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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2011 February 1.
Published in final edited form as:
PMCID: PMC2930259
NIHMSID: NIHMS230760

Direct innervation and modulation of orexin neurons by lateral hypothalamic LepRb neurons

Abstract

Leptin, the adipose-derived hormonal signal of body energy stores, acts via the leptin receptor (LepRb) on neurons in multiple brain regions. We previously identified LepRb neurons in the lateral hypothalamic area (LHA), which are distinct from neighboring leptin-regulated melanin concentrating hormone (MCH)- or orexin (OX)-expressing cells. Neither the direct synaptic targets of LHA LepRb neurons nor their potential role in the regulation of other LHA neurons have been determined, however. We thus generated several adenoviral and transgenic systems in which cre recombinase promotes the expression of the tracer, wheat germ agglutinin (WGA), and utilized these in combination with LepRbcre mice to determine the neuronal targets of LHA LepRb neurons. This analysis revealed that, while some LHA LepRb neurons project to dopamine neurons in the ventral tegmental area (VTA), LHA LepRb neurons also densely innervate the LHA where they directly synapse with OX, but not MCH, neurons. Indeed, few other LepRb neurons in the brain project to the OX-containing region of the mouse LHA, and direct leptin action via LHA LepRb neurons regulates gene expression in OX neurons. These findings thus reveal a major role for LHA leptin action in the modulation of OX neurons, suggesting the importance of LHA LepRb neurons in the regulation of OX signaling that is crucial to leptin action and metabolic control.

Keywords: Leptin receptor, lateral hypothalamus, orexin, mesolimbic system, WGA, GFP

Introduction

Leptin, a polypeptide hormone that is produced by adipocytes in proportion to fat content, signals the repletion of body energy stores to modulate neural processes linked to energy balance (Elmquist et al., 2005; Friedman, 2002; Morton et al., 2006; Myers et al., 2009). Circulating leptin decreases feeding and permits energy expenditure by activating the long-form of its receptor (LepRb) in the brain (Cohen et al., 2001; de Luca et al., 2005). Numerous brain regions contain LepRb-expressing, leptin-responsive neurons- including the hypothalamus, where large numbers of LepRb neurons reside in the arcuate (ARC), ventromedial (VMH), dorsomedial (DMH), and ventral premammillary (PMv) nuclei, as well as the preoptic (POA) and lateral hypothalamic (LHA) areas (Dhillon et al., 2006; Donato et al., 2009; Elmquist et al., 1998; Myers et al., 2009; Scott et al., 2009). LepRb neurons are also found in midbrain sites including the ventral tegmental area (VTA) and dorsal raphe (DR), and brainstem nuclei, such as the nucleus of the solitary tract (NTS) (Figlewicz et al., 2006; Fulton et al., 2006; Grill, 2006; Hommel et al., 2006; Myers et al., 2009; Scott et al., 2009). While LepRb neurons in the well-studied ARC, including POMC- and AgRP-expressing cells, contribute importantly to energy balance, these neurons mediate only a fraction of overall leptin action (Balthasar et al., 2004; Dhillon et al., 2006; Myers et al., 2009; van de et al., 2008), suggesting the importance of the many non-ARC LepRb neurons throughout the brain.

While the medial basal hypothalamus (including the ARC) acts in conjunction with the brainstem to modulate satiety (Myers, et al., 2009; Grill, 2006), a variety of data suggest roles for the LHA in controlling the incentive to feed, including by regulating the mesolimbic dopamine (DA) reward system (DiLeone et al., 2003; Harris et al., 2005; Kelley et al., 2005; Nestler, 2005). Indeed, several populations of LHA neurons have been implicated in the regulation of feeding and the mesolimbic DA system. In addition to GABAergic LHA LepRb neurons, which modulate the expression of tyrosine hydroxylase (Th; the enzyme that catalyzes the rate-limiting step in DA production) in the mesolimbic DA system in response to leptin (Leinninger et al., 2009), the LHA contains populations of widely-projecting neurons that express the neuropeptides orexin (OX) or melanin concentrating hormone (MCH). Among their other roles, LHA OX neurons project to the VTA, where OX controls drug and food reward; MCH neurons project to the striatum to modulate similar parameters (DiLeone et al., 2003; Harris et al., 2005; Sharf et al., 2010). Leptin decreases MCH action by inhibiting Mch expression and blunting endocannabinoid-mediated depolarization-induced suppression of inhibition on MCH neurons (Jo et al., 2005; Qu et al., 1996). The modulation of OX neurons by leptin appears more complex: On one hand, leptin inhibits the firing of OX neurons in slice preparations and blocks the fasting-induced activation of OX neurons detected by c-fos immunostaining in vivo (Funato et al., 2009; Mieda and Yanagisawa, 2002; Yamanaka et al., 2003). Conversely, leptin promotes Ox mRNA expression, and OX signaling via the OX2R contributes to leptin action on energy balance (Funato et al., 2009; Mieda and Yanagisawa, 2002; Tritos et al., 2001; Yamanaka et al., 2003). Thus, while leptin inhibits the activity of OX neurons, leptin also promotes Ox expression and chronic OX signaling functions in concert with leptin action.

