*Edited by Ghousia Begum*

This is a good book on upcoming areas of Ecotoxicology. The first chapter describes genotoxicity of heavy metals in plants. The second chapter offer views on chromatographic methodologies for the estimation of mycotoxin. Chapter three is on effects of xenobiotics on benthic assemblages in different habitats of Australia. Laboratory findings of genotoxins on small mammals are presented in chapter four. The fifth chapter describes bioindicators of soil quality and assessment of pesticides used in chemical seed treatments. European regulation REACH in marine ecotoxicology is described in chapter six. X-ray spectroscopic analysis for trace metal in invertebrates is presented in chapter seven. The last chapter is on alternative animal model for toxicity testing. In conclusion, this book is an excellent and well organized collection of up dated information on Ecotoxicology. The data presented in it might be a good starting point to develop research in the field of ECOTOXICOLOGY.

ISBN 978-953-51-0027-0

Ecotoxicology

Photo by Yuriyve / iStock


*3.1. E*ff*ect of the Solid*/*Liquid Ratio on Extraction Yield and Extract Antioxidant Activity*

The influence of the solvent to walnut leaves ratio on the extraction yield and extract FRAP antioxidant activity is shown in Table 1. The extraction yield was positively influenced by decreasing S/L from 1/5 to 1/7.5. However, a further decrease to 1/10 didn't improve the extraction yield significantly. On the other hand, the extract FRAP antioxidant activity increased with decreasing the S/L ratio in the whole range analyzed. These results were in agreement with previous research on the extraction of phenolic compounds from various plant materials [7,16,17] and can be explained by analyzing the concentration gradient of phenolics between walnut leaves and the solvent. The lower the S/L ratio, the greater the concentration gradient and consequently the extraction rate. Therefore, a S/L ratio of 1/10 was selected for subsequent experiments on analyzing the impact of temperature, time, and ethanol concentration on the extraction of phenolic compounds from walnut leaves. A greater decrease in the S/L ratio was not considered since it would mean not only a higher solvent consumption but also a higher energy cost in the extraction and solvent recovery stages.


**6. Conclusions**

In this paper, we have presented a coupling integral method in order to model thin magnetic and conductive regions which can be coupled with an external electric circuit. This coupling enables the model of conductors with complex shapes, and various skin effects across the thickness of the thin shell are taken into account. Two numerical examples have been presented and the results highlighted the accuracy of the solution provided by our proposed method. In our suggestions, the problem dense matrix and the full memory need could be solved with the compression algorithms.

**Author Contributions:** Conceptualization, Methodology, Validation: T.L.-D. and G.M.; Investigation: T.L.-D.; Data Curation: T.L.-D.; Writing—Original Draft Preparation, Writing—Review & Editing: T.L.-D. and G.M; Funding Acquisition: T.L.-D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ministry of Education and Training, Vietnam, under grant number B2018-BKA-11-CtrVL.

**Conflicts of Interest:** The authors declare that there are no conflicts of interest regarding the publication of this paper.


**The Main Components Content, Rheology Properties and Lipid Profile of Wheat-Soybean Flour**

Nada Nikolić and Miodrag Lazić *University of Niš, Faculty of Technology, Leskovac Serbia*

#### **1. Introduction**

80 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

Oxen, P., & Knorr, D. (1993). Baroprotective effect of high solute concentration against

Park, K. H., Kim, Y. M., & Lee, C. W. (1988). Thermal inactivation kinetics of potato tuber

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Riaz, M. N. (1988). Soybeans as functional foods. *Cereal Foods World*, 44, pp. 88–92, ISSN:

Seyderhelm, I., Boluslawski, S., Michaelis, G., & Knorr, D. (1996). Pressure induced

Steinke, F. H., Waggle, D. H., & Volgarev, M. N. (1992). *New Protein Foods in Human Health: Nutrition, Prevention and Therapy*. CRC Press, Inc., ISBN:<PHONE_NUMBER>, Florida Svensson, S. G., & Eriksson, C. E. (1974). Thermal inactivation of lipoxygenase from peas

Ueno, S., Shigematsu, T., Watanabe, T., Nakajima, K., Murakami, M., Hayashi, M., & Fujii, T.

Utsumi, S., Matsuda, Y., & Mori, T. (1998). Structure-functions relationships of soy proteins.

