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Morphological features of the medial superior olive in autism
BR A I N R ES E A RC H 1 2 0 0 ( 2 00 8 ) 1 3 2 –13 7
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Morphological features of the medial superior olive in autism Randy J. Kulesza⁎, Kathleen Mangunay Lake Erie College of Osteopathic Medicine, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
Autism is a psychosocial disorder clinically characterized by social difficulties, impairment in
Accepted 3 January 2008
communication skills and repetitive behavioral patterns. Despite the increasing reported
Available online 14 January 2008
incidence of autism, the neurobiology of this disorder is poorly understood. However, researchers have uncovered numerous structural anomalies in the brainstem, cerebellum and
Keywords:
forebrain of autistic individuals and there is substantial support for the association of hearing
Hearing
deficits with autism. In an effort to discover an anatomical correlate for the functional auditory
Superior olivary complex
deficits found in autism, we examined the SOC, a group of brainstem nuclei that function in
Morphometry
sound source localization, in post-mortem brain tissue from autistic individuals. The neurons of the medial superior olive (MSO), an SOC nucleus, display a precise geometric organization essential for detection of timing differences between the two ears. We examined the architecture of the MSO in five autistic brains (ages 8 to 32 years) and two age-matched controls (ages 26 and 29 years) and found a significant disruption in the morphology of MSO neurons in autistic brains, involving cell body shape and orientation. The results from this study provide evidence on the cellular level that may help to explain the hearing difficulties associated with autism. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Autism is a developmental disorder characterized by impairment of communication skills and social interaction, sensory abnormalities and a host of stereotypical behaviors (American Psychiatric Association, 1994). Recent estimates of the incidence of autism indicate that it affects approximately 1 in every 152 children (MMWR Surveillance Summary, 2007). There is ample support for neuroanatomical alterations in this disorder, including differences in neuronal packing density, reduced neuronal cell body size, less extensive dendritic arborization and in some brain regions a reduction in the amount of GABA, an inhibitory neurotransmitter (Gaffney et al., 1988; Ritvo et al., 1986; Piven et al., 1992; Palmen et al., 2004; Bauman and Kemper, 2005; Blatt, 2005). In addition, there is evidence that hearing deficiencies affect the vast majority of autistic individuals (Greenspan and Weider, 1997; Tomchek and Dunn, 2007). The auditory dysfunc-
tion observed in autism seems to affect multiple aspects of hearing, including deafness, increased thresholds to tones, intolerance for ordinary sound levels (hyperacusis) and difficulty listening in the presence of background noise (Rosenhall et al., 1999; Roper et al., 2003; Alcantara et al., 2004; Khalfa et al., 2004; Szelag et al., 2004; Kellerman et al., 2005; Lepisto et al., 2005; Teder-Salejarvi et al., 2005; Gravel et al., 2006; Tharpe et al., 2006). Moreover, results from studies examining the auditory brainstem response (ABR) from autistic individuals seem to implicate functional deficits in the lower auditory brainstem (Rosenhall et al., 2003; Tas et al., 2007; Kwon et al., 2007). The human auditory system consists of peripherally located ear structures (pinna, tympanic membrane, ossicles), mechanoreceptive hair cells and a multitude of ascending and descending neuronal circuits within the brainstem and forebrain. The superior olivary complex (SOC) is an aggregation of auditory nuclei situated at the pons-medullary junction. The SOC is a
⁎ Corresponding author. Lake Erie College of Osteopathic Medicine, Auditory Research Center, 1858 West Grandview Boulevard, Erie, PA 16509, USA. Fax: +1 814 866 8411. E-mail address: [email protected] (R.J. Kulesza). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.01.009
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Fig. 1 – The normal human MSO is composed of a highly ordered stack of fusiform and stellate neurons. The MSO is shown in control tissue sectioned in the transverse plane and stained for Nissl substance. Neurons of the MSO are arranged in a narrow column that is tilted slightly towards the midline. Primary dendrites extend into the neuropil on both the medial and lateral sides of the nucleus, but only the initial dendritic trunks are stained in this material. P = posterior, L = lateral; the scale bar is equal to 100 μm.
major site of convergence of auditory information and is the first major station where information from both ears is compared. The human SOC contains eight distinct nuclei, and each of these cell groups forms a unique neuronal circuit and subserves different aspects of auditory processing (Schofield and Cant, 1991; Schofield, 2002; Kulesza, 2007). The human SOC includes two principle nuclei, the medial and lateral superior olives (MSO and LSO, respectively) that play essential roles in localization of sound sources. The human MSO consists of an elongated
column of fusiform and stellate neurons that are aligned in a precise geometric pattern: the cell bodies are elongated in the coronal plane and stacked from anterior to posterior (Kulesza, 2007). Moreover, in vivo electrode recordings in cat provide evidence that the laminar organization of MSO neurons contributes to the laminar representation of sound frequencies within the nucleus (Guinan et al., 1972). The hearing deficits identified through psychoacoustic testing and examination of the ABR in autistic individuals seem to indicate disruption of the lower auditory brainstem. Indeed, study of a single autistic brain after autopsy revealed a complete absence of the SOC (Rodier et al., 1996). Thus, the working hypothesis for this study is that the hearing deficits observed in autistic individuals are the result of a disruption of the normal cytoarchitecture in the lower auditory brainstem, namely the SOC and cochlear nucleus (CN). The goal of this study is to examine the cytoarchitecture of the human MSO in autistic brains and to compare these findings to those from control specimens. The MSO was chosen because of the important functional role it serves and its prominence within the SOC; the MSO is the largest and most densely populated nucleus in the human SOC.
