Understanding the Pathophysiology of Congenital Vestibular Disorders: Current Challenges and Future Directions.

In congenital vestibular disorders (CVDs), children develop an abnormal inner ear before birth and face postnatal challenges to maintain posture, balance, walking, eye-hand coordination, eye tracking, or reading. Only limited information on inner ear pathology is acquired from clinical imaging of the temporal bone or studying histological slides of the temporal bone. A more comprehensive and precise assessment and determination of the underlying mechanisms necessitate analyses of the disorders at the cellular level, which can be achieved using animal models. Two main criteria for a suitable animal model are first, a pathology that mirrors the human disorder, and second, a reproducible experimental outcome leading to statistical power. With over 40 genes that affect inner ear development, the phenotypic abnormalities resulting from congenital vestibular disorders (CVDs) are highly variable. Nonetheless, there is a large subset of CVDs that form a common phenotype of a sac-like inner ear with the semicircular canals missing or dysplastic, and discrete abnormalities in the vestibular sensory organs. We have focused the review on this subset, but to advance research on CVDs we have added other CVDs not forming a sac-like inner ear. We have included examples of animal models used to study these CVDs. Presently, little is known about the central pathology resulting from CVDs at the cellular level in the central vestibular neural network, except for preliminary studies on a chick model that show significant loss of second-order, vestibular reflex projection neurons.

Implementing the chick embryo model to study vestibular developmental disorders.

Children with congenital vestibular disorders show delayed motor development and challenges in maintaining posture and balance. Computed tomography images reveal that these children have abnormal inner ears in the form of a sac, with the semicircular canals missing or truncated. Little is known about how this inner ear abnormality affects central vestibular development. At present, mice with the chromodomain helicase DNA-binding protein 7 mutation are the most common model for studying congenital vestibular disorders, despite forming multiple diverse inner ear phenotypes and inducing abnormal cerebellar and visual system development. To identify the effects of a sac-like inner ear on central vestibular development, we have designed and implemented a new model, the anterior-posterior axis rotated otocyst (ARO) chick, which forms a sac-like inner ear in 85% of cases. The ARO chick is produced by anterior-posterior rotation of the otocyst at embryonic day 2. Here, we describe for the first time the 15% of ARO chicks that form three small semicircular canals and rename the ARO chicks forming sacs (ARO/s chicks). The basic features of the vestibular sensory organs in ARO/s chicks are similar to those found in patients' sacs, and ARO/s hatchlings experience balance and walking problems like patients. Thus, ARO/s chicks have a reproducible inner ear phenotype without abnormalities in vestibular-related structures, making the model a relatively simple one to evaluate the relationship between the sac-like inner ear pathology and formation of the central vestibular neural circuitry. Here, we describe unpublished details on the surgical approaches to produce ARO chicks, including pitfalls and difficulties to avoid.NEW & NOTEWORTHY This paper describes simple techniques for chick otocyst rotation resulting in a sac-like inner ear (85%), the common phenotype in congenital vestibular disorders. We now describe anterior-posterior axis rotated otocyst chicks, which form three small canals (15%), and rename chicks forming a sac (ARO/s chicks). Basic protocols and potential complications of otocyst rotation are described. With the use of ARO/s chicks, it will be possible to determine how the vestibular neural circuit is modified by sac-like inner ear formation.

A New Model for Congenital Vestibular Disorders.

