We used an antibody to choline acetyltransferase (ChAT) to label cholinergic cells in guinea pig brainstem. ChAT-immunoreactive (ChAT-IR) cells comprise several prominent groups, including the pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus, and parabigeminal nucleus, as well as the cranial nerve somatic motor and parasympathetic nuclei. Additional concentrations are present in the parabrachial nuclei and superior colliculus.Among auditory nuclei, the majority of ChAT-IR cells are in the superior olive, particularly in and around the lateral superior olive, the ventral nucleus of the trapezoid body and the superior paraolivary nucleus. A discrete group of ChAT-IR cells is located in the sagulum, and additional cells are scattered in the nucleus of the brachium of the inferior colliculus. A group of ChAT-IR cells lies dorsal to the dorsal nucleus of the lateral lemniscus. A few ChAT-IR cells are found in the cochlear nucleus and the ventral nucleus of the lateral lemniscus.

We combined retrograde tracing with immunohistochemistry for choline acetyltransferase to identify the source of cholinergic input to the inferior colliculus (IC). Injection of a retrograde tracer into one IC labeled cells in many brainstem nuclei. Retrogradely-labeled cells that were also immunoreactive for choline acetyltransferase were identified in two nuclei in the midbrain tegmentum: the pedunculopontine tegmental nucleus (PPT) and the laterodorsal tegmental nucleus (LDT). More PPT and LDT cells project ipsilaterally than contralaterally to the IC and, on both sides, there are more projecting cells in the PPT than in the LDT. Double-labeled cells were not found in any other brainstem nucleus. A common feature of cholinergic cells in PPT and LDT is collateral projections to multiple targets. We placed different retrograde tracers into each IC to identify cells in PPT and LDT that project to both ICs. In both PPT and LDT, a substantial proportion (up to 57%) of the immunoreactive cells that contained tracer from the contralateral IC also contained tracer from the ipsilateral IC. We conclude that acetylcholine in the IC originates from the midbrain tegmental cholinergic nuclei: PPT and LDT. These nuclei are known to participate in arousal, the sleep/wake cycle and prepulse inhibition of acoustic startle. It is likely that the cholinergic input to the IC is directly associated with these functions.

Acetylcholine (ACh) is a neuromodulator that is likely to play a role in plasticity as well as other phenomena at many sites in the auditory system. The auditory cortex receives cholinergic innervation from the basal forebrain, whereas the cochlea receives cholinergic innervation from the superior olivary complex. Much of the remainder of the auditory pathways receives innervation from the pedunculopontine and laterodorsal tegmental nuclei, two nuclei referred to collectively as the pontomesencephalic tegmentum (PMT). The PMT provides the major source of ACh to the auditory thalamus and the midbrain, and is a substantial source (in addition to the superior olivary complex) of ACh in the cochlear nucleus. Individual cholinergic cells in the PMT often have axon branches that innervate multiple auditory nuclei, including nuclei on both sides of the brain as well as nuclei at multiple levels of the auditory system. The auditory cortex has direct axonal projections to the PMT cells, including cholinergic cells that project to the inferior colliculus or cochlear nucleus. The divergent projections of PMT cholinergic cells suggest widespread effects on the auditory pathways. These effects are likely to include plasticity as well as novelty detection, sensory gating, reward behavior, arousal and attention. Descending projections from the forebrain, including the auditory cortex, are likely to provide a high level of cognitive input to these cholinergic effects. Dysfunction associated with the cholinergic system may play a role in disorders such as tinnitus and schizophrenia.

A common method for identifying collateral projections is to inject different retrograde tracers into two targets and examine labeled cells for the presence of both tracers. Double-labeled cells are considered to have collateral projections to the two injection sites. This method is widely considered to underestimate the extent of collaterals. To test the efficiency of double-labeling, we mixed equal volumes of two tracers, injected them into one site in a guinea-pig brain, and counted the resulting labeled cells. Ideally, the tracers would have precisely overlapping injection sites and all labeled cells would contain both tracers. We tested several combinations of tracers: 1) Fast Blue and fluorescein dextran; 2) fluorescein dextran and FluoroGold; 3) fluorescein dextran and FluoroRuby; 4) FluoroGold and green beads; 5) FluoroGold and red beads; 6) FluoroRuby and green beads; and, 7) green beads and red beads. For each combination, a mixture was injected into the left inferior colliculus. After 1 week to allow for transport, labeled cells were counted in the right inferior colliculus and the left temporal cortex. For each mixture, the results were similar for the two areas. The percentage of cells that were double-labeled varied from 0% to 100%, depending on tracer combination. The highest efficiencies (>96%) were observed with red beads and green beads or with FluoroRuby and fluorescein dextran. The limited efficiency of other mixtures could be accounted for only in part by incomplete overlap of the two tracers at the injection site. The results indicate that the specific combination of tracers used to search for collateral projections can greatly affect the findings.

