Rhodamine backfilling and confocal microscopy as a tool for the unambiguous identification of neuronal cell types: A study of the neurones of the rat cochlear nucleus

Abstract

Adequate interpretation of the functional data characterising the projection neurones of the cochlear nucleus (CN) is impossible without the unequivocal classification of these cell types at the end of the experiments. In this study, morphological criteria applicable for unambiguous identification of CN neurones have been sought. The neurones were labelled with rhodamine from incisions severing the projection pathways of the individual cell types, allowing their selective labelling and morphological characterisation. Confocal microscopy was employed for the investigation of the rhodamine-filled cells whose morphology was assessed after reconstructing the three-dimensional images of the cell bodies and proximal processes. The diameters of the somata and the number of processes originating from the cell bodies were also determined. In most of the cases, unambiguous identification of the bushy, octopus and Purkinje-like cells was relatively straightforward. On the other hand, precise classification of the pyramidal cells was often difficult, especially because giant cells could easily possess morphological features resembling pyramidal neurones. Occasionally, giant cells also mimicked the appearance of octopus neurones, which may be another important source of identification error, especially as these two cell types are often situated close to each other in the CN. It is concluded that morphological criteria defined in the present work may be effectively applied for the unambiguous identification of the projection neurones of the CN, even following functional measurements, when the correct cell classification is essential for the interpretation of the experimental data. Moreover, the present study also confirmed that Purkinje-like cells project to the cerebellum. [6,7,10,16,[21][22][23]33,39,42], in which the projection neurones in the posterior part of the ventral CN (pVCN); giant cells are 38 scattered in both the VCN and the dorsal CN (DCN); whereas 39 pyramidal neurones and Purkinje-like cells (PLCs) are found 40 in the DCN only. The activity of neuronal networks consisting 41 of the projection cells and the numerous interneurones ensures 42 that the incoming acoustic information is not merely distributed, 43 but processed in the CN. Consequently, the projection neurones 44 actively contribute to the decoding of the acoustic information. 45 The axons of these cells form the efferent pathways targeting 46 various structures in the brain stem, and carry information about 47 different aspects of the sound stimuli (amplitude, onset, duration, 48 etc.). 49In a study aiming at the description and understanding of 50 the activity of such a complex neuronal structure as the CN, 51 it is imperative to clearly define the functional contribution of The experiments were conducted on 1-month-old Wistar rats (from both 111 sexes) following a protocol that was authorised by the Ethical Committee of the 112 University of Debrecen and was in accordance with the appropriate international 113 and Hungarian laws. The animals we...

Octopus Cells in the Posteroventral Cochlear Nucleus Provide the Main Excitatory Input to the Superior Paraolivary Nucleus

“…The presence of labeled thick fibers that curve around the inferior cerebellar peduncle and circumvent the spinal tract of the trigeminal nerve is compatible with the intermediate acoustic stria (IAS; Figure 2B; Smith et al, 2005). Retrogradely labeled neurons were found in the PVCN with a clear contralateral predominance (Figure 2B), and they were readily identified as octopus cells based on their distinct dendritic shape (Figure 2C; Harrison and Irving, 1966; Saldaña et al, 1987; Pocsai et al, 2007; Bazwinsky et al, 2008). These cells had large, irregularly shaped cell bodies (~ 20 μm in diameter) with three to five thick primary dendrites emerging from only one side of the cell body (Figure 2C).…”

Differences in developmental changes in GABAergic response between bushy and stellate cells in the rat anteroventral cochlear nucleus

“…Bushy cells typically have one or two primary dendrites. Two dendritic processes usually originate from the opposite poles of the cell body and proceed away from each other (Paolini and Clark, 1998; Pocsai et al, 2007). The dendrites of bushy cells characteristically end in a spray of thinner and shorter braches.…”

Growth behavior of cochlear nucleus neuronal cells on semiconductor substrates

“…Varieties of culture conditions for dissociated cell culture of C.N. on extracellular matrix compounds and biomaterials describing neuron morphology as well as setups for electrophysiological studies have been reported 15–19…”