The neural mechanisms by which leptin modulates LHA neurons, such as those expressing OX and MCH, and the potential interaction of LHA LepRb neurons with these neurons (as well as neurons in the VTA and elsewhere) remain unclear. To illuminate these issues, we generated and utilized several genetic tools to specifically examine LepRb neurons and their synaptic contacts. We show that LHA LepRb neurons directly innervate local OX, but not MCH neurons, and regulate gene expression in OX neurons.

Materials and Methods

Materials

Leptin was the generous gift of Amylin Pharmaceuticals, Inc. (San Diego, CA).

Experimental animals

The generation of Leprcre/cre (LepRbCre) mice has been described previously; these animals were produced by intercrossing homozygous animals within our facility. Leprcre/cre mice were bred with Gt(ROSA)26-Sortm2Sho mice purchased from Jackson Laboratory to generate double homozygous Leprcre/cre;Gt(ROSA)26-Sortm2Sho/tm2Sho (LepRbEGFP) mice, which were propagated by intercrossing (Leshan, 2009). C57Bl/6 and Lepob/ob animals were purchased from Jackson Laboratory.

For the generation of iZ/WAP mice, the coding region for wheat germ agglutinin (WGA) was PCR-amplified from pBluescript II SK-WGA (the generous gift of Dr. Yoshihiro Yoshihara, RIKEN Brain Science Institute, Japan (Hanno et al., 2003)) and inserted into the pCALL2-IRES-hAP/cg vector (iZ/AP) (the generous gift of Dr. Corrine Lobe, Toronto, ON (Allen et al., 2006)) downstream of the CMV promoter-driven floxed β-geo cassette and upstream of an IRES-alkaline phosphatase (AP) sequence. The resulting pCALL2-WGA/AP (iZ/WAP) plasmid was submitted to University of Michigan transgenic core for production of transgenic embryonic stem cell clones. Four hundred and eighty clones were screened for single copy number by qPCR for neo sequences (Soliman et al., 2007) and also screened for β-gal expression via immunocytochemical staining (Roche). Five ES clones were expanded and rescreened, and three positive ES clones were injected into blastocysts and implanted into foster mothers. The resulting chimeric male progeny were bred to C57/Bl6 females for the determination of germline transmission (by brown coat color) and confirmed via Southern blotting for the Neo cassette. Several F1 generation iZ/WAP mice from each ES clone were perfused and screened for CNS β-gal expression by immunofluorescent staining using antibodies against β–gal, as detailed below. One iZ/WAP line was determined to express the transgene throughout the CNS, and was chosen for further study. While similar in genesis to previously reported transgenic animals (Braz et al., 2002), this new line demonstrates broader transgene expression in the brain- including the hypothalamus. Subsequent iZ/WAP litters were genotyped by conventional PCR utilizing oligos derived from the original WGA sequence (Forward: AATGAGAAAGATGATGAGCACC; Reverse: AGGTTGTTCGGGCATAGCTT). iZ/WAP animals were bred with LepRbEGFP mice (described above) to generate Leprcre/cre;ROSA26EGFP/EGFP;iZ/WAP (LepRbEGFP/WGA) animals for study.

Animals were housed in our colony in 12h light/dark cycles and given ad libitum access to food and water. All care and procedures for mice were according to guidelines approved by the University of Michigan Committee on the Use and Care of Animals (UCUCA).

