Research ArticleAuditory Midbrain Hypoplasia and Dysmorphology after Prenatal Valproic Acid Exposure
Introduction
Autism spectrum disorder (ASD) is a developmental disorder associated with neurological dysfunction and significant abnormalities in social, communicative and behavioral domains (Allen, 1988, Wing, 1997, American Psychiatric Association, 2013, CDC.gov. 2018). The most recent estimates indicate that by age 8, 1 in 59 children is diagnosed with ASD and the vast majority of these children are male (CDC.gov., 2018). Hypersensitivity to sensory stimuli is a key sign of ASD (CDC.gov., 2018) and can include adverse reactions to touch, smell, taste, light and sound. Furthermore, the majority of individuals with ASD have some manner of difficulty hearing and understanding speech and vocalizations despite normal or even lower thresholds to non-speech sounds (Greenspan and Wieder, 1997, Tomchek, 2007, Gomes et al., 2008, Bolton et al., 2012). Severity of auditory dysfunction in ASD spans a wide spectrum and can range from deafness to hyperacusis, but commonly includes difficulties listening in the presence of background noise and localizing sound sources (Roper and Arnold, 2003, Alcántara et al., 2004, Khalfa et al., 2004, Szelag et al., 2004, Lepistö et al., 2005, Teder-Salejarvi et al., 2005, Gravel et al., 2006, Tharpe et al., 2006, Russo et al., 2009). Consistent with these functional deficits, individuals with ASD have significantly fewer auditory brainstem neurons and surviving neurons are dysmorphic and abnormally organized (Kulesza and Mangunay, 2008, Kulesza et al., 2011, Lukose et al., 2015). Further, there is evidence from a number of non-invasive tests and clinical observations directly supporting auditory brainstem dysfunction in ASD (Ornitz et al., 1985, Khalfa et al., 2001, Kwon et al., 2007, Tas et al., 2007, Lukose et al., 2013, Erturk and Korkmaz, 2016, Bennetto et al., 2017) and studies of the auditory brainstem response indicate abnormal brainstem processing in ASD (Skoff et al., 1980, Gillberg et al., 1983, Rumsey et al., 1984, Martineau et al., 1987, Rosenblum et al., 1980, McClelland and Eyre, 1992, Klin, 1993, Maziade et al., 2000, Rosenhall and Nordin, 2003, Roth and Muchnik, 2012, Azouz et al., 2014, Källstrand et al., 2010, Miron and Ari-Even Roth, 2016, Santos et al., 2017). Together, these studies support our primary hypothesis that abnormal brainstem structure and function are the root cause of auditory processing issues in ASD.
The precise developmental events that lead to the ASD phenotype are poorly understood. At present, the majority of ASD cases are idiopathic, but many are comorbid with other neurodevelopmental disorders such as Fragile X syndrome (Brown et al., 1982). Additionally, maternal exposure to teratogenic drugs can significantly impact fetal brain development and such exposures are likely responsible for a small proportion of ASD cases. In particular, there is a clear association between ASD and prenatal exposure to the anti-epileptic valproic acid (VPA; Christianson and Chesler, 1994, Rodier et al., 1996). VPA is indicated for acute treatment of manic episodes, complex partial seizures and migraines. VPA usage is not advised during pregnancy as this drug is known to cause neurological side effects in the mother and is associated with facial deformities, hypospadias and neural tube defects in the offspring (DiLiberti et al., 1984). Furthermore, prenatal exposure to VPA is associated with a significant increase in the probability of a later diagnosis of ASD and is included as a risk factor for ASD (Moore et al., 2000, Williams et al., 2001, Rasalam et al., 2005, Koren et al., 2006, Bromley et al., 2013, Christensen et al., 2013). Accordingly, in utero exposure to VPA is used as an animal model of ASD (Rodier et al., 1996).
