PHB-10306; No of Pages 7 Physiology & Behavior xxx (2014) xxx–xxx
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Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice Ruby A. Holland, John J. Leonard, Nicholas A. Kensey, Paavali A. Hannikainen, Bart C. De Jonghe ⁎ Dept. of Biobehavioral Health Sciences School of Nursing, University of Pennsylvania, Philadelphia, PA, 19104, United States
H I G H L I G H T S • • • •
Cisplatin reduces food intake and body weight in mice dose-dependently. 1st study examining cisplatin-induced c-Fos expression in mice. 1st study to examine cisplatin-induced AMPAR and NMDAR subunit gene expression. We present evidence for a novel central circuit of cisplatin-induced nausea.
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Article history: Received 13 December 2013 Received in revised form 17 February 2014 Accepted 23 February 2014 Available online xxxx Keywords: NMDA AMPA NR2B Nausea Cisplatin c-Fos
a b s t r a c t Although rats and mice do not vomit, these species are widely studied as models of energy balance and sickness behavior. Previous work has shown that rats exhibit similar neuroanatomical activation of brain and visceral afferent pathways following cisplatin chemotherapy compared to vomiting species. However, the neural response to cisplatin in mice is understudied. Here, food intake, body weight, and central c-Fos immunofluorescence were analyzed in the hindbrains of male C57BL/6 mice following IP saline or cisplatin (5 mg/kg, and 20 mg/kg doses). As glutamate receptor signaling is classically linked to inhibitory feeding pathways in the rodent, gene expression of selected α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptor subunits were assessed in the dorsal vagal complex (DVC), parabrachial nucleus (PBN), amygdala, and bed nucleus of the stria terminalis (BNST). Our results show dosedependent reductions in food intake and body weight following cisplatin treatment, as well as increases in cisplatin-induced c-Fos in the PBN and throughout the DVC. Quantitative PCR analysis shows cisplatin-induced increases in NMDA receptor subunit expression, particularly NR2B, in the DVC, PBN, BNST, and amygdala. In addition, upregulation of AMPA receptor subunits (GluA1 and/or GluA2) were observed in all regions examined except the amygdala. Taken together, these results suggest similar neural pathways mediating cisplatin effects in mice compared to other well-studied species, which are likely mediated by central upregulation of AMPA and NMDA receptors. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Cisplatin chemotherapy is perhaps the most potent and widely studied emetogenic antineoplastic drug [1,2], having been used in the treatment of solid tumors for nearly 35 years. Without anti-emetic medication, cisplatin produces severe nausea, vomiting, anorexia and cachexia in a variety of vomiting species including ferrets, pigs, dogs, cats, shrews, and humans [3–7]. Although rats and mice do not vomit, these species are widely studied as models for food intake regulation, energy balance, and cisplatin-induced anorexia/sickness behavior [8–10]. Previous reports have shown that rats exhibit similar neuroanatomical activation in brain areas and visceral afferent pathways ⁎ Corresponding author. Tel.: +1 215 898 4901; fax: +1 215 573 3859. E-mail address: [email protected] (B.C. De Jonghe).
compared to vomiting species [11–14]. On the other hand, mice have been relatively understudied for neuronal pathways of sickness behavior caused by emetic stimuli such as cisplatin [15,16]. As the overwhelming majority of animal models used in cancer research involve mouse models, it is essential to study the neural correlates of cancerand/or chemotherapy-induced anorexia, nausea, and sickness in mice. Cisplatin robustly stimulates the vagus nerve via serotonin (5-HT) release from enteroendocrine cells and subsequent binding to 5-HT3 receptors on afferent terminals innervating the gut (for more detailed discussion, see Ref. [17]). In rats, these chemotherapy-induced vagal signals are then transmitted to the brain; first synapsing in the NTS in the caudal hindbrain [17]. Rats exhibit similar distributions and magnitude of hindbrain c-Fos activation following cisplatin injection when compared to vomiting species [18,19]. Here, we are the first to examine c-Fos immunofluorescence in mice to test the hypothesis that cisplatin
http://dx.doi.org/10.1016/j.physbeh.2014.02.038 0031-9384/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
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produces similar hindbrain neuronal activation in mice compared to other, more well-studied animal models. Although c-Fos quantification is a useful tool to identify relevant nuclei activated by cisplatin, it is also essential to ascertain which signaling pathways may be mediating anorectic and noxious behavioral effects of cisplatin. It is well-established that vagal afferent projections to the nucleus of the solitary tract (NTS) are primarily glutamatergic [20,21]. Furthermore, presynaptic 5-HT3 receptor activation in the gut, which occurs following cisplatin treatment, can facilitate glutamate release and synaptic input to the AP, NTS, and dorsal motor nucleus (DMN) neurons, likely leading to activation of AMPA or NMDA receptors within the hindbrain [22–24]. These studies suggest that cisplatin-induced vagal stimulation may trigger central glutamate receptor signaling which could contribute to anorexia and emetic-like behaviors in mice. Therefore, the aims of the following studies are 1) to examine whether cisplatin treatment in mice produces anorexia, body weight suppression, and hindbrain neuronal activation similar to more widely studied animal models of nausea and vomiting, and 2) to assess whether glutamate receptor gene expression (i.e., AMPA and NMDA receptor subunits) in areas of the hindbrain and forebrain implicated in the control of feeding and emetic behaviors is altered following cisplatin treatment in mice.
and 30% sucrose-PBS, each overnight. After sucrose incubation, brains were cut at 30 μm with a cryostat (Leica 3050 s). We collected sections from four locations based previous work in both the rat and the musk shrew [12,18]: 1) the caudal hindbrain [dorsal vagal complex (DVC), including NTS, and AP], 2) PBN. Sections were processed for immunofluorescence using a modified procedure [18] (n = 5/condition). We include here only the important modifications. Sections were incubated at room temperature with a polyclonal goat anti-Fos primary antibody (1:2000, sc-52G, Santa Cruz Biotechnology, Santa Cruz, CA; lot no. B2856) containing 2% normal donkey serum for 20 h. Sections were then incubated in donkey Anti-Goat AlexaFluor 594 secondary antibody (1:500, Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h at room temperature. Sections were mounted onto microscope slides and cover slipped with Vectastain Hard Set Mounting Medium and viewed with a fluorescence microscope (Olympus BX60). Images were captured (Nikon DSU3) and quantified by two authors working independently and blind to experimental conditions; the average of these two assessments is reported here. Counts for three coronal brain sections per animal corresponding to each coordinate examined were averaged for inclusion in statistical analysis.
