Differential cholinergic innervation of lemniscal versus non-lemniscal regions of the inferior colliculus.

The inferior colliculus (IC), a midbrain hub for integration of auditory information, receives dense cholinergic input that could modulate nearly all aspects of hearing. A key step in understanding cholinergic modulation is to identify the source(s) and termination patterns of cholinergic input. These issues have not been addressed for the IC in mice, an increasingly important model for study of hearing. We examined cholinergic inputs to the IC in adult male and female mice. We used retrograde tracing and immunochemistry to identify three sources of cholinergic innervation of the mouse IC: the pedunculopontine tegmental nucleus (PPT), the laterodorsal tegmental nucleus (LDT) and the lateral paragigantocellular nucleus (LPGi). We then used Cre-dependent labeling of cholinergic neurons in normal-hearing ChAT-Cre mice to selectively label the cholinergic projections to the IC from each of the cholinergic sources. Labeling of cholinergic projections from the PPT and LDT revealed cholinergic axons and boutons terminating throughout the IC, with the ipsilateral projection being denser. Electron microscopic examination showed that these cholinergic axons can form traditional synaptic junctions with IC neurons. In separate experiments, selective labeling of cholinergic projections from the LPGi revealed bilateral projections to the IC. The LPGi axons exhibited relatively equal densities on ipsilateral and contralateral sides, but on both sides the terminations were largely restricted to the non-lemniscal regions of the IC (i.e., the dorsal cortex, lateral cortex and intercollicular tegmentum). We conclude first that cholinergic axons can form traditional synapses in the IC. In addition, lemniscal and non-lemniscal regions of the IC receive different patterns of cholinergic innervation. The lemniscal IC (IC central nucleus) is innervated by cholinergic neurons in the PPT and the LDT whereas the non-lemniscal "shell" areas of the IC are innervated by the PPT and LDT and by cholinergic neurons in the LPGi. DATA AVAILABILITY: Data will be made available on request.

Characterization of three cholinergic inputs to the cochlear nucleus.

Acetylcholine modulates responses throughout the auditory system, including at the earliest brain level, the cochlear nucleus (CN). Previous studies have shown multiple sources of cholinergic input to the CN but information about their relative contributions and the distribution of inputs from each source is lacking. Here, we used staining for cholinergic axons and boutons, retrograde tract tracing, and acetylcholine-selective anterograde tracing to characterize three sources of acetylcholine input to the CN in mice. Staining for cholinergic axons showed heavy cholinergic inputs to granule cell areas and the dorsal CN with lighter input to the ventral CN. Retrograde tract tracing revealed that cholinergic cells from the superior olivary complex, pontomesencephalic tegmentum, and lateral paragigantocellular nucleus send projections to the CN. When we selectively labeled cholinergic axons from each source to the CN, we found surprising similarities in their terminal distributions, with patterns that were overlapping rather than complementary. Each source heavily targeted granule cell areas and the dorsal CN (especially the deep dorsal CN) and sent light input into the ventral CN. Our results demonstrate convergence of cholinergic inputs from multiple sources in most regions of the CN and raise the possibility of convergence onto single CN cells. Linking sources of acetylcholine and their patterns of activity to modulation of specific cell types in the CN will be an important next step in understanding cholinergic modulation of early auditory processing.

Neurotransmitter phenotype and axonal projection patterns of VIP-expressing neurons in the inferior colliculus.

Neurons in the inferior colliculus (IC), the midbrain hub of the central auditory pathway, send ascending and descending projections to other auditory brain regions, as well as projections to other sensory and non-sensory brain regions. However, the axonal projection patterns of individual classes of IC neurons remain largely unknown. Vasoactive intestinal polypeptide (VIP) is a neuropeptide expressed by subsets of neurons in many brain regions. We recently identified a class of IC stellate neurons that we called VIP neurons because they are labeled by tdTomato (tdT) expression in VIP-IRES-Cre x Ai14 mice. Here, using fluorescence in situ hybridization, we found that tdT+ neurons in VIP-IRES-Cre x Ai14 mice express Vglut2, a marker of glutamatergic neurons, and VIP, suggesting that VIP neurons use both glutamatergic and VIPergic signaling to influence their postsynaptic targets. Next, using viral transfections with a Cre-dependent eGFP construct, we labeled the axonal projections of VIP neurons. As a group, VIP neurons project intrinsically, within the ipsilateral and contralateral IC, and extrinsically to all the major targets of the IC. Within the auditory system, VIP neurons sent axons and formed axonal boutons in higher centers, including the medial geniculate nucleus and the nucleus of the brachium of the IC. Less dense projections terminated in lower centers, including the nuclei of the lateral lemniscus, superior olivary complex, and dorsal cochlear nucleus. VIP neurons also project to several non-auditory brain regions, including the superior colliculus, periaqueductal gray, and cuneiform nucleus. The diversity of VIP projections compared to the homogeneity of VIP neuron intrinsic properties suggests that VIP neurons play a conserved role at the microcircuit level, likely involving neuromodulation through glutamatergic and VIPergic signaling, but support diverse functions at the systems level through their participation in different projection pathways.

Dense cholinergic projections to auditory and multisensory nuclei of the intercollicular midbrain.