Many developmental disorders of the inner ear are manifested clinically as delayed motor development and challenges in maintaining posture and balance, indicating involvement of central vestibular circuits. How the vestibular circuitry is rewired in pediatric cases is poorly understood due to lack of a suitable animal model. Based on this, our lab designed and validated a chick embryo model to study vestibular development in congenital vestibular disorders. The developing inner ear or "otocyst" on the right side of 2-day-old chick embryos (E2) was surgically rotated 180° in the anterior-posterior axis, forming the "anterior-posterior axis rotated otocyst chick" or ARO chick. The ARO chick has a reproducible pathology of a sac with truncated or missing semicircular canals. A sac is the most common inner ear defect found in children with congenital vestibular disorders. In E13 ARO chicks, the sac contained all three cristae and maculae utriculi and sacculi, but the superior crista and macula utriculi were shortened in anterior-posterior extent. Also, the number of principal cells of the tangential vestibular nucleus, a major avian vestibular nucleus, was decreased 66 % on the rotated side. After hatching, no difference was detected between ARO and normal chicks in their righting reflex times. However, unlike normal chicks, ARO hatchlings had a constant, right head tilt, and after performing the righting reflex, ARO chicks stumbled and walked with a widened base. Identifying the structure and function of abnormally developed brain regions in ARO chicks may assist in improving treatments for patients with congenital vestibular disorder.

Admission profile and discharge outcomes for infants aged less than 6 months admitted to inpatient therapeutic care in 10 countries. A secondary data analysis.

Evidence on the management of acute malnutrition in infants aged less than 6 months (infants <6mo) is scarce. To understand outcomes using current protocols, we analysed a sample of 24 045 children aged 0-60 months from 21 datasets of inpatient therapeutic care programmes in 10 countries. We compared the proportion of admissions, the anthropometric profile at admission and the discharge outcomes between infants <6mo and children aged 6-60 months (older children). Infants <6mo accounted for 12% of admissions. The quality of anthropometric data at admission was more problematic in infants <6mo than in older children with a greater proportion of missing data (a 6.9 percentage point difference for length values, 95% CI: 6.0; 7.9, P < 0.01), anthropometric measures that could not be converted to indices (a 15.6 percentage point difference for weight-for-length z-score values, 95% CI: 14.3; 16.9, P < 0.01) and anthropometric indices that were flagged as outliers (a 2.7 percentage point difference for any anthropometric index being flagged as an outlier, 95% CI: 1.7; 3.8, P < 0.01). A high proportion of both infants <6mo and older children were discharged as recovered. Infants <6mo showed a greater risk of death during treatment (risk ratio 1.30, 95% CI: 1.09; 1.56, P < 0.01). Infants <6mo represent an important proportion of admissions to therapeutic feeding programmes, and there are crucial challenges associated with their care. Systematic compilation and analysis of routine data for infants <6mo is necessary for monitoring programme performance and should be promoted as a tool to monitor the impact of new guidelines on care.

Basic Concepts in Understanding Recovery of Function in Vestibular Reflex Networks during Vestibular Compensation.

Unilateral peripheral vestibular lesions produce a syndrome of oculomotor and postural deficits with the symptoms at rest, the static symptoms, partially or completely normalizing shortly after the lesion due to a process known as vestibular compensation. The symptoms are thought to result from changes in the activity of vestibular sensorimotor reflexes. Since the vestibular nuclei must be intact for recovery to occur, many investigations have focused on studying these neurons after lesions. At present, the neuronal plasticity underlying early recovery from the static symptoms is not fully understood. Here we propose that knowledge of the reflex identity and input-output connections of the recorded neurons is essential to link the responses to animal behavior. We further propose that the cellular mechanisms underlying vestibular compensation can be sorted out by characterizing the synaptic responses and time course for change in morphologically defined subsets of vestibular reflex projection neurons. Accordingly, this review focuses on the perspective gained by performing electrophysiological and immunolabeling studies on a specific subset of morphologically defined, glutamatergic vestibular reflex projection neurons, the principal cells of the chick tangential nucleus. Reference is made to pertinent findings from other studies on vestibular nuclei neurons, but no comprehensive review of the literature is intended since broad reviews already exist. From recording excitatory and inhibitory spontaneous synaptic activity in principal cells, we find that the rebalancing of excitatory synaptic drive bilaterally is essential for vestibular compensation to proceed. This work is important for it defines for the first time the excitatory and inhibitory nature of the changing synaptic inputs and the time course for changes in a morphologically defined subset of vestibular reflex projection neurons during early stages of vestibular compensation.

