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. 2025 Apr 4;96:102138. doi: 10.1016/j.molmet.2025.102138

A key role for parabrachial nucleus CGRP neurons in FGF1-Induced anorexia

Jarrad M Scarlett 1,2, Eunsang Hwang 3, Nicole E Richardson 1, Caeley L Bryan 1, Ingrid Redford 1, Emily Quah 1, Erik Tyr R Odderson 1, Pique P Choi 1, Matthew K Hwang 1, Bao Anh Phan 1, Kelly Kadlec 1, Kimberly M Alonge 1,4, Gregory J Morton 1, Kevin W Williams 3,⁎⁎, Michael W Schwartz 1,⁎
PMCID: PMC12142633  PMID: 40189100

Abstract

In addition to sustained glucose lowering, centrally administered fibroblast growth factor 1 (FGF1) induces a potent but transient anorexia in animal models of type 2 diabetes. To investigate the mechanism(s) underlying this anorexic response, the current work focused on a specific neuronal subset located in the external lateral subdivision of the parabrachial nucleus marked by the expression of calcitonin gene-related peptide (elPBNCGRP neurons). These neurons can be activated by withdrawal of upstream GABAergic inhibitory input and are implicated as mediators of the adaptive response (including anorexia) to a wide range of aversive stimuli. To determine if FGF1-induced anorexia is associated with elPBNCGRP neuron activation, we employed adult male CalcaCre:GFP/+ transgenic mice in which GFP is fused to Cre recombinase driven by the CGRP-encoding gene Calca. Here, we show that FGF1 activates elPBNCGRP neurons, both after intracerebroventricular (icv) injection in vivo and when applied ex vivo in a slice preparation, and that the mechanism underlying this effect depends upon reduced GABAergic input from neurons lying upstream. Consistent with this interpretation, we report that the anorexic response to icv FGF1 is reduced by ∼70% when elPBNCGRP neurons are silenced using chemogenetics. Last, we report that effects of icv FGF1 injection on both elPBNCGRP neuron activity and food intake are strongly attenuated by systemic administration of the GABAA receptor agonist Bretazenil. We conclude that in adult male mice, elPBNCGRP neuron activation is a key mediator of FGF1-induced anorexia, and that this activation response is mediated at least in part by withdrawal of GABAergic inhibition.

Keywords: FGF1, Anorexia, CGRP, Parabrachial nucleus, AGRP

Highlights

  • •

    FGF1 induces anorexia and activates CGRP neurons in the parabrachial nucleus.

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    Reduced presynaptic GABAergic input is required for FGF1 to activate these CGRP neurons.

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    Activation of parabrachial CGRP neurons is required for FGF1-induced anorexia.

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    These FG1 effects are ameliorated by increased GABAA receptor signaling.

1. Introduction

Among peptides investigated for their potential to treat type 2 diabetes (T2D) are members of the fibroblast growth factor (FGF) family, several of which mediate their antidiabetic effects via the central nervous system (CNS) [1]. Our group [[2], [3], [4]] and others [5,6] have shown that in multiple rodent models of T2D, a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) can normalize hyperglycemia for weeks or even months. Translation of this observation to the clinic has been hampered, however, by a potent but transient anorexic response that accompanies icv FGF1 injection [[4], [5], [6], [7], [8]]. The current work was undertaken to investigate mechanisms underlying this anorexic response and determine whether it is amenable to therapeutic intervention.

Although FGF1 activates the hypothalamic melanocortin system [6,9], the mechanism underlying FGF1-induced anorexia does not appear to involve increased melanocortin signaling. Melanocortin signaling is governed by two opposing neuronal populations – AgRP neurons and POMC neurons – situated adjacent to one another in the hypothalamic arcuate nucleus (ARC). AgRP is an inverse agonist that inhibits melanocortin-4 receptor (Mc4r) signaling, thereby reducing melanocortin system activity – an effect that increases food intake. In contrast, food intake decreases when Mc4r is activated by release of a-MSH from POMC neurons [10,11]. Since FGF1 both inhibits AgRP neurons [9] and activates POMC neurons [3,4,6,12], the net effect is to increase melanocortin signaling. Combined with evidence that the effect of icv FGF1 injection to induce sustained glucose lowering is blocked by interventions that disrupt Mc4r signaling, the antidiabetic central action of FGF1 has been deemed ‘melanocortin-dependent’ [13]. Yet neither pharmacological nor genetic disruption of Mc4r signaling affects anorexia following icv FGF1 injection [13]. Unlike its antidiabetic action, therefore, FGF1-induced anorexia involves a melanocortin-independent mechanism.

In light of these considerations, we shifted our attention to an anorexigenic population of neurons that express calcium gene-related peptide (CGRP) and are located in the external lateral subdivision of the parabrachial nucleus (elPBNCGRP neurons). These neurons are well recognized for their role in anorexia and malaise (“sickness behavior”) induced by stimuli ranging from the bacterial endotoxin lipopolysaccharide (LPS) to lithium chloride, GLP1 receptor agonists, and certain forms of cancer [[14], [15], [16], [17]], among others. A role for these neurons in the pathogenesis of anorexia was first reported by Palmiter and colleagues, who demonstrated that anorexia resulting from diphtheria toxin-induced ablation of AgRP neurons is dependent on elPBNCGRP neuron activation [18]. Specifically, the withdrawal of tonic GABAergic inhibition supplied by descending projections from AgRP neurons was implicated in the activation of these elPBNCGRP neurons and associated anorexia [14].