Van Loey, I. A. M., Ludikhuyze, L. R., & Hendrickx, M. E. (1999). Soybean lipoxygenase

Wang, R., Zhou, X., & Chen, Z. (2008). High pressure inactivation of lipoxygenase in soy milk and crude soybean extract. *Food Chemistry*, 106, 603–611, ISSN: 0308-8146 Whitaker, J. R. (1972). *Principles of Enzymology for the Food Sciences*. Marcel and Dekker,

Wolf, W. J., & Cowan, J. C. (1975). *Soybeans as a Food Source*. CRC Press, Inc., ISBN:

*Agricultural and Food Chemistry*, 1999, 47, pp. 2468–2474, ISSN: 0021-8561 Ven, C., Matser, & Berg, R. W. (2005). Inactivation of soybean trypsin inhibitors and

*Food Chemistry*, 125, pp. 423–429, ISSN: 0308-8146

223, ISSN: 0023-6438

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0021-8561

0146-6283

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2902, ISSN: 0021-8561

*Chemistry*, 58, pp. 1208–1213, ISSN: 0021-8561

*Chemistry*, 2005, 53, pp. 1087–1092, ISSN: 0021-8561

Inc., ISBN:<PHONE_NUMBER> , Lancaster

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0878191127, Cleveland

inactivation of Rhodotorula rubra. *LWT-Food Science and Technology*, 26, pp. 220–

lipoxygenase. *Journal of Agricultural and Food Chemistry*, 36, pp. 1012–1015, ISSN:

pressure effects on immunoreactivity and nutritional quality of soybean products.

preserved with high pressure. *Journal of Agricultural and Food Chemistry*, 48, pp.

inactivation of selected food enzymes. *Journal of Food Science*, 61, pp. 308–310,

(*Pisum sativum* L.). III. Activation energy obtained from single heat treatment experiments. *LWT-Food Science and Technology*, 7, pp. 142–144, ISSN: 0023-6438 Tangwongchai, R., Ledward, D. A., & Ames, J. M. (2000). Effect of high-pressure treatment

on lipoxygenase activity. *Journal of Agricultural and Food Chemistry*, 48, pp. 2896–

(2010). Generation of free amino acids and γ-aminobutyric acid in water-soaked soybean by high-hydrostatic pressure processing. *Journal of Agricultural and Food*

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inactivation by pressure at subzero and elevated temperatures. *Journal of*

lipoxygenase by high-pressure processing. *Journal of Agricultural and Food*

Soybean (*Glycine max* (L.) Merr.) is a species of legume, native to a Eastern Asia and an important global crop, today. Soybean is rich in high quality proteins, contains essential amino acids, similar to those found in meat, minerals such as Fe, Zn, Cu, Mn, Ca and Mg as well as phytic acid. The bulk of seed soybean proteins contains albumins and globulins as major components, but there are minor, undesirable components such as inhibitors of trypsyn and chymotrypsin, and sugar-binding lecitins. The inhibitors and lecitins are generally inactivated by heat treatment. A new immunochemical methods can be used for quantitative detection of soybean proteins and production of healthful foods (Brandon & Friedman, 2002). The soybean lipid contains a significant amount of unsaturated acids: linolenic acid, known as omega-3 acid, linoleic, -linolenic and arachidonic acid, known as omega-6, and oleic acids known as omega-9 acid and are very important in human nutrition (Liu, 1997). The soybean lipid also contains saturated acids: palmitic and stearic acid (Bressani, 1972; Olguin et al., 2003; Bond et al., 2005), as well as tocopherols (Ortega-Garcia et.al., 2004, Yoshida et al., 2006). These soybean components make the products with soybean have higher nutritional value.

#### **1.1 Soybean in bread making industry**

In the bread making industry, the soybean is used with the aim to increase the bread protein value and decrease carbohydrate value. In ordinary white bread protein content ranges from 8 to 9% and by including soybean, the protein content can be made up to 16% (Ribotta et al. 2010) and at the same time, the dough and bread are richer with lipoxygenase enzymes preparation which can make the dough physical properties better. The fact is that bread made with soybean costs less and it is especially important in countries where wheat is not a major domestic crop. Bread and products with higher protein content and lower carbohydrate content are more suitable for use in some diets than bread and products formulations currently used (Mohamed et al., 2006). As the main protein component in white bread is gluten, a component which causes celiac disease, the usage of soy is useful for decreasing the gluten content in bread. A portion of 0.3 to 5% of soybean flour portion is usually added (Auerman, 1979), but rational addition for increasing bread protein value is 20-30% of soybean flour. Besides whole soybean flour, different soybean products can also

The Main Components Content,

properties were found.

**2. Experimental 2.1 Soybean seed**

**2.2 Chemicals**

additives.

**2.4 Flour analyses**

Chemicals, Germany).

Rheology Properties and Lipid Profile of Wheat-Soybean Flour 83

wheat flour with other changed these properties. Ribotta et al. 2005, presented data about effect of soybean addition on farinographic properties such as water absorption (WA), dough development time (DT), dough stability (Dst) and dough degree of softening (DSf). The present work has been undertaken with the objective to investigate the effect of whole soybean seed flour (»full-fat soy flour«) portion of 3 to 30% on dough farinographic, but extensographic and amylographic properties, too. Based on the composition of wheat and soybean flour lipids, the aim is to obtain lipid composition in wheat-soybean flour mixtures, and compare it to wheat flour only, with an emphasis on content of total saturated fatty acids (TS), total monounsaturated fatty acids (TMUS), total polyunsaturated fatty acids (TPUS) and total unsaturated fatty acids (TU). In order to value wheat-soybean flour mixtures, they were classified into groups by using statistical analysis and Euclidean distances and the correlations between content of some lipid components and rheological