2.
Results
2.1.
General features
The human MSO is first recognized in the rostral medulla oblongata as a prominent column of neurons within the SOC and extends rostro-caudally nearly 6.0 mm into the mid-pons. When viewed in the transverse plane, the MSO appears as a thin stack of neurons, tilted slightly towards the midline. The MSO cell column is approximately 300 μm wide and extends from anterior to posterior approximately 1000 μm. The human MSO contains nearly equal populations of fusiform and stellate neurons and these cells issue primary dendrites into the neuropil on both sides of the nucleus (Kulesza, 2007). The most notable feature of the human MSO is the strikingly consistent geometric arrangement of the constituent neurons. Both fusiform and stellate
Table 1 – Morphometric features of MSO neurons from control and autistic brains Age/sex Coronal sections Control (n = 43) Aut01 (n = 50) Aut03 (n = 117) Aut06 (n = 114)
Mass (g)
Area (μm2)
Perimeter (μm)
Major (μm)
Minor (μm)
Angle (degrees)
Circularity
91 ± 19 91 ± 45+ 87 ± 56+ 65 ± 57⁎+
.55 ± .11 .73 ± .11⁎ .63 ± .12⁎ .71 ± .14⁎
26/M 26/M 13/M 8/M
1520 1610 1470 1570
232 ± 60 252 ± 76 264 ± 80⁎ 158 ± 47⁎
72 ± 12 66 ± 14⁎ 73 ± 15 54 ± 13⁎
28 ± 6 23 ± 6⁎ 26 ± 6 20 ± 6⁎
11 ± 2 14 ± 2⁎ 13 ± 2⁎ 10 ± 2
Parasagittal sections Control (n = 58) 29/M Aut04 (n = 37) 32/M Aut05 (n = 115) 32/M
1514 1694 1510
201 ± 63 146 ± 70⁎ 105 ± 46⁎
73 ± 16 53 ± 17⁎ 49 ± 14⁎
27 ± 7 18 ± 7⁎ 18 ± 6⁎
9±2 10 ± 2 7 ± 2⁎
106 ± 23 111 ± 32+ 101 ± 45+
.49 ± .14 .66 ± .13⁎ .57 ± .15⁎
Fusiform and stellate neurons were pooled for this analysis; data are presented as average ± standard deviation. Mass refers to the overall brain mass as recorded by the Autism Tissue Program; major and minor refer to the length of the major and minor axes of the cell body. All morphological categories were analyzed with ANOVA; angle data were analyzed with ANOVA and the O'Brien test for homogeneity of variance. Asterisks indicate statistical significance by ANOVA, plus symbols indicate statistical significance in variance (p b 0.05). Comparing the angle data between controls and the autistic group reveals large standard deviations, further indicating the variability in these groups.
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groups is shown in Fig. 2. MSO neurons from AUT03 were statistically different from the control in terms of cell body area, minor axis and circularity (ANOVA, p b 0.05). The orientation angle measurement for AUT03 showed a significant difference in variance (Table 1). MSO neurons from AUT06 were statistically different from control in terms of cell body area, perimeter, major axis and circularity. The orientation angles from AUT06 were significantly different by ANOVA and by variance (p b 0.05). Notably, MSO neurons from all three autistic brains were statistically more circular than those from the control. Finally, there was statistical significance in the differences within the AUT groups in all morphological categories (ANOVA, p b 0.05).
2.3.
Parasagittal sections: two 32-year-old males
Examination of the MSO from the control specimen revealed fusiform and elongated stellate neurons that had their long axis oriented mainly in the transverse plane. In comparing brain mass to the control, the brain from AUT04 weighed 180 g
Fig. 2 – MSO neurons in autistic brains have a variable orientation. Shown in A is the distribution of orientation angles from control (dark black line) and three autistic brains (three gray lines) sectioned in the coronal plane. The vast majority of control MSO neurons have orientation angles between 90 and 110 degrees. The orientation angles from autistic MSO neurons have a much more variable distribution. Shown in B is the distribution of orientation angles from tissue sectioned in the parasagittal plane (control in black, autistic in gray). Compared to the control specimen, neurons from the autistic MSO have a more variable orientation.
neurons have long axes that are oriented perpendicular to the long axis of the nucleus (Fig. 1).
2.2.
