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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 2009 March 10.
Published in final edited form as:
PMCID: PMC2597113
NIHMSID: NIHMS68609

Ablation of AgRP Neurons Activates Fos and Gliosis in Post-synaptic Target Regions

Abstract

We have developed a mouse model in which a specific population of inhibitory neurons can be selectively ablated by the action of diphtheria toxin (DT). The model involves targeting the human diphtheria toxin receptor (DTR) to the agouti-related protein (Agrp) locus so that systemic administration of DT kills all of the AgRP-expressing neurons resulting in starvation of the mice. Ablation of AgRP neurons results in robust (5 to 10 fold) activation of Fos gene expression in many brain regions that are innervated by AgRP neurons, including the arcuate nucleus (ARC), paraventricular nucleus, the medial pre-optic area, lateral septum and nucleus of the solitary tract. As expected, there is robust increase in GFAP staining (astrocytes) as well as IBA1 and CD11b staining (microglia) in the ARC in response to AgRP neuron ablation. There is also dramatic increase of these markers in most, but not all, post-synaptic targets of AgRP axons. We used a genetic approach to reduce melanocortin signaling, which attenuated Fos activation in some brain regions after ablation of AgRP neurons. We suggest that loss of inhibitory signaling onto target neurons results in unopposed excitation that is responsible for the activation of Fos and that dysregulation of these neuronal circuits is responsible for starvation. Furthermore, glial cell activation in target areas of AgRP neurons appears to be due to excitotoxicity.

Keywords: neuronal death, Feeding, Glia, Hypothalamus, Appetite, Network, Hindbrain

Introduction

Neuronal death occurs as a natural process during development and as a consequence of traumatic events in the mature nervous system. The detection, removal and shielding of dying neurons is an integrated process that involves participation by astrocytes and microglia as they attempt to preserve the functional integrity of the remaining viable components. Analysis of signaling events between neurons and glial cells that mediate the smooth transition from cell death to reorganization of remaining components is just beginning to be unraveled (Fields and Stevens-Graham, 2002;Hara and Snyder, 2007).

We have developed a method to kill a specific neuronal population in the adult mouse in a relatively rapid and reproducible manner. We targeted expression of DTR to neurons that express AgRP and then administer DT to adult mice which results in ablation of the neurons over the next few days and starvation by ~ 6 days (Luquet et al., 2005). This method of ablation is specific for AgRP-expressing cells, conditional upon DT administration, convenient in that DT can be administered by any route, and reproducible (Luquet et al., 2005).

AgRP neurons are part of a hypothalamic regulatory system that detects and integrates various nutritional, hormonal and neuronal signals to regulate appetite and metabolism and thereby help maintain energy balance (Broberger et al., 1998). AgRP neurons and the neighboring pro-opiomelanocortin (POMC)-expressing neurons send their axonal projections to the same brain regions. The POMC neurons produce melanocortin (α-melanocyte stimulating hormone, α MSH), which activates melanocortin 4 receptors (MC4R) on post-synaptic cells (Cone, 2005). Activation of this melanocortin pathway inhibits feeding and stimulates metabolism. The AgRP neurons counteract the actions of melanocortin signaling by directly inhibiting POMC neurons via GABA release (Cowley et al., 2001), and counteracting the action of melanocortin on post-synaptic cells: AgRP antagonizes binding of melanocortin to MC4R (Cone, 2005), NPY activates Gαi-coupled receptors (Marsh et al., 1998;Pedrazzini et al., 1998), and GABA activates GABAA receptors. Thus, AgRP neurons promote feeding, at least in part, by counteracting the actions of the melanocortin signaling. Consistent with this arrangement, ablation of AgRP neurons in adult mice leads to starvation (Gropp et al., 2005;Luquet et al., 2005;Luquet et al., 2007;Phillips and Palmiter, 2007).

Here we describe cellular consequences of AgRP neuron ablation that help delineate why the mice starve. Death of AgRP neurons is associated with Fos gene activation, as well as robust gliosis, in the target areas. We suggest that dysregulation of post-synaptic cells is responsible for the failure of the mice to eat. We distinguish whether glial cell activation is due to either axonal degeneration or excessive excitation.

Materials and Methods

Animal Maintenance and Neuron Ablation

Mice were housed in a temperature- and humidity-controlled facility with a 12-hour light/dark cycle. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Washington. AgrpDTR/+ mice were generated by targeting the human diphtheria toxin receptor (heparin-binding epidermal growth factor, HB-EGFR) to the Agrp locus (Luquet et al., 2005). Ay; AgrpDTR/+ mice were generated by mating homozygous AgrpDTR/DTR with heterozygous lethal yellow Ay/a mice obtained from Jackson Laboratory (Bar Harbor, ME). The yellow mice derived from this cross comprise the Ay; AgrpDTR/+ group while the black littermates served as AgrpDTR/+ controls. Mice were group housed with standard chow diet (LabDiet 5053) and water provided ad libitum until the beginning of the experiments. To ablate AgRP neurons in adult mice, intramuscular injection of diphtheria toxin (two injections of 50 μg/kg, two days apart; List Biological Laboratories, Campbell, CA) in six-week-old mice was performed (Luquet et al., 2005).

