We have examined the innervation of specific populations of LHA neurons by LepRb neurons, revealing that, while both OX and MCH neurons receive synaptic contact from LepRb neurons, local LHA LepRb neurons project onto OX, but not MCH, neurons. Consistent with the direct projection of LHA LepRb neurons onto local OX neurons, LHA leptin action robustly modulates gene expression in OX neurons, revealing the functional importance of this local circuit. We previously showed that VTA LepRb neurons densely innervate the central nucleus of the amygdala (CeA), where they innervate and regulate CART-expressing neurons (Leshan, et al., 2010).
The strength with which local LHA leptin promotes Ox
mRNA expression compared with that observed in response to systemic leptin (along with the paucity of projections to the OX field from other populations of LepRb neurons) suggests that LHA LepRb neurons likely represent the main neural mediators of leptin action on OX neurons. While the technical difficulties associated with measuring c-fos (a surrogate for neuronal activity) in the region surrounding a cannula prevented us from examining the regulation of OX neuron activity in response to LHA leptin, the lack of significant LepRb projections to this region from elsewhere and the GABAergic nature of the LHA LepRb neurons (Leinninger, et al., 2009
) prompts us to hypothesize that LHA LepRb neurons might inhibit the activity of OX neurons, as well as controlling their gene expression.
While it is clear from our present observations that MCH neurons lie in synaptic contact with LepRb neurons, the lack of WGA accumulation in MCH neurons following intra-LHA injection of Ad-iN/WED in LepRbcre
mice reveals that the LepRb neurons that project onto MCH neurons likely lie outside of the LHA. The population of LepRb neurons that lie upstream of MCH neurons thus remains unidentified, although a variety of previously published data suggest a potential role for ARC melanocortin neurons in this regulation (Hanada et al., 2000
). Tracing from the larger LHA (not specifically the dorsal perifornical area), Elias, et al., revealed a population of leptin-activated ARC neurons projecting to the LHA (Elias et al., 1998
). In addition, our analysis of FG labeling from the LHA region where MCH neurons are located, lateral to the OX field, revealed potential projections from LepRb populations in the ARC (as well as the NTS and DR). Indeed, Mch
expression is increased in mice overexpressing the melanocortin antagonist, Agouti, (although Ox
is not altered); also, melanocortin agonists/antagonists regulate Mch
expression (Hanada et al., 2000
; Kim et al., 2005
; Morton et al., 2004
; Tritos et al., 2001
). Our present data also reveal that other LHA neurons receive input from LHA LepRb neurons, based upon their accumulation of WGA. While markers are not available to identify the likely subpopulations of these, we speculate based upon the small size of many of these cells that they may represent GABAergic interneurons.
Our present data demonstrating the regulation of OX neurons by LHA LepRb neurons suggests important roles for these LHA LepRb neurons in energy balance and in CNS leptin action. While acute injection of OX into the CNS promotes activity, wakefulness, and hyperphagia, the long-term role of OX is to promote activity and energy expenditure, while decreasing feeding (Funato et al., 2009
; Mieda and Yanagisawa, 2002
; Tritos et al., 2001
; Yamanaka et al., 2003
). Indeed, mice (and humans) null for OX are obese, while widespread overexpression of OX promotes leanness (Chemelli et al., 1999
; Funato et al., 2009
; Hara et al., 2001
; Sakurai et al., 1998
). Much of this OX action on energy balance depends upon the OX2R, as mice null for this receptor demonstrate increased feeding and become more obese than controls on a high fat diet; systemic treatment with an OX2R agonist prevents diet-induced obesity (Funato et al., 2009
). Presumably, therefore, the acute effects of OX may be mimicked by increased activity of OX neurons during food restriction (which is blunted by leptin), while the increased expression of Ox
promoted by leptin (via LHA LepRb neurons) would be expected to promote the kind of chronic OX effects required for leptin action. Indeed, we previously showed that intra-LHA leptin decreased feeding and body weight over 24 hours in Lepob/ob
animals (Leinninger et al., 2009
). Unfortunately, acute treatment with systemic OXR antagonists inhibits movement and blunts feeding (data not shown), and the determination of roles for OX in physiologic actions downstream of the LHA LepRb neurons will require circuit-specific manipulation.
Our previous analysis of LHA LepRb neurons also identified projections from these neurons to the VTA, and revealed that LHA leptin treatment of Lepob/ob
animals promotes VTA Th
expression and increased DA content in the nucleus accumbens (NAc) (Leinninger, et al., 2009
). Our present findings that LHA LepRb neurons project onto and regulate OX neurons, which themselves innervate the VTA to modulate the actions of the mesolimbic DA system (Baldo et al., 2003
; Harris et al., 2005
; Kelley et al., 2005
; Nakamura et al., 2000
; Narita et al., 2006
), suggests that LHA LepRb neurons may modulate the mesolimbic DA system indirectly, via OX neurons, as well as by direct projection to the VTA. While outside the scope of this study, it will be important to dissect the relative contributions of these direct and indirect pathways from LHA LepRb neurons to the mesolimbic DA system and energy balance.