Coronal sections: 26, 13 and 8-year-old males
Examination of the MSO from the control specimen (sectioned in the coronal plane) revealed relatively large, fusiform and elongated stellate neurons neatly arranged in a narrow column (as in Fig. 1). These neurons issued dendrites into the neuropil on both sides of the nucleus, consistent with previous reports (Moore and Moore, 1971; Moore, 1987; Kulesza, 2007). The MSO was identified in the rostral medulla/caudal pons in each of the autistic specimens (AUT01, 03 and 06) sectioned in the coronal plane. However, the neurons within the MSO cell column of the autistic specimens appeared morphologically much different from those in the control brains. In comparing the control and AUT01 (both 26-year-old males; Table 1), the brain from AUT01 weighed 90 g more than the control but MSO neurons were statistically similar between the two brains in terms of cell body area. However, there were statistically significant differences in cell body shape — perimeter, major and minor axes and circularity (ANOVA, p b 0.05). The mean of the orientation angle measurements was statistically similar between the control and AUT01, however a statistical difference was observed in the variance of these two groups (O'Brien Test for homogeneity of variance, p b 0.05). The distribution of orientation angles for all
Fig. 3 – MSO neurons in autistic brains display a significantly different morphology. Shown in A is a group of MSO neurons from the control specimen, sectioned in the parasagittal plane. These neurons are either fusiform (arrows) or stellate (arrowheads) and have a long axis oriented in the coronal plane. Shown in B is a group of MSO neurons from an autistic brain (AUT04). These neurons are much smaller and round and few of them show a preferential orientation; occasional stellate neurons are observed (arrowhead). C = caudal, P = posterior; the scale bar is equal to 15 μm.
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more and the brain from AUT05 weighed 4 g less. MSO neurons from the autistic specimens (AUT04 and AUT05) were significantly different in the majority of morphometric categories (Fig. 3; Table 1). Generally, MSO neurons in the autistic brains were smaller in terms of cell body area, perimeter and major axis and these neurons were significantly more round than in control. In terms of orientation angle, the means between the control and AUT groups were statistically similar (ANOVA, p N 0.05). However, comparing the angle measurements in Fig. 2 indicates that the orientation of MSO neurons in the AUT groups is more heterogeneous than control and these differences in variance were statistically significant (O'Brien test for homogeneity of variance, p b 0.05). Finally, in comparing AUT04 and 05, there were statistical differences in cell body area, minor axis and circularity (ANOVA, p b 0.05).
3.
Discussion
Although there are reports in the literature of alterations of neuronal cell body area, number and dendritic morphology in autism, this is the first report of quantitative alterations in neuronal morphology affecting not only cell body size, but also cell body shape and orientation. We observed a disruption in the morphology of MSO neurons in each of the five autistic brains studied. In the majority of cases examined, there was a significant difference in cell body area. However, the most consistent differences were observed in measures of cell body shape (circularity) and the variability in the orientation of the long axis. These results provide evidence suggesting that the MSO in the autistic brain is not morphologically equivalent to the MSO in non-autistic brains. This finding supports recent reports of abnormal auditory brainstem responses (ABR) in children with autism (Rosenhall et al., 2003; Kwon et al., 2007; Tas et al., 2007). Specifically, these reports (Rosenhall et al., 2003; Kwon et al., 2007; Tas et al., 2007) indicate an increased latency between peaks III and V of the ABR (peak III is associated with the SOC and peak V with the inferior colliculus). Disruption of the normal cytoarchitecture of the MSO (smaller caliber, shorter dendrites and smaller neurons with shorter projections) may contribute to this increased latency, at least in part. Despite our findings of abnormal MSO neuronal morphology in all five cases we examined, it remains possible that the differences we observed were caused by something other than autism. One potential cause of disruption within the auditory pathways would be deafness or other hearing deficiency. However, no such deficits were reported for the five autistic individuals we examined. It is also possible that the differences between the control and AUT06 were due at least in part to the age difference. One of the more likely candidates is medication; it is possible that the medications that these individuals were taking before or at the time of death could have impacted neuronal morphology. Examination of human MSO neurons in silver impregnated material provides evidence that the laminar arrangement of the MSO involves not only cell bodies, but also the dendritic arbors (Kulesza, 2007). Also, the inputs reaching the MSO from the cochlear nuclei are segregated on the dendrites: inputs from the ipsilateral cochlear nucleus innervate the lateral dendrites; inputs from the contralateral cochlear nucleus innervate the medial dendrites (Stotler, 1953; Perkins, 1973; Beckius et al.,
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1999). Moreover, in vivo recordings provide strong evidence of an anterior-to-posterior laminar arrangement of sound frequencies (tonotopic axis) in the MSO of the cat (Guinan et al., 1972). Thus, the morphology of MSO neurons and their strict geometric arrangement are intimately related to the organization of afferent information reaching the nucleus and ultimately to the role of the MSO in encoding low-frequency sounds and sound source localization. So, it seems plausible that the morphological differences discovered in the autistic MSO may indicate a disruption of the excitatory/inhibitory inputs reaching the nucleus and the output of the MSO to the inferior colliculus. This finding may help provide a cellular basis for some of the hearing abnormalities described in this disorder, namely deafness. Although we report significant differences in the morphology of MSO neurons between control and autistic individuals, the morphology of MSO neurons in autistic brains is somewhat variable. In fact, we observed significant differences