In Situ Hybridization

Brains were sectioned (coronal 25 μm) and every 8th section was used for either Nissl staining or in situ hybridization with Npy, Agrp, Fos, Egr1 or Arc using an automated procedure for hybridization and image capture. We present the results obtained with Fos in this study. The results obtained with Agrp were published (Wu et al., 2008); results obtained with Npy in the ARC were identical. The results obtained with Egr1 were similar to those of Fos except that the differential response in the a/a versus the Ay/a genetic background was not apparent after DT injection. The Arc hybridization signals were weak in most regions of interest compared to Fos or Egr1. The hybridization data collected for Egr1 and Arc are available upon request. Materials and procedures concerning this high-throughput data generation process (riboprobe production, tissue processing, in situ hybridization, image capture and processing) have been described (Lein et al., 2007) and are available on the Allen Brain Atlas website (www.brain-map.org).

Immunohistochemistry

Mice were killed by CO2 asphyxiation and perfused transcardially with ice-cold PBS buffer containing 4% paraformaldehyde. Brains were dissected and post-fixed overnight at 4°C in the fixation buffer. Free-floating brain sections (30 μm) were washed in PBS and 0.1% Triton X-100 (PBST buffer) solution 3 × 15 min and then blocked with 3% normal donkey serum in PBST for 2 – 3 hr at room temperature. Rabbit anti-NPY (Peninsula Lab, 1:1000 dilution), rabbit anti-GFAP (Novus Biologicals; 1:3000 dilution), rabbit anti-IBA1 (Wako Chemicals USA; 1:1000 dilution), rabbit anti-Fos (Calbiochem, 1:6000 dilution), rat anti-CD11b (AbD Serotec, 1:1000 dilution), mouse anti-NeuN (Chemicon, 1:100 dilution), and/or mouse anti-ACTH (Santa Cruz Biotech, 1:100 dilution) were applied to the sections for overnight incubation at 4°C, followed by 3 15-min rinses in PBST. Finally, sections were incubated in Cy2- or Cy3-labeled secondary antibody (Jackson Immunolaboratory, 1:500 dilution) before visualization. Images were captured using a digital camera mounted on a TCS SP1 confocal microscope (Leica Microsystems USA); all paired photos were obtained through the same system setting. For each group of mice, at least 8 sections from 4 different mice were analyzed.

Data analysis

Quantification of Fos-positive cells was done using the NIH Image software (National Institutes of Health). Anatomical correlations of brain sections and delineation of individual nuclei were determined by comparing landmarks of Nissl staining images with those given in the Paxinos stereotaxic atlas (Paxinos and Frank, 2001). From the anatomically matched sections, a region of interest of the same size was further defined. Meanwhile, an optimized threshold that can discern round Fos-positive nuclei from partially stained ones as well as background noise was preset for all measurement. The total number of pixels of Fos-positive cells inside the defined region was automatically recorded. The signal intensity of NPY and glial cell markers (GFAP, IBA1, and CD11b) was quantitatively assessed using the NIH Image software under the same exposure and threshold setting to quantify the variance of expression level. Data sets collected from all experiments, unless otherwise stated, were analyzed by one-way ANOVA followed by Student-Newman-Keuls method for statistical significance and plotted as means ± standard error of mean (SEM). Post-hoc analysis was performed when group differences were significant by ANOVA at p < 0.05.

Results

Ablation of AgRP neurons by administration of DT induces Fos and gliosis in the ARC

The generation of AgrpDTR/+ mice with the cDNA encoding the human DTR targeted to the Agrp locus has been described (Luquet et al., 2005). Administration of DT (2 intramuscular injections at 50 μg/kg body weight, 2 days apart) results in reliable loss of appetite such that feeding virtually ceases and body weight falls by 20% by the sixth day (Fig. 1A, Luquet et al., 2007;Phillips and Palmiter, 2007;Wu et al., 2008). We terminated the experiments at that point because of animal care concerns, but we know that the mice would die within another day or so. We showed previously that this DT-treatment protocol results in the nearly complete loss of Agrp hybridization signal in the ARC by day 6 after initiation of DT treatment (Wu et al., 2008), in agreement with previous results using antibodies to either AgRP or NPY (Luquet et al., 2005;Luquet et al., 2007).

