Clinical, tomographic, and postmortem aspects of a rare congenital Dicephalus Monauchenos Iniodymus in a Nelore calf produced in vitro from Brazil - case report

ABSTRACT The aim of this work was to report the occurrence of dicephalus iniodymus monauchenos in a Nellore newborn. A three-days old calf, from in vitro production, with duplication of the head and a history of cesarean birth was attended. On physical examination, the dicephalus, iniodymus and monauchenos, which were almost the same size and shape, had four eyes and four ears. Computed tomography showed the presence of two skulls fused with a common occipital foramen, two nasopharynxes, oropharynxes with the presence of a cleft lip and a cleft palate in the right head, which continued in a single esophagus and a single trachea. At necropsy, the presence of duplication of the cerebrum and cerebellum was observed, with union of the parts in the region of the trapezoid body of the brainstem and continued as a single spinal cord. This study characterizes the clinical, tomographic, and necropsy findings of a dicephalus Nelore neonate.

The Untouchable Ventral Nucleus of the Trapezoid Body: Preservation of a Nucleus in an Animal Model of Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by repetitive behaviors, poor social skills, and difficulties with communication and hearing. The hearing deficits in ASD range from deafness to extreme sensitivity to routine environmental sounds. Previous research from our lab has shown drastic hypoplasia in the superior olivary complex (SOC) in both human cases of ASD and in an animal model of autism. However, in our study of the human SOC, we failed to find any changes in the total number of neurons in the ventral nucleus of the trapezoid body (VNTB) or any changes in cell body size or shape. Similarly, in animals prenatally exposed to the antiepileptic valproic acid (VPA), we failed to find any changes in the total number, size or shape of VNTB neurons. Based on these findings, we hypothesized that the neurotransmitter profiles, ascending and descending axonal projections of the VNTB are also preserved in these neurodevelopmental conditions. We investigated this hypothesis using a combination of immunohistochemistry and retrograde tract tracing. We found no difference between control and VPA-exposed animals in the number of VNTB neurons immunoreactive for choline acetyltransferase (ChAT). Additionally, we investigated the ascending projections from the VNTB to both the central nucleus of the inferior colliculus (CNIC) and medial geniculate (MG) and descending projections to the cochlea. Our results indicate no significant differences in the ascending and descending projections from the VNTB between control and VPA-exposed animals despite drastic changes in these projections from surrounding nuclei. These findings provide evidence that certain neuronal populations and circuits may be protected against the effects of neurodevelopmental disorders.

Multiple Sources of Cholinergic Input to the Superior Olivary Complex

The superior olivary complex (SOC) is a major computation center in the brainstem auditory system. Despite previous reports of high expression levels of cholinergic receptors in the SOC, few studies have addressed the functional role of acetylcholine in the region. The source of the cholinergic innervation is unknown for all but one of the nuclei of the SOC, limiting our understanding of cholinergic modulation. The medial nucleus of the trapezoid body, a key inhibitory link in monaural and binaural circuits, receives cholinergic input from other SOC nuclei and also from the pontomesencephalic tegmentum. Here, we investigate whether these same regions are sources of cholinergic input to other SOC nuclei. We also investigate whether individual cholinergic cells can send collateral projections bilaterally (i.e., into both SOCs), as has been shown at other levels of the subcortical auditory system. We injected retrograde tract tracers into the SOC in gerbils, then identified retrogradely-labeled cells that were also immunolabeled for choline acetyltransferase, a marker for cholinergic cells. We found that both the SOC and the pontomesencephalic tegmentum (PMT) send cholinergic projections into the SOC, and these projections appear to innervate all major SOC nuclei. We also observed a small cholinergic projection into the SOC from the lateral paragigantocellular nucleus of the reticular formation. These various sources likely serve different functions; e.g., the PMT has been associated with things such as arousal and sensory gating whereas the SOC may provide feedback more closely tuned to specific auditory stimuli. Further, individual cholinergic neurons in each of these regions can send branching projections into both SOCs. Such projections present an opportunity for cholinergic modulation to be coordinated across the auditory brainstem.

