Afferents to the orexin neurons of the rat brain

Abstract

Emotions, stress, hunger, and circadian rhythms all promote wakefulness and behavioral arousal. Little is known about the pathways mediating these influences, but the orexin-producing neurons of the hypothalamus may play an essential role. These cells heavily innervate many wake-promoting brain regions, and mice lacking the orexin neurons have narcolepsy and fail to rouse in response to hunger (Yamanaka et al. [2003] Neuron 38:701-713). To identify the afferents to the orexin neurons, we first injected a retrograde tracer into the orexin neuron field of rats. Retrogradely labeled neurons were abundant in the allocortex, claustrum, lateral septum, bed nucleus of the stria terminalis, and in many hypothalamic regions including the preoptic area, dorsomedial nucleus, lateral hypothalamus, and posterior hypothalamus. Retrograde labeling in the brainstem was generally more modest, but labeling was strong in the periaqueductal gray matter, dorsal raphe nucleus, and lateral parabrachial nucleus. Injection of an anterograde tracer confirmed that most of these regions directly innervate the orexin neurons, with some of the heaviest input coming from the lateral septum, preoptic area, and posterior hypothalamus. In addition, hypothalamic regions preferentially innervate orexin neurons in the medial and perifornical parts of the field, but most projections from the brainstem target the lateral part of the field. Inputs from the suprachiasmatic nucleus are mainly relayed via the subparaventricular zone and dorsomedial nucleus. These observations suggest that the orexin neurons may integrate a variety of interoceptive and homeostatic signals to increase behavioral arousal in response to hunger, stress, circadian signals, and autonomic challenges.

Fig. 1

Innervation of the orexin neurons. A: With an injection of BD into the lateral septum, several brown, anterogradely labeled boutons closely apposed a dark blue, orexin-IR neuron. B: This injection labeled fibers in the medial and perifornical parts of the orexin field, with fewer fibers in the lateral part. Scale bars = 50 μm in A; 200 μm in B.

Fig. 2

Distribution of CTB injection sites. The left side of each panel shows the typical distribution of orexin neurons. On the right side, injections into the orexin field are labeled in dark gray, and control injections adjacent to the field are labeled light gray. The distance behind bregma is noted at the bottom right.

Fig. 3

Distribution of retrogradely labeled neurons after an injection of CTB into the perifornical part of the orexin field (case 327). Each dot represents five neurons. The heaviest retrograde labeling was in the infralimbic and dorsal peduncular cortices, lateral septum, bed nucleus of the stria terminalis, amygdala, lateral and medial preoptic areas, hypothalamic regions (anterior hypothalamic area, VMH, DMH, PH), and brainstem regions including the PAG, DR, and pre-coeruleus. This injection extended slightly into the zona incerta and also labeled the cingulate cortex, an area not labeled with most other orexin field injections. sl, cl, el, dl, superior lateral, central lateral, external lateral, and dorsal lateral subnuclei of the LPB.

Fig. 3

Distribution of retrogradely labeled neurons after an injection of CTB into the perifornical part of the orexin field (case 327). Each dot represents five neurons. The heaviest retrograde labeling was in the infralimbic and dorsal peduncular cortices, lateral septum, bed nucleus of the stria terminalis, amygdala, lateral and medial preoptic areas, hypothalamic regions (anterior hypothalamic area, VMH, DMH, PH), and brainstem regions including the PAG, DR, and pre-coeruleus. This injection extended slightly into the zona incerta and also labeled the cingulate cortex, an area not labeled with most other orexin field injections. sl, cl, el, dl, superior lateral, central lateral, external lateral, and dorsal lateral subnuclei of the LPB.

Fig. 4

Distribution of BD injection sites. The anterograde tracer BD was injected into many brain regions thought to innervate the orexin neurons. Each BD injection site has been mapped onto template drawings (Paxinos and Watson, 1997), and to minimize clutter, some injections have been mapped to an adjacent section.

Fig. 4

Distribution of BD injection sites. The anterograde tracer BD was injected into many brain regions thought to innervate the orexin neurons. Each BD injection site has been mapped onto template drawings (Paxinos and Watson, 1997), and to minimize clutter, some injections have been mapped to an adjacent section.

Fig. 5

Anterograde labeling of afferents to the orexin neurons from several brain regions. The BD injection site is indicated by an arrow on the darkfield photomicrograph in each left-hand panel. The right-hand panel shows the distribution of orexin-IR neurons: black circles represent orexin neurons receiving appositions from the corresponding injection and white circles indicate orexin neurons with no apparent appositions. Below each drawing is noted the percent of all orexin neurons on that section that receive appositions. Hypothalamic inputs target orexin neurons in the medial and perifornical parts of the orexin field, whereas lighter inputs from brainstem regions tend to innervate orexin neurons in the lateral half of the field. Scale bars in the left panels = 500 μm.

Fig. 6

Summary of afferents to the orexin neurons. The upper panel shows inputs to orexin neurons in the medial part of the orexin field and the lower panel shows inputs to orexin neurons in the lateral part of the field. The heaviest inputs are from the pre-optic area, dorsomedial nucleus, posterior hypothalamus, lateral septum, and BST. This pattern suggests that the orexin neurons are influenced by signals regulating emotions, autonomic tone, appetite, circadian rhythms, and sleep/wake behavior. Regions labeled in dark, medium, and light gray innervate >45%, 25–44%, or 5–24% of the orexin neurons. Inputs that innervate <5% of the orexin neurons are not included. Line thickness indicates the relative number of retrogradely labeled neurons.