Perfusion, immunohistochemistry and immunofluorescence

Perfusion and immunohistochemistry were performed essentially as described (Munzberg et al., 2007). Briefly, mice were deeply anesthetized with a lethal dose of intraperitoneal (i.p.) pentobarbital (150 mg/kg) and transcardially perfused with sterile PBS then 10% neutral buffered formalin. Brains were removed, postfixed overnight and dehydrated in a 30% sucrose solution. Then brains were sectioned into 30 µm coronal slices, collected in four series and stored at −20 °C in cryoprotectant until further use.

Brain sections were incubated in primary antibodies [goat anti-βgal (1:3000, Biogenesis), chicken anti-GFP (1:1000, Abcam), rabbit anti-MCH (1:1000, Phoenix Pharmaceuticals), goat or rabbit anti-OX (1:1000, Santa Cruz/Calbiochem), goat anti-WGA (1:1000, Vector), mouse anti-TH (1:200, Chemicon), sheep anti-α-MSH (1:5000, Chemicon) or rabbit anti-AgRP (1:1000, Phoenix)] overnight at 4°C, and then visualized by immunofluorescent secondary detection using species-specific Alexa 488 or 568 antibodies (1:200, Invitrogen). Sections were mounted on slides and coverslipped with Prolong antifade mounting medium (Invitrogen).

Adenoviral Tracers

The Ad-iZ/EGPFf adenoviral system to trace long axonal processes has been described previously (Leshan 2009; Leinninger, 2009). For the generation of Ad-iN/WED, we first deleted the β-gal-encoding region of the β-geo fusion in pShuttle/iZ (Leinninger et al., 2009; Leshan et al., 2009) by PCR using the Quikchange kit (Stratagene), generating pShuttle/iN (thereby decreasing the size of the construct to promote proper viral packaging with larger inserts). The coding region of WGA, as above, along with an IRES element and the coding sequence for farnesylated dsRED (dsRedf), was then subcloned into the MCS of pShuttle/iN vector to generate pShuttle-iN/WED. Vector DNA was purified, linearized and utilized to generate the cre-inducible WGA adenoviral vector (Ad-iN/WED). Concentrated adenoviral stocks were generated and purified as previously described (Leshan et al., 2009; Morton et al., 2003). Cre-dependent WGA expression from Ad-iN/WED was verified in vitro by immunocytochemical staining for WGA in infected HEK293 cells transfected with a vector encoding cre recombinase.

Site-specific LepRb tract tracing experiments

For tract tracing experiments, LepRbCre or LepRbEGFP mice were anesthetized using isoflurane and placed in a stereotaxic apparatus. After exposing the skull, a guide cannula with stylet was lowered into LHA coordinates (from bregma) (AP:−1.34; ML: −1.1, DV: −5.1; OX field) or (AP: −1.34; ML: −1.38, DV: −5.1; lateral/MCH field) according to Franklin and Paxinos, 1997. The stylet was removed and replaced by an injector and either 50 nl of 4% fluorogold-equivalent (Sigma) or 200–500 nl of Ad-iZ/EGPFf or Ad-iN/WED was acutely injected, using a 500nl Hamilton syringe at a rate of 100 nl/min. After 10 minutes to allow for absorption of tracer, the injector and cannula were removed and the skull and the incision was sealed and sutured. Mice received pre- and post-surgical analgesia (Buprenex-150µl of 0.01mg/ml) and were individually housed for 2–5 days (FG-mediated retrograde tracing) or 5 days (Ad-iZ/EGPFf and Ad-iN/WED-mediated anterograde tracing) before perfusion and processing. Five EGFPf-injected animals and four Ad-iN/WED-injected animals displayed injection sites confined within the LHA, as well as substantial WGA expression, and were thus selected for further analysis.

Data Collection and analysis

Slides were analyzed via light or fluorescent microscopy using an Olympus BX-51 microscope with filters for Alexa 488 or Alexa 568 and images taken with software as previously described (Munzberg et al., 2007). Using Adobe Photoshop software (Adobe Systems, San Jose CA) images were overlaid in different RGB channels to reveal single- or double-labeled cells. Confocal images were captured with an Olympus FV-500.