Figure 1
Kinetics of feeding response, Fos activation, gliosis, and NPY/AgRP cell death after injecting DT

DT treatment also leads to the activation of Fos and gliosis in various brain regions including the ARC on day 6 after initiation of DT treatment (Wu et al., 2008). To examine the kinetics of Fos activation and initiation of gliosis, the ARC region of the hypothalamus was examined by immunohistochemistry after treating AgrpDTR/+ mice with DT for 2, 4 or 6 days. There was robust staining for Fos (half maximal) within the first 2 days after initiating DT treatment, at a time when there was no apparent loss of NPY neurons (Fig. 1B, D–E, P–Q) and no inhibition of feeding (Fig. 1A). In addition, microglial activation was readily apparent in the vicinity of NPY neurons as revealed by staining with an antibody to IBA1 (ionized calcium binding adapter molecule 1, a microglia marker; product of the Aif1 gene) (Fig. 1B, H–I, H′–I′). By day 4, when feeding began to be affected, there was a small loss of NPY staining in cell bodies within the ARC (Fig. 1B, R). Fos and IBA1 staining were maximal at day 4 and CD11b (integrin αmβ2, another marker of activated macrophages) staining became prominent (Fig. 1B, F, J, J′, L–N). By day 6, the mice consumed very little food and they had lost ~20% of their body weight; NPY staining in the ARC was almost gone, but Fos and microglia staining persisted (Fig. 1B, G, K–K′, O, S). Nissl staining revealed that there was ~25% loss of neurons in the ARC at day 6 (Fig. 2). These data suggest that 2 days of DT treatment affects the function of AgRP neurons leading to the activation of Fos in surrounding neurons. Microglia also start to become activated on day 2, several days before there is obvious loss of NPY/AgRP neurons or an overt effect on feeding behavior.

Figure 2
Nissl staining in ARC and PVN of pair-fed control and AgRP neuron-ablated mice

On day 6 after DT treatment, all POMC neurons were Fos positive, as predicted from the loss of inhibitory input from the AgRP neurons (Wu et al., 2008), but POMC neurons accounted for only ~38% of the Fos-positive cells in the ARC (Fig. 3), indicating that other neuronal populations in the ARC also became activated. Meanwhile, virtually all the Fos-positive cells (>97%) were determined to be neurons, as revealed by NeuN, a neuron-specific cell marker (Fig. 3D).

Figure 3
Co-localization of c-Fos and POMC cells in the ARC after ablation of AgRP neurons

Activation of Fos expression and gliosis in the PVN after ablation of AgRP neurons

The PVN is a primary brain region that integrates opposing signals via projections from AgRP and POMC neurons (Balthasar et al., 2005;Kublaoui et al., 2006;Yang et al., 2006). We examined Fos and microglia markers in the PVN of the same mice that were used to study these markers in the ARC. None of these markers were significantly elevated relative to controls 2 days after DT treatment, but they were all increased 4 days after DT treatment and even more so at 6 days (Fig. 1C). There was no loss of neurons in the PVN as assessed by Nissl staining (Fig. 2). Thus, activation of neurons and microglia in this projection field of AgRP neurons was delayed relative to that observed in the area surrounding the AgRP neuron cell bodies in the ARC.

Induction of Fos and gliosis in the PVN is attenuated in the absence of αMSH signaling

We also examined mice in which the AgrpDTR/+ allele was bred into the Ay genetic background (designated Ay; AgrpDTR/+) to examine the consequences of suppressed melanocortin signaling on Fos gene activation. The Ay mice express agouti protein ectopically in virtually all tissues including the brain (Michaud et al., 1993). Like AgRP, agouti protein antagonizes the action of αMSH binding to MC4R and thus, prevents melanocortin signaling via the Gαs-coupled receptor on post-synaptic cells (Cone, 2005). We reasoned that because AgRP neurons innervate the same post-synaptic targets as POMC neurons, activation of Fos in those post-synaptic cells after ablation of inhibitory AgRP neurons would be reduced if melanocortin signaling was blocked.

A high-throughput protocol was used to allow quantitative comparison of in situ hybridization signals from 3 different genes (Fos, Npy, Agrp) in 160 sections from each of 3 to 6 brains from wild-type, AgrpDTR/+, and Ay; AgrpDTR/+ mice that were either treated with DT or not (Lein et al., 2007). Over 1600 mouse brain sections were examined for this study.

We examined Fos induction in the PVN of AgRP neuron-ablated mice in both wild-type and Ay genetic backgrounds. To detect Fos induction elicited by stress of starvation, control groups including DT-treated wild-type, naïve AgrpDTR/+ and naïve Ay; AgrpDTR/+ mice that were food restricted by pair feeding (PF) to lose the same body weight as the neuron-ablated animals. A DT treated group of wild-type mice was also included to identify DT-associated cellular stress responses.