2. Materials and methods
2.4. Experiment 3: AMPA and NMDA receptor subunit expression in the amygdala, BNST, PBN, and NTS-enriched DVC regions of cisplatin-treated mice
2.1. Animals Thirty, 3-month old male C57BL/6J mice (The Jackson Laboratory) were used for experiments. Mice were individually housed in plastic bins in a temperature and humidity controlled room maintained on a 12-h light/12-h dark cycle (lights on at 0900 h). Animals were maintained ad libitum on pelleted chow (Lab Diet 5001) and water. All protocols and procedures were approved by the institutional care and use committee (IACUC, University of Pennsylvania). 2.2. Experiment 1: Food intake and body weight measurements following intraperitoneal (IP) cisplatin in mice Mice were randomly assigned to one of three conditions and received IP injections of saline (0.15 M NaCl: 4 ml/kg, n = 10, BW = 23.5 g ± 1.3) or cisplatin (5 mg/kg, n = 10, BW = 22.9 g ± 1.0; 20 mg/kg, n = 10, BW = 23.1 g ± 1.1) in a weight-matched, between subjects design. All animals were injected between 0900 h–1030 h. Food intake and body weight measurements were taken as previously described [25] immediately prior to injections and 24 h post-injection. Doses of cisplatin were chosen based on previously published cisplatin studies in mice as well as our experiments in the house musk shrew Suncus murinus (i.e., vomiting animals of similar size to laboratory mice) [15,26]. Cisplatin (Sigma-Aldrich; cis-diamineplatium dichloride, no. P4394) was dissolved in saline (0.15 M NaCl), sonicated until clear, and vortexed prior to injection. Following 24 h measurements, all mice were euthanized as described below to facilitate Experiments 2 and 3. 2.3. Experiment 2: Cisplatin-induced hindbrain c-Fos immunofluorescence in mice Twenty-four hours after cisplatin injection, half of the mice from experiment 1 (n = 5/condition) were deeply anesthetized via intraperitoneal ketamine (90 mg/kg), xylazine (2.7 mg/kg), and acepromazine (0.64 mg/kg), and transcardially perfused with 10 ml of 0.2 M phosphate-buffered saline (PBS; pH 7.4), followed by 10 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (7.4) [11]. The 24 hour time point was chosen based on near maximal c-Fos expression observed in the rat and musk shrew in previous published data [12,18]. Whole brains were removed and placed in 10% sucrose-PBS, followed by 20%
Half of the mice from experiment 1 (n = 5/condition) were euthanized immediately following 24 h food intake and body weight measurements via CO2 asphyxiation. Whole brains were collected and flash frozen in liquid nitrogen according to procedures previously published [27]. Bilateral micropunches of BNST, amygdala, NTSenriched DVC and PBN were collected according to previously published methods [28]. The targeted micropunches for each tissue corresponded to the following starting coordinates, with punches extending rostrally, based on [29] (also see Figs. 4A and C, 5A and C): DVC-enriched hindbrain (B: − 7.76 mm, punch depth 1.0 mm), PBN (B: − 5.52 mm, punch depth 0.5 mm) and BNST (B: − 0.10 mm, punch depth 0.5 mm) and amygdala (B: − 1.58 mm, punch depth 0.75 mm). Regions were chosen based on previous reports [12,18] and our a priori hypothesis implicating these areas in cisplatininduced neuronal activation. Briefly, total RNA was extracted from micropunches using TRIzol (Invitrogen) and the RNeasy kit (Qiagen). cDNA was synthesized from 0.5 μg total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). TaqMan gene expression kits and PCR reagents from Applied Biosystems were used to quantify relative mRNA levels of AMPA receptor subunits GluA1 (Gria1; Mm00433753_m1), GluA2 (Gria2; Mm00442822_m1), and NMDA receptor subunits NR1 (Grin1; Mm00626390_m1), NR2A (Grin2A; Mm00433802_m1), NR2B (Grin2b-Mm00433820_m1) by quantitative real-time PCR. Mouse β-actin (VIC-MGB, #4352341E Applied Biosystems) was used as an internal control. Samples were analyzed using the Eppendorf Mastercycler® ep realplex2. Relative mRNA expression was calculated using the comparative Ct method as described previously [30].
2.5. Statistics All data are expressed as means ± SEM. For all experiments, one way ANOVAs were performed to evaluate group differences using drug treatment (cisplatin) as a main effect for food intake, body weight change, c-Fos immunofluorescence, and real time PCR. Planned comparisons were conducted with least significant difference (LSD) tests. Statistical differences between mean values were calculated using SAS 9.2 (SAS Institute Inc., Cary, NC).
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
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3. Results 3.1. Cisplatin dose-dependently reduces food intake and body weight in mice A significant main effect of cisplatin on food intake [F(2,12) = 8.1, P b 0.01] and body weight [F(2,12) = 12.3, P b 0.001] was observed. As shown in Fig. 1, cisplatin reduced 24 h body weight (A) and food intake (B) with both 5 mg/kg and 20 mg/kg doses (p b 0.05). The magnitude of intake and body weight reduction is consistent with other data observed in mice under acute and chronic cisplatin administration paradigms, as well as studies previously published in the rat [11,26]. 3.2. Cisplatin dose-dependently induces hindbrain c-Fos immunofluorescence in mice Representative immunofluorescence images collected from the DVC in saline and cisplatin treated mice are shown in Fig. 2A. c-Fos cell counts were increased in both the NTS and AP regions following cisplatin treatment [all F(2,12) ≥ 24.1; all Ps ≤ 0.001, main effect for each DVC region analyzed; Fig. 2B]. Post-hoc comparisons revealed that cisplatin produced greater c-Fos cell immunofluorescence than saline treatment in all regions at the 20 mg/kg dose, while 5 mg/kg increased immunofluorescence in the medial (B: −7.48 mm) and more rostral (B: −7.20 mm) NTS sections quantified (all Ps b 0.05). Fig. 3A shows representative images from PBN c-Fos detection, which was dose-dependently increased following cisplatin treatment in the externolateral PBN (PBNel), and increased in the ventrolateral PBN (PBNvl) after the high dose of cisplatin treatment [all F(2,12) ≥ 13.1, P b 0.001; Fig. 3B]. 3.3. Increased hindbrain AMPA and NMDA receptor subunit expression in cisplatin treated mice Fig. 4 shows results of quantitative PCR assessment of a subset of AMPA and NMDA receptor subunits from micropunches corresponding to the NTS-enriched DVC (Fig. 4A) and the PBN (Fig. 4C). In NTSenriched DVC micropunches, there was a significant main effect for cisplatin treatment on GluA1 AMPA receptor subunit, and NR1 and NR2B NMDA receptor subunit expression [all F(2,12) ≥ 3.1; all Ps b 0.01; Fig. 4B], with post hoc testing revealing increased subunit expression following cisplatin 20/mg/kg compared to controls (all Ps b 0.05). GluA2 AMPA and NR2A NMDA subunit expression between treatment groups was not significantly different in NTS-enriched DVC punches (all Ps = ns). In PBN micropunches, a significant main effect of cisplatin was also shown for both GluA1 and G1uA2 AMPA receptor subunit expression [all F(2,12) ≥ 3.9; all Ps b 0.01; Fig. 4D], with 20 mg/kg cisplatin treated mice showing significantly increased expression relative
