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.