The whole soybean seeds (*Glycine max* L.) cultivars ZP Lana, grown in Serbia in summer of 2006 were used. The seeds were purchased in local store "Green Apple" (Leskovac, Serbia) and milled. The particle size was determined by method of insemination via riddles with gaps size from 0.315 to 3.15 mm. The overall particle size was determined using equations:

Chemicals used for oil extraction were high quality chemicals (Centrohem, Serbia) and for HPLC and GC analysis they were analytical grade (Riedel-de Haën, Honeywell Specialty

The wheat flour, Kikinda Mill, Serbia (WF) was bought from the local market. The soybean flour (SF) as »full-fat soy flour« was obtained by soybean seeds milling (IKA Model M120), to an overall particle size ( *dsr* ) of 0.4 mm. Quantities of 291, 285, 270, 240 and 210 g of wheat flour and 9, 15, 30, 60 and 90 g of soybean flour, respectively, were used to make flour mixture with 3, 5, 10, 20 and 30 % (w/w) soybean flour portion, without adding

Flour protein content was determined by the Kjeldahl method (Nx5.95). The moisture content was determined by Scaltec SMO 01 (Scaltec instruments, Germany) instruments: flour (5 g) was put into the disk plate analyzer, dried at 110oC to a constant weight, and the

where *i d* is weight of fraction with appropriate particle size in %, and

where *i*1 *d* is bottom riddle gap size and *i*1 *d* is upper riddle gap size.

**2.3 Wheat and soybean flour and flour mixtures**

*sr i i* 100 / *d d* / *d* (1)

*di* (*di*1 *di*1)/ 2 (2)

be used: defatted soy flour (Mashayekh et al., 2008), physically modified soy flour (Maforimbo et al., 2008), soy flour and durum wheat flour mixture (Sabanis & Tzia, 2009), commercial soy protein isolate (Roccia et al., 2009), diffrerent kinds of soy protein powder (Qian et al., 2006) and part of soy seeds such as a hulls (Anjum et a., 2006). Based on these investigations results, different bread formulations are defined, and soy is used in portion up to 20%. When soy flour in wheat flour was to a level of 10% and in durum wheat flour up to 20%, the produced bread was without any negative effects in quality attributes such as colour, hardness and flavour, promising nutritious and healthy alternative to consumers (Sabanis & Tzia, 2009). By investigating the effect of defatted soy flour on sensory and rheological properties of wheat bread, Mashayekh et al. 2008, concluded that adding 3 and 7% defatted soy flour gives as good a loaf of bread as the 100% wheat bread and acceptable consumer attribute with rheological and sensory characteristics. Adding small quantity of soy protein powder of 3% to wheat flour did not change the sensory properties of bread and a large quantity of soy flour adding, exceeding 7%, can lead to stickiness and leguminous flavour (Roccia et al., 2009). The results of investigation (Anjum et al., 2006) of soy hulls usage showed the content of 4.5% soy hulls combined with wheat flour is acceptable and suitable level by the consumers.

#### **1.2 Wheat-soybean bread manufacturing**

The manufacture of bread from flour without gluten represents considerable technological difficulties (Jong et al., 1968; Schober et al., 2003) because gluten is the most important structure forming protein for making bread (Gujural et al., 2003; Moore et al,*.* 2004) and by using appropriate soy-wheat flour mixture these difficulties can be avoided. When soy was added to wheat, the soy globulins interact with wheat gluten proteins forming aggregates of high molecular weight. As reduction-reoxidation treatment facilitated the interaction of glutenin subunits and soy proteins (11S subunits), interaction probably occurs through the oxidation of SH groups (Maforimbo, 2008). Investigations of the changes in glutenin macro polymer content, protein composition and free sulfhydryl content showed that active soy flour decreased glutenin macro polymer content due to gluten depolymerization and glutenin macro polymer content increased by inactive soy flour because soy proteins became insoluble and precipitated together. Soy proteins were associated to wheat protein through physical interaction and covalent and non-covalent bonds during mixing and resting and these interactions produced large and medium-size polymers. This increased solubility of insoluble gluten proteins, producing a weakening of the gluten network (Perez et al., 2008) and decreasing availability of water to build up in gluten network (Roccia et all. 2009). Physicochemical status of soy protein in the product had a great influence on how wheat-soy proteins will interact (Perez et al., 2008). Incorporation of soy proteins changes the rheolocical and bread properties. The investigations showed that adding soy protein powder depresses loaf volume, gives poor crumb characteristics and decreases acceptability by consumers (Qian et al., 2006.) Ribotta et al. 2010. tested different additive combinations for improved bread quality obtained from soy-wheat flour in ratio of 90:10 w/w and found that the combination with transglutaminase showed a major improving effect on dough rheological properties and crumb uniformity.