Plasticity of spontaneous excitatory and inhibitory synaptic activity in morphologically defined vestibular nuclei neurons during early vestibular compensation.

After unilateral peripheral vestibular lesions, the brain plasticity underlying early recovery from the static symptoms is not fully understood. Principal cells of the chick tangential nucleus offer a subset of morphologically defined vestibular nuclei neurons to study functional changes after vestibular lesions. Chickens show posture and balance deficits immediately after unilateral vestibular ganglionectomy (UVG), but by 3 days most subjects begin to recover, although some remain uncompensated. With the use of whole cell voltage-clamp, spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) and miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) were recorded from principal cells in brain slices 1 and 3 days after UVG. One day after UVG, sEPSC frequency increased on the lesion side and remained elevated at 3 days in uncompensated chickens only. Also by 3 days, sIPSC frequency increased on the lesion side in all operated chickens due to major increases in GABAergic events. Significant change also occurred in decay time of the events. To determine whether fluctuations in frequency and kinetics influenced overall excitatory or inhibitory synaptic drive, synaptic charge transfer was calculated. Principal cells showed significant increase in excitatory synaptic charge transfer only on the lesion side of uncompensated chickens. Thus compensation continues when synaptic charge transfer is in balance bilaterally. Furthermore, excessive excitatory drive in principal cells on the lesion side may prevent vestibular compensation. Altogether, this work is important for it defines the time course and excitatory and inhibitory nature of changing spontaneous synaptic inputs to a morphologically defined subset of vestibular nuclei neurons during critical early stages of recovery after UVG.

Expression of glutamate receptors and potassium channels in vestibular nuclei neurons during development of signal processing.

The principal cells of the chick tangential vestibular nucleus offer a simple neuron model to study signal processing in second-order, vestibular reflex projection neurons. The principal cells represent a relatively uniform population of vestibular nuclei neurons which receive a major input from the primary vestibular fibers and send axons to targets mainly involved in the vestibuloocular reflexes. Here, studies performed on ion channels involved in the emergence and establishment of signal processing in this morphologically-identified subset of vestibular nuclei neurons are reviewed, including the AMPA glutamate receptor subunits GluR1, GluR2, GluR3, and GluR4 and the potassium channel subunits Kv1.1 and Kv1.2.

Dopamine-deprived striatal GABAergic interneurons burst and generate repetitive gigantic IPSCs in medium spiny neurons.

Striatal GABAergic microcircuits modulate cortical responses and movement execution in part by controlling the activity of medium spiny neurons (MSNs). How this is altered by chronic dopamine depletion, such as in Parkinson's disease, is not presently understood. We now report that, in dopamine-depleted slices of the striatum, MSNs generate giant spontaneous postsynaptic GABAergic currents (single or in bursts at 60 Hz) interspersed with silent episodes, rather than the continuous, low-frequency GABAergic drive (5 Hz) observed in control MSNs. This shift was observed in one-half of the MSN population, including both "D(1)-negative" and "D(1)-positive" MSNs. Single GABA and NMDA channel recordings revealed that the resting membrane potential and reversal potential of GABA were similar in control and dopamine-depleted MSNs, and depolarizing, but not excitatory, actions of GABA were observed. Glutamatergic and cholinergic antagonists did not block the GABAergic oscillations, suggesting that they were generated by GABAergic neurons. In support of this, cell-attached recordings revealed that a subpopulation of intrastriatal GABAergic interneurons generated bursts of spikes in dopamine-deprived conditions. This subpopulation included low-threshold spike interneurons but not fast-spiking interneurons, cholinergic interneurons, or MSNs. Therefore, a population of local GABAergic interneurons shifts from tonic to oscillatory mode when dopamine deprived and gives rise to spontaneous repetitive giant GABAergic currents in one-half the MSNs. We suggest that this may in turn alter integration of cortical signals by MSNs.

Presynaptic and postsynaptic ion channel expression in vestibular nuclei neurons after unilateral vestibular deafferentation.

