In view of the relatively dense dopaminergic innervation of the LH/PFA, we undertook an examination of the ability of indirect and direct DA agonists on the activity of orexin neurons, as reflected by induction of Fos, the protein product of the early-
immediate gene c-fos. Because orexin neurons are a small population of LH/PFA cells that are scattered across the dorsal hypothalamus, reporters such as Fos are particularly useful in indicating the impact of a pharmacological treatment12 on neurons identified by their transmitter phenotype; the recent introduction of a transgenic mouse with a GRP reporter under the prepro-orexin promotor13,14 also offers a direct method for assessing the physiological changes in orexin cells.
3.1. Amphetamine and Apomorphine Effects on Fos expression in Orexin cells
We first examined the effects of the indirect DA agonist d-amphetamine on Fos expression in orexin neurons. Amphetamine markedly increases arousal and activity, and potently increases extracellular DA levels in mesocorticolimbic forebrain sties. Amphetamine challenge resulted in a marked increase in the percentage of doublelabeled (Fos + orexin) cells, which were primarily seen medial to the fornix.15
Amphetamine results in a net increase in extracellular norepinephrine (NE) as well as DA, and hence one cannot know if the increase in Fos expression in orexin neurons is due to an increase in DA or NE. We therefore challenged animals with apomorphine, a mixed DA agonist at both D1 (D1 and D5)- and D2 (D2, D3, and D4)-like receptors. Apomorphine markedly activated orexin neurons, with a particularly strong effect medial to the fornix (see Figure 2), indicating that activation of DA receptors can drive orexin neurons.
Figure 2. Dopamine agonist induced Fos expression in orexin cells in the medial and lateral LH/PFA. The D1 agonist A77636 and D2 agonist quinpirole (QUIN), as well as the mixed DA agonist apomorphine (APO), increased the percentage of orexin cells relative to vehicle (VEH) control-treated animals. Note the significantly greater effect of DA agonist in the medial sector of the LH/PFA. p < .01
3.2. Activation of Orexin Neurons by D1 and D2 Agonists
Accordingly, we then tested specific D1- and D2-like dopamine agonists. The full D1 agonist A77636 markedly increased Fos expression in both the medial and lateral LH/PFA, but had a greater effect on orexin neurons medial to a line vertically bisecting the fornix. The D2-like agonist quinpirole also activated orexin neurons, but to a lesser degree than observed in response to the D1 agonist (see Fig. 2).
The differential effect of DA agonists on orexin cells in the medial and lateral LH/PFA, and the greater effect elicited by the D1 agonist suggested that DA receptors may be distributed heterogeneously across the LH/PFA. However, we found only rare
LH/PFA cells that express D2 mRNA, and no LH/PFA cells expressing D3, D4, or D5 mRNAs. Thus despite the potent effect of DA agonists on orexin cells, particularly those in the medial LH/PFA, which is consistent with a dopaminergic innervation of the LH/PFA, DA receptors that would mediate a direct effect of the DA agonists are lacking.
3.3. Mechanisms of Dopamine Agonist-Induced Activation of Orexin Neurons
The striking effects of Dl- and D2-like receptor agonists on Fos expression in orexin cells but the absence of DA receptors suggests several different possibilities that may account for the ability of DA agonists to drive Fos.
The first is that the agonists act on afferents to the orexin neurons. Among such sites could be neurons in the NAS, in which both D1- and D2-mRNA expressing cells are found, and where DA agonists exert potent effects. Acute pharmacological inactivation of the NAS resulted in an activation of orexin neurons in the lateral but not medial LH/PFA,16 consistent with accumbal projections being directed to the LH and not invading the area medial to the fornix.17 The PFC also contains D1- and D2-mRNA expressing cells, and an action of DA agonists on this cortical site might be expected to regulated orexin cells, but again in the lateral part of the LH/PFA. However, the fact that both the NAS and PFC primarily project to the area lateral to the fornix yet DA agonist act medial to the fornix suggests that neither accumbal nor prefrontal cortical neurons are the primary target in DA agonist-induced orexin activation. There are obviously neurons in many other sites that innervate the LH/PFA and express DA receptors, and indeed it is possible that D1-like agonists drive one afferent source and D2-like agonists a different set of afferents.
Another mechanism whereby DA agonists might activate orexin cells is by targeting a non-DA receptor. For example, dopamine has a high nanomolar affinity for the V2C receptor, which has been mapped to the LH. However, amphetamine does not bind to this receptor. Thus, it would appear unlikely that DA agonists act through non-cognate receptors to exert their effects.
A third possibility is that the ability of DA agonists to drive orexin cells is mediated by peripheral signals. For example, the long form of the receptor for leptin is expressed on orexin neurons.18 DA agonists may trans-synaptically promote peripheral sources, such as adipocytes, to alter the release such neuroactive species as orexin, ghrelin, and adiponectin, which in turn would modulate the activity of orexin neurons.
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