Figure 1.
Immunohistochemical detection of dopaminergic neurons in the VTA and the SN of the human and the rat.
Representative low-power images of immunostained sections from an adult male human (A) and rat (D) illustrate the distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) dopaminergic neurons in the ventral tegmental area (VTA) and pars compacta of the substantia nigra (SN). Medium-power images (insets B, C, E and F) reveal that the pars compacta of the SN contains densely-packed dopaminergic neurons (arrows) in both species. In contrast, while dopaminergic neurons are distributed loosely in the human VTA (arrows in B), they exhibit a relatively high regional cell density in the VTA of the rat (arrows in E). Cresyl violet staining in A–C visualizes non-dopaminergic perikarya. Scale bar = 200 µm in A, D and 66 µm in B, C, E, F.
Figure 2.
Orexin B-immunoreactive inputs to dopaminergic and non-dopaminergic neurons in the human and rat VTA and SN.
Images of histological samples from an adult male human (A–H) and rat (I–L) illustrate orexin B (black) and tyrosine hydroxylase (TH; brown) immunoreactivities visualized with the silver-gold intensified nickel-diaminobenzidine and diaminobenzidine chromogens, respectively, in the ventral tegmental area (VTA; A–E, I, J) and the pars compacta of the substantia nigra (SN; F–H, K, L). Cresyl violet was applied in A–H to also reveal non-dopaminergic perikarya in human midbrain sections. Arrows in high power photomicrographs point to the sporadically encountered neuronal appositions between orexin B-immunoreactive (IR) axons and TH-IR (B–D, G, H, J, L) or TH-immunonegative (E) perikarya and dendrites. Dopaminergic neurons of the human VTA (A–E) form a loose cell mass in which TH-IR dopaminergic and Nissl-labeled non-dopaminergic neurons intermingle. Orexin B-IR appositions can only be observed on a relatively small subset of the dopaminergic cell bodies (B) and dendrites (B, D). Framed regions in C are shown in high-power micrographs D and E and illustrate orexinergic contacts on a TH-IR dendrite (D) and a TH-immunonegative Nissl-stained perikaryon (E), respectively. The pars compacta of the human SN exhibits a high density of dopaminergic cell bodies which contain dark brown neuromelanin (nm) granules (F–H). High-power images illustrate the infrequent apposition of orexin B-IR axons to the dendrites (G) and perikarya (H) of a small subset of TH-IR dopaminergic neurons. The VTA of the rat (I) exhibits a higher density of dopaminergic neurons, compared with the human VTA (A). Orexinergic contacts (J) on these neurons are rare. Similarly to the human, dopaminergic neurons of the rat form a compact cell population in the SN (K) and receive orexin B-immunoreactive inputs infrequently (L). Comparison of the above innervation patterns in the two species provides quantitative evidence for significantly heavier input frequencies in the human (Figs. 3 and 4), whereas Fig. 5 illustrates that the vast majority of orexinergic inputs target the dendritic compartment of dopaminergic neurons in both regions of both species. p, TH-IR perikarya; d, TH-IR dendrites; nm, dopaminergic cell bodies containing high levels of neuromelanin pigment. Scale bar = 10 µm in D, E, G, H, 30 µm in B, C, J, L and 130 µm in A, F, I, K.
Figure 3.
Percentages of dopaminergic somata receiving orexinergic innervation in the VTA and the SN.
Bar graph illustrates the low percentages of tyrosine hydroxylase-immunoreactive dopaminergic neuronal cell bodies that receive innervation from orexin B-immunoreactive axons in the ventral tegmental area (VTA) and substantia nigra (SN) of adult male humans and rats. Note that while the axo-somatic innervation is quite sparse in both species, the percentage of dopaminergic cell bodies receiving orexinergic input is 5-times higher in the human compared with the rat VTA and 2.2-times higher in the human compared with the rat SN. *p<0.05; **p<0.01.
Figure 4.
Mean incidences of orexin B-immunoreactive afferent contacts onto individual dopaminergic cell bodies.
The mean incidences of orexin B-immunoreactive afferent contacts onto the cell bodies of dopaminergic neurons in the ventral tegmental area (VTA) and the substantia nigra (SN) are low both in adult male humans and rats. Individual dopaminergic cell bodies receive 5-times more orexinergic appositions in the human compared with the rat VTA and 2.6-times more appositions in the human compared with the rat SN. *p<0.05; **p<0.01.
Figure 5.
Percentages of orexin B-immunoreactive contacts targeting the dendritic compartment of dopaminergic neurons.
The mean percentages of orexin B-immunoreactive contacts that target the dendritic compartment of dopaminergic neurons were calculated from the first randomly encountered one hundred orexin B/TH appositions in each human individual and in each rat. Both in the ventral tegmental area (VTA) and the substantia nigra (SN) and both in humans and rats, the vast majority (86–88%) of orexinergic inputs to dopaminergic neurons is axo-dendritic.