Anna Lysakowski

A regional ultrastructural analysis of the cellular and synaptic architecture in the chinchilla cristae ampullares

The chinchilla crista ampullaris was studied in 10 samples, each containing 32 consecutive ultrathin sections of the entire neuroepithelium. Dissector methods were used to estimate the incidence of various synaptic features, and results were confirmed in completely reconstructed hair cells. There are large regional variations in cellular and synaptic architecture. Type I and type II hair cells are shorter, broader, and less densely packed in the central zone than in the intermediate and peripheral zones. Complex calyx endings are most common centrally. On average, there are 15–20 ribbon synapses and 25–30 calyceal invaginations in each type I hair cell. Synapses and invaginations are most numerous centrally. Central type II hair cells receive considerably fewer afferent boutons than do peripheral type II hair cells, but have similar numbers of ribbon synapses. The numbers are similar because central type II hair cells make more synapses with the outer faces of calyx endings and with individual afferent boutons. Most afferent boutons get one ribbon synapse. Boutons without ribbon synapses were only found peripherally, and boutons getting multiple synapses were most frequent centrally. Throughout the neuroepithelium, there is an average of three to four efferent boutons on each type II hair cell and calyx ending. Reciprocal synapses are rare. Most synaptic ribbons in type I hair cells are spherules; those in type II hair cells can be spherical or elongated and are particularly heterogeneous centrally. Consistent with the proposal that the crista is concentrically organized, the intermediate and peripheral zones are each similar in their cellular and synaptic architecture near the base and near the planum. An especially differentiated subzone may exist in the middle of the central zone. J. Comp. Neurol. 389:419–443, 1997.

Hair cell ribbon synapses

Hearing and balance rely on the faithful synaptic coding of mechanical input by the auditory and vestibular hair cells of the inner ear. Mechanical deflection of their stereocilia causes the opening of mechanosensitive channels, resulting in hair cell depolarization, which controls the release of glutamate at ribbon-type synapses. Hair cells have a compact shape with strong polarity. Mechanoelectrical transduction and active membrane turnover associated with stereociliar renewal dominate the apical compartment. Transmitter release occurs at several active zones along the basolateral membrane. The astonishing capability of the hair cell ribbon synapse for temporally precise and reliable sensory coding has been the subject of intense investigation over the past few years. This research has been facilitated by the excellent experimental accessibility of the hair cell. For the same reason, the hair cell serves as an important model for studying presynaptic Ca2+ signaling and stimulus-secretion coupling. In addition to common principles, hair cell synapses differ in their anatomical and functional properties among species, among the auditory and vestibular organs, and among hair cell positions within the organ. Here, we briefly review synaptic morphology and connectivity and then focus on stimulus-secretion coupling at hair cell synapses.

M-Like K+ Currents in Type I Hair Cells and Calyx Afferent Endings of the Developing Rat Utricle

Type I vestibular hair cells have large K+ currents that, like neuronal M currents, activate negative to resting potential and are modulatable. In rodents, these currents are acquired postnatally. In perforated-patch recordings from rat utricular hair cells, immature hair cells [younger than postnatal day 7 (P7)] had a steady-state K+ conductance (g−30) with a half-activation voltage (V1/2) of −30 mV. The size and activation range did not change in maturing type II cells, but, by P16, type I cells had added a K conductance that was on average fourfold larger and activated much more negatively. This conductance may comprise two components: g−60 (V1/2 of −60 mV) and g−80 (V1/2 of −80 mV). g−80 washed out during ruptured patch recordings and was blocked by a protein kinase inhibitor. M currents can include contributions from KCNQ and ether-a-go-go-related (erg) channels. KCNQ and erg channel blockers both affected the K+ currents of type I cells, with KCNQ blockers being more potent at younger than P7 and erg blockers more potent at older than P16. Single-cell reverse transcription-PCR and immunocytochemistry showed expression of KCNQ and erg subunits. We propose that KCNQ channels contribute to g−30 and g−60 and erg subunits contribute to g−80. Type I hair cells are contacted by calyceal afferent endings. Recordings from dissociated calyces and afferent endings revealed large K+ conductances, including a KCNQ conductance. Calyx endings were strongly labeled by KCNQ4 and erg1 antisera. Thus, both hair cells and calyx endings have large M-like K+ conductances with the potential to control the gain of transmission.