#### **1.3 Aims of investigations**

Dough rheological properties have great relevance in predicting the mixing behaviour, sheeting and baking performance (Dobraszczyk & Morgenstain, 2003) and supplementation wheat flour with other changed these properties. Ribotta et al. 2005, presented data about effect of soybean addition on farinographic properties such as water absorption (WA), dough development time (DT), dough stability (Dst) and dough degree of softening (DSf). The present work has been undertaken with the objective to investigate the effect of whole soybean seed flour (»full-fat soy flour«) portion of 3 to 30% on dough farinographic, but extensographic and amylographic properties, too. Based on the composition of wheat and soybean flour lipids, the aim is to obtain lipid composition in wheat-soybean flour mixtures, and compare it to wheat flour only, with an emphasis on content of total saturated fatty acids (TS), total monounsaturated fatty acids (TMUS), total polyunsaturated fatty acids (TPUS) and total unsaturated fatty acids (TU). In order to value wheat-soybean flour mixtures, they were classified into groups by using statistical analysis and Euclidean distances and the correlations between content of some lipid components and rheological properties were found.


**Meet the editor**

Dr Sadaf Naz obtained her PhD from University of the Punjab, Lahore, where she initiated one of the first studies on determining the genetic underpinnings of hearing loss in Pakistan. After completing different postdoctoral research projects at National Institute on Deafness and other Communication, Disorders, National Institutes of Health, USA, she attained a faculty position at School of

Biological Sciences at University of the Punjab, Lahore. Dr. Naz's laboratory studies molecular genetics of different human disorders and the major focus of her research group is identification of genes involved in moderate to severe deafness.

Contents

**Preface IX**

Josefina Gutierrez

Takahiro Tamesue

Chapter 1 **Technology for Hearing Evaluation 3**

**for Hearing Screening 25**

Chapter 3 **A Prediction Method for Speech Audibility**

Gleich Otto and Strutz Jürgen

Chapter 5 **Families of Children with Hearing Loss**

Bolsoni-Silva Alessandra Turini and Rodrigues Olga Maria Piazentin Rolim

Chapter 7 **Hearing Loss in Minor Head Injury 135**  Lingamdenne Paul Emerson

**Part 2 Childhood Hearing Loss 93**

Zerrin Turan

**Part 3 Injuries & Traumas 133**

Chapter 2 **Contralateral Suppression of Otoacoustic Emissions:**

**Taking Account of Hearing Loss Due to Aging Under Meaningless Noise 51**

Chapter 4 **The Mongolian Gerbil as a Model for the Analysis of**

**and Parental Educational Practices 95**

Chapter 6 **Early Intervention with Children Who Have a Hearing Loss: Role of the Professional and Parent Participation 117**

**Working Towards a Simple Objective Frequency Specific Test**

Nikolaus E. Wolter, Robert V. Harrison and Adrian L. James

**Peripheral and Central Age-Dependent Hearing Loss 67**

**Part 1 Hearing 1**


3.3.1. Spinocerebellar Ataxia Type 7

Spinocerebellar ataxia type 7 (SCA7) is an autosomal-dominant neurodegenerative syndrome that outcomes from polyglutamine expansion of ataxin 7 (ATXN7). Remarkably, although ATXN7 has a widespread expression in SCA7 patients, the pathology primarily hits the cerebellum and the retina [124]. A recently published paper suggested that RELN might be a formerly unidentified factor accountable for the tissue specificity of SCA7 [125].

#### 3.3.2. Autism and the Heterozygous Reeler Mouse

The disorders of the autistic spectrum (ASD), which are characterized by social, behavioral, and language insufficiencies, comprise Asperger syndrome, autism, and pervasive developmental disorder-not otherwise specified (PDD-NOS). Less than 20% of these disorders, acknowledged as "syndromic autism", derives from monogenetic diseases, most commonly fragile X syndrome and tuberous sclerosis. The remaining 80% of ASD cases are classified as "non-syndromic autism" and are widely investigated to find candidate genes that may contribute to pathology [126].

#### Genetics

At present autism cannot be considered, strictly speaking, a genetic disease, as one or more causative gene(s) has (have) not been found yet. The first gene association study implicating *RELN* in autism dates to 2001 [127]. However, subsequent gene population surveys yielded contrasting results [128–131]. Nonetheless, a more recent meta-analysis showed that at least one single nucleotide polymorphism (SNP) in *RELN* could be significantly associated with the risk of autism [132]. Therefore, results of SNP analysis appear to be compatible with the idea that heterozygous mutations in *RELN* may contribute to the onset of the disorder. Genetic studies on autism led to two main outcomes: 1. the more predominant existence of rare or de novo inherited mutations of a number of genes in autistic patients; 2. the discovery of certain common gene variants that contribute to the risk of autism but are also present, albeit at lower frequency, in the normal population [133]. When more than two de novo mutations occur in a gene, the latter becomes a very likely causative candidate of a disorder. There are four unique documented de novo mutations of *RELN* associated with autism [134–136], thus implicating *RELN* as a possible cause of ASD. However, although nonsense mutations are more frequent in autistic patients than in controls after whole-exome sequencing, there is not a striking gross increase of de novo mutations in the former [135]. To study autism heritability, one can also employ a different approach that distinguishes total narrow-sense heritability from that due to common gene variants. By this method, it was concluded that narrow-sense heritability of autism is ∼52.4%, and that the main contribution heritability was due to common gene variants, whereas rare de novo mutations contribute only for about 2.6% of cases, but substantially influence individual liability to the condition [137]. Thus, *RELN* may primarily have a role in the individual *predisposition* to manifest autism rather than being one of the contributory causes of the disorder.