In addition to withdrawal of GABAergic input, elPBNCGRP neurons are activated by excitatory input from ascending hindbrain projections that convey satiety related as well as aversive visceral cues from the GI tract [19]. Thus, elPBNCGRP neuron activity appears to be governed by the balance between inhibitory input from descending AgRP neuron projections and excitatory input from ascending hindbrain neurons. These considerations, together with evidence that FGF1 inhibits AgRP neurons in a sustained manner [9], led us to hypothesize that FGF1-induced anorexia involves elPBNCGRP neuron activation, and that this effect results from withdrawal of inhibitory GABAergic input. Inherent in this formulation is that the feeding and glycemic effects of FGF1 involve distinct neurocircuitries, with only the latter being melanocortin-dependent.

Our findings offer both direct and indirect support for this hypothesis. Using a histochemical approach, we report that elPBNCGRP neurons are robustly activated following icv FGF1 injection, and we further demonstrate that chemogenetic inhibition of these neurons sharply attenuates FGF1-induced anorexia. We also show that both elPBNCGRP neuron activation and anorexia induced by icv FGF1 are suppressed by systemic administration of a GABAA receptor agonist at a dose below that needed to affect food intake on its own. Using slice electrophysiology, we further demonstrate that FGF1 depolarizes a subset of elPBNCGRP neurons via a mechanism involving reduced inhibitory GABAergic input. These data collectively support the conclusion that anorexia elicited by icv FGF1 injection requires elPBNCGRP neuron activation via a mechanism involving withdrawal of GABAergic inhibition, and that both responses can be ameliorated by low-dose GABAA receptor agonism.

2. Results

2.1. elPBNCGRP neuron activation following icv FGF1 injection

As an initial test of the hypothesis that elPBNCGRP neurons are activated following a single icv injection of FGF1, we utilized transgenic CalcaCre:GFP/+ mice to enable visualization of cFos protein, a marker of neuronal activation [20], in GFP-labeled elPBNCGRP neurons. To minimize baseline cFos expression in these neurons, mice were studied after an overnight fast [15]. Five hours after icv injection of either FGF1 (3 μg) or vehicle, mice were euthanized and brains processed for immunohistochemistry to determine the total number of neurons throughout the elPBN that were either CGRP+ (GFP), cFos+, CGRP+/cFos+, or cFos+/CGRP+. Representative images comparing CGRP+/cFos+ elPBNCGRP neurons between icv FGF1- and icv vehicle-injected mice are shown in (Figure 1A). As expected, the total number of CGRP+ neurons was comparable across all mice (Figure 1B), but both the total number of cFos+ cells in the elPBN and the number of CGRP+/cFos+ cells were increased ∼4-fold in icv FGF1-treated mice compared to vehicle-treated controls (Figure 1C,D). Nevertheless, not all CGRP+ cells in the elPBN were cFos+, and cFos was also detected in elPBN cells that do not express CGRP with the percentage of cFos+/CGRP+ cells comparable between icv FGF1- and icv vehicle-injected mice (Figure 1E). These data indicate that icv FGF1 injection activates both a large subset of elPBNCGRP neurons and an as yet unidentified population CGRP- neurons in this brain area. We also note that some elPBNCGRP neurons are not activated by FGF1.

Figure 1.

Figure 1

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Central injection of FGF1 activates CGRP neurons. (A) Immunohistochemical detection of Calca-locus driven GFP (green), cFos (red), and colocalization of GFP and cFos (right panels) in the elPBN of CalcaCre:GFP/+ mice 5 h after icv vehicle (Veh) or FGF1 (3 μg). Quantitation of (B) total CGRP+ cells, (C) total cFos+ cells, (D) percent of CGRP neurons that co-express cFos in the elPBN, and (E) percent of cFos cells that co-express CGRP in the elPBN. Scale bar = 100 μm, n = 4–6 group, mean ± SEM. Unpaired t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

2.2. Electrophysiological response of elPBNCGRP neurons to bath application of FGF1

We next characterized the response of elPBNCGRP neurons to FGF1 ex vivo using whole-cell patch-clamp electrophysiology. While bath application of FGF1 (10 nM) showed no significant change in the resting membrane potential (RMP) of elPBNCGRP neurons as a whole population (Supplemental Fig. 1), it produced heterogeneous effects across different subpopulations of elPBNCGRP neurons. In 7 of 18 neurons, FGF1 depolarized the cells (38.9%, RMP: −50.0 ± 1.7 mV to −46.3 ± 1.6 mV, p = 0.0001, Figure 2A,D). Conversely, 4 neurons were hyperpolarized (22.2%, RMP: −51.3 ± 2.1 mV to −57.7 ± 2.4 mV, p = 0.0617, Figure 2B,D), while 7 neurons exhibited no detectable change in RMP (38.9%, RMP: −51.1 ± 3.9 mV to −51.1 ± 4.0 mV, Figure 2C,D). Consistent with our c-Fos data (Figure 1), these findings demonstrate that FGF1 depolarizes a significant subset of elPBNCGRP neurons, although not all such neurons show this response.

Figure 2.

Figure 2

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Heterogeneous response of PBN CGRP neurons to bath application of FGF1 ex vivo. Representative traces and histograms showing depolarization (n = 7, A), hyperpolarization (n = 4, B), and no change (n = 7, C) in the resting membrane potential (RMP) of distinct subsets of elPBNCGRP neurons after bath application of FGF1 (10 nM). (D) Pie chart showing percentages of depolarization, hyperpolarization, and no change. Error bars indicate SEM. ∗∗∗p < 0.001, paired t-test compared to aCSF.