A GABAergic component has been identified in the projection from the inferior colliculus (IC) to the medial geniculate body (MG) in cats and rats. We sought to determine if this GABAergic pathway exists in guinea pig, a species widely used in auditory research. The guinea pig IC contains GABAergic cells, but their relative abundance in the IC and their relative contributions to tectothalamic projections are unknown. We identified GABAergic cells with immunochemistry for glutamic acid decarboxylase (GAD) and determined that ~21% of IC neurons are GABAergic. We then combined retrograde tracing with GAD immunohistochemistry to identify the GABAergic tectothalamic projection. Large injections of Fast Blue, red fluorescent beads or FluoroGold were deposited to include all subdivisions of the MG. The results demonstrate a GABAergic pathway from each IC subdivision to the ipsilateral MG. GABAergic cells constitute ~22% of this ipsilateral pathway. In addition, each subdivision of the IC had a GABAergic projection to the contralateral MG. Measured by number of tectothalamic cells, the contralateral projection is about 10% of the size of the ipsilateral projection. GABAergic cells constitute about 20% of the contralateral projection. In summary, the results demonstrate a tectothalamic projection in guinea pigs that originates in part from GABAergic cells that project ipsilaterally or contralaterally to the MG. The results show similarities to both rats and cats, and carry implications for the role of GABAergic tectothalamic projections vis-à-vis the presence (in cats) or near absence (in rats and guinea pigs) of GABAergic interneurons in the MG.

These results failed to support previous research, which indicated that amplification of auditory input could benefit balance in individuals with hearing and balance disorders. Further research utilizing randomized controlled trials is needed to resolve the disparity between the current results and those of previous studies.

Acetylcholine affects a variety of cell types in the cochlear nucleus (CN) and is likely to play a role in numerous functions. Previous work in rats suggested that the acetylcholine arises from cells in the superior olivary complex, including cells that have axonal branches that innervate both the CN and the cochlea (i.e., olivocochlear cells) as well as cells that innervate only the CN. We combined retrograde tracing with immunohistochemistry for choline acetyltransferase to identify the source of ACh in the CN of guinea pigs. The results confirm a projection from cholinergic cells in the superior olivary complex to the CN. In addition, we identified a substantial number of cholinergic cells in the pedunculopontine tegmental nucleus (PPT) and the laterodorsal tegmental nucleus (LDT) that project to the CN. On average, the PPT and LDT together contained about 26% of the cholinergic cells that project to CN, whereas the superior olivary complex contained about 74%. A small number of additional cholinergic cells were located in other areas, including the parabrachial nuclei.The results highlight a substantial cholinergic projection from the pontomesencephalic tegmentum (PPT and LDT) in addition to a larger projection from the superior olivary complex. These different sources of cholinergic projections to the CN are likely to serve different functions. Projections from the superior olivary complex are likely to serve a feedback role, and may be closely tied to olivocochlear functions. Projections from the pontomesencephalic tegmentum may play a role in such things as arousal and sensory gating. Projections from each of these areas, and perhaps even the smaller sources of cholinergic inputs, may be important in conditions such as tinnitus as well as in normal acoustic processing. Keywordsarousal; sensory gating; tinnitus; olivocochlear; pedunculopontine tegmental nucleus; laterodorsal tegmental nucleus Cholinergic innervation of the cochlear nucleus (CN) has been demonstrated by many methods, including physiology, receptor binding, enzyme assays, histochemistry and Correspondence: Brett R. Schofield, PhD, NEOUCOM, Department of Anatomy and Neurobiology, PO Box 95, Rootstown, OH 44272 USA,, bschofie@neoucom.edu; Jeffrey G. Mellott, PhD, Address: as above, jmellott@neoucom.edu; Susan D. Motts, PhD, Address: as above, smotts@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 note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2012 April 28. and Whitfield, 1968;Caspary et al., 1983;Osen et al., 1984;Godfrey et al., 1987Godfrey ...

Direct projections from the cochlear nucleus (CN) to the medial geniculate body (MG) mediate a high-speed transfer of acoustic information to the auditory thalamus. Anderson etal. (2006) used anterograde tracers to label the projection from the dorsal CN (DCN) to the MG in guinea pigs. We examined this pathway with retrograde tracers. The results confirm a pathway from the DCN, originating primarily from the deep layers. Labeled cells included a few giant cells and a larger number of small cells of unknown type. Many more labeled cells were present in the ventral CN (VCN). These cells, identifiable as multipolar (stellate) or small cells, were found throughout much of the VCN. Most of the labeled cells were located contralateral to the injection site. The CN to MG pathway bypasses the inferior colliculus (IC), where most ascending auditory information is processed. Anderson etal. (2006) hypothesized that CN-MG axons are collaterals of axons that reach the IC. We tested this hypothesis by injecting different fluorescent tracers into the MG and IC and examining the CN for double-labeled cells. After injections on the same side of the brain, double-labeled cells were found in the contralateral VCN and DCN. Most double-labeled cells were in the VCN, where they accounted for up to 37% of the cells labeled by the MG injection. We conclude that projections from the CN to the MG originate from the VCN and, less so, from the DCN. A significant proportion of the cells send a collateral projection to the IC. Presumably, the collateral projections send the same information to both the MG and the IC. The results suggest that T-stellate cells of the VCN are a major source of direct projections to the auditory thalamus.