Further support for a RELN involvement in autism derived from the detection of reduced expression of the *RELN* transcript and protein in autistic individuals. Decreased RELN levels were apparent in the superior frontal cortex [10] and cerebellum of autistics as compared to controls [10,11,138]. In these areas, the levels of *RELN* mRNA were lower, as was the *DAB1* transcript, whereas *VLDLR* mRNA levels augmented.

#### Imaging

Imaging findings in autism have been recently reviewed [139]. Numerous observations join to prove that there is an atypical development of the brain in autistic children. Early cross-sectional studies demonstrated that the brain of these children had a higher volume than that of regularly developing subjects. However, growth curves in the two groups eventually met at later childhood. More specifically, in the 6–35-year interval, there was an initial period of brain overgrowth, and then growth slowed down or even stopped during early and late infancy to which a phase of fast reduction of the brain volume eventually followed [140]. Neuroimaging data also indicated that differences in the brain of autistic people started to be detectable within the first two years after birth, *before* clinical symptoms became obvious. There are conflicting views about the probability that an accelerated growth rate of the brain in this postnatal window goes together with the occurrence of early neurodevelopmental perturbations [139]. In relation to this, it is relevant that we still do not know when the initial neuropathological signs of autism occur, also from the paucity of studies on autistic children during the first year of life.

The mechanisms at the basis of the abnormal growth of the autistic brain are also unclear. Although most imaging studies have focused onto the gray matter of the cerebral cortex, there are data indicating that an increased amount of cerebrospinal fluid in the subarachnoid space [141] and/or a greater volume of the white matter [142] occurred in parallel to the enlargement of the autistic brain. As regarding the cerebral cortex, its surface, but not thickness, increased in the autistic brain [143].

To summarize, that an initial brain overgrowth may be a reliable biomarker for autism remains highly questionable. Thus, it seems more profitable to focus onto regional brain structural differences in a more effective search for new neuroanatomical findings of clinical relevance [139].

Before entering the description of regional MRI investigations in autism, it is important to stress that, at present, there are no specific and/or causative objective findings for the condition, but, instead, the very same regions altered in autism may be interested in other psychiatric conditions.

The individual constituents of the neural circuitries causal of ASD are well defined. They include regions of the fronto-temporal, fronto-parietal, and dorsolateral prefrontal cortex (PFC); parts of the limbic system; the fronto-striatal circuitry, and the cerebellum. Neuroimaging studies on these regions have employed different approaches such as the definition of a region-of-interest (ROI), voxel- or vertex-vise methods. Traditional ROI studies have reported atypical findings in brain areas that participate to social cognition such as the medial PFC, the anterior cingulate cortex (ACC), the inferior frontal cortex, the superior temporal sulcus, the amygdala, and the anterior insula.

The cerebellum was larger than in controls in several MRI studies on autistic patients older than three years [144], but not in younger children [143]. Differently from the cerebellum, the size of the vermis was smaller [145–147] or larger [146] or did not display any relevant difference [147], and such discrepancies possibly depend from the different clinical presentations of the condition [147,148]. It is also unclear whether there are differences in size of individual vermal lobules, as claimed by some authors [145], but not others [147]. Similarly, there were no differences between cerebellar hemispheres in one study [147], whereas another group has found the hemispheric size as the only significant structural dissimilarity between verbal and nonverbal subjects [149].

The still fuzzy picture emerging from the imaging studies onto the autistic brain makes it very difficult to compare the human and mouse data in the search for common biomarkers. To our knowledge, there are only two MRI studies on the brain of the heterozygous *Reeler* mouse. In the first, Badea and co-worker [29] reported that the total volume of the brain, the ventricular volume and the hippocampal volume correspondingly raised of about 6%, 82%, and 7% compared to normal control mice. However, after statistical analysis, they showed that these volumes were like those of *reln*+/+ normal mice. They also measured the areas of different parts of the brain in comparison with wild-type mice and found no differences in hippocampus and cerebellum, but an enlargement of the lateral ventricles. A more recent paper confirmed the ventricular enlargement, but found a reduction of the cerebellar volume, whereas the volume of the motor cortex as well as its thickness was unchanged [150]. Therefore, given the paucity of data in mouse and the still unclear MRI pattern in human autistic subjects, one can only conclude that, at present, the cerebellum could be a part of the brain deserving further imaging investigations for translational purposes.

