Heterogeneous Calretinin Expression in the Avian Cochlear Nucleus Angularis

Abstract

Multiple calcium-binding proteins (CaBPs) are expressed at high levels and in complementary patterns in the auditory pathways of birds, mammals, and other vertebrates, but whether specific members of the CaBP family can be used to identify neuronal subpopulations is unclear. We used double immunofluorescence labeling of calretinin (CR) in combination with neuronal markers to investigate the distribution of CR-expressing neurons in brainstem sections of the cochlear nucleus in the chicken (Gallus gallus domesticus). While CR was homogeneously expressed in cochlear nucleus magnocellularis, CR expression was highly heterogeneous in cochlear nucleus angularis (NA), a nucleus with diverse cell types analogous in function to neurons in the mammalian ventral cochlear nucleus. To quantify the distribution of CR in the total NA cell population, we used antibodies against neuronal nuclear protein (NeuN), a postmitotic neuronspecific nuclear marker. In NA neurons, NeuN label was variably localized to the cell nucleus and the cytoplasm, and the intensity of NeuN immunoreactivity was inversely correlated with the intensity of CR immunoreactivity. The percentage of CR+neurons in NA increased from 31 % in embryonic (E)17/18 chicks, to 44 % around hatching (E21), to 51 % in postnatal day (P) 8 chicks. By P8, the distribution of CR + neurons was uniform, both rostrocaudal and in the tonotopic (dorsoventral) axis.Immunoreactivity for the voltage-gated potassium ion channel Kv1.1, used as a marker for physiological type, showed broad and heterogeneous postsynaptic expression in NA, but did not correlate with CR expression. These results suggest that CR may define a subpopulation of neurons within nucleus angularis.

Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems

“…However, even though we applied strict criteria to prevent weak NeuN + cells from being identified as NeuN − cells, substantial portions of CB + , CR + , and PV + neurons were still NeuN − while all SMI + neurons were NeuN + . This result is supported by a recent report that some CR + are not NeuN + , and CR and NeuN immunoreactivities have a negative correlation in the avian brainstem (Bloom et al, 2014). Likewise, in our experiments on human visual association cortex, cells with strong immunoreactivity against calcium-binding protein markers were frequently negative or very weakly positive for NeuN.…”

Intrinsic physiological properties underlie auditory response diversity in the avian cochlear nucleus

“…The NA neuron population exhibits heterogeneous immunostaining to K v 1.1, the low-threshold K ϩ channel pore-forming subunit (Bloom et al 2014;Fukui and Ohmori 2003), and Tonic neurons exhibit sensitivity to dendrotoxin, a potent I LT blocker, which increases Tonic firing rate (Fukui and Ohmori 2003). Because NA neurons are thought to encode sound intensity with increasing firing rate, we asked whether I LT could influence neuronal input-output functions.…”

“…We observed no relationship between cell type and location within NA, although tonotopic and anatomical differences were beyond the scope of this study, so they cannot be ruled out. Tonotopic differences in intrinsic, synaptic, morphologi-cal, and neurochemical markers have been observed in NM (Akter et al 2018;Fukui and Ohmori 2004;Hong et al 2018aHong et al , 2018bKöppl 1994;Kuba and Ohmori 2009;Oline et al 2016;Oline and Burger 2014;Wang et al 2017) and NA (Ahn and MacLeod 2016;Bloom et al 2014;Carr and Boudreau 1991;Levin et al 1997;Soares and Carr 2001), so it is possible that tonotopic gradients exist.…”

“…The lower input resistance may be related to a greater expression of I LT . I LT is an outward positive current partially active at resting potential, with variable expression in NA neurons (Bloom et al 2014;Fukui and Ohmori 2003). Partial activation at rest would be expected to result in low input resistance.…”

“…Whether NA's auditory response diversity is a product of intrinsic mechanisms (i.e., ion channels), synaptic mechanisms, or a combination is unclear. NA neurons exhibit diverse responses to current injection (Ahn et al 2014;Fukui and Ohmori 2003;Kreeger et al 2012;Soares et al 2002) and show variation in K ϩ channels (Bloom et al 2014;Fukui and Ohmori 2003). They also show variation in synaptic properties like short-term plasticity (MacLeod et al 2007;MacLeod and Horiuchi 2011) and dendritic morphology (Soares et al 2002;Soares and Carr 2001).…”