Afferent connections of the laterodorsal and the pedunculopontine tegmental nuclei in the rat: A retro‐ and antero‐grade transport and immunohistochemical study

Abstract

Increasingly strong evidence suggests that cholinergic neurons in the mesopontine tegmentum play important roles in the control of wakefulness and sleep. To understand better how the activity of these neurons is regulated, the potential afferent connections of the laterodorsal (LDT) and pedunculopontine tegmental nuclei (PPT) were investigated in the rat. This was accomplished by using retrograde and anterograde axonal transport methods and NADPH‐diaphorase histochemistry. Immunohistochemistry was also used to identify the transmitter content of some of the retrogradely identified afferents. Following injections of the retrograde tracer wheatgerm agglutinin‐conjugated horseradish peroxidase (WGA‐HRP) into either the LDT or the PPT, labelled neurons were seen in a number of limbic forebrain structures. The medial prefrontal cortex and lateral habenula contained more retrogradely labelled neurons from the LDT, whereas in the bed nucleus of the stria terminalis and central nucleus of the amygdala, more cells were labelled from the PPT. Moderate numbers of neurons were seen in the magnocellular regions of the basal forebrain, and many labelled neurons were observed in the lateral hypothalamus, the zona incerta, and the midbrain central gray from both the LDT and the PPT. Accessory oculomotor nuclei in the midbrain as well as eye movement‐related structures in the lower brainstem contained some neurons labelled from the LDT, and fewer neurons from the PPT. A few labelled neurons were seen in somatosensory and other sensory relay nuclei in the brainstem and the spinal cord. Retrograde labelling was seen in a number of extrapyramidal structures, including the globus pallidus, entopenduncular and subthalamic nuclei, and substantia nigra following PPT injections; with LDT injections, labelling was similar in density in the substantia nigra but virtually absent in the entopeduncular and subthalamic nuclei. Data with the fluorescent retrograde tracer fluorogold combined with immunofluorescence indicated that many neurons in the zona incerta‐lateral hypothalamic region that were retrogradely labelled from the LDT contained α‐melanocyte‐stimulating hormone. Numerous neurons were labelled throughout the reticular formation of the brainstem following either LDT or PPT injections. Many neurons retrogradely labelled in the LDT and PPT, the dorsal and median raphe nuclei, and the locus ceruleus contained choline acetyltransferase, serotonin, and tyrosine hydroxylase, respectively. The anterograde tracers WGA‐HRP and phaseolus vulgaris leucoagglutinin were used to confirm some of the projections indicated by the retrograde labelling data; anterograde labelling was seen in the LDT and PPT following injections of one of these tracers into the medial prefrontal cortex, lateral hypothalamus, and the contralateral LDT. NADPH‐diaphorase histochemistry combined with immunohistochemistry indicated that serotonin and tyrosine hydroxylase‐immunoreactive fibers bearing occasional swellings were closely associated with NADPH‐diaphorase‐positive, cholinergic neurons in the LDT and PPT. These data suggest that potential afferents to the LDT and the PPT arise in many common, but also some separate structures in the forebrain, brainstem, and spinal cord. Both the LDT and the PPT appear to receive heavy inputs from three structures: brainstem reticular formation, midbrain central gray, and lateral hypothalamus‐zona incerta region. Other, less prominent afferents originate in a number of limbic, oculomotor, extrapyramidal, and sensory structures, with some degrees of preference for the LDT or PPT. Some of the reticular connections with the LDT and PPT are serotoninergic or noradrenergic. The reticuloreticular network also includes commissural and rostrocaudal cholinergic interconnections between the LDT and PPT. The apparent similarities in both the afferent and efferent connections of the LDT and the PPT are consistent with the working hypothesis that cholinergic neurons in the mesopontine tegmentum represent a single functional unit. The potential afferent connections identified in the present study also suggest that the PPT and LDT are involved in the regulation of behavioral state.