2.3. Electrophysiological response of elPBNCGRP neurons following icv FGF1 injection in vivo

As a complement to the above studies (Figure 1, Figure 2), we performed whole-cell patch clamp recordings on elPBNCGRP neurons 5 h after a single icv injection of either FGF1 (3 μg) or saline. In mice that received icv saline injection, the RMP and action potential frequency (APF) of elPBNCGRP neurons were −50.0 ± 1.3 mV and 0.7 ± 0.2 Hz (n = 23, Figure 3A,C, and D), respectively. By comparison, elPBNCGRP neurons were significantly depolarized following icv FGF1 injection (n = 25, RMP: −45.4 ± 1.0 mV, p = 0.0082, Figure 3B,C), and while the APF was also increased, this effect did not achieve statistical significance (APF: 0.96 ± 0.2 Hz, p = 0.82, Figure 3D), presumably owing to heterogeneity inherent in the response.

Figure 3.

Figure 3

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Impact of a single FGF1 icv injection on the cellular activity of PBN CGRP neurons. Representative traces of resting membrane potential (RMP) and action potential frequency (APF) of elPBNCGRP neurons recorded 5 h after injection into the lateral cerebral ventricle of either saline (A) or FGF1 (3 μg) (B). Histogram summarizing effects on the RMP (C) and APF (D) shown in Panels A and B. Error bars indicate SEM. ∗P < 0.05, unpaired t-test compared to Saline group. The number of neurons studied for each group is in parentheses.

To investigate whether the depolarizing effect of icv FGF1 on elPBNCGRP neurons involves either an increase in excitatory or a decrease of inhibitory synaptic activity [17,21], we monitored EPSPs and IPSPs in these neurons (Figure 4). Unexpectedly, we observed a tendency for the EPSC frequency of elPBNCGRP neurons to be lower following icv FGF1 compared to saline injection, although the effect did not achieve statistical significance (Saline: 6.0 ± 0.6 Hz, n = 18 vs. FGF1: 4.3 ± 0.6 Hz, n = 23, p = 0.0573, Figure 4E), nor did the small decrease of EPSC amplitude (Saline: 15.9 ± 0.6 pA, n = 18, vs. FGF1: 15.5 ± 0.7 pA, n = 23, p = 0.69, Figure 4F). In contrast, a significant decrease in IPSC frequency was observed in elPBNCGRP neurons receiving icv FGF1 injection vs. icv saline injected mice (Saline: 1.4 ± 0.3 Hz, n = 19 vs. FGF1: 0.6 ± 0.1 Hz, n = 22; p = 0.0085; Figure 4G). This finding identifies reduced GABAergic synaptic activity as a potential mediator of the effect of centrally administered FGF1 to depolarize elPBNCGRP neurons. In addition, however, IPSC amplitude was not significantly altered (Saline: 20.8 ± 1.2 pA vs. FGF1: 22.5 ± 1.6 pA, p = 0.44, Figure 4H).

Figure 4.

Figure 4

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Impact of a single icv FGF1 injection on synaptic activity of elPBNCGRP neurons. Voltage clamp recording of excitatory postsynaptic currents (EPSCs) observed in elPBNCGRP neurons after icv injection of either saline (A) or FGF1 (B). Voltage clamp recording of inhibitory postsynaptic currents (IPSCs) observed in the same neurons after icv saline (C) or FGF1 (3 μg) (D) injection. Histograms showing the mean EPSC frequency (E) and amplitude (F) or mean IPSC frequency (G) and amplitude (H) of elPBNCGRP neurons following either icv saline (black) or FGF1 (red) injection. Data are expressed as mean ± SEM. ∗∗ p < 0.01, unpaired t-test compared to Saline group. The number of neurons studied for each group is in parentheses. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

2.4. Dependence of elPBNCGRP neuron depolarization on reduced GABAergic input

As a further test of the hypothesis that a decrease of fast ionotropic GABAergic activity contributes to FGF1-mediated depolarization of elPBNCGRP neurons, we pretreated brain slices from CGRP reporter mice with the GABAA receptor antagonist picrotoxin (50 μM). Bath application of FGF1 in the presence of picrotoxin resulted in a significant hyperpolarization of the RMP in 4 of 14 elPBNCGRP neurons (28.6%, RMP: −46.6 ± 2.3 mV to −51.6 ± 2.0 mV, p = 0.0054; Figure 5A,C) but remained unchanged in a majority of these neurons (71.4%, RMP: −50.3 ± 2.0 mV to −50.4 ± 2.0 mV, p = 0.65, Figure 5B,C). Notably, the previously noted effect of FGF1 to depolarize a subset of these neurons (Figure 2) was not observed in brain slices pre-treated with picrotoxin. Following FGF1 bath application, therefore, depolarization of a subset of elPBNCGRP neurons appears to require reduced presynaptic GABAergic input from upstream neurons; in contrast, the inhibitory effect of FGF1 application on a small subset of elPBNCGRP neurons is mediated independently of GABAergic activity. These data collectively indicate that reduced presynaptic GABAergic input is required for FGF1-induced activation of elPBNCGRP neurons.

Figure 5.