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to controls (all Ps b 0.05). NR2B subunit expression was dosedependently increased in the PBN [F(2,12) ≥ 6.3; P b 0.001, main effect for cisplatin treatment; Fig. 4D], with both 5 mg/kg and 20 mg/kg doses increasing expression relative to saline treated mice. No group differences in NR1 and NR2A subunit gene expression were observed in the PBN of the mice (all Ps = ns). 3.4. Increased forebrain AMPA and NMDA receptor subunit expression in cisplatin treated mice In micropunches directed at the amygdala (Fig. 5A), no change in AMPA receptor subunits were observed (all Ps = ns), however all NMDA receptor subtypes measured were increased in cisplatin treated mice [all F(2,12) ≥ 4.8; all Ps b 0.01, main effect for treatment; Fig. 5B], with post-hoc analysis showing increased expression of NR1, NR2A and NR2B with 5 mg/kg, and NR2A and N2B with 20 mg/kg relative to controls (all Ps b 0.05). In BNST micropunches [Fig. 5C], a main effect of cisplatin was noted for GluA1 and NR2B subunit expression [all F(2,12) ≥ 3.9; all Ps b 0.01], with post hoc analysis revealing significantly increased expression in cisplatin 20 mg/kg treated mice compared to saline treated mice [Fig. 5D]. No additional differences were noted in the BNST (all Ps = ns). 4. Discussion The current set of studies sought to examine behavioral, neuronal, and molecular indicators of cisplatin-induced anorexia/sickness in mice. We believe this to be the first report of quantification and analysis of cisplatin-induced neuronal activation, via c-Fos immunofluorescence detection, in this species which is widely used as an animal model in the study of energy balance and cancer treatment. In addition, this the first study to examine the expression of central AMPA and NMDA receptor subunits following systemic cisplatin treatment in mice. Our results show robust activation of c-Fos in the NTS, AP and lateral PBN of cisplatin-treated mice at doses which potently reduced food intake and body weight. These data indicate similarity of cisplatin-induced DVC and PBN activation between mice and other small animal species [12,31]. Importantly, we also show evidence for increases in expression of AMPA and NMDA subunit expression, in particular the NR2B NMDA receptor subunit, within the DVC, PBN, BNST, and amygdala following cisplatin treatment. Involvement of NR2B upregulation and enhanced action is consistent with an emerging literature suggesting NR2B subunits and glutamate receptor signaling routed through the PBN act as mediators of a central anorectic and/or nauseogenic circuit [32,33]. As a whole, our findings are indicative of widespread neural activation highlighting the potential for glutamatergic signaling within several
Fig. 1. Cisplatin dose-dependently reduces food intake and body weight in mice. Cisplatin reduced 24 h body weight change (A) and food intake (B) with both 5 mg/kg and 20 mg/kg doses (*P b 0.05, saline vs. cisplatin).
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
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Fig. 2. Cisplatin dose-dependently induces hindbrain c-Fos immunofluorescence in mice. A: Representative immunofluorescence images collected from the DVC in saline and cisplatin treated mice. Coordinates correspond to distance from Bregma according to Paxinos and Franklin [29]. B: Cisplatin produces greater c-Fos immunofluorescence than saline treatment in the NTS and AP (*P b 0.05, saline vs. cisplatin).
key brain nuclei as a mediator of nauseogenic and/or anorectic signaling caused by cisplatin chemotherapy. As mentioned previously, the vagal mediation of cisplatin-induced emetic signals is well established. This activation can be blocked with 5-HT3 receptor antagonists and has been shown in electrophysiological, surgical, and behavior paradigms to be primarily mediated by the common hepatic branch of the vagus [11,34–36]. Along these lines, an intact vagus is likely necessary to observe a maximal emetic response to cisplatin as total subdiaphragmatic vagal ablation can block cisplatininduced vomiting [37,38]. Thus, while great strides have been made in the understanding of the peripheral mechanisms behind cisplatin action [39], the central mechanisms of nausea sensations, and the neurotransmitter signaling by which cisplatin elicits these symptoms, remain understudied and are the focus of the current studies. Here, we show cisplatin-induced central c-Fos activation in the NTS and AP of the caudal brainstem, suggesting stimulation of hindbrain emetic circuits by cisplatin. NTS expression of c-Fos is likely due in large part to 5-HT3 mediated vagal activation, as complete vagotomy
can drastically reduce or block NTS c-Fos expression and/or emetic behavior [18,19,40]. Circulating humoral factors such as plasma 5-HT or substance P which are elevated following cisplatin treatment [41,42] may account for AP neuronal activation following cisplatin, as previous reports have shown no change in cisplatin-induced AP c-Fos following vagotomy or systemic pretreatment with a 5-HT3 antagonist [40]. In addition, direct application of Substance P or 5-HT to the AP can activate AP neurons [43]. There is also the strong potential for cisplatininduced cytokines [44] to act via both vagal and humoral means [45] to activate central pathways of sickness, however this hypothesis requires direct testing. Clear evidence also exists for substance P/NK-1 [46], and 5-HT/5-HT3 [33] signaling endemic to the NTS as potential mediators of emetic behavior and anorexia. Thus, it is possible that cisplatin-induced NTS Fos-positive neurons are direct and/or indirect mediators of multiple neurotransmitter signaling pathways simultaneously. No dorsal motor nucleus (DMN) expression was observed following cisplatin treatment, a finding similar to data from the rat [12].
Fig. 3. A: Representative immunofluorescence images collected from the PBN in saline and cisplatin treated mice. B: Cisplatin produces greater c-Fos immunofluorescence than saline treatment in the PBNel and PBNvl. (*P b 0.05, saline vs. cisplatin).
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
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Fig. 4. Increased hindbrain AMPA and NMDA receptor subunit expression in cisplatin treated mice. Micropunch coordinates for NTS-enriched DVC (4A) and the PBN (4C). Black circles correspond to micropunch diameter and approximate location. B: GluA1 AMPA and NR1 and NR2B NMDA receptor subunit expressions were increased following cisplatin injection compared to controls in NTS-enriched DVC punches. D: GluA1 and G1uA2 AMPA and NR2B NMDA subunit expression was increased in the PBN (*P b 0.05, saline vs. cisplatin).
Additionally, as many caudal NTS neurons project to the DMN to control gastric emptying [47], our relative lack of cisplatin-induced caudal NTS Fos, compared to rostral regions, would be consistent with a mechanism involving minimal vagal efferent activation. This finding, however, contrasts with previously published work in the vomiting musk shrew,
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Fig. 5. Increased forebrain AMPA and NMDA receptor subunit expression in cisplatin treated mice. Micropunch coordinates for amygdala (A) and BNST (C). Black circles correspond to micropunch diameter and approximate location. B: Expression of NR1, NR2A, and NR2B NMDA receptor subunits was increased in cisplatin treated mice, D: Expression of GluA1 AMPA and NR2B NMDA subunits was increased in cisplatin treated mice compared to saline controls (*P b 0.05, saline vs. cisplatin).