Ablation of AgRP neurons by DT treatment of AgrpDTR/+ mice dramatically activated Fos expression in the PVN in comparison with two different PF control groups (Fig. 4C, C′ compared to A, A′ or B, B′, images from 2 different mice; data from ≥4 mice are quantified in Fig. 4R). There was no appreciable Fos signal in untreated WT mice (data not shown). Fos mRNA increased >5 fold in the PVN of DT-treated AgrpDTR/+ mice relative to the pair-fed or DT-treated, wild-type controls (p < 0.01). Chronic blockade of the MC4R signaling pathway by ectopic expression of agouti in Ay; AgrpDTR/+ mice, significantly attenuated Fos response in the PVN relative to AgrpDTR/+ mice (Fig. 4E, E′ vs. C, C′ p < 0.01). Moreover, the data indicate that the induction of Fos was not due to stress of starvation or non-specific DT toxicity because PF and DT treatment in control mice had only small effects on Fos expression (Fig. 4 A, A′, B, B′, D, D′). These data indicate that neuronal activity in the PVN is greatly enhanced after removal of inhibitory AgRP input and that blockade of MC4R signaling ameliorated the excitability of the PVN.

Figure 4
Blockade of MC4R signaling counteracts Fos induction and ameliorates the hypersensitivity of glial cells in the PVN of AgRP-ablated mice

Data consistency is illustrated in the duplicate figures (Fig. 4 A–E and A′–E′) and was verified by quantitative comparison of Fos mRNA level in anatomically matched brain sections of the cortex from control and experimental groups; this brain region is not known to be involved in regulation of energy balance (Supplemental Fig. 1A–G). No statistical difference of signal intensity was found across control and neuron-ablated groups (Supplemental Fig. 1H; ANOVA, p > 0.13), suggesting that performing a direct comparison of Fos activity from mice of different genotypes is valid for identifying any difference at AgRP target areas.

We postulated that either loss of axon terminals due to DT treatment of AgrpDTR/+ mice or the resulting excessive neuronal activation in post-synaptic targets of AgRP neurons could result in excitotoxicity and neuroinflammation -cascade of events characterized by glial cell activation (Raivich et al., 1999). GFAP (glial fibrillary acidic protein, an astrocyte marker) activation in the PVN of the DT-treated AgrpDTR/+ mice and the Ay, AgrpDTR/+ mice relative to the 4 control groups is shown in Fig. 4 F–K. Activation is revealed as an apparent increase in the immunostaining and change in morphology of the astrocytes; see Fig. 4 H′ and I′ for magnified views of the regions that are boxed in Fig. 4 H and I, respectively. The activation of microglia was assessed by immunostaining with an antibody against IBA1 in an adjacent set of sections from the 6 groups of mice (Fig. 4 L–Q and magnified in Fig. 4 N′ and O′). Quantitative analysis revealed numerous activated glial cells with compact, bushy processes in the PVN of DT-treated AgrpDTR/+ mice, whereas the majority of glial cells in pair-fed and DT-treated wild-type mice were resting with fine ramifications of their processes (Fig. 4H′ and I′ or 4N′ and O′; quantified in Fig. 4R). Ablation-induced activation of both astrocytes and microglial cells was attenuated in DT-treated Ay; AgrpDTR/+ mice relative to AgrpDTR/+ mice (Fig. 4R), suggesting that glial cells in the PVN are sensitive to the antagonistic action of agouti on MC4R-expressing neurons. These results indicate that glial cell markers provide an additional tool for assessing relevant AgRP target nuclei.

Two distinct classes of AgRP targets are discerned by comparing the effects of AgRP ablation in AgrpDTR/+ and Ay; AgrpDTR/+ mice

Because our strategy recapitulated the functional relationship between AgRP neurons and target cells in the PVN, we proceeded to evaluate Fos expression in other brain regions that receive projections from AgRP neurons (Broberger et al., 1998;Haskell-Luevano et al., 1999). To minimize impact from the small variations underlying the automated process of in situ hybridization and image acquisition, we restricted our analysis to those brain regions where there was >50% increase in Fos mRNA in DT-treated AgrpDTR/+ mice relative to PF, DT-treated wild-type mice. Six different regions fell within our screening criteria, including three hypothalamic nuclei [the ARC, the dorsal medial hypothalamus (DMH), and the medial pre-optic area (MPO)] and three extra-hypothalamic regions [the lateral septum (LS), the hippocampal dentate gyrus (DG), and the nucleus tractus solitarius (NTS)].