Vestibular compensation refers to the recovery of function occurring after unilateral vestibular deafferentation, but some patients remain uncompensated. Similarly, more than half of the operated chickens compensate three days after unilateral vestibular ganglionectomy (UVG), but the rest remain uncompensated. This review focuses on the studies performed on the principal cells of the chick tangential nucleus after UVG. The tangential nucleus is a major avian vestibular nucleus whose principal cells are all second-order, vestibular reflex projection neurons participating in the vestibuloocular and vestibulocollic reflexes controlling posture, balance, and eye movements. Using whole-cell patch-clamp approach in brain slice preparations, spontaneous spike firing, ionic conductances, and spontaneous excitatory postsynaptic currents (sEPSCs) are recorded in principal cells from controls and operated chickens three days after UVG. In compensated chickens, the proportion of spontaneous spike firing principal cells and their spike discharge rate are symmetric on the lesion and intact sides, with the rates increased over controls. However, in the uncompensated chickens, the spike discharge rate increases on the lesion side, but not on the intact side, where only silent principal cells are recorded. In all the experimental groups, including controls, silent principal cells are distinguished from spontaneous spiking cells by smaller persistent sodium conductances and higher activation thresholds for the fast sodium channel. In addition, silent principal cells on the intact side of uncompensated chickens have larger dendrotoxin-sensitive potassium conductances, with a higher ratio of immunolabeling for surface/cytoplasmic expression of a dendrotoxin-sensitive, potassium channel subunit, Kv1.1. Finally, in compensated chickens, sEPSC frequency is symmetric bilaterally, but in uncompensated chickens sEPSC frequency increased only on the lesion side, where the expression of Kv1.2 decreased in synaptotagmin-labeled terminal profiles on the principal cell bodies. Altogether, the specific sodium and potassium channels important for the development of spike firing pattern and/or presynaptic glutamate release on vestibular reflex projection neurons may be critically involved in changing postsynaptic neuron excitability after vestibular deafferentation.

Emergence of action potential generation and synaptic transmission in vestibular nucleus neurons.

Principal cells of the chick tangential nucleus are vestibular nucleus neurons in the hindbrain. Although detailed information is available on the morphogenesis of principal cells and synaptogenesis of primary vestibular fibers, this is the first study of their early functional development, when vestibular terminals emerge at embryonic days 10 and 13 (E10 and E13). At E10, 60% of principal cells generated spikes on depolarization, whereas 50% exhibited excitatory postsynaptic currents (EPSCs) on vestibular-nerve stimulation. The frequency was 0.2 Hz for glutamatergic spontaneous EPSCs (sEPSCs) at -60 mV, and 0.6 Hz for spontaneous inhibitory postsynaptic current (sIPSC) at +10 mV and completely GABAergic. All of these synaptic events were TTX-insensitive, miniature events. At E13, 50% of principal cells generated spikes on depolarization and 82% exhibited EPSCs on vestibular-nerve stimulation. The frequency was 0.7 Hz for sEPSCs at -60 mV, and 0.8 Hz for sIPSCs at +10 mV. Most principal cells had sIPSCs composed of both GABAergic (75%) and glycinergic (25%) events, but a few cells had only GABAergic sIPSCs. TTX decreased the frequency of EPSCs by 12%, and the IPSCs by 17%. In summary, at E10, some principal cells generated immature spikes on depolarization and EPSCs on vestibular-nerve stimulation. At E10, GABAergic events predominated, AMPA events had low frequencies, and glycinergic activity was absent. By E13, glycinergic events first appeared. This data were compared systematically to that obtained from the late-term embryo and hatchling to reveal the long-term sequence of changes in synaptic events and excitability and offer a broader understanding of how the vestibular system is assembled during development.

Interneurons targeting similar layers receive synaptic inputs with similar kinetics.