Figure 5

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The effect of FGF1 on the resting membrane potential of PBN CGRP neurons in presence of picrotoxin. Representative traces and histograms showing hyperpolarization (n = 4, A) and no response (n = 10, B) in the resting membrane potential (RMP) of the elPBNCGRP neurons following bath application of FGF1 (10 nM) in presence of picrotoxin (50 μM). (D) Pie chart showing percentages of hyperpolarization and no change. Error bars indicate SEM. ∗∗p < 0.01, paired t-test compared to bath application of Picrotoxin (PX) alone.

2.5. Role of elPBNCGRP neuron activation in FGF1-induced anorexia and weight loss

To determine if activation of elPBNCGRP neurons is required for icv FGF1 to induce anorexia and weight loss, we determined the effect of chemogenetic inhibition of these neurons on anorexia and weight loss induced by icv FGF1 injection. CalcaCre:GFP/+ transgenic mice underwent bilateral injections of either an AAV containing a Cre-dependent hM4Di-mCherry transgene [22] or mCherry control at the time of lateral ventricular cannula placement. After allowing 2 weeks for surgical recovery and viral transgene expression, each mouse received an ip injection of either CNO (1 mg/kg) or saline, followed 2 h later by a single icv injection of FGF1. For this study, we selected a lower dose of FGF1 (1 μg vs. the 3 μg dose used in neuron activation studies) that retains its anorexigenic activity with fewer, potentially confounding illness-like behaviors such as lethargy. As previously reported [2,4,6,13], icv injection of FGF1 induced robust anorexia and weight loss (measured 12 h later) and, as expected, these FGF1-induced responses were not altered any of the control groups (including hM4Di or mCherry mice receiving ip saline and mCherry controls receiving ip CNO). In contrast, anorexia and weight loss induced by icv FGF1 injection were reduced by ∼70% following chemogenetic inhibition of elPBNCGRP neurons (Figure 6A,B). Thus, elPBNCGRP neuron activation is required for FGF1-induced anorexia and weight loss.

Figure 6.

Figure 6

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Chemogenetic inhibition of CGRP neurons prevents weight loss and anorexia in response to icv FGF1.CalcaCre:GFP/+ mice were studied following bilateral microinjection of an AAV containing either hM4Di-mCherry or mCherry control into the elPBN. After a 2 wk recovery period, each mouse received an ip injection of either saline or CNO, followed 2 h later by a single icv injection of FGF1 (1 μg). Food intake and body weight were measured over the next 12 h. In mice receiving CNO, chemogenetic inhibition of elPBNCGRP neurons ameliorated the potent reductions of both (A) body weight and (B) food intake induced by central FGF1 administration. Error bars represent mean ± SEM. 2-way ANOVA with Bonferroni's multiple comparisons test.

To confirm CNO-induced inhibition of elPBNCGRP neurons expressing the Cre-dependent hM4Di-mCherry transgene, we quantified the total number of CGRP neurons throughout the elPBN of these mice that were either mCherry+, cFos+ (imaged in Alexa Fluor 647 far-red dye and pseudocolored green), or cFos+/mCherry+ (Figure 7A). As expected, the total number of mCherry+ neurons in the elPBN (representing elPBNCGRP neurons expressing Cre-dependent hM4Di-mCherry transgene or mCherry control) did not differ significantly between groups (Figure 7B). In contrast, the effect of icv FGF1 injection on both the total number of cFos+ cells and the number of cFos+/mCherry+ cells in the elPBN was reduced by chemogenetic inhibition of elPBNCGRP neurons (induced by ip administration of CNO; Figure 7C,D). As expected, therefore, elPBNCGRP neurons expressing the Cre-dependent hM4Di-mCherry transgene are inhibited by administration of CNO.

Figure 7.

Figure 7

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Chemogenetic inhibition of CGRP blocks activation of CGRP neurons in response to icv FGF1. (A) Immunohistochemical detection of CGRP neurons expressing mCherry (red), cFos (green), and colocalization of mCherry and cFos (right merge panel) in the elPBN of CalcaCre:GFP/+ mice. Mice that had received bilateral injections of either an AAV containing hM4Di-mCherry or mCherry control were euthanized 5 h after icv FGF1 (1 μg). 2 h before icv injection, mice received an ip injection of either vehicle (Veh) or CNO. Quantitation of (B) total number of CGRP neurons expressing mCherry in the elPBN, (C) total number of cFos+ neurons and (D) percent of CGRP+ neurons expressing mCherry that co-express cFos. Scale bar = 100 μm, n = 4–6 group, error bars represent mean ± SEM. 2-way ANOVA with Bonferroni's multiple comparisons test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

2.6. Effects of a GABAA receptor agonist on both FGF1-induced anorexia and elPBNCGRP neuron activation

Based on prior work demonstrating that GABA release from AgRP neurons onto neurons in the lateral PBN potently impacts food intake and body weight [18], and on our observation that AgRP neuron activity is rapidly and durably inhibited following icv FGF1 injection [9], we next asked whether either FGF1-induced elPBNCGRP neuron activation or the associated anorexia and weight loss are blunted by systemic administration of bretazenil, a GABAA receptor partial agonist. To this end, CalcaCre:GFP/+ transgenic mice were implanted subcutaneously with Alzet minipumps to deliver either bretazenil (750 ng/h rip) or vehicle for 14 days. As previously reported [18], chronic subcutaneous infusion of bretazenil at this dose had no effects on food intake or body weight prior to icv FGF1 injection (Supplemental Figure 2, A and B). On day 14, mice received a single icv injection of FGF1 (1 μg) or vehicle and food intake and body weight were monitored over a 24-h period to determine the impact of systemic bretazenil administration on FGF1-induced weight loss and anorexia. As predicted, systemic delivery of bretazenil significantly blunted both weight loss and anorexia following icv FGF1 injection (Supplemental Figure 2, C and D). Although this effect is modest compared to the ∼70%-reduction of FGF1-induced anorexia resulting from chemogenetic inhibition of elPBNCGRP neurons, the results nevertheless support a role for reduced GABAergic signaling in the effect of icv FGF1 injection on food intake.