DT-treated AgrpDTR/+ mice showed significant Fos induction in all six regions (~7 fold in the ARC, ~2.5 fold in the DMH and the MPO, ~3 fold in the LS, ~2 fold in the DG, ~4 fold in the NTS) in comparison to pair-fed, DT-treated wild-type mice and pair-fed AgrpDTR/+ mice (Fig. 5A–F; Supplemental Figs. 2–7). However, comparing the Fos induction in DT-treated AgrpDTR/+ mice and DT-treated Ay; AgrpDTR/+ mice revealed different responses depending on the brain region examined. Fos expression was significantly reduced (55 to 85%) in the ARC, DMH, MPO and the LS when the MC4R signaling was blocked by agouti (Fig. 5A–D; Supplemental Figs. 2–5; p < 0.01). In contrast, the intensity of Fos signal was the same in the DG and the NTS when comparing the effect of DT in mice with or without Ay gene (Fig. 5E, F; Supplemental Figs. 6, 7; p > 0.59). These results suggest that POMC neurons are opposed by AgRP signaling in the ARC, DMH, MPO and LS as they are in the PVN. The observation that chronic blockade of MC4R by agouti elicited virtually no impact on neuron hyperactivity in the DG and the NTS suggests that melanocortin signaling in those regions does not contribute to neuronal activation.

Figure 5
Fos expression and glial cell activation are differentially regulated in diverse targets of AgRP neurons

We also examined the response of glial cells in these target brain regions. The data reveal that astrocytes and microglia were activated to various extents in the ARC, DMH, MPO, and LS of DT-treated AgrpDTR/+ mice as compared to WT and pair-fed control groups, implying that ablation of AgRP neurons caused significant neuron activation and/or damage in these downstream areas (Fig. 5A–D; Supplemental Figs. 8–15). However, differing from the attenuated glial response shown in the PVN of DT-treated Ay; AgrpDTR/+ mice, the astrocytes and microglia in the ARC, DMH, MPO, and LS of the Ay; AgrpDTR/+ mice remained abnormally active, except for a reduced IBA1 immunoreactivity in the DMH. DT treatment elicited comparable glial reactivity in the DG of wild-type, AgrpDTR/+, and Ay; AgrpDTR/+ mice, which may be due to non-specific, DT-mediated toxicity; nevertheless, astrocytes, but not microgila, in the NTS exhibited more robust reactivity in neuron-ablated mice as compared to DT-treated wild-type mice (Fig. 5E, F; Supplemental Figs. 16–19).

Some post-synaptic targets of AgRP neurons manifest neither Fos activation nor gliosis

While most brain regions that are established targets of AgRP neurons revealed robust activation of Fos gene expression and glia, others such as the bed nucleus of the stria terminalis (BST), the ventral tegmental area (VTA), and the paranigral nucleus (PN), did not. Examples of brain regions that receive extensive innervation from AgRP neurons but did not show any significant Fos induction or activation of astrocytes or microglia are shown in Figure 6. The innervation of these regions by AgRP neurons, based on immunohistochemistry by Broberger et al (1998), are as extensive as other regions (e.g. the MPO and LS) in which Fos was robustly induced. These results indicate that loss of AgRP axons does not invariably lead to Fos activation or gliosis.

Figure 6
Some post-synaptic targets of AgRP neurons show neither Fos activation nor gliosis

Discussion

Mice with targeted ablation of hypothalamic AgRP-expressing neurons provided a unique opportunity to study the activation of neural circuits that presumably mediate the starvation phenotype. We found that ablation of AgRP neurons leads to a dramatic activation of Fos in most, but not all, known targets of POMC and AgRP neurons. Activated microglia and astrocytes were also detected in most regions displaying elevated Fos expression. Chronic blockade of melanocortin signaling by ectopic expression of agouti, attenuated Fos expression in all brain regions except for the DG and the NTS, but did not prevent starvation (Wu et al., 2008). We hypothesize that the dysregulation of neuronal signaling as indicated by activation of Fos expression mediates the starvation phenotype.

A relatively complete profile of AgRP- and POMC-immunoreactive fibers projected from the ARC has been characterized in rodents (Broberger et al., 1998;Haskell-Luevano et al., 1999;Watson et al., 1978;Jacobowitz and O’Donohue, 1978;Nilaver et al., 1979). However, the functional significance of the overlap in POMC and AgRP-positive fibers in target areas has not been examined. We used the semi-automated, whole brain, in situ hybridization approach to gain functional information about the relative importance of various signaling pathways. Fos expression throughout the brain was examined with special attention to the sites that receive projections from AgRP neurons. We suspect that activation of Fos reflects unbalanced excitatory and inhibitory inputs to the Fos-expressing cells. Most of the excitatory input presumably comes from glutamatergic afferents along with activation by various Gαs- and Gαq-coupled receptors, whereas the most of the inhibitory inputs come from GABAergic activation of GABAA receptors and activation of Gαi- and Gαo-coupled receptors. Thus, ablation of AgRP neurons is predicted to remove several inhibitory inputs to post-synaptic neurons resulting in unopposed excitation and consequent induction of Fos gene expression. Most post-synaptic neurons probably receive excitatory inputs in addition to those from POMC neurons. Thus, whether Fos is activated or not and whether it is affected by blockade of melanocortin signaling would likely depend on the relative strengths of the various inputs.