## Histopathology

A series of histological alterations of the whole brain occur in the autistic brain. In the first histological surveys, the only cortical area showing qualitative structural abnormalities was the ACC that, in autistic patients, lacked architectural refinement and had only a coarse lamination [151]. However, in the following decades substantial amounts of data have been collected and the list of cerebral structures displaying histopathological changes in ASD has grown substantially to include a series of cortical regions, the amygdala, the cerebellum and the brainstem, see e.g., [152,153]. Below we will briefly summarize the most significant histopathological findings in human patients and compare them to those in the heterozygous *Reeler* mouse. However, the interpretation of both human and mouse finding needs often much caution, because not all studies used sound quantitative approaches and/or proper stereological procedures.

#### a) Changes Affecting the Whole Brain

The diffuse alterations observed in the brains of autistic subjects at postmortem included cortical dysplasia and neuronal heterotopia, with the formation of aggregates of neuronal cell bodies in anomalous positions [154]. Other alterations, i.e., differences in size of the neuronal nucleus and perikaryon, occurred at the cytological level. These differences started being evident in young children and became more apparent in adults, but then tended to re-equilibrate with time [155]. Remarkably, there might be some compensations between different areas, as in some parts of the brain neurons were bigger, but smaller in others.

In the autistic brain there was also an increase of the neuropil extension in certain but not all cortical areas that have been investigated so far [156]. It is unclear which neuropil component(s) is (are) responsible of these volumetric variations. Fewer dendrites were, in fact, immunostained for microtubule-associated protein 2 (MAP2) in the PFC [157] and a reduction of dendritic spines was reported in hippocampus [158], but other studies reached completely opposite conclusions after examination of the pyramidal neurons from layers 2 and 5 in the frontal, temporal, and parietal cortex [159]. The issue of dendrite and dendritic spines density is quite important in the general framework of this discussion, because these parameters have been widely investigated, primarily aiming to validate the heterozygous *Reeler* mouse as a translational model.

Alterations in neuronal differentiation and migration may also occur in the autistic nervous system and, thus, the consequences of a dysregulation of these processes may be at the basis of whole brain changes in autism [154]. In spite of this, there are only a few investigations on the expression of RELN in the brain of autistic patients and, after quantitative analysis, there was no alteration in the density of layer 1 RELN+ neurons in the superior temporal lobe of the autistic brain, although these neurons represent about 70% of the total layer 1 population [160].

#### b) Brain Regional Changes

#### Forebrain

Most of the histopathological observations on the brain of autistic patients focused on the cerebral cortex and hippocampus and, broadly speaking, alterations were almost exclusively restricted to neurons. The parameters considered have been size, number, and density of the different neuronal populations, often in relation to the cortical layers or the hippocampal subfields. A point of attention in considering these studies is that, in several cases, comparative brain volume evaluations between autistic patients and controls are missing, while they are, instead, essential to settle whether modifications in cell density reflect true differences in total cell counts.

#### Cerebral Cortex

The autistic pathology affects several regions of the cerebral cortex.

The PFC, which plays a major role in cognitive control, displayed a general overgrowth with an increase in the number of neurons, whereas glial cells were apparently unaffected. Among the GABAergic interneurons, there was a numerical increase of the PV-immunoreactive chandelier neurons, whereas the calbindin- and calretinin-expressing neurons were unaltered [153,161–163]. However, after qRT-PCR, the levels of the RELN and GAD67 mRNAs diminished in the post-mortem PFC from autistic patients in comparison to healthy controls [164].

In the inferior frontal cortex, changes affected the small-sized pyramidal neurons that did not display numerical alterations but were of smaller size [163].

The fusiform gyrus, which intervenes in facial recognition and social interactions, had a reduced neuronal density in layer 3, whereas neurons in layers 2, 5, and 6 were less numerous, being also smaller in layers 5 and 6 [165], but these alterations were not confirmed in [166].

In the frontoinsular cortex and ACC, both intervening in emotional regulation and self and others awareness, lamination was rudimental. In the former, von Economo neurons (VENs) of layer 5 increased in number [167–169], whereas in ACC neuronal density augmented in layers 1–2 of area 24a of the left hemisphere but diminished in layers 5–6 of area 24c; size, instead, diminished in all layers of area 24b [151,170].

The anterior midcingulate cortex also displayed a numerical increase of VENs, as well as of the pyramidal neurons of layer 5 that, however, were of smaller size [171].

Lastly, the entorhinal cortex, which has a role in memory, navigation and perception of time, displayed characteristic terminal swellings, referred to as spheroids [172] that were also observed in all hippocampal subfields (see below).

