Enhanced Activation of HCN Channels Reduces Excitability and Spike-Timing Regularity in Maturing Vestibular Afferent Neurons.

Vestibular ganglion neurons (VGNs) transmit information along parallel neuronal pathways whose signature distinction is variability in spike-timing; some fire at regular intervals while others fire at irregular intervals. The mechanisms driving timing differences are not fully understood but two opposing (but not mutually exclusive) hypotheses have emerged. In the first, regular-spiking is inversely correlated to the density of low-voltage-gated potassium currents (IKL). In the second, regular spiking is directly correlated to the density of hyperpolarization-activated cyclic nucleotide-sensitive currents (IH). Supporting the idea that variations in ion channel composition shape spike-timing, VGNs from the first postnatal week respond to synaptic-noise-like current injections with irregular-firing patterns if they have IKL and with more regular firing patterns if they do not. However, in vitro firing patterns are not as regular as those in vivo Here we considered whether highly-regular spiking requires IH currents and whether this dependence emerges later in development after channel expression matures. We recorded from rat VGN somata of either sex aged postnatal day (P)9-P21. Counter to expectation, in vitro firing patterns were less diverse, more transient-spiking, and more irregular at older ages than at younger ages. Resting potentials hyperpolarized and resting conductance increased, consistent with developmental upregulation of IKL Activation of IH (by increasing intracellular cAMP) increased spike rates but not spike-timing regularity. In a model, we found that activating IH counter-intuitively suppressed regularity by recruiting IKL Developmental upregulation in IKL appears to overwhelm IH These results counter previous hypotheses about how IH shapes vestibular afferent responses.SIGNIFICANCE STATEMENT Vestibular sensory information is conveyed on parallel neuronal pathways with irregularly-firing neurons encoding information using a temporal code and regularly-firing neurons using a rate code. This is a striking example of spike-timing statistics influencing information coding. Previous studies from immature vestibular ganglion neurons (VGNs) identified hyperpolarization-activated mixed cationic currents (IH) as driving highly-regular spiking and proposed that this influence grows with the current during maturation. We found that IH becomes less influential, likely because maturing VGNs also acquire low-voltage-gated potassium currents (IKL), whose inhibitory influence opposes IH Because efferent activity can partly close IKL, VGN firing patterns may become more receptive to extrinsic control. Spike-timing regularity likely relies on dynamic ion channel properties and complementary specializations in synaptic connectivity.

Early postnatal maturation in vestibulospinal pathways involved in neck and forelimb motor control.

To assess the organization and functional development of vestibulospinal inputs to cervical motoneurons (MNs), we have used electrophysiology (ventral root and electromyographic [EMG] recording), calcium imaging, trans-synaptic rabies virus (RV) and conventional retrograde tracing and immunohistochemistry in the neonatal mouse. By stimulating the VIIIth nerve electrically while recording synaptically mediated calcium responses in MNs, we characterized the inputs from the three vestibulospinal tracts, the separate ipsilateral and contralateral medial vestibulospinal tracts (iMVST/cMVST) and the lateral vestibulospinal tract (LVST), to MNs in the medial and lateral motor columns (MMC and LMC) of cervical segments. We found that ipsilateral inputs from the iMVST and LVST were differentially distributed to the MMC and LMC in the different segments, and that all contralateral inputs to MMC and LMC MNs in each segment derive from the cMVST. Using trans-synaptic RV retrograde tracing as well as pharmacological manipulation of VIIIth nerve-elicited synaptic responses, we found that a substantial proportion of inputs to both neck and forelimb extensor MNs was mediated monosynaptically, but that polysynaptic inputs were also significant. By recording EMG responses evoked by natural stimulation of the vestibular apparatus, we found that vestibular-mediated motor output to the neck and forelimb musculature became more robust during the first 10 postnatal days, concurrently with a decrease in the latency of MN discharge evoked by VIIIth nerve electrical stimulation. Together, these results provide insight into the complexity of vestibulospinal connectivity in the cervical spinal cord and a cogent demonstration of the functional maturation that vestibulospinal connections undergo postnatally. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1061-1077, 2016.

Maturation of glutamatergic transmission in the vestibulo-olivary pathway impacts on the registration of head rotational signals in the brainstem of rats.

