Percentages of orexin B-immunoreactive contacts targeting the dendritic compartment of dopaminergic neurons.

<p>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.</p

Mean incidences of orexin B-immunoreactive afferent contacts onto individual dopaminergic cell bodies.

<p>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.</p

Immunohistochemical detection of dopaminergic neurons in the VTA and the SN of the human and the rat.

<p>Representative low-power images of immunostained sections from an adult male human (<b>A</b>) and rat (<b>D</b>) 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>B</b>, <b>C</b>, <b>E</b> and <b>F</b>) 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>B</b>), they exhibit a relatively high regional cell density in the VTA of the rat (arrows in <b>E</b>). Cresyl violet staining in <b>A–C</b> visualizes non-dopaminergic perikarya. Scale bar = 200 µm in <b>A</b>, <b>D</b> and 66 µm in <b>B, C, E, F</b>.</p

Percentages of dopaminergic somata receiving orexinergic innervation in the VTA and the SN.

<p>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; <b>**</b>p<0.01.</p

Orexin B-immunoreactive inputs to dopaminergic and non-dopaminergic neurons in the human and rat VTA and SN.

<p>Images of histological samples from an adult male human (<b>A–H</b>) and rat (<b>I–L</b>) 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; <b>A–E</b>, <b>I</b>, <b>J</b>) and the pars compacta of the substantia nigra (SN; <b>F–H</b>, <b>K</b>, <b>L</b>). Cresyl violet was applied in <b>A–H</b> 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>B–D, G, H, J, L</b>) or TH-immunonegative (<b>E</b>) perikarya and dendrites. Dopaminergic neurons of the human VTA (<b>A–E</b>) 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>B</b>) and dendrites (<b>B, D</b>). Framed regions in <b>C</b> are shown in high-power micrographs <b>D</b> and <b>E</b> and illustrate orexinergic contacts on a TH-IR dendrite (<b>D</b>) and a TH-immunonegative Nissl-stained perikaryon (<b>E</b>), respectively. The pars compacta of the human SN exhibits a high density of dopaminergic cell bodies which contain dark brown neuromelanin (nm) granules (<b>F–H</b>). High-power images illustrate the infrequent apposition of orexin B-IR axons to the dendrites (<b>G</b>) and perikarya (<b>H</b>) of a small subset of TH-IR dopaminergic neurons. The VTA of the rat (<b>I</b>) exhibits a higher density of dopaminergic neurons, compared with the human VTA (<b>A</b>). Orexinergic contacts (<b>J</b>) on these neurons are rare. Similarly to the human, dopaminergic neurons of the rat form a compact cell population in the SN (<b>K</b>) and receive orexin B-immunoreactive inputs infrequently (<b>L</b>). 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 <b>D</b>, <b>E</b>, <b>G</b>, <b>H</b>, 30 µm in <b>B</b>, <b>C</b>, <b>J</b>, <b>L</b> and 130 µm in <b>A</b>, <b>F</b>, <b>I</b>, <b>K</b>.</p

Protocol for anxiety-related studies.

<p>Fed rats were injected with saline or ghrelin directly into the amygdala at time zero. In the FOOD ACCESS paradigm, rats were allowed access to food during the first hour after injection whereas food access was denied in the FOOD WITHHELD paradigm. After this, all rats underwent tests exploring anxiety-like behaviour, first in the EPM test (5 min) and then in the open field test (40 min). Afterwards all the rats were returned to their home cages and post-test food intake measured for 1 hr (corresponding to time 2–3 hr after injection).</p

<i>In situ</i> hybridization detection of GHSR mRNA in the rat amygdala.

<p>The autoradiographic detection of the isotopic <i>in situ</i> hybridization probe to GHSR mRNA revealed GHSR mRNA expression in distinct neurons of the amygdala. Note in A that the highest levels of expression could be observed in the ventrolateral (LaVL) and ventromedial (LaVM) parts of the lateral amygdaloid nucleus and in the posteroventral part of the medial amygdaloid nucleus (MePV). The parcellation and nuclear structure of the amygdala is shown in B. Also note the heavy GHSR mRNA signal in the arcuate (ARC) and ventromedial (VMH) nuclei. High-power photomicrographs (C–E) were amplified from various regions of the amygdala shown in A (cresyl violet counterstaining). Scale bars = 500 µm in A and B and 13 µm in C–E.</p

Histological verification of the location of the injection cannula in the lateral amygdaloid nucleus.

<p>A: Photomicrograph of a 40 µm counterstained coronal section of rat brain at level Bregma −3.3, illustrating the injection site. B: Schematic representation of the amygdala according to the rat brain atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046321#pone.0046321-Paxinos1" target="_blank">[37]</a>. The shadow area outlines the region defined as the lateral amygdaloid nucleus. Scale bar = 1 mm. Abbreviations: BLA (basolateral amygdaloid nucleus, anterior), BLP (basolateral amygdaloid nucleus, posterior), BMP (basomedial amygdaloid nucleus, posterior), BMA (basomedial amygdaloid nucleus, anterior), CeC (central amygdaloid nucleus, central), CeL (central amygdaloid nucleus, lateral), LaDL (lateral amygdaloid nucleus, lateral), LaVL (lateral amygdaloid nucleus, ventrolateral), LaVM (lateral amygdaloid nucleus, ventromedial), OT (optic tract).</p

Effects of intra-amygdala administration of ghrelin on anxiety-like behavior in rats given access to food.

<p>In rats given access to food during the first hour after intra-amygdala injection (FOOD ACCESS), ghrelin increased food intake relative to saline controls (g of chow), both during this hour and during the 1 hr measurement taken after the anxiety tests (A). In this paradigm there was no effect of ghrelin (relative to saline controls) on anxiety-like behavior in either the EPM test (time spent in the open arm; B) or the open field test (central activity or central rearing; C, D respectively). *P<0.05 **P<0.01, vs. saline. Independent samples t-test, SPSS.</p

Effects of intra-amygdala administration of ghrelin on anxiety-like behavior in rats denied access to food.

<p>In this “FOOD WITHHELD" paradigm rats were denied access to food during the first hour after intra-amygdala injection. (A) An orexigenic response to intra-amygdala ghrelin injection was detected when animals were returned to their home cages after the anxiety testing. Intra-amygdala ghrelin injection decreased anxiety-like behavior relative to saline controls, reflected by an increase in the amount of time spent in the open arms in the EPM test (B) and by the increase in central activity (C) and central rearings (D) in the open field test. *P<0.05 **P<0.01, ***P<0.001, vs. saline. Independent samples t-test, SPSS.</p