Another issue of interest is the possibility that there are alterations in the minicolumnar organization of the cerebral cortex in early age onset autism [156], as it might be the case in *Reeler* mice. Specifically, it appeared that minicolumns were smaller, more numerous and with lower neuronal density in several cortical areas of autistic (and Asperger's syndrome) patients, although these observations still need to be confirmed in full [156]. Under this perspective, it may be useful to here recall some of the results on the localization of the neural cell adhesion molecule 2 (NCAM2) in the *Reeler* mouse because the molecule has also been proposed as a predisposition gene for the development of autism [173]. In mutants, NCAM2 immunopositive and negative patches formed a mosaic filled with dendritic aggregates originating from two different populations of neurons in a fashion suggestive of a minicolumnar organization [174,175]. However, one must consider these findings with much caution, as that minicolumns are indeed the fundamental modular units of neocortical organization is currently still a matter of debate, see e.g., [176] for review.

Whereas the human cerebral cortex has been widely investigated in autism, investigations on the cerebral cortex of the heterozygous *Reeler* mouse have been relatively few but reported a reduction in the levels of GAD67 [53,177] like that observed in humans. Since the studies on hippocampus led to highly comparable results in the two species, it would be of importance to undertake rigorous investigations on the number and density of the cortical neurons in *reln*+/− mice, with attention to the different neurochemical populations of inhibitory interneurons. The results of these studies will be relevant, from one side, to validate the mouse model and, from the other, to confirm some numerical observations in humans that, as mentioned at beginning of this section, need validation using approaches more reliable than those often employed at histopathology.

#### Hippocampus and Amygdala

In humans, beside the widespread occurrence of spheroids, other general changes in hippocampus [151,158,172,178,179] consisted in a reduction of neuronal size and dendritic arbors, and these smaller neurons appeared to be more densely packed. There were also a series of specific modifications affecting the excitatory pyramidal neurons that were more numerous in CA1, but less abundant in all other adjacent hippocampal regions. The GABAergic inhibitory interneurons, instead, displayed a higher density, specifically the calbindin-immunoreactive neurons in the dentate gyrus, the parvalbumin-immunoreactive neurons in CA1 and CA3, and the calretinin-immunoreactive neurons in CA1 [151,158,172,178,179].

In the heterozygous *Reeler* mouse the hippocampus displayed reduced levels of GAD67, [53,177] that could be somewhat restored after stereotaxic injections of Reln [67,180]. These experiments indicated the existence of a causal link between the decrease in GAD67 expression and Reln haplodeficiency. In keeping with such a possibility, Reln supplementation could, at least partly, reverse such a decrease. Other experiments, in line with this interpretation, have confirmed that the decrease in the levels of GAD67 in heterozygous mice can be overturned, e.g., after administration of nicotine, which reduces the GAD67 promoter methylation and increases its transcription [177].

In heterozygous mice, pyramidal neurons displayed a reduction in the average length and width of their apical and basal dendritic spines [52], consistent with the decrease in the spread of the dendritic arbor of the same population of neurons in humans. Additionally, Reln supplementation was effective in promoting a full (apical) or partial (basal shaft) spine recovery [180]. These morphological observations are in line with a previous report showing that, in the forebrain, spines were hypertrophic in mice conditionally overexpressing Reln [66]. At electrophysiological recordings CA1 pyramidal neurons displayed reduced spontaneous inhibitory postsynaptic currents [181], an observation that was fully coherent with the reduction of the inhibitory input from the GABAergic interneurons observed histologically.

Synaptic plasticity is fundamental for hippocampal function. In CA1 of heterozygous mice, LTP was impaired [182] as well as long-term depression (LTD) [181], which returned to normal levels after the administration of Reln [180]. Additionally, in *reln*+/− and *reln*−/− mice, post tetanic potentiation (PTP), a form of short-term plasticity that depends on neurotransmitter release, was reduced in CA1 [180,183] and could be reversed by Reln [180].

Collectively these data indicate that the experimental administration of the glycoprotein was able to reverse the morphological, neurochemical, and physiological hippocampal deficits consequent to a reduction of brain Reln. Translationally, they are very important because they offer some cues for further investigations onto the autistic brain. It would be of interest to map the post-mortem distribution of hippocampal RELN in patients compared to healthy controls, to ascertain whether the pattern of immunoreactivity will be consistent between humans and mice.

In the autistic patients, several studies reported that the amygdala, which is involved in emotional learning, increased in size and displayed an augmented density of neurons within the medial, central, and cortical nuclei [151,184–187]. Neurons were, however, less numerous, although numerical variations could be age dependent. To our knowledge, Boyle et al have investigated in detail the amygdala of the homozygous *Reeler* mouse with a marker-based phenotypic approach [188], but there are no data on heterozygous mice.

#### Cerebellum

Analysis of cerebellar alterations in autism has attracted many efforts of the basic researchers and clinicians. The most consistent anatomic findings in autistic patients were a reduction in size of certain lobules of the cerebellar vermis (but see 3.3.2b Imaging) and a decrease in the number [186,189–192] and size [193,194] of the Purkinje neurons. The inhibitory GABAergic basket and stellate interneurons that connect with these neurons did not show quantitative differences compared to normal cerebella [195]. This observation is indicative of a late developmental death of the Purkinje neurons, as they differentiate well before the interneurons. In addition to structural observations, Western blots demonstrated a reduction of about 40% in the level of expression of RELN in autistic patients related to age and sex corresponding controls [11]. Quantitative RT-PCR also showed a drop of RELN and GAD67 mRNAs in the post-mortem autistic brain [164].