in most morphological categories within the five autistic brains we examined. Rodier et al. (1996) reported on the cytoarchitecture of numerous brainstem nuclei from a single autistic brain. In this particular specimen, based on their description and figures, the SOC appears to be absent or greatly reduced; the MSO cell column is not discernable in their figures. In contrast, we were able to identify the MSO in all five of our autistic cases. These observations, taken together, suggest that there is great variability in the brainstem abnormalities in autism. Perhaps the brain examined by Rodier et al. was a brain that was severely affected by autism. This variability in morphology is likely related to function: individuals with more severe anatomical abnormalities are likely to have more noticeable functional deficits. Despite recent reports of anatomical differences in the autistic brain, many regions remain unexplored. It must be noted that the MSO is but one small component of the entire auditory pathway. Other brain regions, such as the cochlear nucleus or the inferior colliculi, were not examined. Thus, it remains possible that many other components of the auditory pathway, even other nuclei within the SOC are also affected. This is an attractive speculation, given the role of the SOC in forming the olivocochlear bundle, an efferent pathway that functions in gain control of the organ of Corti and the deficits observed in autism (increased thresholds to tones, hyperacusis and difficulty listening in background noise). Additionally, it remains possible that lower regions of the auditory brainstem are affected even more severely than the MSO. Many new questions have arisen from this study regarding the size and course of the major brainstem auditory tracts (i.e. trapezoid body, posterior acoustic stria, lateral lemniscus), the arrangement and complexity of dendritic arbors of MSO neurons and neurochemistry of these cells (i.e. are these cells using the appropriate neurotransmitters?). Although there is a tremendous literature on autism, the neurobiology of this disorder remains somewhat elusive. It is unclear what developmental or maturational process has gone awry, if any. Have these MSO neurons been denied the appropriate growth factors, or have they not been contacted or innervated by the correct number or population of growth cones, or has there been an over-pruning of inputs later in development, perhaps even postnatally? Obviously many questions
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remain about the direct cause of autism and the time period during which it develops. Even more intriguing is why some brain regions are affected and others appear normal. Although data are accumulating about the functional deficits in autism, the anatomical deficits are not completely documented. If we are ever to completely understand this disorder, we must be fully aware of all the brain regions involved and to what degree they are affected. An in-depth understanding of the neuroanatomical changes that occur consistently in autism will be essential in understanding this disorder and may, in the future, aid in the development of instructional or therapeutic measures for individuals with this disorder.
4.
Experimental procedures
4.1.
Tissue sections
Histological slides from autistic and age-matched control brains were kindly loaned by the Autism Tissue Program (ATP; http://www.brainbank.org). Celloidin embedded brains were cut at a thickness of 200 μm and stained with cresyl violet. This report is based on data from five autistic cases (with no reported hearing abnormalities) and two agematched control cases. Specimens were divided into two groups based on plane of section (since neuronal morphology varies depending on plane of section). The first group includes specimens sectioned in the coronal plane; this group includes tissue from three individuals diagnosed with autism (AUT01 [26year-old male], AUT03 [13-year-old male] and AUT06 [8-year-old male]) and one control (26-year-old male). The second group includes specimens sectioned in the parasagittal plane; this group includes two individuals diagnosed with autism (AUT04 [32-year-old male] and AUT05 [32-year-old male also diagnosed with Fragile X]) and one control (29-year-old male). The series of sections provided by the ATP were incomplete; no specimen included the entire extent of the MSO. As a result, estimates of neuronal number and packing density could not be reliably calculated.
4.2.
Morphometric analysis
Tissue sections were examined with an Olympus BX45 microscope and photographed with an Olympus DP12 digital camera. For morphometric analyses, sections were randomly selected throughout the rostro-caudal extent of the MSO and only neurons with visible nucleoli and cell bodies that were completely within the tissue section were included (to prevent the inclusion of incomplete neuronal profiles). Cell bodies were traced while focusing (to most accurately determine the cell body contour) with the aid of a camera lucida attachment (Olympus; using a 40× objective with a final on paper magnification of 675×) and these tracings were digitized into jpeg format using a flatbed scanner. The digitized tracings were imported into ImageJ software (available at http://rsb.info.nih. gov/ij/) and measurements of cell body area, perimeter, length of the major and minor axis, circularity and orientation of the long axis were made using the “Analyze” feature. ImageJ was calibrated to a standard scale bar for each trial. An index of
circularity was calculated for each soma using the following equation: h i Circularity ¼ 4π⁎ Area=Perimeter2 This calculation yields an estimate of soma shape independent of size (Yin et al., 1990). Using this formula a perfectly circular soma yields a value of “1”, while increasingly irregular or elliptical profiles yield decreasing values. Measurements were also made of the orientation (angle) of the long axis of individual neurons using ImageJ. For tissue sectioned in the coronal plane, angle measurements were made in reference to the vertical midline of the tissue. So in any section, neurons with a long axis parallel to midline will have an angle measurement of 90°; and neurons with a long axis parallel to horizontal will have a measurements of 0°. For consistency, angle measurements were taken only from neurons on the right side of the brain. Statistical comparisons of all morphometric data were made using the data analysis feature in Excel (Microsoft) or JMP (SAS).