The recognition of head orientation in the adult involves multi-level integration of inputs within the central vestibular circuitry. How the different inputs are recruited during postnatal development remains unclear. We hypothesize that glutamatergic transmission at the vestibular nucleus contributes to developmental registration of head orientations along the vestibulo-olivary pathway. To investigate the maturation profile by which head rotational signals are registered in the brainstem, we used sinusoidal rotations on the orthogonal planes of the three pairs of semicircular canals. Fos expression was used as readout of neurons responsive to the rotational stimulus. Neurons in the vestibular nucleus and prepositus hypoglossal nucleus responded to all rotations as early as P4 and reached adult numbers by P21. In the reticular formation and inferior olive, neurons also responded to horizontal rotations as early as P4 but to vertical rotations not until P21 and P25, respectively. Neuronal subpopulations that distinguish between rotations activating the orthogonally oriented vertical canals were identifiable in the medial and spinal vestibular nuclei by P14 and in the inferior olivary subnuclei IOß and IOK by P25. Neonatal perturbation of glutamate transmission in the vestibular nucleus was sufficient to derange formation of this distribution in the inferior olive. This is the first demonstration that developmental refinement of glutamatergic synapses in the central vestibular circuitry is essential for developmental registration of head rotational signals in the brainstem.

Morphologically mixed chemical-electrical synapses formed by primary afferents in rodent vestibular nuclei as revealed by immunofluorescence detection of connexin36 and vesicular glutamate transporter-1.

Axon terminals forming mixed chemical/electrical synapses in the lateral vestibular nucleus of rat were described over 40 years ago. Because gap junctions formed by connexins are the morphological correlate of electrical synapses, and with demonstrations of widespread expression of the gap junction protein connexin36 (Cx36) in neurons, we investigated the distribution and cellular localization of electrical synapses in the adult and developing rodent vestibular nuclear complex, using immunofluorescence detection of Cx36 as a marker for these synapses. In addition, we examined Cx36 localization in relation to that of the nerve terminal marker vesicular glutamate transporter-1 (vglut-1). An abundance of immunolabeling for Cx36 in the form of Cx36-puncta was found in each of the four major vestibular nuclei of adult rat and mouse. Immunolabeling was associated with somata and initial dendrites of medium and large neurons, and was absent in vestibular nuclei of Cx36 knockout mice. Cx36-puncta were seen either dispersed or aggregated into clusters on the surface of neurons, and were never found to occur intracellularly. Nearly all Cx36-puncta were localized to large nerve terminals immunolabeled for vglut-1. These terminals and their associated Cx36-puncta were substantially depleted after labyrinthectomy. Developmentally, labeling for Cx36 was already present in the vestibular nuclei at postnatal day 5, where it was only partially co-localized with vglut-1, and did not become fully associated with vglut-1-positive terminals until postnatal day 20-25. The results show that vglut-1-positive primary afferent nerve terminals form mixed synapses throughout the vestibular nuclear complex, that the gap junction component of these synapses contains Cx36, that multiple Cx36-containing gap junctions are associated with individual vglut-1 terminals and that the development of these mixed synapses is protracted over several postnatal weeks.

Postnatal expression of TrkB receptor in rat vestibular nuclear neurons responsive to horizontal and vertical linear accelerations.

We examined the maturation expression profile of tyrosine kinase B (TrkB) receptor in rat vestibular nuclear neurons that were activated by sinusoidal linear acceleration along the horizontal or vertical axis. The otolithic origin of Fos expression in these neurons was confirmed with labyrinthectomized controls and normal controls, which showed only sporadically scattered Fos-labeled neurons in the vestibular nucleus. In P4-6 test rats, no Fos-labeled neurons were found in the vestibular nucleus, but the medial and spinal vestibular neurons showed weak immunoreactivity for TrkB. The intensity of TrkB immunoreactivity in vestibular nuclear neurons progressively increased in the second postnatal week but remained low in adults. From P7 onward, TrkB-expressing neurons responded to horizontal or vertical otolithic stimulation with Fos expression. The number of Fos-labeled vestibular nuclear neurons expressing TrkB increased with age, from 13-43% in P7 rats to 85-90% in adult rats. Our results therefore suggest that TrkB/neurotrophin signaling plays a dominant role in modulating vestibular nuclear neurons for the coding of gravity-related horizontal head movements and for the regulation of vestibular-related behavior during postnatal development.

Molecular identity, ontogeny, and cAMP modulation of the hyperpolarization-activated current in vestibular ganglion neurons.

