Combination sensitivity in central auditory neurons is a form of spectrotemporal integration in which excitatory responses to sounds at one frequency are facilitated by sounds within a distinctly different frequency band. Combination-sensitive neurons respond selectively to acoustic elements of sonar echoes or social vocalizations. In mustached bats, this response property originates in high frequency representations of the inferior colliculus (IC) and depends on low- and high-frequency-tuned glycinergic inputs. To identify source of these inputs, we combined glycine immunohistochemistry with retrograde tract-tracing. Tracers were deposited at high-frequency (>56 kHz), combination-sensitive recording sites in IC. Most glycine-immunopositive, retrogradely labeled cells were in ipsilateral ventral and intermediate nuclei of the lateral lemniscus (VNLL and INLL), with some double-labeling in ipsilateral lateral and medial superior olivary nuclei (LSO and MSO). Generally, double-labeled cells were in expected high-frequency tonotopic areas, but some VNLL and INLL labeling appeared to be in low-frequency representations. To test whether these nuclei provide low-frequency-tuned input to the high-frequency IC, we combined retrograde tracing from IC combination-sensitive sites with anterograde tracing from low-frequency-tuned sites in the anteroventral cochlear nucleus (AVCN). Only VNLL and INLL contained retrogradely labeled cells near (≤50 µm) anterogradely labeled boutons. These cells likely receive excitatory low-frequency input from AVCN. Results suggest that combination-sensitive facilitation arises through convergence of high-frequency glycinergic inputs from VNLL, INLL, or MSO and low-frequency glycinergic inputs from VNLL or INLL. This work establishes an anatomical basis for spectrotemporal integration in the auditory midbrain and a functional role for monaural nuclei of the lateral lemniscus.

In the intermediate nucleus of the lateral lemniscus (INLL), some neurons display a form of spectral integration in which excitatory responses to sounds at their best frequency are inhibited by sounds within a frequency band at least one octave lower. Previous work showed that this response property depends on low-frequency-tuned glycinergic input. To identify all sources of inputs to these INLL neurons, and in particular the low-frequency glycinergic input, we combined retrograde tracing with immunohistochemistry for the neurotransmitter glycine. We deposited a retrograde tracer at recording sites displaying either high best frequencies (>75 kHz) in conjunction with combination-sensitive inhibition, or at sites displaying low best frequencies (23)(24)(25)(26)(27)(28)(29)(30). Most retrogradely labeled cells were located in the ipsilateral medial nucleus of the trapezoid body (MNTB) and contralateral anteroventral cochlear nucleus. Consistent labeling, but in fewer numbers, was observed in the ipsilateral lateral nucleus of the trapezoid body (LNTB), contralateral posteroventral cochlear nucleus, and a few other brainstem nuclei. When tracer deposits were combined with glycine immunohistochemistry, most double-labeled cells were observed in the ipsilateral MNTB (84%), with fewer in LNTB (13%). After tracer deposits at combination-sensitive recording sites, a striking result was that MNTB labeling occurred in both medial and lateral regions. This labeling appeared to overlap the MNTB labeling that resulted from tracer deposits in low-frequency recording sites of INLL. These findings suggest that MNTB is the most likely source of low-frequency glycinergic input to INLL neurons with high best frequencies and combination-sensitive inhibition. This work establishes an anatomical basis for frequency integration in the auditory brainstem. Keywords combination sensitivity; spectral integration; glycine; mustached bat; INLL; Pteronotus parnelliiAlthough the initial stages of the auditory system create and maintain a spectral decomposition of sounds, acoustically guided behavior and perception are based on integration of information across spectral elements. Whether the task is localization of sounds (Hebrank and Wright, 1974, Knudsen andKonishi 1979;Middlebrooks, 1992;Populin and Yin, 1998), the analysis of sonar echoes (Simmons et al., 2004; Genzel and © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.Corresponding author: Jeffrey Wenstrup, Ph.D., Department of Anatomy and Neurobiology, Northeastern Ohio Universities Colleges of Medicine and Pharmacy, 4209 State Route 44, PO Box 95, Rootstown, Ohio 44272, Telephone: (330) Fax: (330) 325-5916, jjw@neoucom.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please n...