Acknowledgments This work was supported in part by a grant from the Deafness Research Foundation. The authors would like to thank Dr Jerzy Weigel and the Autism Tissue Program for kindly providing the tissue and the LECOM Research Collaborative for their continued support. We would also like to thank Dr Jack Caldwell for critically reading an earlier version of this manuscript.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Morphological features of the medial superior olive in autism Randy J. Kulesza⁎, Kathleen Mangunay Lake Erie College of Osteopathic Medicine, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
Autism is a psychosocial disorder clinically characterized by social difficulties, impairment in
Accepted 3 January 2008
communication skills and repetitive behavioral patterns. Despite the increasing reported
Available online 14 January 2008
incidence of autism, the neurobiology of this disorder is poorly understood. However, researchers have uncovered numerous structural anomalies in the brainstem, cerebellum and
Keywords:
forebrain of autistic individuals and there is substantial support for the association of hearing
Hearing
deficits with autism. In an effort to discover an anatomical correlate for the functional auditory
Superior olivary complex
deficits found in autism, we examined the SOC, a group of brainstem nuclei that function in
Morphometry
sound source localization, in post-mortem brain tissue from autistic individuals. The neurons of the medial superior olive (MSO), an SOC nucleus, display a precise geometric organization essential for detection of timing differences between the two ears. We examined the architecture of the MSO in five autistic brains (ages 8 to 32 years) and two age-matched controls (ages 26 and 29 years) and found a significant disruption in the morphology of MSO neurons in autistic brains, involving cell body shape and orientation. The results from this study provide evidence on the cellular level that may help to explain the hearing difficulties associated with autism. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Autism is a developmental disorder characterized by impairment of communication skills and social interaction, sensory abnormalities and a host of stereotypical behaviors (American Psychiatric Association, 1994). Recent estimates of the incidence of autism indicate that it affects approximately 1 in every 152 children (MMWR Surveillance Summary, 2007). There is ample support for neuroanatomical alterations in this disorder, including differences in neuronal packing density, reduced neuronal cell body size, less extensive dendritic arborization and in some brain regions a reduction in the amount of GABA, an inhibitory neurotransmitter (Gaffney et al., 1988; Ritvo et al., 1986; Piven et al., 1992; Palmen et al., 2004; Bauman and Kemper, 2005; Blatt, 2005). In addition, there is evidence that hearing deficiencies affect the vast majority of autistic individuals (Greenspan and Weider, 1997; Tomchek and Dunn, 2007). The auditory dysfunc-
tion observed in autism seems to affect multiple aspects of hearing, including deafness, increased thresholds to tones, intolerance for ordinary sound levels (hyperacusis) and difficulty listening in the presence of background noise (Rosenhall et al., 1999; Roper et al., 2003; Alcantara et al., 2004; Khalfa et al., 2004; Szelag et al., 2004; Kellerman et al., 2005; Lepisto et al., 2005; Teder-Salejarvi et al., 2005; Gravel et al., 2006; Tharpe et al., 2006). Moreover, results from studies examining the auditory brainstem response (ABR) from autistic individuals seem to implicate functional deficits in the lower auditory brainstem (Rosenhall et al., 2003; Tas et al., 2007; Kwon et al., 2007). The human auditory system consists of peripherally located ear structures (pinna, tympanic membrane, ossicles), mechanoreceptive hair cells and a multitude of ascending and descending neuronal circuits within the brainstem and forebrain. The superior olivary complex (SOC) is an aggregation of auditory nuclei situated at the pons-medullary junction. The SOC is a
⁎ Corresponding author. Lake Erie College of Osteopathic Medicine, Auditory Research Center, 1858 West Grandview Boulevard, Erie, PA 16509, USA. Fax: +1 814 866 8411. E-mail address: [email protected] (R.J. Kulesza). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.01.009
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Fig. 1 – The normal human MSO is composed of a highly ordered stack of fusiform and stellate neurons. The MSO is shown in control tissue sectioned in the transverse plane and stained for Nissl substance. Neurons of the MSO are arranged in a narrow column that is tilted slightly towards the midline. Primary dendrites extend into the neuropil on both the medial and lateral sides of the nucleus, but only the initial dendritic trunks are stained in this material. P = posterior, L = lateral; the scale bar is equal to 100 μm.
major site of convergence of auditory information and is the first major station where information from both ears is compared. The human SOC contains eight distinct nuclei, and each of these cell groups forms a unique neuronal circuit and subserves different aspects of auditory processing (Schofield and Cant, 1991; Schofield, 2002; Kulesza, 2007). The human SOC includes two principle nuclei, the medial and lateral superior olives (MSO and LSO, respectively) that play essential roles in localization of sound sources. The human MSO consists of an elongated
column of fusiform and stellate neurons that are aligned in a precise geometric pattern: the cell bodies are elongated in the coronal plane and stacked from anterior to posterior (Kulesza, 2007). Moreover, in vivo electrode recordings in cat provide evidence that the laminar organization of MSO neurons contributes to the laminar representation of sound frequencies within the nucleus (Guinan et al., 1972). The hearing deficits identified through psychoacoustic testing and examination of the ABR in autistic individuals seem to indicate disruption of the lower auditory brainstem. Indeed, study of a single autistic brain after autopsy revealed a complete absence of the SOC (Rodier et al., 1996). Thus, the working hypothesis for this study is that the hearing deficits observed in autistic individuals are the result of a disruption of the normal cytoarchitecture in the lower auditory brainstem, namely the SOC and cochlear nucleus (CN). The goal of this study is to examine the cytoarchitecture of the human MSO in autistic brains and to compare these findings to those from control specimens. The MSO was chosen because of the important functional role it serves and its prominence within the SOC; the MSO is the largest and most densely populated nucleus in the human SOC.