Molecular Microdomains in a Sensory Terminal, the Vestibular Calyx Ending

Many primary vestibular afferents form large cup-shaped postsynaptic terminals (calyces) that envelope the basolateral surfaces of type I hair cells. The calyceal terminals both respond to glutamate released from ribbon synapses in the type I cells and initiate spikes that propagate to the afferents central terminals in the brainstem. The combination of synaptic and spike initiation functions in these unique sensory endings distinguishes them from the axonal nodes of central neurons and peripheral nerves, such as the sciatic nerve, which have provided most of our information about nodal specializations. We show that rat vestibular calyces express an unusual mix of voltage-gated Na and K channels and scaffolding, cell adhesion, and extracellular matrix proteins, which may hold the ion channels in place. Protein expression patterns form several microdomains within the calyx membrane: a synaptic domain facing the hair cell, the heminode abutting the first myelinated internode, and one or two intermediate domains. Differences in the expression and localization of proteins between afferent types and zones may contribute to known variations in afferent physiology.

Morphophysiological and ultrastructural studies in the mammalian cristae ampullares

There are three kinds of afferent terminations in the cristae ampullares. Calyx units innervate a few neighboring type I hair cells. Bouton units contact several type II hair cells. Dimorphic units innervate both kinds of receptors. Axon diameters are largest for calyx fibers and smallest for bouton fibers. Dimorphic units supply all parts of the sensory epithelium. Calyx units are confined to the central zone of the crista and bouton units to its peripheral zone. Intra-axonal labeling was used to determine the innervation patterns of physiologically characterized afferents. Calyx units are irregularly discharging. Dimorphic units in the central zone have a more irregular discharge than those in the peripheral zone. Bouton units, which have also been identified by their slow conduction velocities, are regularly discharging. An afferents discharge regularity, sensitivity to externally applied galvanic currents and response dynamics are more closely related to its epithelial location than to its branching pattern or to the types and number of hair cells it contacts. Of the various discharge properties studied, only the rotational gains seemed closely related to terminal morphology. Afferents innervating the central and peripheral zones differ in their innervation patterns and discharge properties. A preliminary ultrastructural study indicates that there also are regional variations in synaptic organization. Type II hair cells in the peripheral zone are contacted by many more afferent boutons than those in the central zone. Individual central boutons have multiple ribbon synapses with type II hair cells, whereas each peripheral bouton usually has a single synaptic contact. Synapses between type II hair cells and calyx endings are common centrally, but not peripherally. Two synaptic features did not vary regionally: 1) type I hair cells usually make 10-20 ribbon synapses with their calyx endings; and 2) each type II hair cell is contacted by 2-6 efferent endings. The number of efferent boutons in contact with each calyx ending declines slightly from the peripheral zone to the central zone. Reciprocal synapses were rare.

An investigation of collateral projections of the dorsal lateral geniculate nucleus and other subcortical structures to cortical areas V1 and V4 in the macaque monkey: a double label retrograde tracer study

SummaryPrevious anterograde studies in the macaque monkey have shown that, in addition to the projection to striate cortex (V1), the dorsal lateral geniculate nucleus (DLG) has a sparse, horizontally segregated projection to layers IV and V of prestriate cortex (V4). However, the distribution and degree of axon collateralization of DLG cells which give rise to these projections are unknown. This study was designed to answer these questions. The DLG (along with the pulvinar and other subcortical regions) was examined for the presence of single- or doublelabeled cells after injections of two different (fluorescent or HRP) retrograde tracers into corresponding retinotopic points in visual cortical areas V1 and V4. In the DLG, it was found that cells projecting to V4, which reside in or near the tectorecipient interlaminar zones of the DLG, do not project to V1 and thus represent a separate population of cells. The organization of the macaque geniculo-prestriate projection thus seems quite different from that of carnivores. Both single- and double-labeled cells were found in other subcortical areas, e.g., single-labeled cells were found in the claustrum, hypothalamus and lateral pulvinar, and a double-labeled cell population was found in the inferior pulvinar.

Mechanisms of Efferent-Mediated Responses in the Turtle Posterior Crista

To study the cellular mechanisms of efferent actions, we recorded from vestibular-nerve afferents close to the turtle posterior crista while efferent fibers were electrically stimulated. Efferent-mediated responses were obtained from calyx-bearing (CD, calyx and dimorphic) afferents and from bouton (B) afferents distinguished by their neuroepithelial locations into BT units near the torus and BM units at intermediate sites. The spike discharge of CD units is strongly excited by efferent stimulation, whereas BT and BM units are inhibited, with BM units also showing a postinhibitory excitation. Synaptic activity was recorded intracellularly after spikes were blocked. Responses of BT/BM units to single efferent shocks consist of a brief depolarization followed by a prolonged hyperpolarization. Both components reflect variations in hair-cell quantal release rates and are eliminated by pharmacological antagonists of α9/α10 nicotinic receptors. Blocking calcium-dependent SK potassium channels converts the biphasic response into a prolonged depolarization. Results can be explained, as in other hair-cell systems, by the sequential activation of α9/α10 and SK channels. In BM units, the postinhibitory excitation is based on an increased rate of hair-cell quanta and depends on the preceding inhibition. There is, in addition, an efferent-mediated, direct depolarization of BT/BM and CD fibers. In CD units, it is the exclusive efferent response. Nicotinic antagonists have different effects on hair-cell efferent actions and on the direct depolarization of CD and BT/BM units. Ultrastructural studies, besides confirming the efferent innervation of type II hair cells and calyx endings, show that turtle efferents commonly contact afferent boutons terminating on type II hair cells.