We present evidence indicating that only some of the established AgRP-immunoreactive sites displayed elevated Fos induction after AgRP neuron ablation, including the ARC, PVN, DMH, MPO, and LS, whereas other areas did not manifest enhanced Fos expression. AgRP target areas were evaluated based on screening results with statistical difference between food-restricted animals and neuron-ablated animals. Brain areas that had Fos expression below an appreciable level or with insignificant change relative to food-restricted animals were excluded from our analysis. For example, the paraventricular nucleus of the thalamus (PVT) receives sparse innervations of AgRP fibers, yet displayed a comparable, prominent Fos expression in food-restricted, control mice and DT-treated AgrpDTR/+ mice (data not shown). Other regions that are clearly innervated by AgRP fibers, such as the red nucleus (RN), the VTA, and the PAG, showed minimal or no Fos activity (Fig. 6 and data not shown). Several possibilities may underlie that lack of Fos induction in some brain regions after loss of AgRP neuron innervation. First, inhibition from AgRP neurons may be a small fraction of total inhibitory inputs. Second, some targets of AgRP neurons may not receive strong excitatory inputs. Third, induction of Fos requires multiple cellular signals (for example, calcium- and cAMP-mediated signals) and some of these signals may be absent. Four, we only examined Fos expression thoroughly at day 6, so some early changes may have resolved by then. Therefore, the AgRP-projection areas that we studied here represent a subset of those that could be important in mediating starvation.

Six days after DT treatment, there were few remnants of AgRP neurons in the ARC as assessed by in situ hybridization or immunohistochemistry. Nearly all of the neighboring POMC neurons expressed Fos mRNA, as expected, but many other unidentified neurons in the ARC were also Fos positive. We observed strong glial cell activation in the ARC 6 days after commencing DT treatment. Activated microglia and astrocytes underwent dramatic morphological changes including enlargement of the cell soma and thickening of proximal process (Ladeby et al., 2005). Interestingly, the activation of Fos and the microglial marker IBA1 was apparent in the ARC by day 2, prior to any loss of neurons or an effect on feeding. DT acts by inhibiting protein synthesis (Collier, 1975), so we presume that AgRP neuron function is initially impaired and progresses to cell death a few days later. The activation of Fos presumably reflects loss of inhibitory inputs onto POMC and other local neurons from impaired AgRP neurons while the microglial activation may represent responses to distress signals from either the impaired AgRP neurons or overly excited post-synaptic cells. By day 4, the CD11b microglical marker became prominent, which coincides with the time when AgRP neuron markers began to disappear; hence, CD11b may reflect a more advanced stage of microglial activation, perhaps associated with phagocytosis of dying cells.

Glial cell activation in post-synaptic targets of AgRP neurons was also robust and was associated with Fos induction in the DMH, PVN, MPO and LS. Such glial cell activation could reflect responses to axonal degeneration, but glutamate-mediated activation of astrocytes is also possible (Garden and Moller, 2006;Raivich et al., 1999;Hanisch and Kettenmann, 2007). Astrocytes normally express glutamate receptors, whereas microglial cells only express glutamate receptors after they become activated; both cell types can be killed by glutamate (Matute et al., 2006) and gliosis can precede neuronal degeneration is some situations (Kielar et al., 2007). The parallel amelioration of glial and Fos activation by agouti expression in the PVN, under conditions where axonal degeneration was the same, provides an example where something other than axon degeneration, perhaps glutamate, modulates glial cell activation. In some brain regions AgRP neuron terminals degenerate but there was no activation of Fos or glial markers. This observation suggests that axonal degeneration is insufficient to activate microglia and favors an excitotoxic interpretation. The observations that glial cell activation occurs only in brain regions that are direct targets of AgRP neurons indicate that activation is not propagated to downstream neurons. It is unlikely that gliosis and subsequent release of cytokines is directly responsible for starvation in this model because ablation of POMC neurons, which reside and project to virtually all the same brain regions as AgRP neurons, does not result in starvation (Gropp et al., 2005). Quantitative analysis of neuronal loss in target areas based on Nissl staining did not reveal evidence of pyknotic nuclei or reduced neuronal density in any of the brain regions that received the highest glial cell activation scores, except for the ARC where there was obvious loss of neurons. Thus, we tentatively suggest that glial cell activation in target areas reflects a neuroprotective role of these cells rather than contributing to neurotoxicity.