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
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signals as it receives dense projections from the NTS and AP and has outputs to amygdala and BNST among other nuclei [49–53]. The NTS distribution of cisplatin-induced Fos observed in our studies shows that the rostral NTS exhibits greater expression, relative to caudal regions. Given that dense projections to the PBN originate from the rostral NTS [47], we speculate that our findings implicate activation of NTS neurons connecting monosynaptically with the PBN. With this in mind, it may be considered that a known subpopulation of these ascending PBN projections are glutamatergic [48], consistent with our gene expression data described below. Interestingly, in rats with PBN lesions, kaolin intake (an indicator of nausea) is increased dramatically [31]. Although this finding has not been followed up systematically, it is clear that the PBN neurons and/or PBN connections may exert a gating or inhibitory effect the intake of kaolin and gastric dysfunction associated with cisplatin treatment. In this context, calcitonin gene-related peptide-expressing neurons within the external lateral PBN have recently been suggested to play a crucial role in mediating anorectic circuits [54]. What is perhaps most notably underrepresented in the literature is examination of the central neurotransmitters mediating nausea and sickness behavior rodent models. The glutamatergic nature of vagal afferents [20,21] and serotonergic facilitation of glutamate signaling [22–24] in the brainstem, point to glutamate receptor signaling as a candidate pathway contributing to the emetic effects of cisplatin. Our results show AMPA (GluA1) and NMDA (NR1 and NR2B) receptor subunit upregulation in NTS-enriched DVC tissues from cisplatin treated mice. Cisplatin also elicits release of inflammatory cytokines, including TNF-α [44], which can increase glutamate release and NMDA receptor signaling [55]. Thus, it may be that TNF-α and/or other circulating cytokines also play a role in cisplatin-induce NMDA receptor upregulation cisplatin by amplifying vagal input signals [45]. In addition to the NTS, our results also show upregulation of AMPA receptors subunits and NR2B NMDA receptor unit in the PBN. As mentioned above, the PBN plays an integrative role in the mediating cisplatin-induced activation of the neuroaxis. Along these lines, recent evidence from Palmiter and colleagues points to a major role for glutamate signaling in the NTS and the PBN in the mediation of a central anorectic circuit [33]. Intriguingly, NR2B NMDA receptor subunits within the PBN may be responsible for enhanced PBN activation and engagement of a multisite “starvation” network [32,54]. Thus, we may interpret these findings together as evidence for a common nauseogenic/ anorectic pathway of NMDA-mediated processing of anorectic and noxious signals by PBN neurons. In the forebrain, we observe increased expression of NMDA receptor subunits in the amygdala as well as the NR2B subunit in the BNST. Similarly, cisplatin has been shown to induce c-Fos expression in the amygdala of the rat and musk shrew [12,18]. Thus, it is possible that activation of NMDA receptors within the amygdala and BNST represent stimulation of a nausea circuit. Lithium chloride, a potent inducer of visceral illness, has also been shown to stimulate neurons in the lateral PBN that project to the amygdala [32]. Future research should determine whether this circuit is also involved in mediation of cisplatin-induced anorexia/emesis. Finally, glutamate activity in the amygdala is linked to the transmission and learning related to visceral information in the formation of a conditioned taste aversion, a commonly used proxy for nausea in the rodent [56]. Taken together, the amygdala and BNST are both physiologically and anatomically connected to brainstem nuclei to potentially mediate neuroendocrine signals involved in feeding and nausea via glutamatergic circuits linked to the PBN. 5. Conclusions In summary, the transmission of cisplatin-induced nauseogenic and emetic information across the neuroaxis of mice extends to multiple sites and neurotransmitters in a broad network of activation. These current data suggest similar neural pathways mediating cisplatin effects
in mice compared to other well-studied species, which are likely mediated by central upregulation of AMPA and NMDA receptors. In future, the use of mouse models will be advantageous to study nausea and sickness behavior on cancerous backgrounds, in combination with emetogenic treatments, to provide a greater understanding of chemotherapy-induced anorexia and nausea/vomiting. Acknowledgments We wish to thank Amber L. Alhadeff for a critical reading of the manuscript. This manuscript is based on work presented during the 2013 Annual Meeting of the Society for the Study of Ingestive Behavior, July 30 - August 3, 2013. Grants This work was supported by: The McCabe Fund, The Biobehavioral Research Center of the University of Pennsylvania School of Nursing, the University of Pennsylvania Research Foundation, and the Center for Undergraduate Research at The University of Pennsylvania. References [1] Percie du Sert N, Rudd JA, Apfel CC, Andrews PL. Cisplatin-induced emesis: systematic review and meta-analysis of the ferret model and the effects of 5-HT(3) receptor antagonists. Cancer Chemother Pharmacol 2011;67:667–86. [2] Andrews PL, Horn CC. Signals for nausea and emesis: implications for models of upper gastrointestinal diseases. Auton Neurosci 2006;125:100–15. [3] Florczyk AP, Schurig JE, Bradner WT. Cisplatin-induced emesis in the Ferret: a new animal model. Cancer Treat Rep 1982;66:187–9. [4] Gylys JA, Doran KM, Buyniski JP. Antagonism of cisplatin induced emesis in the dog. Res Commun Chem Pathol Pharmacol 1979;23:61–8. [5] Matsuki N, Ueno S, Kaji T, Ishihara A, Wang CH, Saito H. Emesis induced by cancer chemotherapeutic agents in the Suncus murinus: a new experimental model. Jpn J Pharmacol 1988;48:303–6. [6] McCarthy LE, Borison HL. Antiemetic activity of N-methyllevonantradol and nabilone in cisplatin-treated cats. J Clin Pharmacol 1981;21:30S–7S. [7] Szelenyi I, Herold H, Gothert M. Emesis induced in domestic pigs: a new experimental tool for detection of antiemetic drugs and for evaluation of emetogenic potential of new anticancer agents. J Pharmacol Toxicol Methods 1994;32:109–16. [8] Horn CC, Kimball BA, Wang H, Kaus J, Dienel S, Nagy A, et al. Why can't rodents vomit? A comparative behavioral, anatomical, and physiological study. PLoS One 2013;8:e60537. [9] Horn CC. The medical implications of gastrointestinal vagal afferent pathways in nausea and vomiting. Curr Pharm Des 2013. [10] Schwartz GJ, Zeltser LM. Functional organization of neuronal and humoral signals regulating feeding behavior. Annu Rev Nutr 2013;33:1–21. [11] De Jonghe BC, Horn CC. Chemotherapy-induced pica and anorexia are reduced by common hepatic branch vagotomy in the rat. Am J Physiol Regul Integr Comp Physiol 2008;294:R756–65. [12] Horn CC, Ciucci M, Chaudhury A. Brain Fos expression during 48 h after cisplatin treatment: neural pathways for acute and delayed visceral sickness. Auton Neurosci 2007;132:44–51. [13] Vera G, Chiarlone A, Martin MI, Abalo R. Altered feeding behaviour induced by longterm cisplatin in rats. Auton Neurosci 2006;126–127:81–92. [14] Malik NM, Moore GB, Smith G, Liu YL, Sanger GJ, Andrews PL. Behavioural and hypothalamic molecular effects of the anti-cancer agent cisplatin in the rat: a model of chemotherapy-related malaise? Pharmacol Biochem Behav 2006;83:9–20. [15] Liu YL, Malik N, Sanger GJ, Friedman MI, Andrews PL. Pica — a model of nausea? Species differences in response to cisplatin. Physiol Behav 2005;85:271–7. [16] Yamamoto K, Matsunaga S, Matsui M, Takeda N, Yamatodani A. Pica in mice as a new model for the study of emesis. Methods Find Exp Clin Pharmacol 2002;24:135–8. [17] Rudd JA, Andrews PLR. In: Hesketh PJ, editor. Management of nausea and vomiting in cancer and cancer treatment. Jones and Bartlett; 2005. p. 15–65. [18] De Jonghe BC, Horn CC. Chemotherapy agent cisplatin induces 48-h Fos expression in the brain of a vomiting species, the house musk shrew (Suncus murinus). Am J Physiol Regul Integr Comp Physiol 2009;296:R902–11. [19] Horn CC. Brain Fos expression induced by the chemotherapy agent cisplatin in the rat is partially dependent on an intact abdominal vagus. Auton Neurosci 2009;148:76–82. [20] McCann MJ, Rogers RC. Functional and chemical anatomy of a gastric vago-vagal reflex. In: Tache Y, Wingate DL, Burks TF, editors. Innervation of the gut: pathophysiological implications. Boca Raton, FL: CRC; 1994. p. 81–92. [21] Sykes RM, Spyer KM, Izzo PN. Demonstration of glutamate immunoreactivity in vagal sensory afferents in the nucleus tractus solitarius of the rat. Brain Res 1997;762:1–11. [22] Jeggo RD, Kellett DO, Wang Y, Ramage AG, Jordan D. The role of central 5-HT3 receptors in vagal reflex inputs to neurones in the nucleus tractus solitarius of anaesthetized rats. J Physiol 2005;566:939–53. [23] Funahashi M, Mitoh Y, Matsuo R. Activation of presynaptic 5-HT3 receptors facilitates glutamatergic synaptic inputs to area postrema neurons in rat brain slices. Methods Find Exp Clin Pharmacol 2004;26:615–22.