Animals exposed to VPA in utero demonstrate many autistic-like behaviors including reduced social explorations and predilection for repetitive behaviors (reviewed by Nicolini and Fahnestock, 2018) and demonstrate ataxia on gait tasks (Main and Kulesza, 2017). Additionally, there are numerous gross and neuropathological changes associated with in utero VPA exposure in rodents. VPA-exposed animals have smaller bodies and brains, and delayed gross development of the eye and external ear (Zimmerman et al., 2018). Consistent with clinical and neuropathological observations in ASD, animals exposed to VPA have marked auditory dysfunction (Gandal et al., 2010, Engineer et al., 2014, Anomal et al., 2015, Dubiel and Kulesza, 2016) and hypoplasia of auditory hindbrain centers (Lukose et al., 2011, Zimmerman et al., 2018). More specifically, VPA-exposed animals have significantly fewer neurons in the cochlear nucleus (CN) and superior olivary complex (SOC), fewer calbindin-immunopositive (CB+) neurons and reduced dopaminergic inputs to CN and SOC neurons (Lukose et al., 2011, Zimmerman et al., 2018). Additionally, VPA-exposed animals have abnormal activation patterns after exposure to pure tone stimuli (Dubiel and Kulesza, 2016). Specifically, when control animals are exposed to pure tone stimuli, c-Fos immunolabeling is found in narrow tonotopic bands within many auditory nuclei. However, similar stimulation to VPA-exposed animals results in significantly more c-Fos immunolabeled neurons in the CN, SOC and central nucleus of the inferior colliculus (CNIC) and these neurons extend well beyond the characteristic tonotopic bands observed in control animals. Further, in vivo recordings from the auditory cortex in VPA-exposed animals have revealed increased response latencies, decreased phase-locking capabilities (Gandal et al., 2010), abnormal mapping of sound frequencies (Anomal et al., 2015) and abnormal responses to vocalizations (Engineer et al., 2014). Together, these results indicate that prenatal VPA exposure in rodents results in significant alterations in structure and function in the central auditory pathways.
These previous studies have focused on the hindbrain and the auditory cortex and have neglected several large auditory centers in the pons, midbrain and thalamus, namely the nuclei of the lateral lemniscus (NLL), the IC and the medial geniculate complex. Consequently, the impact of prenatal VPA exposure on these auditory centers has not been studied. The IC is an important relay and processing center for both ascending and descending auditory information. In fact, previous estimates of neuronal number in rats indicate that the IC outnumbers subcollicular auditory centers more than five to one (Kulesza et al., 2002). Based on the structural and functional changes that occur in the CN, SOC and auditory cortex after VPA exposure, we hypothesize that the NLL and CNIC are significantly impacted by VPA exposure. Herein, we examine this hypothesis in a repeated in utero exposure model using morphometric techniques, estimates of neuronal number, histochemistry for perineuronal nets (PNNs) and immunofluorescence for the calcium-binding protein calbindin (CB) and tyrosine hydroxylase (TH).
Section snippets
Animals
All animal handling procedures were approved by the LECOM IACUC (protocol #16-02). Sprague–Dawley rats were raised and maintained in the laboratory vivarium on a 12-h light/dark cycle with unrestricted access to food and water.
Exposure to VPA was done as previously described (Main and Kulesza, 2017, Zimmerman et al., 2018). Briefly, animals engaged in timed mating and pregnant females were randomly assigned to control or VPA-exposed groups. Pregnant females were fed meals of 3.1 g of peanut
VPA exposure resulted in fewer CNIC neurons
Consistent with previous reports (Main and Kulesza, 2017, Zimmerman et al., 2018) we found significantly lower brain weights after VPA exposure (at P50). In control animals, the brainstem weighed 0.56 ± 0.02 g. After VPA exposure, brainstems weighed only 0.51 ± 0.02 g (t10 = 3.37, p < .01; Fig. 1B).
VPA exposure resulted in a notable reduction in the size of the CNIC and reduced neuronal packing density (Fig. 2A, B). However, there were significantly larger neurons in the CNIC after VPA exposure
Discussion
This report provides the first evidence that in utero VPA exposure results in significantly fewer neurons, dysmorphology, decreased CB-immunolabeling and reduced dopaminergic input to the auditory midbrain. These findings are consistent with previous reports that VPA impacts structure and function of the auditory hindbrain and cortex (Lukose et al., 2011, Gandal et al., 2010, Engineer et al., 2014, Anomal et al., 2015, Dubiel and Kulesza, 2016, Zimmerman et al., 2018). Together, these studies
Acknowledgments
The authors would like to thank the Lake Erie College of Osteopathic Medicine Research Collective, Dr Diana Speelman for constructive comments, Mary Petro for technical assistance and Jerome McGraw (Penn State Behrend) for technical assistance with confocal microscopy. This work was supported by the Lake Erie College of Osteopathic Medicine and the Lake Erie Consortium for Osteopathic Medical Training.
Disclosure of potential conflicts of interest
The authors declare that they have no conflicts of interest.
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