2.
Results
2.1.
General features
The human MSO is first recognized in the rostral medulla oblongata as a prominent column of neurons within the SOC and extends rostro-caudally nearly 6.0 mm into the mid-pons. When viewed in the transverse plane, the MSO appears as a thin stack of neurons, tilted slightly towards the midline. The MSO cell column is approximately 300 μm wide and extends from anterior to posterior approximately 1000 μm. The human MSO contains nearly equal populations of fusiform and stellate neurons and these cells issue primary dendrites into the neuropil on both sides of the nucleus (Kulesza, 2007). The most notable feature of the human MSO is the strikingly consistent geometric arrangement of the constituent neurons. Both fusiform and stellate
Table 1 – Morphometric features of MSO neurons from control and autistic brains Age/sex Coronal sections Control (n = 43) Aut01 (n = 50) Aut03 (n = 117) Aut06 (n = 114)
Mass (g)
Area (μm2)
Perimeter (μm)
Major (μm)
Minor (μm)
Angle (degrees)
Circularity
91 ± 19 91 ± 45+ 87 ± 56+ 65 ± 57⁎+
.55 ± .11 .73 ± .11⁎ .63 ± .12⁎ .71 ± .14⁎
26/M 26/M 13/M 8/M
1520 1610 1470 1570
232 ± 60 252 ± 76 264 ± 80⁎ 158 ± 47⁎
72 ± 12 66 ± 14⁎ 73 ± 15 54 ± 13⁎
28 ± 6 23 ± 6⁎ 26 ± 6 20 ± 6⁎
11 ± 2 14 ± 2⁎ 13 ± 2⁎ 10 ± 2
Parasagittal sections Control (n = 58) 29/M Aut04 (n = 37) 32/M Aut05 (n = 115) 32/M
1514 1694 1510
201 ± 63 146 ± 70⁎ 105 ± 46⁎
73 ± 16 53 ± 17⁎ 49 ± 14⁎
27 ± 7 18 ± 7⁎ 18 ± 6⁎
9±2 10 ± 2 7 ± 2⁎
106 ± 23 111 ± 32+ 101 ± 45+
.49 ± .14 .66 ± .13⁎ .57 ± .15⁎
Fusiform and stellate neurons were pooled for this analysis; data are presented as average ± standard deviation. Mass refers to the overall brain mass as recorded by the Autism Tissue Program; major and minor refer to the length of the major and minor axes of the cell body. All morphological categories were analyzed with ANOVA; angle data were analyzed with ANOVA and the O'Brien test for homogeneity of variance. Asterisks indicate statistical significance by ANOVA, plus symbols indicate statistical significance in variance (p b 0.05). Comparing the angle data between controls and the autistic group reveals large standard deviations, further indicating the variability in these groups.
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groups is shown in Fig. 2. MSO neurons from AUT03 were statistically different from the control in terms of cell body area, minor axis and circularity (ANOVA, p b 0.05). The orientation angle measurement for AUT03 showed a significant difference in variance (Table 1). MSO neurons from AUT06 were statistically different from control in terms of cell body area, perimeter, major axis and circularity. The orientation angles from AUT06 were significantly different by ANOVA and by variance (p b 0.05). Notably, MSO neurons from all three autistic brains were statistically more circular than those from the control. Finally, there was statistical significance in the differences within the AUT groups in all morphological categories (ANOVA, p b 0.05).
2.3.
Parasagittal sections: two 32-year-old males
Examination of the MSO from the control specimen revealed fusiform and elongated stellate neurons that had their long axis oriented mainly in the transverse plane. In comparing brain mass to the control, the brain from AUT04 weighed 180 g
Fig. 2 – MSO neurons in autistic brains have a variable orientation. Shown in A is the distribution of orientation angles from control (dark black line) and three autistic brains (three gray lines) sectioned in the coronal plane. The vast majority of control MSO neurons have orientation angles between 90 and 110 degrees. The orientation angles from autistic MSO neurons have a much more variable distribution. Shown in B is the distribution of orientation angles from tissue sectioned in the parasagittal plane (control in black, autistic in gray). Compared to the control specimen, neurons from the autistic MSO have a more variable orientation.
neurons have long axes that are oriented perpendicular to the long axis of the nucleus (Fig. 1).
2.2.