Leptin treatment to assess changes in LHA gene expression

Two cohorts of adult male Lepob/ob mice (n=20 each cohort) were obtained: the first cohort for systemic (i.p) treatment with sterile PBS or leptin (5mg/kg) and the second for the chronic implantation of cannulae into the LHA (AP: −1.30; ML: −1.12; DV: −4.15), as described previously (Leinninger et al., 2009). Briefly, cannulae with dummy injector were stereotaxically inserted and affixed into the LHA site. After surgery, mice were single housed and checked daily for food intake and body weight to monitor their recovery. After 7–10 days of recovery, mice were treated with 250nl of either sterile PBS or leptin (0.001 ng/nl, = 0.25ng). Both cohorts were treated with either PBS or leptin every 12 h for 24 h, with the final dose given 2h before sacrificing for a total of three injections (for a total of 0.75 ng leptin) over 26hr of treatment. We previously selected this dosing regimen to approximate physiologic leptin concentrations in the volume of distribution of the LHA, to avoid the detectable spread of leptin to other hypothalamic regions, and to provide long enough treatment to permit the detection of changes in gene expression (Leinninger, et al., 2009). Following treatment, specific nuclei of the brain were microdissected as described previously (Leinninger et al., 2009).

RNA extraction and analysis of gene expression by qPCR

LHA and ARC were microdissected and snap frozen for later processing and analysis of mRNA expression by quantitative RT-PCR (as previously described) (Leinninger et al., 2009). Briefly, RNA was extracted using TRIzol (Invitrogen) and converted to cDNA using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). cDNA was analyzed in triplicate via qRT-PCR for Gapdh and neuropeptide gene expression (as previously described (Bates et al., 2003), or as supplied by Applied Biosystems) using an Applied Biosystems 7500. Gene expression was normalized to Gapdh expression and relative mRNA expression was calculated via the 2−ΔΔCt method. Cannulated mice were also normalized to the contralateral (unperturbed) side. Statistical significance was determined by student’s t test as computed with Excel.

Results

Molecular tools to examine neural mechanisms of leptin action

As the identification of LepRb neurons by direct detection of LepRb remains problematic, we previously generated LepRbEGFP reporter mice, in which Leprcre-driven cre recombinase expression within LepRb neurons mediates the expression of enhanced green fluorescent protein (EGFP) from the ROSA26-EGFP allele to reveal LepRb neurons (Leinninger et al., 2009; Leshan et al., 2009; Myers et al., 2009) (Figure 1A–C). While EGFP expression in LepRbEGFP reporter mice reveals that LHA LepRb neurons are distinct from MCH and OX neurons (Figure 1B,C)(Leinninger et al., 2009), leptin is known to modulate the activity and gene expression of MCH and OX neurons (Funato et al., 2009; Jo et al., 2005; Mieda and Yanagisawa, 2002; Qu et al., 1996; Tritos et al., 2001; Yamanaka et al., 2003), suggesting that LepRb neurons synapse with these LHA neurons to regulate them. To permit the examination of projections from LepRb neurons and the identification of their synaptic targets, we thus generated transgenic animals and adenoviral systems to promote the expression of farnesylated EGFP (EGFPf; which is targeted to the membrane and thus more robustly reveals axonal projections than standard cytoplasmic EGFP (Zylka et al., 2005)) or wheat germ agglutinin (WGA; a lectin that passes transynaptically to accumulate in target neurons (Braz et al., 2002; Hanno et al., 2003)) (Figure 1A).

Figure 1
LepRbcre-dependent transgenic and adenoviral tracers to study LepRb-expressing cells in the LHA

LepRbEGFP/WGA mice reveal LHA neurons that lie in synaptic contact with LepRb neurons

To examine whether LepRb neurons lie in synaptic contact with MCH and OX neurons in the LHA, we generated the iZ/WAP transgenic mouse line, in which cre recombinase promotes the expression of WGA in cre-expressing neurons. We bred iZ/WAP to the Leprcre;ROSA26-EGFP (LepRbEGFP) background to produce LepRbEGFP/WGA animals in which LepRb neurons express EGFP and WGA, and in which WGA accumulates in non-EGFP-expressing neurons that lie in synaptic contact with LepRb neurons (Figures 1A, ,2).2). While an internal ribosome entry site (IRES) and the sequence encoding alkaline phosphatase (AP) was inserted into our construct downstream of WGA, the IRES-mediated expression of AP proved to be insufficient to mediate its detection in the CNS (data not shown), necessitating the use of cre-inducible EGFP to label primary LepRb neurons.

