompson AP, Thomson S, Tryfon A, Tsiantis J, Van Engeland H, Vincent JB, Volkmar F, Wallace S, Wang K, Wang Z, Wassink TH, Webber C, Weksberg R, Wing K, Wittemeyer K, Wood S, Wu J, Yaspan BL, Zurawiecki D, Zwaigenbaum L, Buxbaum JD, Cantor RM, Cook EH, Coon H, Cuccaro ML, Devlin B, Ennis S, Gallagher L, Geschwind DH, Gill M, Haines JL, Hallmayer J, Miller J, Monaco AP, Nurnberger Jr JI, Paterson AD, Pericak-Vance MA, Schellenberg GD, Szatmari P, Vicente AM, Vieland VJ, Wijsman EM, Scherer SW, Sutcliffe JS, Betancur C (2010) Functional impact of global rare copy number variation in autism spectrum disorders. Nature 466:368–372. Piven J, Nehme E, Simon J, Barta P, Pearlson G, Folstein SE (1992) Magnetic resonance imaging in autism: measurement of the cerebellum, pons, and fourth ventricle. Biol Psychiatry 31(5):491–504. Raymond GV, Bauman ML, Kemper TL (1996) Hippocampus in autism: a Golgi analysis. Acta Neuropathol 91(1):117–119. Repetto GM, White LM, Bader PJ, Johnson D, Knoll JH (1998) Interstitial duplications of chromosome region 15q11q13: clinical and molecular characterization. Am J Med Genet 79(2):82–89.