Notably many of the histological alterations in the human autistic cerebellum are like those described in *reln*+/− mice. These animals displayed a progressive loss of Purkinje neurons already during the first weeks of life [35], and inferior numbers of these cells were observed in adult subjects as well [196]. Human MRI studies did not allow, at present, to ascertain whether the cerebellar vermis is hit by the pathology in its entirety or, rather, only at specific lobuli. Therefore, our group has, at first, focused its attention on five different lobules, which receive diverse types of afferent functional inputs, to analyze the number and topological organization of the Purkinje neurons in *reln*+/+ and *reln*+/− adult mice of both sexes [197]. We have thus shown that the Purkinje neurons: 1. Displayed a lower density in *reln*+/− males (14.37%) and *reln*+/− females (17.73%) compared to *reln*+/+ males; 2. Were larger in *reln*+/− males than in the other phenotypes under study, and smaller in females (regardless of the *reln* genetic background) than in *reln*+/+ males; 3. Were more messily arranged along the YZ axis of the vermis in *reln*+/− males than in *reln*+/+ males and, except in central lobule, *reln*+/− females.

Very recently, as many observations have associated a number of synapse-related genes in the genesis of autism and other neuropsychiatric conditions [198,199], we have examined the expression of synaptophysin 1 (SYP1) and contactin 6 (CNTN6) in the vermis of *reln*+/− and *reln*+/+ adult mice of both genders [200].

SYP1 is a pre-synaptic marker and CNTN6 is a marker of the synapses made by the parallel fibers onto the Purkinje neurons' dendrites. Notably, there is evidence, although still to be validated in full, that SYP1 is involved in the structural alterations of the autistic synapses [189,201], and very recent observations have shown that copy number variations [202] or a truncating variant [203] of

*CNTN6* are found in autistic patients. In addition, *CNTN6* mutations may be a risk factor for several neurodevelopmental and neuropsychiatric disorders [150,199,204].

In line with these human studies, we have demonstrated that *reln*+/− mouse males displayed a statistically significant drop of 11.89% in SYP1 compared to sex-matched normal animals, whereas no modifications were detected comparing *reln*+/+ and *reln*+/− females [200]. In *reln*+/− male and female mice, reductions in SYP1 levels were particularly evident in the molecular layer, whereas in heterozygous mice of both sexes a reduction in CNTN6 occurred in all the three cortical layers of the vermis. In addition, alterations in the levels of expression of SYP1 in the molecular layer of male *reln*+/− mice ensued across all lobules except lobule VII, but they were limited to lobule II for the granular layer and lobule VII for the Purkinje cell layer.

Thus, the widespread reduction of SYP1 and of CNTN6 in the molecular layer of *reln*+/− male mice well matched with the autistic phenotype in humans [150].

In the vermis (and the whole cerebellum), there is proof for a topographic segregation of the areas controlling motion versus those connected to cognitive and affective functions, and the diverse lobules are coupled with precise zones of the brain and spinal cord [205]. The CNS areas that handle sensorimotor inputs are directly or indirectly connected with the anterior lobe (lobules I–V of the vermis), lobule VIII, and, to a lesser grade, with lobule VI; on the contrary, cortical association areas that collect non-motor responses are linked to lobules VI and VII. Existing clinical data indicate that the vermis is the chief target of the limbic system, and physiological and behavioral observations implicate the vermis in the regulation of emotions [206]. Therefore, the neurochemical modifications of the cerebellar cortex in heterozygous mice are fully in line with the possibility that the social and communication aberrations typical of autism rest on anomalies of the limbic system and its connections [207,208].

At post-mortem, a numerical reduction of the Purkinje neurons in the posterior cerebellum was long ago described in autistic subjects [184,209], but it did not appear to disturb the vermis [189]. Hypoplasia in lobules VI and VII was initially detected in vivo using MRI [145], but subsequent observations proved the existence of two distinct autistic subtypes related to vermian hypoplasia or hyperplasia [146]. A systematic review and meta-analysis of the accounts of structural MRI has then established that the reduction in size of lobules VI–X (i.e., the lobules included in the posterior cerebellum) showed a remarkable heterogeneity that associated to differences in time of life and intelligence quotient (IQ) merely in lobules VI–VII [210]. Other observations showed that the posterior/inferior vermis, i.e., lobules VII, VIIIb (left), and IX, was more prone to pathological deviations [211], with a decrease of the gray matter after quantitative MRI [190,212,213]. Therefore, it appears that the cerebellar phenotype of the heterozygous *Reeler* mouse is fully compatible with that in humans and that a deeper structural and neurochemical characterization may be useful to direct the discovery of new biomarkers of translational interest.