Cholinergic axons from the pedunculopontine tegmental nucleus (PPT) innervate the inferior colliculus where they are positioned to modulate both excitatory and inhibitory circuits across the central nucleus and adjacent cortical regions. More rostral regions of the auditory midbrain include the nucleus of the brachium of the inferior colliculus (NBIC), the intercollicular tegmentum (ICt) and the rostral pole of the inferior colliculus (ICrp). These regions appear especially important for multisensory integration and contribute to orienting behavior and many aspects of auditory perception. These regions appear to receive cholinergic innervation but little is known about the distribution of cholinergic axons in these regions or the cells that they contact. The present study used immunostaining to examine the distribution of cholinergic axons and then used chemically-specific viral tracing to examine cholinergic projections from the PPT to the intercollicular areas in male and female transgenic rats. Staining with antibodies against vesicular acetylcholine transporter revealed dense cholinergic innervation throughout the NBIC, ICt and ICrp. Deposits of viral vector into the PPT labeled cholinergic axons bilaterally in the NBIC, ICt and ICrp. In each area, the projections were denser on the ipsilateral side. The axons appeared morphologically similar across the three areas. In each area, en passant and terminal boutons from these axons appeared in the neuropil and also in close apposition to cell bodies. Immunostaining with a marker for GABAergic cells suggested that the cholinergic axons likely contact both GABAergic and non-GABAergic cells in the NBIC, ICt and ICrp. Thus, the cholinergic axons could affect multisensory processing by modulating excitatory and inhibitory circuits in the NBIC, ICt and ICrp. The similarity of axons and their targets suggests there may be a common function for cholinergic innervation across the three areas. Given what is known about the PPT, such functions could be associated with arousal, sleep-wake cycle, reward and plasticity.

Cholinergic Projections From the Pedunculopontine Tegmental Nucleus Contact Excitatory and Inhibitory Neurons in the Inferior Colliculus.

The inferior colliculus processes nearly all ascending auditory information. Most collicular cells respond to sound, and for a majority of these cells, the responses can be modulated by acetylcholine (ACh). The cholinergic effects are varied and, for the most part, the underlying mechanisms are unknown. The major source of cholinergic input to the inferior colliculus is the pedunculopontine tegmental nucleus (PPT), part of the pontomesencephalic tegmentum known for projections to the thalamus and roles in arousal and the sleep-wake cycle. Characterization of PPT inputs to the inferior colliculus has been complicated by the mixed neurotransmitter population within the PPT. Using selective viral-tract tracing techniques in a ChAT-Cre Long Evans rat, the present study characterizes the distribution and targets of cholinergic projections from PPT to the inferior colliculus. Following the deposit of viral vector in one PPT, cholinergic axons studded with boutons were present bilaterally in the inferior colliculus, with the greater density of axons and boutons ipsilateral to the injection site. On both sides, cholinergic axons were present throughout the inferior colliculus, distributing boutons to the central nucleus, lateral cortex, and dorsal cortex. In each inferior colliculus (IC) subdivision, the cholinergic PPT axons appear to contact both GABAergic and glutamatergic neurons. These findings suggest cholinergic projections from the PPT have a widespread influence over the IC, likely affecting many aspects of midbrain auditory processing. Moreover, the effects are likely to be mediated by direct cholinergic actions on both excitatory and inhibitory circuits in the inferior colliculus.

Perineuronal nets and subtypes of GABAergic cells differentiate auditory and multisensory nuclei in the intercollicular area of the midbrain.

The intercollicular region, which lies between the inferior and superior colliculi in the midbrain, contains neurons that respond to auditory, visual, and somatosensory stimuli. Golgi studies have been used to parse this region into three distinct nuclei: the intercollicular tegmentum (ICt), the rostral pole of the inferior colliculus (ICrp), and the nucleus of the brachium of the IC (NBIC). Few reports have focused on these nuclei, especially the ICt and the ICrp, possibly due to lack of a marker that distinguishes these areas and is compatible with modern methods. Here, we found that staining for GABAergic cells and perineuronal nets differentiates these intercollicular nuclei in guinea pigs. Further, we found that the proportions of four subtypes of GABAergic cells differentiate intercollicular nuclei from each other and from adjacent inferior collicular subdivisions. Our results support earlier studies that suggest distinct morphology and functions for intercollicular nuclei, and provide staining methods that differentiate intercollicular nuclei and are compatible with most modern techniques. We hope that this will help future studies to further characterize the intercollicular region.

Shaping of discrete auditory inputs to extramodular zones of the lateral cortex of the inferior colliculus.

The multimodal lateral cortex of the inferior colliculus (LCIC) exhibits a modular-extramodular micro-organization that is evident early in development. In addition to a set of neurochemical markers that reliably highlight its modular-extramodular organization (e.g. modules: GAD67-positive, extramodular zones: calretinin-positive, CR), mature projection patterns suggest that major LCIC afferents recognize and adhere to such a framework. In adult mice, distinct afferent projections appear segregated, with somatosensory inputs targeting LCIC modules and auditory inputs surrounding extramodular fields. Currently lacking is an understanding regarding the development and shaping of multimodal LCIC afferents with respect to its emerging modular-extramodular microarchitecture. Combining living slice tract-tracing and immunocytochemical approaches in GAD67-GFP knock-in mice, the present study characterizes the critical period of projection shaping for LCIC auditory afferents arising from its neighboring central nucleus (CNIC). Both crossed and uncrossed projection patterns exhibit LCIC extramodular mapping characteristics that emerge from initially diffuse distributions. Projection mismatch with GAD-defined modules and alignment with encompassing extramodular zones becomes increasingly clear over the early postnatal period (birth to postnatal day 12). CNIC inputs terminate almost exclusively in extramodular zones that express CR. These findings suggest multimodal LCIC inputs may initially be sparse and intermingle, prior to segregation into distinct processing streams. Future experiments are needed to determine the likely complex interactions and mechanisms (e.g. activity-dependent and independent) responsible for shaping early modality-specific LCIC circuits.