To determine if FGF1-induced elPBNCGRP neuron activation is also blunted by bretazenil administration, separate cohorts of mice underwent the same treatment and were euthanized 5 h after icv injection for histochemical analysis. As before (Figure 1), we quantified the total number of neurons throughout the elPBN that were either CGRP+ (GFP), cFos+, or cFos+/CGRP+ neurons between icv FGF1-injected mice receiving either subcutaneous vehicle or bretazenil (Figure 8A). Neither the total number of CGRP+ neurons nor the number of cFos+ cells in the elPBN of mice receiving icv vehicle were significantly impacted by bretazenil infusion (Figure 8C and D). In contrast, while icv FGF1 injection elicited the expected, robust increase in the total of cFos+ cells and the number of cFos+/CGRP+ cells in the elPBN compared to icv vehicle-treated controls, this activation response was largely prevented by bretazenil infusion (Figure 8C,D). This finding supports a model wherein elPBNCGRP neuron activation induced by icv FGF1 requires decreased GABAA receptor signaling.

Figure 8.

Figure 8

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Administration of bretazenil blunts activation of CGRP neurons following icv FGF1 injection. Immunohistochemical analysis of the elPBN of mice studied in Figure 8 that had received continuous subcutaneous administration of either Vehicle (Veh) or bretazenil by osmotic minipump for 14 d. Detection of (A) Calca-locus driven GFP (green), cFos (red), and colocalization of GFP and cFos (right merge panel) in the elPBN of CalcaCre:GFP/+ mice 5 h after icv injection of either FGF1 (1 μg) or vehicle. Quantitation of (B) total CGRP+ cells, (C) total cFos+ cells and (D) percent of CGRP neurons that co-express cFos. Scale bar = 100 μm, n = 4–6 group, error bars represent mean ± SEM. 2-way ANOVA with Bonferroni's multiple comparisons test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

A caveat to these findings is that the effect of bretazenil to inhibit FGF1-induced activation of elPBNCGRP neurons appears to be far more robust than the associated suppression of FGF1-induced anorexia. This observation supports the hypothesis that the latter effect involves activation of neurons additional to elPBNCGRP neurons via a mechanism that does not require reduced GABAergic input.

3. Discussion

A unique aspect of the antidiabetic action of FGF1 is its duration: following a single icv injection, hyperglycemia is ameliorated for weeks or months in multiple rodent models of T2D. Based on part on the transient nature of the effect of icv FGF1 injection on food intake and body weight [3,4,6,8,12], we hypothesized that mechanisms underlying the effects of FGF1 on feeding and glucose homeostasis are distinct from one another. The anorexic response elicited by icv FGF1 injection lasts several days in both normal and diabetic rodents. The anorexic and aversive properties of centrally administered FGF1 were first reported in rats in 1996 [23]. In a subsequent pilot study performed in Rhesus macaque monkeys, intracisternal FGF1 administration was shown to induce anxiety, immobilization, anorexia and reduced water intake lasting 2–3 days (unpublished data). Extending these findings is a large literature demonstrating that across multiple mouse models, icv FGF1 injection reliably elicits a robust anorexic response that is transient, lasting no more than one week [[2], [3], [4], [5], [6],8,9,12,24]. The current studies were undertaken to investigate mechanisms underlying this transient anorexic response.

Based on published evidence that anorexic induced by icv FGF1 injection is not impacted by genetic or pharmacological interventions that disrupt MC4R signaling [13], we shifted our focus onto melanocortin-independent mechanisms. Among potential candidates are elPBNCGRP neurons, based on evidence that activation of these neurons rapidly inhibits food intake, and that anorexia and malaise induced by several different aversive stimuli can be alleviated by silencing these neurons [14,15,19]. Thus, we first asked whether elPBNCGRP neurons are activated following central FGF1 administration. To this end, we performed a series of studies in CalcaCre:GFP/+ transgenic mice in which GFP is fused to Cre recombinase driven by the CGRP-encoding gene Calca [15]. Using both histochemical and electrophysiological methods, we report that a subset of elPBNCGRP neurons is robustly activated following icv FGF1 injection. We also demonstrate that the stimulatory effect of FGF1 on these neurons requires reduced GABAergic input, and that both elPBNCGRP neuron activation and food intake suppression following icv injection of FGF1 are blunted by systemic administration of the GABAA receptor agonist bretazenil (at a dose below that needed to affect food intake in control mice).

To investigate whether elPBNCGRP neuron activation is required for FGF1-induced anorexia, we employed a chemogenetic silencing approach. Our finding that anorexia induced by icv injection of FGF1 is almost fully blocked by DREADD-mediated inhibition of elPBNCGRP neurons indicates that activation of this neuronal subset is required for this response. An alternative interpretation of these data is that elPBNCGRP neuron inhibition stimulates food intake, and that this effect offsets an action of FGF1 mediated elsewhere. However, one of us (MWS) co-authored a publication showing that while functional inactivation of elPBNCGRP neurons alters meal patterning in mice, it has no net effect on either daily food intake or body weight [19]. Based on these collective findings, we therefore conclude that FGF1-induced anorexia is dependent on elPBNCGRP neuron activation, and that the underlying mechanism involves withdrawal of inhibitory input from GABAergic neurons lying upstream.