The Cl- channel TMEM16A controls the generation of cochlear Ca2+ waves and promotes the refinement of auditory brainstem networks

Before hearing onset (postnatal day 12 in mice), inner hair cells (IHC) spontaneously fire action potentials thereby driving pre-sensory activity in the ascending auditory pathway. The rate of IHC action potential bursts is modulated by inner supporting cells (ISC) of Kölliker's organ through the activity of the Ca2+ activated Cl- channel TMEM16A. Here we show that conditional deletion of Tmem16a in mice disrupts the generation of Ca2+ waves within Köllike's organ, reduces the burst firing activity and the frequency-selectivity of auditory brainstem neurons in the medial nucleus of the trapezoid body (MNTB), and also impairs the refinement of MNTB projections to the lateral superior olive (LSO). These results reveal the importance of the activity of Köllike's organ for the refinement of central auditory connectivity. In addition, our study suggests a mechanism for the generation of Ca2+ waves, which may also apply to other tissues expressing TMEM16A.

Cytoarchitecture of the medial nucleus of trapezoid body of three neotropical species of bats (Noctilio leporinus, Phyllostomus hastatus, and Carollia perspicillata) with different foraging behavior

The present study was taken to test the hypothesis that the medial nucleus of the trapezoid body (MNTB) of echolocating neotropical bats with different foraging behavior will exhibit morphological variations in relative size, degree of complexity and spatial distribution. The brains were collected from six male adult bats of each species: Noctilio leporinus (fish-eating), Phyllostomus hastatus (carnivorous/ omnivorous) and Carollia perspicillata (fruit-eating) and were double-embedded and transverse serial sections were cut and stained with cresyl fast violet. The results showed that the MNTB is well developed in all the bats in general and the mean length of the MNTB was 1160 ± 124 µm in N. leporinus, 400 ± 59 µm in P. hastatus and 320 ± 25µm in C. perspicillata. The body and brain weight do not reflect proportionately on the size of the MNTB in the present study. The hearing frequency spectrum did not covary with the size of the MNTB among the bats studied. The MNTB is clearly demarcated from the ventral nucleus of the trapezoid body (VNTB) only in P. hastatus. The MNTB comprised mainly three types of cells in all three bats: dense-staining multipolar cells (12.5 µm and 25.0 µm diameter); light-staining multipolar cells measuring (12.5 µm and 25.0 µm diameter) and light-staining round cells (5.0 µm diameter). The large sized MNTB was observed in N. leporinus, which suggests that it relies heavily on echolocation whereas P. hastatus and C. perspicillata use echolocation as well but also rely on hearing, smell and vision.

Using cortical neuron markers to target cells in the dorsal cochlear nucleus

The dorsal cochlear nucleus (DCN) is the first auditory region that integrates somatosensory and auditory inputs. The region is of particular interest for auditory research due to the large incidence of somatic tinnitus and increased aberrant activity in other forms of tinnitus. Yet, the lack of useful genetic markers for in vivo manipulations hinders the elucidation of the DCN contribution to tinnitus pathophysiology. In this work, we assessed whether adeno-associated viral vectors (AAV) containing the calcium/calmodulin-dependent protein kinase 2 alpha (CaMKIIα) promoter and our mouse line of nicotinic acetylcholine receptor alpha 2 subunit (Chrna2)-Cre can be used to target specific DCN populations. The CaMKIIα promoter is usually applied in studies of principal neurons of neo and paleocortex while Chrna2-cre mice express Cre recombinase in cortical dendrite inhibiting interneurons. We found that CaMKIIα cannot be used to specifically target excitatory fusiform DCN neurons. EYFP expression driven by the CaMKIIα promoter was stronger in the fusiform layer but labelled cells showed a diverse morphology indicating that they belong to different classes of DCN neurons. Light stimulation after driving Channelrhodopsin2 (ChR2) by the CaMKIIα promoter generated spikes in some units but firing rate decreased when light stimulation coincide with sound presentation. Expression and activation of eArch3.0 (CaMKIIα driven) in the DCN produced spike inhibition in some units but, most importantly, sound-driven spikes were delayed by concomitant light stimulation. We explored the existence of Cre+ cells in the DCN of Chrna2-Cre mice by hydrogel embedding technique (CLARITY). There were almost no Cre+ cell bodies in the DCN; however, we observed profuse projections arising from the ventral cochlear nucleus (VCN). Anterograde labeling Cre dependent AAV injected in the VCN revealed two main projections: one arising in the ipsilateral superior olive and the contralateral medial nucleus of the trapezoid body (bushy cells) and a second bundle terminating in the DCN, suggesting the latter to be excitatory Chrna2+ T-stellate cells). Stimulating ChR2 expressing terminals (light applied on the DCN) of VCN Chrna2+ cells increased firing of sound responding and nonresponding DCN units. This work shows that molecular tools intensively used in cortical studies may be useful for manipulating the DCN especially in tinnitus studies.