229

Rineer S, Finucane B, Simon EW (1998) Autistic symptoms among children and young adults with isodicentric chromosome 15. Am J Med Genet 81:428–433. Ritvo ER, Freeman BJ, Scheibel AB, Duong T, Robinson H, Guthrie D, Ritvo A (1986) Lower Purkinje cell counts in the cerebella of four autistic subjects: initial findings of the UCLA-NSAC Autopsy Research Report. Am J Psychiatry 143(7):862–866. Rodier PM, Ingram JL, Tisdale B, Nelson S, Romano J (1996) Embryological origin for autism: developmental anomalies of the cranial nerve motor nuclei. J Comp Neurol 370(2):247–261. Roberts SE, Dennis NR, Browne CE, Willatt L, Woods G, Cross I, Jacobs PA, Thomas S (2002) Characterisation of interstitial duplications and triplications of chromosome 15q11–q13. Hum Genet 110(3):227–234. Roper L, Arnold P, Monteiro B (2003) Co-occurrence of autism and deafness: diagnostic considerations. Autism 7(3):245–253. Rosenhall U, Nordin V, Sandstro¨m M, Ahlse´n G, Gillberg C (1999) Autism and hearing loss. J Autism Dev Disord 29(5):349–357. Rosenhall U, Nordin V, Brantberg K, Gillberg C (2003) Autism and auditory brain stem responses. Ear Hear 24(3):206–214. Roth DA, Muchnik C, Shabtai E, Hildesheimer M, Henkin Y (2012) Evidence for atypical auditory brainstem responses in young children with suspected autism spectrum disorders. Dev Med Child Neurol 54(1):23–29. Rumsey JM, Grimes AM, Pikus AM, Duara R, Ismond DR (1984) Auditory brainstem responses in pervasive developmental disorders. Biol Psychiatry 19(10):1403–1418. Russo N, Zecker S, Trommer B, Chen J, Kraus N (2009) Effects of background noise on cortical encoding of speech in autism spectrum disorders. J Autism Dev Disord 39(8):1185–1196. Sebat J, Lakshmi B, Malhotra D, Troge J, Lese-Martin C, Walsh T, Yamrom B, Yoon S, Krasnitz A, Kendall J, Leotta A, Pai D, Zhang R, Lee YH, Hicks J, Spence SJ, Lee AT, Puura K, Lehtima¨ki T, Ledbetter D, Gregersen PK, Bregman J, Sutcliffe JS, Jobanputra V, Chung W, Warburton D, King MC, Skuse D, Geschwind DH, Gilliam TC, Ye K, Wigler M (2007) Strong association of de novo copy number mutations with autism. Science 316:445–449. Schinzel A, Niedrist D (2001) Chromosome imbalances associated with epilepsy. Am J Med Genet Semin Med Genet 106:119–124. Schroer RJ, Phelan MC, Michaelis RC, Crawford EC, Skinner SA, Cuccaro M, Simensen RJ, Bishop J, Skinner C, Fender D, Stevenson RE (1998) Autism and maternally derived aberrations of chromosome 15q. Am J Med Genet 76(4):327–336. Schumann CM, Amaral DG (2006) Stereological analysis of amygdala neuron number in autism. J Neurosci 26:7674–7679. Shao Y, Cuccaro ML, Hauser ER, Raiford KL, Menold MM, Wolpert CM, Ravan SA, Elston L, Decena K, Donnelly SL, Abramson RK, Wright HH, DeLong GR, Gilbert JR, Pericak-Vance MA (2003) Fine mapping of autistic disorder to chromosome 15q11q13 by use of phenotypic subtypes. Am J Hum Genet 72(3):539–548. Simic M, Turk J (2004) Autistic spectrum disorder associated with partial duplication of chromosome 15; three case reports. Eur Child Adolesc Psychiatry 13(6):389–393. Skoff BF, Mirsky AF, Turner D (1980) Prolonged brainstem transmission time in autism. Psychiatry Res 2(2):157–166. Smith SE, Zhou YD, Zhang G, Jin Z, Stoppel DC, Anderson MP (2011) Increased gene dosage of Ube3a results in autism traits and decreased glutamate synaptic transmission in mice. Sci Translat Med 3(103):103ra97. Stoner R, Chow ML, Boyle MP, Sunkin SM, Mouton PR, Roy S, Wynshaw-Boris A, Colamarino SA, Lein ES, Courchesne E (2014) Patches of disorganization in the neocortex of children with autism. N Engl J Med 370(13):1209–1219. Szelag E, Kowalska J, Galkowski T, Po¨ppel E (2004) Temporal processing deficits in high-functioning children with autism. Br J Psychol 95(Pt 3):269–282. Teder-Sa¨leja¨rvi WA, Pierce KL, Courchesne E, Hillyard SA (2005) Auditory spatial localization and attention deficits in autistic adults. Brain Res Cogn Brain Res 23(2–3):221–234.

