Cholinergic and non-cholinergic projections from the pedunculopontine and laterodorsal tegmental nuclei to the medial geniculate body in Guinea pigs

Abstract

The midbrain tegmentum is the source of cholinergic innervation of the thalamus and has been associated with arousal and control of the sleep/wake cycle. In general, the innervation arises bilaterally from the pedunculopontine tegmental nucleus (PPT) and the laterodorsal tegmental nucleus (LDT). While this pattern has been observed for many thalamic nuclei, a projection from the LDT to the medial geniculate body (MG) has been questioned in some species. We combined retrograde tracing with immunohistochemistry for choline acetyltransferase (ChAT) to identify cholinergic projections from the brainstem to the MG in guinea pigs. Double-labeled cells (retrograde and immunoreactive for ChAT) were found in both the PPT (74%) and the LDT (26%). In both nuclei, double-labeled cells were more numerous on the ipsilateral side. About half of the retrogradely labeled cells were immunonegative, suggesting they are non-cholinergic. The distribution of these immunonegative cells was similar to that of the immunopositive ones: more were in the PPT than the LDT and more were on the ipsilateral than the contralateral side. The results indicate that both the PPT and the LDT project to the MG, and suggest that both cholinergic and non-cholinergic cells contribute substantially to these projections.

Keywords: GABA; acetylcholine; arousal; auditory; glutamate; sleep; thalamus.

Figure 1

Photomicrographs of medial geniculate body (MG) subdivisions and representative tracer injection sites in transverse sections. (A) Section stained with cytochrome oxidase (CO) showing the subdivisions of MG: dl, dorsolateral; m, medial; s, shell; sg, suprageniculate; v, ventral. (B) Injection of red beads that is centered in the ventral MG (v) and extends into the dorsolateral subdivision (dl). This photomicrograph is of the right MG, but was flipped to make comparisons between the cases easier. GP 595R. (C) Injection of green beads confined to the ventral subdivision. The image is an overlay of a fluorescence image to visualize the GB and a brightfield image to show the CO counterstain. GP 587L. (D) An injection of red beads within the suprageniculate subdivision (sg). This photomicrograph is of the right MG, but was flipped to make comparisons between the cases easier. GP 586R. Scale bars = 0.5mm for A–D.

Figure 2

Photomicrographs of labeled cells in the PMT after injections in MG. In each panel the left image shows cells labeled with retrograde tracer [green beads for A–C (GP 585L), FluoroGold for D (GP 484L)]. The right image shows the same field of view filtered for ChAT immunolabel. Arrowheads indicate cells labeled with both tracer and immunolabel; arrows indicate cells labeled with tracer only. (A,B) PPT cells projecting to the ipsilateral MG. (C) PPT cell projecting to the contralateral MG. (D) LDT cell projecting to contralateral MG. All photomicrographs are taken from transverse sections. Scale bar = 10μm.

Figure 3

Plots of labeled cells in the PMT after large (A) or small (B,C) injections in the thalamus. (A) Drawings of transverse sections showing a large FluoroGold injection (blue shading, sections 1–7 [left side of section only]) in the left thalamus and labeled cells in the PMT nuclei (sections 13–17). An entire midbrain section is shown (bottom of column) for orientation of brainstem sections. Only the portion of tegmentum that included the PPT and the LDT (indicated by box) are shown in the remaining sections. The legend indicates the type of label in all panels; each symbol represents at least one labeled cell. GP 604L. (B) Drawing through the center of a green bead injection site confined to the ventral subdivision of the MG. Labeled cells are shown in the PMT at 3 rostro-caudal levels. GP 587L. (C) Drawing through the center of a red bead injection in the MG suprageniculate subdivision, and resulting label in the PMT. The drawings were flipped to make comparisons between the cases easier. GP 586R. In all panels, the sections are numbered from rostral to caudal with each whole number representing a 300-μm spacing. The extent of the PPT and LDT nuclei are indicated by the distribution of the ChAT immunopositive cells (green triangles). The ChAT immunopositive cells within the periaqueductal gray (PAG) are in the LDT; the ChAT immunopositive cells outside the PAG are in the PPT. The superior cerebellar peduncle is shown in gray as it is a helpful landmark for the PPT. APT, anterior pretectal nucleus; Aq, aqueduct; CG, central gray; dl, dorsolateral subdivision of MG; DLL, dorsal nucleus of lateral lemniscus; Ha, habenula; IC, inferior colliculus; LDT, laterodorsal tegmental nucleus; lfp; longitudinal fasciculus of the pons; LG, lateral geniculate body; LP, lateral posterior nucleus; m, medial subdivision of MG; mlf, medial longitudinal fasciculus; Mo5, motor nucleus of 5^th nerve; PAG, periaqueductal gray; PBG, parabigeminal nucleus; PnO, pontine reticular nucleus, oral part; Po, posterior nucleus of the thalamus; PPT, pedunculopontine tegmental nucleus; Pul, pulvinar nucleus; RtTg, reticulotegmental nucleus of the pons; s, shell subdivision of MG; SC, superior colliculus; scp, superior cerebellar peduncle; sg, suprageniculate subdivision of MG; SN, substantia nigra; v, ventral subdivision of MG; xscp, decussation of scp; ZI, zona incerta.

Figure 4

Graphs to illustrate the contribution of ipsilateral and contralateral PPT and LDT to the overall projection to MG. Tracer-labeled cells were counted in every sixth section and tallied according to whether they were ChAT+ or ChAT−. The results are expressed as a percentage of the total number of tracer-labeled cells. (A) Results from a very large injection (GP604L). (B) Aggregate results from nine cases with small injections located almost entirely within the MG. Error bars indicate standard error of the mean. cLDT, contralateral laterodorsal tegmental nucleus; cPPT, contralateral pedunculopontine tegmental nucleus; iLDT, ipsilateral laterodorsal tegmental nucleus; iPPT, ipsilateral pedunculopontine tegmental nucleus.