Figure 2
LepRbEGFP/WGA mice reveal LepRb-expressing cells and their projection targets in the LHA

Staining for WGA in LepRbEGFP mice or in iZ/WAP mice not containing a cre allele revealed no detectable WGA-immunoreactivity (-IR) (data not shown), but numerous strongly WGA-IR (as well as GFP-IR) neurons were detected in the hypothalamus of LepRbEGFP/WGA mice, including in the LHA (Figure 2A–L). Costaining for GFP/LepRb and WGA revealed neurons within the LHA that contained both GFP- and WGA-IR neurons, representing WGA-expressing LHA LepRb neurons (Figure 2A–D, arrows). Additionally, we detected many WGA-IR cells that were devoid of GFP-IR, representing neurons that lie in synaptic contact with LepRb neurons, but which are not themselves LepRb neurons (Figure 2A–D, arrowheads). Also, in utilizing these LepRbEGFP/WGA mice, we explored whether LepRb neurons communicate with MCH and/or OX neurons by examining the colocalization of WGA with either MCH-IR or OX-IR neurons (Figure 2 E–H, I–L, respectively). This analysis revealed that approximately 10% of MCH and 30% of OX neurons also contained strong WGA-IR. Thus, while MCH and OX neurons in the LHA do not express LepRb to directly respond to leptin, they represent synaptic targets of LepRb neurons, consistent with the previously-described regulation of OX and MCH neurons by leptin (Qu et al., 1996; Yamanaka et al., 2003). Since WGA expression in LepRbEGFP/WGA mice is driven from a standard transgenic promoter, it is not clear whether the finding that not all of these neurons contain WGA indicates mosaic WGA expression in the LepRb neurons, or whether LepRb neurons only innervate a subset of OX and MCH neurons. Furthermore, since LepRb neurons throughout the brain express WGA, the analysis of LepRbEGFP/WGA mice does not identify the specific population of LepRb neurons that project onto MCH or OX neurons.

Few extra-LHA LepRb neurons project to the dorsal perifornical LHA

To identify brain regions containing LepRb neurons that might project onto neurons in the LHA, we injected the retrograde tracer, fluorogold (FG), into the LHA of LepRbEGFP mice (Figure 3A), and analyzed these brains for regions containing FG- and GFP-IR (Figure 3B–F). FG was specifically injected into the dorsal perifornical LHA in the region containing LHA LepRb neurons (Figure 3A). The majority of LHA LepRb neurons lie in the mid- and caudal portion of the dorsal perifornical region of the LHA, and are largely co-distributed with OX neurons (Figure 1C). Several brain regions contained both LHA-projecting (FG-IR) and LepRb (GFP-IR neurons), including the VTA (Fig 3C,D) and the POA (Figure 3E,F), although only a few LepRb/GFP neurons in the VTA accumulated FG from the dorsal perifornical LHA. Little or no FG from this region of the LHA accumulated in ARC neurons (Figure 3B). Consistently, few neuropeptide-IR projections from ARC POMC and AgRP neurons are detected in the main OX field, but rather densely innervate the surrounding area (Supplemental Figure 1). We furthermore tested FG tracing from the region of the LHA lateral to the OX field, where MCH neurons are typically located (Supplemental Figure 2A). These injections yielded FG tracing to LepRb neurons in the NTS, DR, and the ARC (Supplemental Figure 2B–D). Overall, while LepRb neurons (including those from the ARC) innervate some portions of the LHA, these data reveal that few extra-LHA LepRb neurons project to the dorsal perifornical region of the LHA that contains LepRb and OX neurons, suggesting that LHA LepRb neurons might represent the primary LepRb neurons that innervate this area and the OX neurons contained therein.