Two brain regions (the DG and NTS) showed Fos activation after DT treatment that was not attenuated in the Ay genetic background. In both of these regions the glial cell activation was observed in all groups treated with DT, even in wild-type mice. Thus, glial cell activation in these brain regions could reflect enhanced sensitivity to DT. Because AgRP-immunoreactive fibers have not been reported in the DG (Haskell-Luevano et al., 1999) activation of Fos there probably reflects activation from some other brain region as a consequence of AgRP neuron ablation. Excitotoxicity in the hippocampus in response to seizures results in DG neuron cell death and activation of Npy gene expression (Cole et al., 2000;Palmiter et al., 1998;Vezzani et al., 1999), but we did not observe any significant cell death or changes in Npy mRNA among control and AgRP-ablated experimental groups (data not shown), suggesting that the activation was mild relative to that which occurs during seizures. Because the DG has never been implicated in regulation of feeding behavior and it is not a direct target of AgRP neurons, we discount the significance of Fos activation there.

The other brain region where loss of MC4R signaling did not affect Fos induction was the NTS; however, the identity of the Fos-positive cells in the NTS is currently unknown. The NTS is traditionally perceived as a center for detection and transduction of satiety signals from vagal afferents, and gut peptides, like cholecystokinin (Badman and Flier, 2005). The NTS also responds to glucose, leptin, melanocortin and urocortin (Grill and Kaplan, 2002). There is general agreement that activation of caudal brain stem is necessary for feeding (Grill, 2006). A reciprocal crosstalk between the NTS and hypothalamic cell groups including the AgRP and POMC neurons may underlie the NTS mediation of feeding activity (Cone, 2005). We hypothesize that dysregulation within the NTS mediates the inhibition of food intake measured by intra-oral infusion of food directly into the mouth of AgRP-neuron ablated mice (Wu et al., 2008). However, it is not established whether activation of Fos in the NTS is a due to loss of direct AgRP projections to the NTS (Broberger et al., 1998) or an indirect consequence of dysregulation in other brain regions that send projections to the NTS.

Supplementary Material

Supp1

Acknowledgments

We thank Glenda Froelick and Betty Li for help with histology, Aundrea Rainwater for help with mouse breeding, and the staff of the Allen Brain Research Institute for performing the in situ hybridization studies. We appreciate the helpful comments on the manuscript by Drs. Nephi Stella and Tomas Möller. This work was supported in part by NIH DA024908 to R.D.P.