While the identify of these upstream GABAergic neurons remains to be determined, AgRP neurons sit atop our list of potential candidates, for the following reasons: 1) AgRP neurons are inhibited in a durable manner following icv FGF1 injection [9], 2) the ARC (where AgRP neurons are located) is a particularly sensitive site for anorexia elicited by FGF1 [8], 3) AgRP neurons send inhibitory GABAergic projections from the ARC to CGRP neurons in the elPBN [18], and 4) AgRP neuron ablation causes severe, life-threatening anorexia by disinhibiting and thereby activating elPBNCGRP neurons [18]. Taken together, these findings support a model wherein FGF1-induced anorexia involves a mechanism whereby AgRP neuron inhibition leads to reduced GABAergic inhibition of elPBNCGRP neurons.

This interpretation, however, does not exclude a role for other mechanisms, and several observations are compatible with this possibility. For one, we report that although FGF1-induced anorexia is potently suppressed by chemogenetic silencing of these neurons (by ∼70%), a modest residual anorexia persists. While this residual effect could reflect incomplete viral targeting of elPBNCGRP neurons, we cannot exclude the possibility that non-CGRP neurons are also engaged by FGF1 and contribute to FGF1-induced anorexia. Our findings using bretazenil (Figure 8) are also consistent with this possibility. Specifically, whereas FGF1-induced elPBNCGRP neuron activation was potently suppressed by bretazenil administration (as judged by cFos induction), its ability to prevent FGF1-induced anorexia was much more modest. Together, these findings suggest a role for as yet unidentified neuronal subsets in the mechanism underlying FGF1-induced anorexia – presumably, via a mechanism that does not require reduced GABAergic signaling.

Aversive GI manifestations are commonly encountered in clinical settings. For example, the use of GLP1 receptor agonist (GLP1RA) drugs such as semaglutide is commonly associated with GI side effects including nausea, vomiting and malaise. These drugs work by activating GLP1 receptors expressed by diverse cell types throughout the body [16,25,26], including neurons in hindbrain areas such as the area postrema and nucleus of the solitary tract [27,28]. Subsets of these GLP1-responsive hindbrain neurons send excitatory projections to the PBN, where they activate elPBNCGRP neurons [25] as part of an ascending visceral pathway that conveys afferent ‘gut-brain’ signals elaborated by the GI tract upon (or in anticipation of) food consumption. Thus, the most common side effects of these popular drugs likely involve elPBNCGRP neuron activation.

The same ascending pathway is also implicated in the pathogenesis of anorexia, nausea and GI distress associated with chemotherapeutic agents such as cis-platinum [29], systemic infection/inflammation (modeled by LPS injection), certain forms of cancer [14], and conditions associated with elevated circulating levels of the peptide GDF-15 (in uremia [30] and hyperemesis gravidarum [31]). In this context, our finding that FGF1-induced activation of elPBNCGRP neurons can be prevented by GABAergic agonist by drugs raises a clinically relevant question: can commonly encountered aversive GI manifestations be ameliorated by GABAergic drugs that blunt elPBNCGRP neuron activation? Future studies are warranted to investigate this question.

4. Methods

4.1. Sex as a biological variable

Sex was not considered as a biological variable in these studies, as our previous work reveals no sexual dimorphism in the anorexic response to icv FGF1 injection in rodents. Thus, male mice were used in all studies; the expectation that our findings apply to both males and females will be explored in future studies.

4.2. Animals

Animals were housed individually under specific pathogen-free conditions in a temperature-controlled environment with ad libitum access to water and standard laboratory chow (LabDiet, St Louis, MO). Adule male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Male CalcaCre:GFP/+ transgenic mice are a generous gift from Dr. Richard Palmiter [15]. Until stereotaxic surgery mice were group-housed; thereafter, they were housed individually.

4.3. Stereotaxic surgeries

Lateral ventricle (LV) cannulations (8IC315GAS5SC, 26-ga, Plastics One, Roanoke, VA) were performed under isoflurane anesthesia using the following stereotaxic coordinates based on the Mouse Brain Atlas: −0.7 mm posterior to bregma; 1.3 mm lateral, and 1.95 mm below the skull surface [32]. For viral injections either the inhibitory DREADD (hM4Di) or mCherry control AAV was injected bilaterally into the elPBN (antero-posterior (AP), −4.9 mm; medio-lateral (ML), 1.4 mm; dorso-ventral (DV), 3.8 mm) at a rate of 0.2 μl min−1 for 2.5 min (0.5 μl total volume). Mice were treated peri-operatively with Buprenorphine SR (1 mg/mL; 0.1 mL SQ per 25 g; 1 dose for 72 h) and Carprofen (1.3 mg/mL; 0.3 mL SQ per 25 g; 1 dose per 24 h; 3 days). Afterward, the mice were given a 2 wk recovery period to allow for virally-transduced gene expression and to acclimate to handling and experimental paradigms prior to study.