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
R.A. Holland et al. / Physiology & Behavior xxx (2014) xxx–xxx [24] Wang Y, Ramage AG, Jordan D. Presynaptic 5-HT3 receptors evoke an excitatory response in dorsal vagal preganglionic neurones in anaesthetized rats. J Physiol 1998;509(Pt 3):683–94. [25] De Jonghe BC, Hayes MR, Zimmer DJ, Kanoski SE, Grill HJ, Bence KK. Food intake reductions and increases in energetic responses by hindbrain leptin and melanotan II are enhanced in mice with POMC-specific PTP1B deficiency. Am J Physiol Endocrinol Metab 2012;303:E644–51. [26] Liu YL, Malik NM, Sanger GJ, Andrews PL. Ghrelin alleviates cancer chemotherapyassociated dyspepsia in rodents. Cancer Chemother Pharmacol 2006;58:326–33. [27] Hayes MR, Skibicka KP, Bence KK, Grill HJ. Dorsal hindbrain 5′-adenosine monophosphate-activated protein kinase as an intracellular mediator of energy balance. Endocrinology 2009;150:2175–82. [28] Hayes MR, Skibicka KP, Leichner TM, Guarnieri DJ, DiLeone RJ, Bence KK, et al. Endogenous leptin signaling in the caudal nucleus tractus solitarius and area postrema is required for energy balance regulation. Cell Metab 2010;11:77–83. [29] Paxinos G, Franklin KBJ. Paxinos and Franklin's the mouse brain in stereotaxic coordinates. 4th ed. Amsterdam: Boston: Elsevier/Academic Press; 2013. [30] Bence KK, Delibegovic M, Xue B, Gorgun CZ, Hotamisligil GS, Neel BG, et al. Neuronal PTP1B regulates body weight, adiposity and leptin action. Nat Med 2006;12:917–24. [31] Horn CC, De Jonghe BC, Matyas K, Norgren R. Chemotherapy-induced kaolin intake is increased by lesion of the lateral parabrachial nucleus of the rat. Am J Physiol Regul Integr Comp Physiol 2009;297:R1375–82. [32] Wu Q, Zheng R, Srisai D, McKnight GS, Palmiter RD. NR2B subunit of the NMDA glutamate receptor regulates appetite in the parabrachial nucleus. Proc Natl Acad Sci U S A 2013;110:14765–70. [33] Wu Q, Clark MS, Palmiter RD. Deciphering a neuronal circuit that mediates appetite. Nature 2012;483:594–7. [34] Wang FB, Powley TL. Vagal innervation of intestines: afferent pathways mapped with new en bloc horseradish peroxidase adaptation. Cell Tissue Res 2007;329:221–30. [35] Horn CC, Richardson EJ, Andrews PL, Friedman MI. Differential effects on gastrointestinal and hepatic vagal afferent fibers in the rat by the anti-cancer agent cisplatin. Auton Neurosci 2004;115:74–81. [36] Phillips RJ, Baronowsky EA, Powley TL. Afferent innervation of gastrointestinal tract smooth muscle by the hepatic branch of the vagus. J Comp Neurol 1997;384:248–70. [37] Fukui H, Yamamoto M, Sato S. Vagal afferent fibers and peripheral 5-HT3 receptors mediate cisplatin-induced emesis in dogs. Jpn J Pharmacol 1992;59:221–6. [38] Andrews PL, Davis CJ, Bingham S, Davidson HI, Hawthorn J, Maskell L. The abdominal visceral innervation and the emetic reflex: pathways, pharmacology, and plasticity. Can J Physiol Pharmacol 1990;68:325–45. [39] Hesketh PJ. Chemotherapy-induced nausea and vomiting. N Engl J Med 2008;358:2482–94. [40] Reynolds DJ, Barber NA, Grahame-Smith DG, Leslie RA. Cisplatin-evoked induction of c-fos protein in the brainstem of the ferret: the effect of cervical vagotomy and the anti-emetic 5-HT3 receptor antagonist granisetron (BRL 43694). Brain Res 1991;565:231–6.
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[41] Dey D, Abad J, Ray AP, Darmani NA. Differential temporal changes in brain and gut substance P mRNA expression throughout the time-course of cisplatin-induced vomiting in the least shrew (Cryptotis parva). Brain Res 2010;1310:103–12. [42] Higa GM, Auber ML, Altaha R, Piktel D, Kurian S, Hobbs G, et al. 5-Hydroxyindoleacetic acid and substance P profiles in patients receiving emetogenic chemotherapy. J Oncol Pharm Pract 2006;12:201–9. [43] Carpenter DO, Briggs DB, Strominger N. Responses of neurons of canine area postrema to neurotransmitters and peptides. Cell Mol Neurobiol 1983;3:113–26. [44] Ramesh G, Reeves WB. TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity. J Clin Invest 2002;110:835–42. [45] Hermann GE, Rogers RC. TNFalpha: a trigger of autonomic dysfunction. Neuroscientist 2008;14:53–67. [46] Ray AP, Chebolu S, Ramirez J, Darmani NA. Ablation of least shrew central neurokinin NK1 receptors reduces GR73632-induced vomiting. Behav Neurosci 2009;123:701–6. [47] Grill HJ, Hayes MR. Hindbrain neurons as an essential hub in the neuroanatomically distributed control of energy balance. Cell Metab 2012;16:296–309. [48] Gill CF, Madden JM, Roberts BP, Evans LD, King MS. A subpopulation of neurons in the rat rostral nucleus of the solitary tract that project to the parabrachial nucleus express glutamate-like immunoreactivity. Brain Res 1999;821:251–62. [49] Jia HG, Rao ZR, Shi JW. An indirect projection from the nucleus of the solitary tract to the central nucleus of the amygdala via the parabrachial nucleus in the rat: a light and electron microscopic study. Brain Res 1994;663:181–90. [50] Krukoff TL, Harris KH, Jhamandas JH. Efferent projections from the parabrachial nucleus demonstrated with the anterograde tracer Phaseolus vulgaris leucoagglutinin. Brain Res Bull 1993;30:163–72. [51] Papas S, Ferguson AV. Electrophysiological characterization of reciprocal connections between the parabrachial nucleus and the area postrema in the rat. Brain Res Bull 1990;24:577–82. [52] Voshart K, van der Kooy D. The organization of the efferent projections of the parabrachial nucleus of the forebrain in the rat: a retrograde fluorescent doublelabeling study. Brain Res 1981;212:271–86. [53] Norgren R. Taste pathways to hypothalamus and amygdala. J Comp Neurol 1976;166:17–30. [54] Carter ME, Soden ME, Zweifel LS, Palmiter RD. Genetic identification of a neural circuit that suppresses appetite. Nature 2013;503:111–4. [55] Jara JH, Singh BB, Floden AM, Combs CK. Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK-dependent death. J Neurochem 2007;100:1407–20. [56] Miranda MI, Ferreira G, Ramirez-Lugo L, Bermudez-Rattoni F. Glutamatergic activity in the amygdala signals visceral input during taste memory formation. Proc Natl Acad Sci U S A 2002;99:11417–22.