Figure 3
Retrograde tracing from the LHA OX field in LepRbEGFP mice to determine potential presynaptic LepRb populations

Innervation of the VTA and LHA by LHA LepRb neurons

To more closely examine the potential innervation of the perifornical LHA by LHA LepRb neurons, we first injected the previously-described Ad-iZ/EGFPf (Leshan, 2009; Leinninger, 2009) into LepRbcre mice (Figures 1A, ,4),4), thus mediating the expression of EGFPf specifically in cre-expressing (i.e., LepRb) neurons. Since EGFPf is targeted to the membrane, it robustly reveals the axonal architecture of EGFPf-expressing cells. We thus injected Ad-iZ/EGFPf into the LHA of LepRbcre mice (Figure 4A), promoting the expression of EGFPf in LHA LepRb neurons, and analyzed the GFP-IR neurites within the LHA (Figure 4B,C). Many of these neurites demonstrated the “beads on a string” appearance consistent with the presence of synapses, similar to the appearance of LHA LepRb axons in more distant regions, such as the VTA (Figure 4D–F). These data thus suggest that LHA LepRb neurons may innervate local, as well as distant, neurons. Furthermore, in analyzing the LHA for GFP-IR and OX-IR, we detect EGFP/LepRb fibers in close contact with OX neurons (Figure 4G–L), suggesting that LHA LepRb neurons may innvervate local OX neurons.

Figure 4
Ad-iZ/EGFPf injection to the LHA of LepRbcre mice provides evidence for intra-LHA as well as intra-VTA synapses on projections from LHA LepRb neurons

To reveal specific populations of neurons that lie in synaptic contact with LHA LepRb neurons, we generated the transsynaptic adenoviral tracer, Ad-iN/WED, to mediate the expression of a WGA/EGFP fusion protein in cre-expressing cells (Figure 1A). Thus, site-specific injection of Ad-iN/WED to cre-expressing animals is expected to promote the expression of WGA/EGFP in anatomically-restricted populations of cre-expressing neurons around the injection site, and also effect WGA/EGFP accumulation in the synaptic targets of these cre-expressing neurons. While Ad-iN/WED was also designed to express dsREDf to reveal the neurites of the cre-expressing cells, IRES-mediated dsRedf expression was insufficient to enable its detection in in vitro tests or in vivo (data not shown).

We administered Ad-iN/WED into the LHA of LepRbcre mice and perfused them for the detection of WGA-IR after 5 days. Intra-LHA administration of Ad-iN/WED produced WGA expression in the LHA of LepRbcre mice (Figure 5A,B); no WGA-IR was detected following the injection of Ad-iN/WED in non-cre-expressing mice, however (data not shown). We analyzed WGA-IR neurons in four LepRbcre mice in which the injection sites were restricted to the dorsal perifornical area of the LHA (Supplemental Figure 3), where LHA OX and LepRb neurons are concentrated, and in which robust WGA expression was observed. To determine the neurons innervated by LHA LepRb neurons, we initially examined WGA accumulation in the LHA and the major extra-LHA projection site of LHA LepRb neurons, the VTA (Figure 5C–F)(Leinninger et al., 2009). While this analysis demonstrated the presence of copious WGA-IR neurons in the LHA, it revealed very few WGA-IR neurons in the VTA. Some of these VTA WGA-IR neurons colocalized with TH, suggesting that at least some LHA LepRb neurons directly innervate VTA DA neurons. Other distant regions, including the ARC, similarly contained few or no WGA-IR neurons (data not shown). While these data are consistent with a direct synaptic connection between some LHA LepRb neurons and VTA DA targets, the paucity of detectable WGA-IR VTA neurons suggests that the WGA/EGFP transgene expressed by Ad-iN/WED may poorly traverse synapses far removed from the primary WGA/EGFP-expressing neurons (Leinninger et al., 2009).

Figure 5
Modest accumulation of WGA in VTA relative to LHA neurons following Ad-iN/WED injection to the LHA of LepRbcre mice

LHA LepRb neurons synapse with OX, but not MCH, neurons

To examine the potential innervation of local LHA neurons by LHA LepRb neurons, we examined the accumulation of WGA by OX and MCH neurons in the LHA of four LepRbcre mice following the intra-LHA injection of Ad-iN/WED. This analysis revealed the accumulation of WGA-IR in numerous OX-IR neurons (Figure 6 E–H), but not MCH-IR neurons (Figure 6 A–D)(see also Supplemental Figure 4). Thus, LHA LepRb neurons lie in synaptic contact with OX neurons, but not MCH neurons. These data thus reveal that non-LHA LepRb neurons must mediate the effects of leptin on MCH neurons, and also suggest a role for LHA LepRb neurons in the control of OX neurons by leptin.