GABAergic interneurons play diverse and important roles in controlling neuronal network dynamics. They are characterized by an extreme heterogeneity morphologically, neurochemically, and physiologically, but a functionally relevant classification is still lacking. Present taxonomy is essentially based on their postsynaptic targets, but a physiological counterpart to this classification has not yet been determined. Using a quantitative analysis based on multidimensional clustering of morphological and physiological variables, we now demonstrate a strong correlation between the kinetics of glutamate and GABA miniature synaptic currents received by CA1 hippocampal interneurons and the laminar distribution of their axons: neurons that project to the same layer(s) receive synaptic inputs with similar kinetics distributions. In contrast, the kinetics distributions of GABAergic and glutamatergic synaptic events received by a given interneuron do not depend upon its somatic location or dendritic arborization. Although the mechanisms responsible for this unexpected observation are still unclear, our results suggest that interneurons may be programmed to receive synaptic currents with specific temporal dynamics depending on their targets and the local networks in which they operate.

Spontaneous synaptic activity is primarily GABAergic in vestibular nucleus neurons of the chick embryo.

The principal cells of the chick tangential nucleus are vestibular nucleus neurons participating in the vestibular reflexes. In 16-day embryos, the application of glutamate receptor antagonists abolished the postsynaptic responses generated on vestibular-nerve stimulation, but spontaneous synaptic activity was largely unaffected. Here, spontaneous synaptic activity was characterized in principal cells from brain slices at E16 using whole cell voltage-clamp recordings. With KCl electrodes, the frequency of spontaneous inward currents was 3.1 Hz at -60 mV, and the reversal potential was +4 mV. Cs-gluconate pipette solution allowed the discrimination of glycine/GABA(A) versus glutamate receptor-mediated events according to their different reversal potentials. The ratio for spontaneous excitatory to inhibitory events was about 1:4. Seventy-four percent of the outward events were GABA(A), whereas 26% were glycine receptor-mediated events. Both pre- and postsynaptic GABA(B) receptor effects were shown, with presynaptic GABA(B) receptors inhibiting 40% of spontaneous excitatory postsynaptic currents (sEPSCs) and 53% of spontaneous inhibitory postsynaptic currents (sIPSCs). With TTX, the frequency decreased approximately 50% for EPSCs and 23% for IPSCs. These data indicate that the spontaneous synaptic activity recorded in the principal cells at E16 is primarily inhibitory, action potential-independent, and based on the activation of GABA(A) receptors that can be modulated by presynaptic GABA(B) receptors.

Paradoxical anti-epileptic effects of a GluR5 agonist of kainate receptors.

Kainate generates in adult hippocampal neurons a seizure but also a massive excitation of interneurons and a dramatic increase of the inhibitory drive that impinges on principal cells. This "overinhibition" is largely mediated by GluR5-containing kainate receptors that are enriched on interneurons. Here, using the neonatal intact hippocampus in vitro and the triple chamber, we first show that this mechanism is fully operative in neonatal neurons. We then report that application to one hippocampus of (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid-a relatively selective agonist of GluR5 containing kainate receptors-depresses the propagation of seizure generated in the opposite hippocampus by a convulsive agent. We conclude that the selective excitation of interneurons by GluR5-containing kainate receptor agonists opens a new therapeutic approach for the epilepsies.

Quantal release of glutamate generates pure kainate and mixed AMPA/kainate EPSCs in hippocampal neurons.

The relative contribution of kainate receptors to ongoing glutamatergic activity is at present unknown. We report the presence of spontaneous, miniature, and minimal stimulation-evoked excitatory postsynaptic currents (EPSCs) that are mediated solely by kainate receptors (EPSC(kainate)) or by both AMPA and kainate receptors (EPSC(AMPA/kainate)). EPSC(kainate) and EPSC(AMPA/kainate) are selectively enriched in CA1 interneurons and mossy fibers synapses of CA3 pyramidal neurons, respectively. In CA1 interneurons, the decay time constant of EPSC(kainate) (circa 10 ms) is comparable to values obtained in heterologous expression systems. In both hippocampal neurons, the quantal release of glutamate generates kainate receptor-mediated EPSCs that provide as much as half of the total glutamatergic current. Kainate receptors are, therefore, key players of the ongoing glutamatergic transmission in the hippocampus.