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
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Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice Ruby A. Holland, John J. Leonard, Nicholas A. Kensey, Paavali A. Hannikainen, Bart C. De Jonghe ⁎ Dept. of Biobehavioral Health Sciences School of Nursing, University of Pennsylvania, Philadelphia, PA, 19104, United States
H I G H L I G H T S • • • •
Cisplatin reduces food intake and body weight in mice dose-dependently. 1st study examining cisplatin-induced c-Fos expression in mice. 1st study to examine cisplatin-induced AMPAR and NMDAR subunit gene expression. We present evidence for a novel central circuit of cisplatin-induced nausea.
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Article history: Received 13 December 2013 Received in revised form 17 February 2014 Accepted 23 February 2014 Available online xxxx Keywords: NMDA AMPA NR2B Nausea Cisplatin c-Fos
a b s t r a c t Although rats and mice do not vomit, these species are widely studied as models of energy balance and sickness behavior. Previous work has shown that rats exhibit similar neuroanatomical activation of brain and visceral afferent pathways following cisplatin chemotherapy compared to vomiting species. However, the neural response to cisplatin in mice is understudied. Here, food intake, body weight, and central c-Fos immunofluorescence were analyzed in the hindbrains of male C57BL/6 mice following IP saline or cisplatin (5 mg/kg, and 20 mg/kg doses). As glutamate receptor signaling is classically linked to inhibitory feeding pathways in the rodent, gene expression of selected α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptor subunits were assessed in the dorsal vagal complex (DVC), parabrachial nucleus (PBN), amygdala, and bed nucleus of the stria terminalis (BNST). Our results show dosedependent reductions in food intake and body weight following cisplatin treatment, as well as increases in cisplatin-induced c-Fos in the PBN and throughout the DVC. Quantitative PCR analysis shows cisplatin-induced increases in NMDA receptor subunit expression, particularly NR2B, in the DVC, PBN, BNST, and amygdala. In addition, upregulation of AMPA receptor subunits (GluA1 and/or GluA2) were observed in all regions examined except the amygdala. Taken together, these results suggest similar neural pathways mediating cisplatin effects in mice compared to other well-studied species, which are likely mediated by central upregulation of AMPA and NMDA receptors. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Cisplatin chemotherapy is perhaps the most potent and widely studied emetogenic antineoplastic drug [1,2], having been used in the treatment of solid tumors for nearly 35 years. Without anti-emetic medication, cisplatin produces severe nausea, vomiting, anorexia and cachexia in a variety of vomiting species including ferrets, pigs, dogs, cats, shrews, and humans [3–7]. Although rats and mice do not vomit, these species are widely studied as models for food intake regulation, energy balance, and cisplatin-induced anorexia/sickness behavior [8–10]. Previous reports have shown that rats exhibit similar neuroanatomical activation in brain areas and visceral afferent pathways ⁎ Corresponding author. Tel.: +1 215 898 4901; fax: +1 215 573 3859. E-mail address: [email protected] (B.C. De Jonghe).
compared to vomiting species [11–14]. On the other hand, mice have been relatively understudied for neuronal pathways of sickness behavior caused by emetic stimuli such as cisplatin [15,16]. As the overwhelming majority of animal models used in cancer research involve mouse models, it is essential to study the neural correlates of cancerand/or chemotherapy-induced anorexia, nausea, and sickness in mice. Cisplatin robustly stimulates the vagus nerve via serotonin (5-HT) release from enteroendocrine cells and subsequent binding to 5-HT3 receptors on afferent terminals innervating the gut (for more detailed discussion, see Ref. [17]). In rats, these chemotherapy-induced vagal signals are then transmitted to the brain; first synapsing in the NTS in the caudal hindbrain [17]. Rats exhibit similar distributions and magnitude of hindbrain c-Fos activation following cisplatin injection when compared to vomiting species [18,19]. Here, we are the first to examine c-Fos immunofluorescence in mice to test the hypothesis that cisplatin
http://dx.doi.org/10.1016/j.physbeh.2014.02.038 0031-9384/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
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produces similar hindbrain neuronal activation in mice compared to other, more well-studied animal models. Although c-Fos quantification is a useful tool to identify relevant nuclei activated by cisplatin, it is also essential to ascertain which signaling pathways may be mediating anorectic and noxious behavioral effects of cisplatin. It is well-established that vagal afferent projections to the nucleus of the solitary tract (NTS) are primarily glutamatergic [20,21]. Furthermore, presynaptic 5-HT3 receptor activation in the gut, which occurs following cisplatin treatment, can facilitate glutamate release and synaptic input to the AP, NTS, and dorsal motor nucleus (DMN) neurons, likely leading to activation of AMPA or NMDA receptors within the hindbrain [22–24]. These studies suggest that cisplatin-induced vagal stimulation may trigger central glutamate receptor signaling which could contribute to anorexia and emetic-like behaviors in mice. Therefore, the aims of the following studies are 1) to examine whether cisplatin treatment in mice produces anorexia, body weight suppression, and hindbrain neuronal activation similar to more widely studied animal models of nausea and vomiting, and 2) to assess whether glutamate receptor gene expression (i.e., AMPA and NMDA receptor subunits) in areas of the hindbrain and forebrain implicated in the control of feeding and emetic behaviors is altered following cisplatin treatment in mice.
and 30% sucrose-PBS, each overnight. After sucrose incubation, brains were cut at 30 μm with a cryostat (Leica 3050 s). We collected sections from four locations based previous work in both the rat and the musk shrew [12,18]: 1) the caudal hindbrain [dorsal vagal complex (DVC), including NTS, and AP], 2) PBN. Sections were processed for immunofluorescence using a modified procedure [18] (n = 5/condition). We include here only the important modifications. Sections were incubated at room temperature with a polyclonal goat anti-Fos primary antibody (1:2000, sc-52G, Santa Cruz Biotechnology, Santa Cruz, CA; lot no. B2856) containing 2% normal donkey serum for 20 h. Sections were then incubated in donkey Anti-Goat AlexaFluor 594 secondary antibody (1:500, Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h at room temperature. Sections were mounted onto microscope slides and cover slipped with Vectastain Hard Set Mounting Medium and viewed with a fluorescence microscope (Olympus BX60). Images were captured (Nikon DSU3) and quantified by two authors working independently and blind to experimental conditions; the average of these two assessments is reported here. Counts for three coronal brain sections per animal corresponding to each coordinate examined were averaged for inclusion in statistical analysis.