Coronal sections: 26, 13 and 8-year-old males
Examination of the MSO from the control specimen (sectioned in the coronal plane) revealed relatively large, fusiform and elongated stellate neurons neatly arranged in a narrow column (as in Fig. 1). These neurons issued dendrites into the neuropil on both sides of the nucleus, consistent with previous reports (Moore and Moore, 1971; Moore, 1987; Kulesza, 2007). The MSO was identified in the rostral medulla/caudal pons in each of the autistic specimens (AUT01, 03 and 06) sectioned in the coronal plane. However, the neurons within the MSO cell column of the autistic specimens appeared morphologically much different from those in the control brains. In comparing the control and AUT01 (both 26-year-old males; Table 1), the brain from AUT01 weighed 90 g more than the control but MSO neurons were statistically similar between the two brains in terms of cell body area. However, there were statistically significant differences in cell body shape — perimeter, major and minor axes and circularity (ANOVA, p b 0.05). The mean of the orientation angle measurements was statistically similar between the control and AUT01, however a statistical difference was observed in the variance of these two groups (O'Brien Test for homogeneity of variance, p b 0.05). The distribution of orientation angles for all
Fig. 3 – MSO neurons in autistic brains display a significantly different morphology. Shown in A is a group of MSO neurons from the control specimen, sectioned in the parasagittal plane. These neurons are either fusiform (arrows) or stellate (arrowheads) and have a long axis oriented in the coronal plane. Shown in B is a group of MSO neurons from an autistic brain (AUT04). These neurons are much smaller and round and few of them show a preferential orientation; occasional stellate neurons are observed (arrowhead). C = caudal, P = posterior; the scale bar is equal to 15 μm.
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more and the brain from AUT05 weighed 4 g less. MSO neurons from the autistic specimens (AUT04 and AUT05) were significantly different in the majority of morphometric categories (Fig. 3; Table 1). Generally, MSO neurons in the autistic brains were smaller in terms of cell body area, perimeter and major axis and these neurons were significantly more round than in control. In terms of orientation angle, the means between the control and AUT groups were statistically similar (ANOVA, p N 0.05). However, comparing the angle measurements in Fig. 2 indicates that the orientation of MSO neurons in the AUT groups is more heterogeneous than control and these differences in variance were statistically significant (O'Brien test for homogeneity of variance, p b 0.05). Finally, in comparing AUT04 and 05, there were statistical differences in cell body area, minor axis and circularity (ANOVA, p b 0.05).
3.
Discussion
Although there are reports in the literature of alterations of neuronal cell body area, number and dendritic morphology in autism, this is the first report of quantitative alterations in neuronal morphology affecting not only cell body size, but also cell body shape and orientation. We observed a disruption in the morphology of MSO neurons in each of the five autistic brains studied. In the majority of cases examined, there was a significant difference in cell body area. However, the most consistent differences were observed in measures of cell body shape (circularity) and the variability in the orientation of the long axis. These results provide evidence suggesting that the MSO in the autistic brain is not morphologically equivalent to the MSO in non-autistic brains. This finding supports recent reports of abnormal auditory brainstem responses (ABR) in children with autism (Rosenhall et al., 2003; Kwon et al., 2007; Tas et al., 2007). Specifically, these reports (Rosenhall et al., 2003; Kwon et al., 2007; Tas et al., 2007) indicate an increased latency between peaks III and V of the ABR (peak III is associated with the SOC and peak V with the inferior colliculus). Disruption of the normal cytoarchitecture of the MSO (smaller caliber, shorter dendrites and smaller neurons with shorter projections) may contribute to this increased latency, at least in part. Despite our findings of abnormal MSO neuronal morphology in all five cases we examined, it remains possible that the differences we observed were caused by something other than autism. One potential cause of disruption within the auditory pathways would be deafness or other hearing deficiency. However, no such deficits were reported for the five autistic individuals we examined. It is also possible that the differences between the control and AUT06 were due at least in part to the age difference. One of the more likely candidates is medication; it is possible that the medications that these individuals were taking before or at the time of death could have impacted neuronal morphology. Examination of human MSO neurons in silver impregnated material provides evidence that the laminar arrangement of the MSO involves not only cell bodies, but also the dendritic arbors (Kulesza, 2007). Also, the inputs reaching the MSO from the cochlear nuclei are segregated on the dendrites: inputs from the ipsilateral cochlear nucleus innervate the lateral dendrites; inputs from the contralateral cochlear nucleus innervate the medial dendrites (Stotler, 1953; Perkins, 1973; Beckius et al.,
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1999). Moreover, in vivo recordings provide strong evidence of an anterior-to-posterior laminar arrangement of sound frequencies (tonotopic axis) in the MSO of the cat (Guinan et al., 1972). Thus, the morphology of MSO neurons and their strict geometric arrangement are intimately related to the organization of afferent information reaching the nucleus and ultimately to the role of the MSO in encoding low-frequency sounds and sound source localization. So, it seems plausible that the morphological differences discovered in the autistic MSO may indicate a disruption of the excitatory/inhibitory inputs reaching the nucleus and the output of the MSO to the inferior colliculus. This finding may help provide a cellular basis for some of the hearing abnormalities described in this disorder, namely deafness. Although we report significant differences in the morphology of MSO neurons between control and autistic individuals, the morphology of MSO neurons in autistic brains is somewhat variable. In fact, we observed significant differences in most morphological categories within the five autistic brains we examined. Rodier et al. (1996) reported on the cytoarchitecture of numerous brainstem nuclei from a single autistic brain. In this particular