References

  • Badman MK, Flier JS. The gut and energy balance: visceral allies in the obesity wars. Science. 2005;307:1909–1914. [PubMed]
  • Balthasar N, Dalgaard LT, Lee CE, Yu J, Funahashi H, Williams T, Ferreira M, Tang V, McGovern RA, Kenny CD, Christiansen LM, Edelstein E, Choi B, Boss O, Aschkenasi C, Zhang CY, Mountjoy K, Kishi T, Elmquist JK, Lowell BB. Divergence of melanocortin pathways in the control of food intake and energy expenditure. Cell. 2005;123:493–505. [PubMed]
  • Broberger C, Johansen J, Johansson C, Schalling M, Hokfelt T. The neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice. Proc Natl Acad Sci U S A. 1998;95:15043–15048. [PubMed]
  • Cole TB, Robbins CA, Wenzel HJ, Schwartzkroin PA, Palmiter RD. Seizures and neuronal damage in mice lacking vesicular zinc. Epilepsy Res. 2000;39:153–169. [PubMed]
  • Collier RJ. Diphtheria toxin: mode of action and structure. Bacteriol Rev. 1975;39:54–85. [PMC free article] [PubMed]
  • Cone RD. Anatomy and regulation of the central melanocortin system. Nat Neurosci. 2005;8:571–578. [PubMed]
  • Cowley MA, Smart JL, Rubinstein M, Cerdan MG, Diano S, Horvath TL, Cone RD, Low MJ. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature. 2001;411:480–484. [PubMed]
  • Fields RD, Stevens-Graham B. New insights into neuron-glia communication. Science. 2002;298:556–562. [PMC free article] [PubMed]
  • Garden GA, Moller T. Microglia biology in health and disease. J Neuroimmune Pharmacol. 2006;1:127–137. [PubMed]
  • Grill HJ. Distributed neural control of energy balance: contributions from hindbrain and hypothalamus. Obesity. 2006;14(Suppl 5):216S–221S. [PubMed]
  • Grill HJ, Kaplan JM. The neuroanatomical axis for control of energy balance. Front Neuroendocrinol. 2002;23:2–40. [PubMed]
  • Gropp E, Shanabrough M, Borok E, Xu AW, Janoschek R, Buch T, Plum L, Balthasar N, Hampel B, Waisman A, Barsh GS, Horvath TL, Bruning JC. Agouti-related peptide-expressing neurons are mandatory for feeding. Nat Neurosci. 2005;8:1289–1291. [PubMed]
  • Hanisch UK, Kettenmann H. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci. 2007;10:1387–1394. [PubMed]
  • Hara J, Beuckmann CT, Nambu T, Willie JT, Chemelli RM, Sinton CM, Sugiyama F, Yagami K, Goto K, Yanagisawa M, Sakurai T. Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity. Neuron. 2001;30:345–354. [PubMed]
  • Hara MR, Snyder SH. Cell signaling and neuronal death. Annu Rev Pharmacol Toxicol. 2007;47:117–141. [PubMed]
  • Haskell-Luevano C, Chen P, Li C, Chang K, Smith MS, Cameron JL, Cone RD. Characterization of the neuroanatomical distribution of agouti-related protein immunoreactivity in the rhesus monkey and the rat. Endocrinology. 1999;140:1408–1415. [PubMed]
  • Jacobowitz DM, O’Donohue TL. alpha-Melanocyte stimulating hormone: immunohistochemical identification and mapping in neurons of rat brain. Proc Natl Acad Sci U S A. 1978;75:6300–6304. [PubMed]
  • Kielar C, Maddox L, Bible E, Pontikis CC, Macauley SL, Griffey MA, Wong M, Sands MS, Cooper JD. Successive neuron loss in the thalamus and cortex in a mouse model of infantile neuronal ceroid lipofuscinosis. Neurobiol Dis. 2007;25:150–162. [PMC free article] [PubMed]
  • Kublaoui BM, Holder JL, Jr, Tolson KP, Gemelli T, Zinn AR. SIM1 overexpression partially rescues agouti yellow and diet-induced obesity by normalizing food intake. Endocrinology. 2006;147:4542–4549. [PubMed]
  • Ladeby R, Wirenfeldt M, Garcia-Ovejero D, Fenger C, ssing-Olesen L, Dalmau I, Finsen B. Microglial cell population dynamics in the injured adult central nervous system. Brain Res Brain Res Rev. 2005;48:196–206. [PubMed]
  • Lein ES, et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature. 2007;445:168–176. [PubMed]
  • Luquet S, Perez FA, Hnasko TS, Palmiter RD. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates. Science. 2005;310:683–685. [PubMed]
  • Luquet S, Phillips CT, Palmiter RD. NPY/AgRP neurons are not essential for feeding responses to glucoprivation. Peptides. 2007;28:214–225. [PubMed]
  • Marsh DJ, Hollopeter G, Kafer KE, Palmiter RD. Role of the Y5 neuropeptide Y receptor in feeding and obesity. Nat Med. 1998;4:718–721. [PubMed]
  • Matute C, Domercq M, Sanchez-Gomez MV. Glutamate-mediated glial injury: mechanisms and clinical importance. Glia. 2006;53:212–224. [PubMed]
  • Michaud EJ, Bultman SJ, Stubbs LJ, Woychik RP. The embryonic lethality of homozygous lethal yellow mice (Ay/Ay) is associated with the disruption of a novel RNA-binding protein. Genes Dev. 1993;7:1203–1213. [PubMed]
  • Nilaver G, Zimmerman EA, Defendini R, Liotta AS, Krieger DT, Brownstein MJ. Adrenocorticotropin and beta-lipotropin in the hypothalamus. Localization in the same arcuate neurons by sequential immunocytochemical procedures. J Cell Biol. 1979;81:50–58. [PMC free article] [PubMed]
  • Paxinos G, Frank KB. The Mouse Brain in Sterotaxic Coordinates. San Diego: Academic Press; 2001.
  • Pedrazzini T, Seydoux J, Kunstner P, Aubert JF, Grouzmann E, Beermann F, Brunner HR. Cardiovascular response, feeding behavior and locomotor activity in mice lacking the NPY Y1 receptor. Nat Med. 1998;4:722–726. [PubMed]
  • Phillips CT, Palmiter RD. Role of AgRP-expressing Neurons in Lactation. Endocrinology 2007 [PubMed]
  • Raivich G, Bohatschek M, Kloss CU, Werner A, Jones LL, Kreutzberg GW. Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function. Brain Res Brain Res Rev. 1999;30:77–105. [PubMed]
  • Vezzani A, Sperk G, Colmers WF. Neuropeptide Y: emerging evidence for a functional role in seizure modulation. Trends Neurosci. 1999;22:25–30. [PubMed]
  • Watson SJ, Akil H, Richard CW, III, Barchas JD. Evidence for two separate opiate peptide neuronal systems. Nature. 1978;275:226–228. [PubMed]
  • Wu Q, Howell MP, Cowley MA, Palmiter RD. Starvation after AgRP neuron ablation is independent of melanocortin signaling. Proc Natl Acad Sci U S A. 2008;105:2687–2692. [PubMed]
  • Yang C, Gagnon D, Vachon P, Tremblay A, Levy E, Massie B, Michaud JL. Adenoviral-mediated modulation of Sim1 expression in the paraventricular nucleus affects food intake. J Neurosci. 2006;26:7116–7120. [PubMed]