2. Materials and methods
2.4. Experiment 3: AMPA and NMDA receptor subunit expression in the amygdala, BNST, PBN, and NTS-enriched DVC regions of cisplatin-treated mice
2.1. Animals Thirty, 3-month old male C57BL/6J mice (The Jackson Laboratory) were used for experiments. Mice were individually housed in plastic bins in a temperature and humidity controlled room maintained on a 12-h light/12-h dark cycle (lights on at 0900 h). Animals were maintained ad libitum on pelleted chow (Lab Diet 5001) and water. All protocols and procedures were approved by the institutional care and use committee (IACUC, University of Pennsylvania). 2.2. Experiment 1: Food intake and body weight measurements following intraperitoneal (IP) cisplatin in mice Mice were randomly assigned to one of three conditions and received IP injections of saline (0.15 M NaCl: 4 ml/kg, n = 10, BW = 23.5 g ± 1.3) or cisplatin (5 mg/kg, n = 10, BW = 22.9 g ± 1.0; 20 mg/kg, n = 10, BW = 23.1 g ± 1.1) in a weight-matched, between subjects design. All animals were injected between 0900 h–1030 h. Food intake and body weight measurements were taken as previously described [25] immediately prior to injections and 24 h post-injection. Doses of cisplatin were chosen based on previously published cisplatin studies in mice as well as our experiments in the house musk shrew Suncus murinus (i.e., vomiting animals of similar size to laboratory mice) [15,26]. Cisplatin (Sigma-Aldrich; cis-diamineplatium dichloride, no. P4394) was dissolved in saline (0.15 M NaCl), sonicated until clear, and vortexed prior to injection. Following 24 h measurements, all mice were euthanized as described below to facilitate Experiments 2 and 3. 2.3. Experiment 2: Cisplatin-induced hindbrain c-Fos immunofluorescence in mice Twenty-four hours after cisplatin injection, half of the mice from experiment 1 (n = 5/condition) were deeply anesthetized via intraperitoneal ketamine (90 mg/kg), xylazine (2.7 mg/kg), and acepromazine (0.64 mg/kg), and transcardially perfused with 10 ml of 0.2 M phosphate-buffered saline (PBS; pH 7.4), followed by 10 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (7.4) [11]. The 24 hour time point was chosen based on near maximal c-Fos expression observed in the rat and musk shrew in previous published data [12,18]. Whole brains were removed and placed in 10% sucrose-PBS, followed by 20%
Half of the mice from experiment 1 (n = 5/condition) were euthanized immediately following 24 h food intake and body weight measurements via CO2 asphyxiation. Whole brains were collected and flash frozen in liquid nitrogen according to procedures previously published [27]. Bilateral micropunches of BNST, amygdala, NTSenriched DVC and PBN were collected according to previously published methods [28]. The targeted micropunches for each tissue corresponded to the following starting coordinates, with punches extending rostrally, based on [29] (also see Figs. 4A and C, 5A and C): DVC-enriched hindbrain (B: − 7.76 mm, punch depth 1.0 mm), PBN (B: − 5.52 mm, punch depth 0.5 mm) and BNST (B: − 0.10 mm, punch depth 0.5 mm) and amygdala (B: − 1.58 mm, punch depth 0.75 mm). Regions were chosen based on previous reports [12,18] and our a priori hypothesis implicating these areas in cisplatininduced neuronal activation. Briefly, total RNA was extracted from micropunches using TRIzol (Invitrogen) and the RNeasy kit (Qiagen). cDNA was synthesized from 0.5 μg total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). TaqMan gene expression kits and PCR reagents from Applied Biosystems were used to quantify relative mRNA levels of AMPA receptor subunits GluA1 (Gria1; Mm00433753_m1), GluA2 (Gria2; Mm00442822_m1), and NMDA receptor subunits NR1 (Grin1; Mm00626390_m1), NR2A (Grin2A; Mm00433802_m1), NR2B (Grin2b-Mm00433820_m1) by quantitative real-time PCR. Mouse β-actin (VIC-MGB, #4352341E Applied Biosystems) was used as an internal control. Samples were analyzed using the Eppendorf Mastercycler® ep realplex2. Relative mRNA expression was calculated using the comparative Ct method as described previously [30].
2.5. Statistics All data are expressed as means ± SEM. For all experiments, one way ANOVAs were performed to evaluate group differences using drug treatment (cisplatin) as a main effect for food intake, body weight change, c-Fos immunofluorescence, and real time PCR. Planned comparisons were conducted with least significant difference (LSD) tests. Statistical differences between mean values were calculated using SAS 9.2 (SAS Institute Inc., Cary, NC).