specimen, based on their description and figures, the SOC appears to be absent or greatly reduced; the MSO cell column is not discernable in their figures. In contrast, we were able to identify the MSO in all five of our autistic cases. These observations, taken together, suggest that there is great variability in the brainstem abnormalities in autism. Perhaps the brain examined by Rodier et al. was a brain that was severely affected by autism. This variability in morphology is likely related to function: individuals with more severe anatomical abnormalities are likely to have more noticeable functional deficits. Despite recent reports of anatomical differences in the autistic brain, many regions remain unexplored. It must be noted that the MSO is but one small component of the entire auditory pathway. Other brain regions, such as the cochlear nucleus or the inferior colliculi, were not examined. Thus, it remains possible that many other components of the auditory pathway, even other nuclei within the SOC are also affected. This is an attractive speculation, given the role of the SOC in forming the olivocochlear bundle, an efferent pathway that functions in gain control of the organ of Corti and the deficits observed in autism (increased thresholds to tones, hyperacusis and difficulty listening in background noise). Additionally, it remains possible that lower regions of the auditory brainstem are affected even more severely than the MSO. Many new questions have arisen from this study regarding the size and course of the major brainstem auditory tracts (i.e. trapezoid body, posterior acoustic stria, lateral lemniscus), the arrangement and complexity of dendritic arbors of MSO neurons and neurochemistry of these cells (i.e. are these cells using the appropriate neurotransmitters?). Although there is a tremendous literature on autism, the neurobiology of this disorder remains somewhat elusive. It is unclear what developmental or maturational process has gone awry, if any. Have these MSO neurons been denied the appropriate growth factors, or have they not been contacted or innervated by the correct number or population of growth cones, or has there been an over-pruning of inputs later in development, perhaps even postnatally? Obviously many questions
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remain about the direct cause of autism and the time period during which it develops. Even more intriguing is why some brain regions are affected and others appear normal. Although data are accumulating about the functional deficits in autism, the anatomical deficits are not completely documented. If we are ever to completely understand this disorder, we must be fully aware of all the brain regions involved and to what degree they are affected. An in-depth understanding of the neuroanatomical changes that occur consistently in autism will be essential in understanding this disorder and may, in the future, aid in the development of instructional or therapeutic measures for individuals with this disorder.
4.
Experimental procedures
4.1.
Tissue sections
Histological slides from autistic and age-matched control brains were kindly loaned by the Autism Tissue Program (ATP; http://www.brainbank.org). Celloidin embedded brains were cut at a thickness of 200 μm and stained with cresyl violet. This report is based on data from five autistic cases (with no reported hearing abnormalities) and two agematched control cases. Specimens were divided into two groups based on plane of section (since neuronal morphology varies depending on plane of section). The first group includes specimens sectioned in the coronal plane; this group includes tissue from three individuals diagnosed with autism (AUT01 [26year-old male], AUT03 [13-year-old male] and AUT06 [8-year-old male]) and one control (26-year-old male). The second group includes specimens sectioned in the parasagittal plane; this group includes two individuals diagnosed with autism (AUT04 [32-year-old male] and AUT05 [32-year-old male also diagnosed with Fragile X]) and one control (29-year-old male). The series of sections provided by the ATP were incomplete; no specimen included the entire extent of the MSO. As a result, estimates of neuronal number and packing density could not be reliably calculated.
4.2.
Morphometric analysis
Tissue sections were examined with an Olympus BX45 microscope and photographed with an Olympus DP12 digital camera. For morphometric analyses, sections were randomly selected throughout the rostro-caudal extent of the MSO and only neurons with visible nucleoli and cell bodies that were completely within the tissue section were included (to prevent the inclusion of incomplete neuronal profiles). Cell bodies were traced while focusing (to most accurately determine the cell body contour) with the aid of a camera lucida attachment (Olympus; using a 40× objective with a final on paper magnification of 675×) and these tracings were digitized into jpeg format using a flatbed scanner. The digitized tracings were imported into ImageJ software (available at http://rsb.info.nih. gov/ij/) and measurements of cell body area, perimeter, length of the major and minor axis, circularity and orientation of the long axis were made using the “Analyze” feature. ImageJ was calibrated to a standard scale bar for each trial. An index of
circularity was calculated for each soma using the following equation: h i Circularity ¼ 4π⁎ Area=Perimeter2 This calculation yields an estimate of soma shape independent of size (Yin et al., 1990). Using this formula a perfectly circular soma yields a value of “1”, while increasingly irregular or elliptical profiles yield decreasing values. Measurements were also made of the orientation (angle) of the long axis of individual neurons using ImageJ. For tissue sectioned in the coronal plane, angle measurements were made in reference to the vertical midline of the tissue. So in any section, neurons with a long axis parallel to midline will have an angle measurement of 90°; and neurons with a long axis parallel to horizontal will have a measurements of 0°. For consistency, angle measurements were taken only from neurons on the right side of the brain. Statistical comparisons of all morphometric data were made using the data analysis feature in Excel (Microsoft) or JMP (SAS).
Acknowledgments This work was supported in part by a grant from the Deafness Research Foundation. The authors would like to thank Dr Jerzy Weigel and the Autism Tissue Program for kindly providing the tissue and the LECOM Research Collaborative for their continued support. We would also like to thank Dr Jack Caldwell for critically reading an earlier version of this manuscript.
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