Inositol 1,4,5‐trisphosphate receptors: Immunocytochemical localization in the dorsal cochlear nucleus

Abstract

In the cochlear nucleus of mammals, the relatively homogeneous responses of auditory nerve fibers are transformed into a variety of different response patterns by the different classes of resident neurons. The spectrum of these responses is hypothesized to depend on the types and distribution of receptors, ion channels, G proteins, and second messengers that form the signaling capabilities in each cell class. In the present study, we examined the immunocytochemical distribution of the inositol 1,4,5-triphosphate (IP3) receptor in the dorsal cochlear nucleus to better understand how this second messenger might be involved in shaping the neural signals evoked by sound. Affinity-purified polyclonal antibodies directed against the IP3 receptor labeled a homogeneous population of neurons in the dorsal cochlear nucleus of rats, guinea pigs, mustache bats, cats, New World owl monkeys, rhesus monkeys, and humans. These cells were all darkly immunostained except in the human where the labeling was less intense. Immunoblots of dorsal cochlear nucleus tissue from the rat revealed a single band of protein of molecular weight approximately 260 kD, which is the same size as the purified receptor, indicating that our antibodies reacted specifically with the IP3 receptor. These immunolabeled neurons were identified as cartwheel cells on the basis of shared characteristics across species, including cell body size and distribution, the presence of a highly invaginated nucleus, and a well-developed system of cisternae. Reaction product was localized along the membranes of rough and smooth endoplasmic reticulum, subsurface cisternae, and the nuclear envelope. This label was distributed throughout the cartwheel cell body and dendritic shafts but not within dendritic spines, axons, or axons terminals. The regular pattern of immunolabeling across mammals suggests that IP3 and cartwheel cells are conserved in evolution and that both play an important but as yet unknown role in hearing.

Bidirectional synaptic plasticity in the cerebellum-like mammalian dorsal cochlear nucleus

“…Group 1 mGluRs have been shown to activate phospholipase C and increase the level of IP 3 , which in turn allows Ca 2ϩ to be released from intracellular stores through IP 3 receptors. Cartwheel, but not fusiform, cells are labeled by antibodies to IP 3 receptors (30). The presence of vesicles in spines of cartwheel cells indicates that Ca 2ϩ released from intracellular stores could act within individual spines (30,31).…”

“…Cartwheel, but not fusiform, cells are labeled by antibodies to IP 3 receptors (30). The presence of vesicles in spines of cartwheel cells indicates that Ca 2ϩ released from intracellular stores could act within individual spines (30,31). Group 3 mGluRs, activated by L-AP4, act presynaptically in the DCN (14).…”

“…Like Purkinje cells, cartwheel cells fire complex action potentials (10,12), contain mGluR1␣ in their spines (16,17), IP 3 in their dendrites (30), and PEP19 (32), cerebellin (33), and glutamic acid decarboxylase (34). Genetic mutations affect Purkinje cells and cartwheel cells similarly (35).…”

Primary innervation of the avian and mammalian cochlear nucleus

“…Both inputs are excitatory and run orthogonally to one another. In the auditory system, they run longitudinally along the surface of the DCN, perpendicular to the isofrequency contours where they form excitatory synapses with the apical dendrites of pyramidal and cartwheel cells [47,121,173,218]. In this way, granule cells exert their influence upon pyramidal neurons that provide the main output for the nucleus [44,106,222].…”

Revealing the molecular layer of the primate dorsal cochlear nucleus

“…Vibratome slices followed 2 cycles of freezing and thawing on dry ice, washed in 0.1M PB and were either stained for Nissl or incubated with the corresponding primary antibody [calbindin-D28k (1:3,000, Sigma, Saint Louis, MO), IP3r (inositol 1,4,5-triphosphate receptor; 0.1 µg/ml, Sharp et al, 1993;Ryugo et al, 1995); PDE10A (phosphodiesterase 10A; 1.2 µg/ ml, Seeger et al, 2003); GluR1 (1.5 µg, Wenthold, 1992;Douyard et al, 2007), GluR2/3 (1.5 µg; Wenthold, 1992;Douyard et al, 2007), GluR4 (1.5 µg, Wenthold, 1992;Douyard et al, 2007), delta ½ (1.0 µg /ml; Mayat et al, 1995;Rubio and Wenthold, 1997), and VGLUT1 (1:2,000; Synaptic System, Göttingen, Germany)] in 0.1M PB for 48 hours at 4°C. All the antibodies used were polyclonal with the exception of calbindin and PDE10A.…”