Our use of LepRb-specific genetic and adenoviral systems reveals a limited set of direct interactions between LepRb neurons and brain regions of the mesolimbic DA system: The midbrain contains LepRb neurons as well as receiving projections from LepRb neurons of the LHA (Leinninger et al., 2009
), and, to a lesser extent, the PAG and POA. Most LepRb projections into the amygdala and striatum target the CeA and IPAC components of the extCeA, where leptin regulates neuronal activity, as assessed by pCREB-IR. These LepRb projections to the extCeA derive from the midbrain, including the VTA (), and VTA LepRb neurons project solely to the extCeA. Within the CeA, LepRb projections synapse with CART neurons, and regulate their gene expression. The LepRb neurons that originate in the midline nuclei of the midbrain (e.g., RLi) send a few projections to the NAc in addition to densely innervating the extended central amygdala. The CeA and IPAC receive the vast majority of projections from both VTA and midline midbrain LepRb neurons, however. This specificity of projections from LepRb VTA neurons fits well with other recent findings that describe discrete patterns of projection, gene expression, and functional properties for subsets of VTA DA neurons (Ikemoto, 2007
;Margolis et al., 2008
;Lammel et al., 2008
LepRb neurons originating in the midbrain have specific and circumscribed targets in striatal projection regions
As for all experimental tools, the Ad-iZ/EGFPf and LepRbEGFPf systems possess inherent limitations. The expression of EGFPf in the brains of transgenic LepRbEGFPf mice is modest compared to that mediated by the higher copy number and stronger promoter system of the Ad-iZ/EGFPf, rendering it difficult to detect the relatively weak innervation of the NAc by LepRb neurons that could be observed with midline midbrain injection of Ad-iZ/EGFPf. Overall, the LepRbEGFPf mice clearly reveal the much greater density of LepRb projections into the extended amygdala than the NAc, however.
For Ad-iZ/EGFPf studies, the necessity of utilizing mice (specifically, transgenic animals with cre recombinase expression in LepRb neurons) with their small brains and resultant close spacing among midbrain nuclei limits the extent to which it is possible to isolate VTA relative to RLi LepRb labelling. The advantages of these systems, however, include the strict specificity for LepRb neurons, and the prospective interrogation of projections from LepRb neurons or anatomically-defined subpopulations of LepRb neurons. In this case, the use of the Ad-iZ/EGFPf system revealed the heretofore unsuspected dominant innervation of the CeA and IPAC by midbrain LepRb neurons, which we then confirmed by standard tracing methods.
While others have previously demonstrated the existence of LepRb-expressing VTA neurons (Figlewicz et al., 2006
;Hommel et al., 2006
;Fulton et al., 2006
), the potential manner(s) in which LepRb VTA neurons might differ from other VTA neurons was not clear. Here, we demonstrate the virtually exclusive innervation of the CeA and IPAC by LepRb VTA neurons, which contrasts with the predominant innervation of the NAc by the larger general population of VTA neurons. Although Fulton, et al., previously suggested that midbrain LepRb neurons innervate the NAc, the location of the LepRb neurons in question was not clear from the data shown (Fulton et al., 2006
). Based upon our present results, we surmise that the NAc-projecting LepRb midbrain neurons identified lie within the RLi or other midline areas of the midbrain, rather than the VTA DA LepRb neurons.
The laboratory of DiLeone has directly examined the role for midbrain LepRb action in long-term energy balance: Direct bilateral application of leptin to the midbrain of normal rats decreased food intake over 24 hours, and AAV-RNAi-mediated knockdown of midbrain LepRb in rats increased food intake, activity, and sucrose preference without altering body weight (Hommel et al., 2006
). These data suggest a role for midbrain LepRb neurons in the modulation of feeding and activity. Our present findings regarding the projection patterns of VTA and midbrain LepRb neurons suggest the possibility that these LepRb neurons may also control behaviors not previously examined, however, and that previous data regarding the function of these neurons should be considered in this light.
That leptin promotes extCeA CREB phosphorylation and modulates CeA Cart
expression suggests the functional relevance of the LepRb VTA→extCeA projections for CeA physiology. In addition to promoting incentive salience, midbrain neurons in general (and VTA DA neurons specifically) also function in the modulation of anxiety behaviors and in learning related to aversive stimuli, and aversive signals from the VTA may be conveyed by specific subsets of midbrain neurons (Matsumoto and Hikosaka, 2009
). The well-known role of the CeA in anxiety and the behavioral response to aversive stimuli suggests a potential role for amygdala-projecting VTA neurons in such reactions. Indeed, leptin decreases anxiety-like behaviors in leptin-deficient and normal animals (Liu et al., 2009
). Additionally, evidence that CeA-projecting LepRb neurons (i.e., VTA LepRb neurons) synapse with CeA CART neurons and regulate their Cart
expression suggests that leptin may modulate CART-associated behaviors in the amygdala. Increased CeA Cart
expression correlates with anxiety, depression, and stress responses under a variety of conditions (Dandekar et al., 2009
;Dandekar et al., 2008b
;Dandekar et al., 2008a
;Hunter et al., 2007
). The majority (95% +/−1%) of CeA CART neurons express Gad1
, which produces GABA (Supplemental Figure 5
); hence, GABA signaling by CeA CART neurons also likely participates in the action of VTA LepRb neurons.
While the effect of leptin on the firing and gene expression in LepRb VTA neurons specifically remains unclear, due to the inability to record specifically from LepRb neurons, Hommel, et al., suggested that leptin hyperpolarizes VTA DA neurons (Hommel et al., 2006
;Roseberry et al., 2007
). Indeed, our finding that leptin decreases Cart
expression in the CeA not only suggests the functional relevance of this circuit, but is consistent with the notion that leptin decreases DA efflux into the CeA, since DA promotes Cart
expression in the CeA and elsewhere (Fagergren and Hurd, 1999
). One reasonable hypothesis thus suggests that leptin action via VTA LepRb neurons regulates the extCeA and CeA-directed behaviors. A great deal more work will be required to fully examine this issue, however.
Previous data also demonstrate that leptin promotes the expression of TH and increases vesicular DA stores in the VTA and NAc (Fulton et al., 2006
;Roseberry et al., 2007
); recent data suggest that leptin action via LHA LepRb neurons plays a major role in these effects, however (Leinninger et al., 2009
), consistent with our present finding that the LHA contributes substantial LepRb projections into the VTA. The modulation of DA production and content in the VTA by this leptin-controlled pathway may contribute to the modulation of incentive value, the response to drugs of abuse, and other dopamine-dependent behaviors.
Clearly, leptin also controls the mesolimbic DA system by less direct means, involving additional synapses. Indeed, lateral hypothalamic melanin concentrating hormone (MCH) and orexin (OX) neurons project to the NAc and VTA, respectively, and modulate the mesolimbic DA system and feeding (Bartke et al., 2001
). Neither of these leptin-inhibited populations of LHA neurons express LepRb (Leinninger et al., 2009
), however, and leptin must act trans-synaptically to regulate MCH and OX neurons.
Overall, our data reveal a specific and circumscribed set of projections from LepRb neurons into the extCeA, and that these projections stem primarily from midbrain (especially VTA) LepRb neurons. Based upon these data and our finding that leptin controls the activity and Cart gene expression in CeA neurons, midbrain leptin action likely controls an amygdala-specific subset of the functions ascribed to the larger mesolimbic DA system.