4.4. Intracerebroventricular injection

Body weight, food intake and blood glucose measurements were taken every morning at 10 AM (CST) beginning at least 2 days prior to icv injections. Groups were matched for mean levels of blood glucose, food intake and body weight for DREADD and bretazenil studies. Animals received a single 2 or 3 μL injection into the LV of either murine FGF1 (0.5 or 1.0 μg/μL; a generous gift from Novo Nordisk) or 0.9% saline vehicle using a 33-gauge needle (Plastics One, Roanoke, VA) inserted 0.8 mm beyond the tip of the LV cannula.

4.5. Viral injections

Chemogenetic inhibition of elPBNCGRP neurons was achieved by microinjection of an adeno-associated virus (AAV)1 containing Cre-dependent cassette for the inhibitory DREADD, pAAV1-hSyn-DIO-hM4D(Gi)-mCherry (Addgene plasmid # 44362) [22] or mCherry control pAAV1-hSyn-DIO-mCherry (Addgene plasmid # 50459) [22]. Activation of the DREADD receptor was induced by intraperitoneal (ip) administration of the agonist, clozapine N-oxide (CNO, 1 mg/kg) (#C0832, Sigma–Aldrich, St. Louis, MO).

4.6. Drug treatment

Alzet 14-day micro-osmotic pumps (model 1002, Durect, Cupertino, CA, USA) were filled with 100 μL of either the GABAA receptor agonist bretazenil (3 mg/mL in saline plus 10% DMSO; SigmaAldrich, St Louis, MO, USA) or vehicle (saline plus 10% DMSO) and implanted subcutaneously between the scapulae of anesthetized mice 3 days before receiving a single icv FGF1 injection. Based on an infusion rate of 0.25 μL/h, minipumps delivered bretazenil subcutaneously at a rate of 750 ng/h, a dose below that needed to independently impact food intake or body weight [18].

4.7. Immunofluorescence

For immunohistochemical studies, mice were anesthetized with ketamine and xylazine and perfused with ice-cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M PBS, after which brains were removed. Anatomically-matched free-floating coronal sections (30 μm thickness) from the rostral to caudal extent of the outer external lateral subdivision of the PBN from approximately Bregma −4.90 to −5.50 were collected, washed in PBS at room temperature, permeabilized in 0.1% Triton X-100 and 3% BSA, blocked in freshly prepared 5% normal serum (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania) and incubated overnight at 4 °C with rabbit anti-cFos antibody (1:10,000; PC38; Oncogene Research Products, Boston, MA), followed by incubation in donkey anti-rabbit Alexa 555 or donkey anti-rabbit Alexa 647 (1:1,000; Invitrogen, Waltham, MA). Sections were washed, re-blocked in normal serum, and then incubated with chicken anti-GFP antibody (1:5,000; ab13970; Abcam, Cambridge, MA), followed by incubation in goat anti-chicken Alexa 488 (1:1,000; Jackson ImmunoResearch Laboratories, West Grove, PN). Sections were then washed overnight in PBS and mounted on super-frost plus microscope slides. Immunofluorescence images were captured using a Leica SP8X Scanning Confocal microscope (Buffalo Grove, IL) with a HC FLUOTAR L 25X/0.95 W objective. Quantification of total elPBNCGRP and cFos cell count was performed using Qupath https://qupath.github.io. Quantification of co-localization of cFos with elPBNCGRP neurons and mCherry with elPBNCGRP neurons was performed by: (i) exporting Qupath images to ImageJ (Fiji, NIH) and converting to binary images, (ii) using Image Calculator to multiply matched binary images to identify elPBNCGRP neurons that contain cFos or mCherry, and (iii) quantification of elPBNCGRP neurons that were cFos or mCherry positive using the Analyze Particles feature [33].

4.8. Electrophysiology: brain slice preparation

Brain slices were prepared from mice as previously described [9,[34], [35], [36]]. Briefly, male mice were deeply anesthetized with i.p. injection of 7% chloral hydrate and transcardially perfused with a modified ice-cold artificial CSF (ACSF) (described below). The mice were then decapitated, and the entire brain was removed and immediately submerged in ice-cold, carbogen-saturated (95% O2 and 5% CO2) ACSF (126 mM NaCl, 2.8 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 5 mM glucose). Coronal sections (250 μm) were cut with a Leica VT1000S Vibratome and then incubated in oxygenated ACSF (32 °C–34 °C) for at least 1 h before recording. The slices were bathed in oxygenated ACSF (32 °C–34 °C) at a flow rate of ∼2 mL/min. All electrophysiology recordings were performed at room temperature.

4.9. Electrophysiology: whole-cell recordings

The pipette solution for whole-cell recordings was as follows: 125 mM K-gluconate, 2–10 mM KCl, 10 mM HEPES, 5–10 mM EGTA, 1 mM CaCl2, 1 mM MgCl2, and 1–2 mM MgATP, 0–5 HEPES, 0.03 mM Alexa Fluor 350 hydrazide dye, pH 7.3. Electrophysiological recordings were performed similar to previous reports [9,[34], [35], [36]]. Briefly, epifluorescence was used to target fluorescent cells, at which time the light source was switched to infrared differential interference contrast imaging to obtain the whole-cell recording (Zeiss Axioskop FS2 Plus equipped with a fixed stage and a QuantEM:512SC electron-multiplying charge-coupled device camera). Electrophysiological signals were recorded using an Axopatch 700B amplifier (Molecular Devices); low-pass filtered at 2–5 kHz and analyzed offline on a PC with patch-clamp (pCLAMP) electrophysiology data acquisition and analysis program (Molecular Devices). Membrane potentials and firing rates were determined from CGRP neurons in brain slices. For acute drug administration, we targeted a CGRP neuron in 1 slice, and after recording switched to another slice to target the next CGRP neuron. Recording electrodes showed resistances of 2.5–5 MΩ when filled with the K-gluconate internal solution.

