Background Precise timing of action potentials in neural circuitry is often critical for accurate processing of sensory information. This is especially true for the auditory brainstem nuclei responsible for localizing sound on the azimuth. Sound localization is determined by comparing the difference in arrival time of sound to both ears, called interaural time differences (ITDs). In mammals, this computation takes place in the medial superior olive (MSO), which receives bilateral excitation from both cochlear nuclei. The MSO also receives an inhibitory input from the medial nucleus of the trapezoid body (MNTB), which is essential for accurate sound localization. The MNTB receives a large calyceal input from the contralateral cochlear nucleus. This synapse is specialized to relay the precise timing of action potentials from the cochlear nucleus to other brainstem auditory nuclei, including the MSO. Recent evidence demonstrates that the MNTB also receives inhibitory input; however, the source of this inhibition is not known. We hypothesized that this inhibition might be coming from the MNTB itself. Neurons in the MNTB were filled with a neural tracer by electroporation, then the tissue was imaged in order to map the axonal projection of MNTB cells. The presence of MNTB axons terminating in the MNTB would suggest a recurrent source of inhibition.
Study Design and Methods Explanted brainstems of Mongolian gerbils were injected with rhodamine and incubated in a physiologic solution for 4 hours. Then brainstems were fixed and sliced on a vibratome at 60 μm and counterstained using green fluorescent NISSL. The sections were then imaged using confocal microscopy.
Results The injections were well localized in the MNTB and were refined so that a relatively few number of neurons were filled. Unfortunately, the large calyceal synapses on the MNTB cells took up the neuronal tracer as well, and we were unable to follow MNTB axons through the tissue due to the prevalence of labeled calyces and their axons. However, this method did produce a large number of labeled calyces, which can be used in the future for immunohistochemical studies to localize specific proteins to the calyx.
Conclusions This method was inadequate to trace axons of MNTB cells. However, this technique fills calyces in MNTB nicely and may be useful in the future for immunohistochemical studies.
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