Nitric oxide synthase localized in a subpopulation of vestibular efferents with NADPH diaphorase histochemistry and nitric oxide synthase immunohistochemistry.

Efferent innervation of the vestibular labyrinth is known to be cholinergic. More recent studies have also demonstrated the presence of the neuropeptide calcitonin gene‐related peptide in this system. Nitric oxide is one of a new class of neurotransmitters, the gaseous transmitters. It acts as a second messenger and neurotransmitter in diverse physiological systems. We decided to investigate the anatomical distribution of the synthetic enzyme for nitric oxide, nitric oxide synthase (NOS), to clarify the role of nitric oxide in the vestibular periphery. NADPH diaphorase histochemical and NOS I immunohistochemical studies were done in the adult chinchilla and rat vestibular brainstem; diaphorase histochemistry was done in the chinchilla periphery. Retrograde tracing studies to verify the presence of NOS in brainstem efferent neurons were performed in young chinchillas. Our light microscopic results show that NOS I, as defined mainly by the presence of NADPH diaphorase, is present in a subpopulation of both brainstem efferent neurons and peripheral vestibular efferent boutons. Our ultrastructural results confirm these findings in the periphery. NADPH diaphorase is also present in a subpopulation of type I hair cells, suggesting that nitric oxide might be produced in and act locally upon these cells and other elements in the sensory epithelium. A hypothesis about how nitric oxide is produced in the vestibular periphery and how it may interact with other elements in the vestibular sensory apparatus is presented in the discussion . J. Comp. Neurol. 427:508–521, 2000.

Hair cells in mammalian utricles

Two morphological classes of mechanosensory cells have been described in the vestibular organs of mammals, birds, and reptiles: type I and type II hair cells. Type II hair cells resemble hair cells in other organs in that they receive bouton terminals from primary afferent neurons. In contrast, type I hair cells are enveloped by large cuplike afferent terminals called calyces. Type I and II cells differ in other morphological respects: cell shape, hair bundle properties, and more subtle ultrastructural features. Understanding the functional significance of these strikingly different morphological features has proved to be a challenge. Experiments that correlated the response properties of primary vestibular afferents with the morphologies of their afferent terminals suggested that the synapse between the type I hair cell and calyx ending is lower gain than that between a type II hair cell and a bouton ending. Recently, patch-clamp experiments on isolated hair cells have revealed that type I hair cells from diverse species have a large potassium conductance that is activated at the resting potential. As a consequence, the voltage responses generated by the type I hair cells in response to injected currents are smaller than those generated by type II hair cells. This may contribute to the lower gain of type I inputs to primary afferent neurons. Studies of neonatal mouse utricles show that the type I-specific potassium conductance is not present at birth but emerges during the first postnatal week, a period of morphological differentiation of type I and type II hair cells.

Peripherin immunoreactivity labels small diameter vestibular ‘bouton’ afferents in rodents

Recent morphophysiological studies have described three different subpopulations of vestibular afferents. The purpose of this study was to determine whether peripherin, a 56-kDa type III intermediate filament protein present in small sensory neurons in dorsal root ganglion and spiral ganglion cells, would also label thin vestibular afferents. Peripherin immunohistochemistry was done on vestibular sensory organs (cristae ampullares, utriculi and sacculi) of chinchillas, rats, and mice. In these sensory organs, immunoreactivity was confined to the extrastriolar region of the utriculus and the peripheral region of the crista. The labelled terminals were all boutons, except for an occasional calyx. In vestibular ganglia, immunoreactivity was restricted to small vestibular ganglion cells with thin axons. The immunoreactive central axons of vestibular ganglion cells form narrow bundles as they pass through the caudal spinal trigeminal tract. As they exit this tract, several bundles coalesce to form a single, narrow bundle passing caudally through the ventral part of the lateral vestibular nucleus. Finally, we conclude that all labelled axons and terminals were vestibular afferents rather than efferents, as no immunoreactivity in the vestibular efferent nucleus of the brainstem was observed.