Properties, developmental regulation, and cAMP modulation of the hyperpolarization-activated current (I(h)) were investigated by the whole cell patch-clamp technique in vestibular ganglion neurons of the rat at two postnatal stages (P7-10 and P25-28). In addition, by RT-PCR and immunohistochemistry the identity and distribution of hyperpolarization-activated and cyclic nucleotide-gated channel (HCN) isoforms that generate I(h) were investigated. I(h) current density was larger in P25-28 than P7-10 rats, increasing 410% for small cells (<30 pF) and 200% for larger cells (>30 pF). The half-maximum activation voltage (V(1/2)) of I(h) was -102 mV in P7-10 rats and in P25-28 rats shifted 7 mV toward positive voltages. At both ages, intracellular cAMP increased I(h) current density, decreased its activation time constant (τ), and resulted in a rightward shift of V(1/2) by 9 mV. Perfusion of 8-BrcAMP increased I(h) amplitude and speed up its activation kinetics. I(h) was blocked by Cs(+), zatebradine, and ZD7288. As expected, these drugs also reduced the voltage sag caused with hyperpolarizing pulses and prevented the postpulse action potential generation without changes in the resting potential. RT-PCR analysis showed that HCN1 and HCN2 subunits were predominantly amplified in vestibular ganglia and end organs and HCN3 and HCN4 to a lesser extent. Immunohistochemistry showed that the four HCN subunits were differentially expressed (HCN1 > HCN2 > HCN3 ≥ HCN4) in ganglion slices and in cultured neurons at both P7-10 and P25-28 stages. Developmental changes shifted V(1/2) of I(h) closer to the resting membrane potential, increasing its functional role. Modulation of I(h) by cAMP-mediated signaling pathway constitutes a potentially relevant control mechanism for the modulation of afferent neuron discharge.

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.

Intrinsic membrane properties of central vestibular neurons in rodents.

Numerous studies in rodents have shown that the functional efficacy of several neurotransmitter receptors and the intrinsic membrane excitability of central vestibular neurons, as well as the organization of synaptic connections within and between vestibular nuclei can be modified during postnatal development, after a lesion of peripheral vestibular organs or in vestibular-deficient mutant animals. This review mainly focuses on the intrinsic membrane properties of neurons of the medial vestibular nuclei of rodents, their postnatal maturation, and changes following experimental or congenital alterations in vestibular inputs. It also presents the concomitant modifications in the distribution of these neurons into different neuron types, which has been based on their membrane properties in relation to their anatomical, biochemical, or functional properties. The main points discussed in this review are that (1) the intrinsic membrane properties can be used to distinguish between two dominant types of neurons, (2) the system remains plastic throughout the whole life of the animal, and finally, (3) the intracellular calcium concentration has a major effect on the intrinsic membrane properties of central vestibular neurons.

Developmental distribution of vestibular nuclear neurons responsive to different speeds of horizontal translation.

To examine whether subgroups of vestibular nuclear neurons encode different frequency oscillation of horizontal linear motion, Fos immunohistochemistry was used to document neuronal subpopulations that were functionally activated by such otolithic stimulations. Conscious rats at P7, P14 and adult were subjected to sinusoidal linear acceleration along the transverse axis on the horizontal plane. Labyrinthectomized and/or stationary controls showed only sporadically scattered Fos-labeled neurons in the vestibular nuclei, confirming otolithic origin of c-fos expression. In each age group, Fos-labeled neurons responsive to high frequency stimulation (>1.5 Hz) were clustered in the lateral region of the medial vestibular nucleus while those to low frequency stimulation (0.5-1.0 Hz) were found in the medial portion of the medial vestibular nucleus. The number of these neurons increased with age. No apparent frequency-related distribution pattern of Fos-labeled neurons was observed in other vestibular nuclei and subgroups. Our findings therefore reveal subpopulations of central vestibular neurons responsive to different stimulus frequencies that correspond to head motions ranging from tilt to translation.

Identification of vestibuloocular projection neurons in the developing chicken medial vestibular nucleus.

Biocytin was injected into the oculomotor, trochlear, or abducens nucleus on one side using isolated chicken brainstem preparations or brain slices to identify the medial vestibular nucleus (MVN) neurons projecting to these targets. Oculomotor nucleus injections produced retrogradely labeled neurons in the contralateral ventrolateral MVN (MVN(VL)), with few labeled neurons in the ipsilateral MVN(VL) and rarely in the dorsomedial MVN on either side. Labeled MVN(VL) neurons were identified as stellate (95%) and elongate (5%) cells. Trochlear nucleus injections produced a similar pattern of MVN neuron labeling. Abducens nucleus injections resulted in retrogradely labeled stellate (87%) and elongate (13%) neurons in the MVN(VL), which had smaller cell bodies than those projecting to the oculomotor nucleus. Anteroposteriorly, labeled MVN(VL) neurons were coextensive with the tangential nucleus, with neurons projecting to the oculomotor nucleus distributed lateral to and intermixed with the more medially situated neurons projecting to the abducens nucleus. The fundamental pattern of vestibuloocular projecting neurons was similar at both embryonic ages studied, E16 and E13. In contrast to the case in mammals, where most vestibuloocular projection neurons reside within the MVN, most retrogradely labeled neurons in these chicken preparations were found within the ventrolateral vestibular, descending vestibular, and tangential nuclei. The morphological identification and mapping of vestibuloocular projection neurons in the chicken MVN described here represents the first step in a systematic evaluation of the relationship between avian vestibuloocular neuron structure and function.