4.10. Analysis and statistics

Results are expressed as mean ± SEM. Significance was set at ∗p < 0.05 for all statistical measures. For immunohistochemical experiments, data are shown as dot plots representing data from individual animals and bar graphs representing average ± SEM. Statistical analyses using unpaired Student's t-test was performed using R [37]. For DREADD experiments, two-way repeated measures ANOVA detected significant interaction of viral genotype x pharmacological treatment for food intake (F3,11 = 21.00, P ≤ 0.0001) and body weight (F3,16 = 13.37, P = 0.0001). Bonferroni post-hoc test was used to detect significant differences between hM4Di-transduced animals and mCherry-transduced control animals, as indicated. For Bretazenil experiments, two-way repeated measures ANOVA detected significant interaction of icv treatment x mini-pump pharmacological treatment for food intake (F3,15 = 97.30, P < 0.0001) and body weight (F3,15 = 29.80, P < 0.0001). Bonferroni post-hoc test was used to detect significant differences between groups. For bath application studies: A change in membrane potential was required to be at least 2 mV in amplitude. Membrane potential values were not compensated to account for junction potential (−8 mV). All graphs and figures were generated using either GraphPad Prism 10.0 software (Graphpad Software Inc, Boston, MA), or CorelDraw X8 (Corel Corp, Ottawa, ON). All data from different groups were analyzed using an unpaired, paired, or multiple unpaired t-test as well as two-way ANOVA where appropriate. Results are reported as the mean ± SEM unless indicated otherwise, as indicated in each figure legend; where n represents the number of cells studied.

4.11. Study approval

All experiments were performed in accordance with the guidelines established by the Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011) and were approved by the University of Washington and the University of Texas Institutional Animal Care and Use Committees.

CRediT authorship contribution statement

Jarrad M. Scarlett: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Eunsang Hwang: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Nicole E. Richardson: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Caeley L. Bryan: Writing – review & editing, Investigation, Formal analysis, Data curation, Conceptualization. Ingrid Redford: Writing – review & editing, Software, Investigation, Formal analysis, Data curation. Emily Quah: Writing – review & editing, Investigation, Formal analysis, Data curation, Conceptualization. Erik Tyr R. Odderson: Writing – review & editing, Investigation, Formal analysis, Data curation. Pique P. Choi: Writing – review & editing, Formal analysis, Data curation. Matthew K. Hwang: Writing – review & editing, Investigation, Formal analysis, Data curation. Bao Anh Phan: Writing – review & editing, Investigation, Formal analysis, Data curation. Kelly Kadlec: Writing – review & editing, Investigation, Formal analysis, Data curation. Kimberly M. Alonge: Writing – review & editing, Supervision, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Gregory J. Morton: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Kevin W. Williams: Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation. Michael W. Schwartz: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Authorship note

JMS and EH are co-first authors. KWW and MWS are co–senior authors.

Funding

This work was supported by grants to: J.M.S. (K08 DK114474, R03 DK128383, and DoD W81XWH2110635), E.H. (National Research Foundation of Korea – NRF 2021R1A6A3A14044733), KA (DP2 AI171150 and R21 AG074152), G.J.M. (R01 DK089056, R01 DK124238 and ADA 1-19-IBS-192), K.W.W. (R01 DK119169, R56 DK135501, and PO1 DK119130-03), M.W.S (R01 DK101997 and R01 DK083042), the NIDDK-funded Nutrition Obesity Research Center (DK035816) and the Diabetes, Obesity and Metabolism Training Grant (DK007247; N.E.R., P.P.Q) at the University of Washington. Funding in support of these studies was also provided to M.W.S. through an agreement with Novo Nordisk (CMS-431104).

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Michael W. Schwartz reports financial support was provided by National Institute of Diabetes and Digestive and Kidney Diseases. Jarrad Scarlett reports financial support was provided by National Institute of Diabetes and Digestive and Kidney Diseases. Eunsang Hwang reports financial support was provided by National Research Foundation of Korea. Jarrad Scarlett reports financial support was provided by US Department of Defense. Kimberly Alonge reports financial support was provided by National Institute of Allergy and Infectious Diseases. Kimberly Alonge reports financial support was provided by National Institute on Aging. Gregory Morton reports financial support was provided by National Institute of Diabetes and Digestive and Kidney Diseases. Kevin Williams reports financial support was provided by National Institute of Diabetes and Digestive and Kidney Diseases. Pique Choi reports financial support was provided by National Institute of Diabetes and Digestive and Kidney Diseases. Nicole Richardson reports was provided by National Institute of Diabetes and Digestive and Kidney Diseases. Michael Schwartz reports a relationship with Novo Nordisk Inc that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

The authors gratefully acknowledge Dr. Richard Palmiter (University of Washington) for providing the CGRP-Cre:GFP mice and Vincent Damian for generating mice and maintaining mouse colonies.

Footnotes

Supplementary data to this article can be found online at https://doi.org/10.1016/j.molmet.2025.102138.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Multimedia component 1
mmc1.pdf (165.6KB, pdf)
Multimedia component 2
mmc2.pdf (265.6KB, pdf)

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
mmc1.pdf (165.6KB, pdf)
Multimedia component 2
mmc2.pdf (265.6KB, pdf)

Data Availability Statement

Data will be made available on request.

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