230

R. Lukose et al. / Neuroscience 286 (2015) 216–230

Tharpe AM, Bess FH, Sladen DP, Schissel H, Couch S, Schery T (2006) Auditory characteristics of children with autism. Ear Hear 27(4):430–441. Thompson CK, Brenowitz EA (2005) Seasonal change in neuron size and spacing but not neuronal recruitment in a basal ganglia nucleus in the avian song control system. J Comp Neurol 481(3):276–283. Tomchek SD, Dunn W (2007) Sensory processing in children with and without autism: a comparative study using the short sensory profile. Am J Occup Ther 61(2):190–200. Tuchman RF, Rapin I (2002) Epilepsy in autism. Lancet Neurol 1:352–358. Urraca N, Cleary J, Brewer V, Pivnick EK, McVicar K, Thibert RL, Schanen NC, Esmer C, Lamport D, Reiter LT (2013) The interstitial duplication 15q11.2–q13 syndrome includes autism mild facial anomalies and a characteristic EEG signature. Autism Res 6(4):268–279. Valvo G, Baldini S, Brachini F, Apicella F, Cosenza A, Ferrari AR, Guerrini R, Muratori F, Romano MF, Santorelli FM, Tancredi R, Sicca F (2013) Somatic overgrowth predisposes to seizures in autism spectrum disorders. PLoS One 8(9):e75015. Veenstra-VanderWeele J, Gonen D, Leventhal BL, Cook Jr EH (1999) Mutation screening of the UBE3A/E6-AP gene in autistic disorder. Mol Psychiatry 4(1):64–67. Vorstman JA, Staal WG, van Daalen E, van Engeland H, Hochstenbach PF, Franke L (2006) Identification of novel autism candidate regions through analysis of reported cytogenetic abnormalities associated with autism. Mol Psychiatry 11(1):18–28. Wagoner JL, Kulesza RJ (2009) Topographical and cellular distribution of perineuronal nets in the human cochlear nucleus. Hear Res 254(1–2):42–53. Weigiel J, Kuchna I, Nowicki K, Imaki H, Wegiel J, Marchi E, Ma SY, Chauhan A, Chauhan V, Wierzba Bobrowicz T, de Leon M, Saint Louis LA, Cohen IL, London E, Brown WT, Wisniewski T (2010) The neuropathology of autism: defects of neurogenesis and neuronal migration, and dysplastic changes. Acta Neuropathol 119:755–770. Wegiel J, Frackowiak J, Mazur-Kolecka B, Schanen NC, Cook Jr EH, Sigman M, Brown WT, Kuchna I, Wegiel J, Nowicki K, Imaki H,

Ma SY, Chauhan A, Chauhan V, Miller DL, Mehta PD, Flory M, Cohen IL, London E, Reisberg B, de Leon MJ, Wisniewski T (2012a) Abnormal intracellular accumulation and extracellular Ab deposition in idiopathic and Dup15q11.2–q13 autism spectrum disorders. PLoS One 7(5):e35414. Wegiel J, Schanen NC, Cook EH, Sigman M, Brown WT, Kuchna I, Nowicki K, Wegiel J, Imaki H, Ma SY, Marchi E, WierzbaBobrowicz T, Chauhan A, Chauhan V, Cohen IL, London E, Flory M, Lach B, Wisniewski T (2012b) Differences between the pattern of developmental abnormalities in autism associated with duplications 15q11.2–q13 and idiopathic autism. J Neuropathol Exp Neurol 71(5):382–397. Wegiel J, Flory M, Kuchna I, Nowicki K, Ma SY, Imaki H, Wegiel J, Cohen IL, London E, Brown WT, Wisniewski T (2014) Brainregion-specific alterations of the trajectories of neuronal volume growth throughout the lifespan in autism. Acta Neuropathol Commun 2(1):28. Wing L (1997) The autistic spectrum. Lancet 350(9093):1761–1766. Wisniewski L, Hassold T, Heffelfinger J, Higgins JV (1979) Cytogenetic and clinical studies in five cases of inv dup(15). Hum Genet 50(3):259–270. Whitney ER, Kemper TL, Bauman ML, Rosene DL, Blatt GJ (2008) Cerebellar Purkinje cells are reduced in a subpopulation of autistic brains: a stereological experiment using calbindin-D28k. Cerebellum 7(3):406–416. Wolpert C, Pericak-Vance MA, Abramson RK, Wright HH, Cuccaro ML (2000) Autistic symptoms among children and young adults with isodicentric chromosome 15. Am J Med Genet 96(1):128–129. Yin TC, Carney LH, Joris PX (1990) Interaural time sensitivity in the inferior colliculus of the albino cat. J Comp Neurol 295(3):438–448. Yip J, Soghomonian JJ, Blatt GJ (2007) Decreased GAD67 mRNA levels in cerebellar Purkinje cells in autism: pathophysiological implications. Acta Neuropathol 113(5):559–568. Yip J, Soghomonian JJ, Blatt GJ (2009) Decreased GAD65 mRNA levels in select subpopulations of neurons in the cerebellar dentate nuclei in autism: an in situ hybridization study. Autism Res 2:50–59.

(Accepted 13 November 2014) (Available online 5 December 2014)