Figure 6
Local tracing of LepRb neurons and their synaptic targets in the LHA after Ad-iN/WED injection to the LHA of LepRbcre mice

LHA leptin action regulates gene expression in OX neurons

To determine the potential role for LHA LepRb neurons in the control of OX neurons in response to leptin, we examined the ability of LHA leptin to regulate these neurons. Previous data have demonstrated that leptin modulates OX neurons in multiple manners: systemic leptin administration promotes Ox mRNA expression (Figure 7A), but also inhibits the fasting-stimulated activation of OX neurons, as detected by c-fos-IR (Funato et al., 2009; Mieda and Yanagisawa, 2002; Tritos et al., 2001; Yamanaka et al., 2003). Since our previous experience reveals that intra-LHA cannulation interferes with the examination of LHA c-fos-IR (data not shown), we chose to focus on the modulation of OX neuron gene expression in response to intra-LHA leptin infusion. We thus studied the regulation of ARC and LHA gene expression in response to systemic (5 mg/kg i.p.; Figure 7A) or intra-LHA (0.25 ng; Figure 7B) leptin in Lepob/ob animals, in which the absence of endogenous leptin permits the sensitive examination of leptin action. We previously demonstrated the confinement of leptin action to the LHA using this small dose of intra-LHA leptin (Leinninger et al., 2009). Animals were treated with vehicle or leptin for 26 hours before sacrifice for the harvesting of microdissected ARC and LHA tissue and the preparation of RNA for the analysis of gene expression by qPCR. Leptin induces Socs3 expression in a cell-autonomous manner (Banks et al., 2000; Bjorbaek et al., 2000) as well as directly promoting Pomc expression in ARC POMC neurons (Elmquist et al., 2005; Friedman, 2002; Morton et al., 2006; Myers et al., 2009); changes in Socs3 and Pomc mRNA thus serve as a marker of direct leptin action in the ARC (Baskin 2000; Elias 1999). Systemic leptin treatment significantly increased the expression of Pomc and Socs3 in the ARC, as well as increasing the expression of Ox (by 2-fold) and Nptx2 (neuronal activity-regulated pentraxin; a.k.a., Narp), which is coexpressed with OX in the LHA (Reti 2002; Blouin 2005; Crocker 2005)) (by 20%) in the LHA (Figure 7A). In contrast to the effects of systemic leptin, intra-LHA leptin failed to significantly modulate ARC Pomc or Socs3 gene expression, consistent with the predicted site-specificity of intra-LHA leptin action and the lack of projections from LHA LepRb neurons to the ARC (Figure 3B & 7B). Furthermore, intra-LHA leptin treatment failed to change LHA Mch expression. In contrast, intra-LHA leptin promoted a dramatic increase in LHA Ox (approximately 25-fold) and Nptx2 (approximately 4-fold) mRNA expression (Figure 7B). Thus, leptin action via LHA LepRb neurons robustly modulates gene expression in OX neurons, specifically.

Figure 7
Changes in gene expression after systemic and local leptin treatment in Lepob/ob (ob/ob) mice

Discussion

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 and Narp 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.

Supplementary Material

Supp1

Acknowledgements

Supported by NIH DK057768 and DK078056, grants from the American Diabetes Association and American Heart Association, and the Marilyn H. Vincent Foundation (to MGM), and the Reproductive Studies Program training grant at the University of Michigan (NIHT32 HD7048) (GWL). We thank members of the Myers lab, especially Drs. Rebecca Leshan and Christa Patterson, for discussions and support throughout. We thank Yusong Gong for excellent technical assistance in the development of mouse models and adenoviral tracers. We thank ZZ Zhang for excellent technical assistance; we thank Amylin Pharmaceuticals for the generous gift of leptin; we thank Dr. Yoshihiro Yoshihara, RIKEN Brain Science Institute, Japan for the gift of the WGA plasmid and Dr. Corrinne Lobe, Toronto, Canada for the iZAP plasmid. Core support was provided by The University of Michigan Cancer Center, NIH CA46592 and the University of Michigan Diabetes Research and Training Center, NIH DK20572.

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