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
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3. Results 3.1. Cisplatin dose-dependently reduces food intake and body weight in mice A significant main effect of cisplatin on food intake [F(2,12) = 8.1, P b 0.01] and body weight [F(2,12) = 12.3, P b 0.001] was observed. As shown in Fig. 1, cisplatin reduced 24 h body weight (A) and food intake (B) with both 5 mg/kg and 20 mg/kg doses (p b 0.05). The magnitude of intake and body weight reduction is consistent with other data observed in mice under acute and chronic cisplatin administration paradigms, as well as studies previously published in the rat [11,26]. 3.2. Cisplatin dose-dependently induces hindbrain c-Fos immunofluorescence in mice Representative immunofluorescence images collected from the DVC in saline and cisplatin treated mice are shown in Fig. 2A. c-Fos cell counts were increased in both the NTS and AP regions following cisplatin treatment [all F(2,12) ≥ 24.1; all Ps ≤ 0.001, main effect for each DVC region analyzed; Fig. 2B]. Post-hoc comparisons revealed that cisplatin produced greater c-Fos cell immunofluorescence than saline treatment in all regions at the 20 mg/kg dose, while 5 mg/kg increased immunofluorescence in the medial (B: −7.48 mm) and more rostral (B: −7.20 mm) NTS sections quantified (all Ps b 0.05). Fig. 3A shows representative images from PBN c-Fos detection, which was dose-dependently increased following cisplatin treatment in the externolateral PBN (PBNel), and increased in the ventrolateral PBN (PBNvl) after the high dose of cisplatin treatment [all F(2,12) ≥ 13.1, P b 0.001; Fig. 3B]. 3.3. Increased hindbrain AMPA and NMDA receptor subunit expression in cisplatin treated mice Fig. 4 shows results of quantitative PCR assessment of a subset of AMPA and NMDA receptor subunits from micropunches corresponding to the NTS-enriched DVC (Fig. 4A) and the PBN (Fig. 4C). In NTSenriched DVC micropunches, there was a significant main effect for cisplatin treatment on GluA1 AMPA receptor subunit, and NR1 and NR2B NMDA receptor subunit expression [all F(2,12) ≥ 3.1; all Ps b 0.01; Fig. 4B], with post hoc testing revealing increased subunit expression following cisplatin 20/mg/kg compared to controls (all Ps b 0.05). GluA2 AMPA and NR2A NMDA subunit expression between treatment groups was not significantly different in NTS-enriched DVC punches (all Ps = ns). In PBN micropunches, a significant main effect of cisplatin was also shown for both GluA1 and G1uA2 AMPA receptor subunit expression [all F(2,12) ≥ 3.9; all Ps b 0.01; Fig. 4D], with 20 mg/kg cisplatin treated mice showing significantly increased expression relative
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to controls (all Ps b 0.05). NR2B subunit expression was dosedependently increased in the PBN [F(2,12) ≥ 6.3; P b 0.001, main effect for cisplatin treatment; Fig. 4D], with both 5 mg/kg and 20 mg/kg doses increasing expression relative to saline treated mice. No group differences in NR1 and NR2A subunit gene expression were observed in the PBN of the mice (all Ps = ns). 3.4. Increased forebrain AMPA and NMDA receptor subunit expression in cisplatin treated mice In micropunches directed at the amygdala (Fig. 5A), no change in AMPA receptor subunits were observed (all Ps = ns), however all NMDA receptor subtypes measured were increased in cisplatin treated mice [all F(2,12) ≥ 4.8; all Ps b 0.01, main effect for treatment; Fig. 5B], with post-hoc analysis showing increased expression of NR1, NR2A and NR2B with 5 mg/kg, and NR2A and N2B with 20 mg/kg relative to controls (all Ps b 0.05). In BNST micropunches [Fig. 5C], a main effect of cisplatin was noted for GluA1 and NR2B subunit expression [all F(2,12) ≥ 3.9; all Ps b 0.01], with post hoc analysis revealing significantly increased expression in cisplatin 20 mg/kg treated mice compared to saline treated mice [Fig. 5D]. No additional differences were noted in the BNST (all Ps = ns). 4. Discussion The current set of studies sought to examine behavioral, neuronal, and molecular indicators of cisplatin-induced anorexia/sickness in mice. We believe this to be the first report of quantification and analysis of cisplatin-induced neuronal activation, via c-Fos immunofluorescence detection, in this species which is widely used as an animal model in the study of energy balance and cancer treatment. In addition, this the first study to examine the expression of central AMPA and NMDA receptor subunits following systemic cisplatin treatment in mice. Our results show robust activation of c-Fos in the NTS, AP and lateral PBN of cisplatin-treated mice at doses which potently reduced food intake and body weight. These data indicate similarity of cisplatin-induced DVC and PBN activation between mice and other small animal species [12,31]. Importantly, we also show evidence for increases in expression of AMPA and NMDA subunit expression, in particular the NR2B NMDA receptor subunit, within the DVC, PBN, BNST, and amygdala following cisplatin treatment. Involvement of NR2B upregulation and enhanced action is consistent with an emerging literature suggesting NR2B subunits and glutamate receptor signaling routed through the PBN act as mediators of a central anorectic and/or nauseogenic circuit [32,33]. As a whole, our findings are indicative of widespread neural activation highlighting the potential for glutamatergic signaling within several
Fig. 1. Cisplatin dose-dependently reduces food intake and body weight in mice. Cisplatin reduced 24 h body weight change (A) and food intake (B) with both 5 mg/kg and 20 mg/kg doses (*P b 0.05, saline vs. cisplatin).
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
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Fig. 2. Cisplatin dose-dependently induces hindbrain c-Fos immunofluorescence in mice. A: Representative immunofluorescence images collected from the DVC in saline and cisplatin treated mice. Coordinates correspond to distance from Bregma according to Paxinos and Franklin [29]. B: Cisplatin produces greater c-Fos immunofluorescence than saline treatment in the NTS and AP (*P b 0.05, saline vs. cisplatin).
key brain nuclei as a mediator of nauseogenic and/or anorectic signaling caused by cisplatin chemotherapy. As mentioned previously, the vagal mediation of cisplatin-induced emetic signals is well established. This activation can be blocked with 5-HT3 receptor antagonists and has been shown in electrophysiological, surgical, and behavior paradigms to be primarily mediated by the common hepatic branch of the vagus [11,34–36]. Along these lines, an intact vagus is likely necessary to observe a maximal emetic response to cisplatin as total subdiaphragmatic vagal ablation can block cisplatininduced vomiting [37,38]. Thus, while great strides have been made in the understanding of the peripheral mechanisms behind cisplatin action [39], the central mechanisms of nausea sensations, and the neurotransmitter signaling by which cisplatin elicits these symptoms, remain understudied and are the focus of the current studies. Here, we show cisplatin-induced central c-Fos activation in the NTS and AP of the caudal brainstem, suggesting stimulation of hindbrain emetic circuits by cisplatin. NTS expression of c-Fos is likely due in large part to 5-HT3 mediated vagal activation, as complete vagotomy
can drastically reduce or block NTS c-Fos expression and/or emetic behavior [18,19,40]. Circulating humoral factors such as plasma 5-HT or substance P which are elevated following cisplatin treatment [41,42] may account for AP neuronal activation following cisplatin, as previous reports have shown no change in cisplatin-induced AP c-Fos following vagotomy or systemic pretreatment with a 5-HT3 antagonist [40]. In addition, direct application of Substance P or 5-HT to the AP can activate AP neurons [43]. There is also the strong potential for cisplatininduced cytokines [44] to act via both vagal and humoral means [45] to activate central pathways of sickness, however this hypothesis requires direct testing. Clear evidence also exists for substance P/NK-1 [46], and 5-HT/5-HT3 [33] signaling endemic to the NTS as potential mediators of emetic behavior and anorexia. Thus, it is possible that cisplatin-induced NTS Fos-positive neurons are direct and/or indirect mediators of multiple neurotransmitter signaling pathways simultaneously. No dorsal motor nucleus (DMN) expression was observed following cisplatin treatment, a finding similar to data from the rat [12].
Fig. 3. A: Representative immunofluorescence images collected from the PBN in saline and cisplatin treated mice. B: Cisplatin produces greater c-Fos immunofluorescence than saline treatment in the PBNel and PBNvl. (*P b 0.05, saline vs. cisplatin).
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
R.A. Holland et al. / Physiology & Behavior xxx (2014) xxx–xxx
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Fig. 4. Increased hindbrain AMPA and NMDA receptor subunit expression in cisplatin treated mice. Micropunch coordinates for NTS-enriched DVC (4A) and the PBN (4C). Black circles correspond to micropunch diameter and approximate location. B: GluA1 AMPA and NR1 and NR2B NMDA receptor subunit expressions were increased following cisplatin injection compared to controls in NTS-enriched DVC punches. D: GluA1 and G1uA2 AMPA and NR2B NMDA subunit expression was increased in the PBN (*P b 0.05, saline vs. cisplatin).
Additionally, as many caudal NTS neurons project to the DMN to control gastric emptying [47], our relative lack of cisplatin-induced caudal NTS Fos, compared to rostral regions, would be consistent with a mechanism involving minimal vagal efferent activation. This finding, however, contrasts with previously published work in the vomiting musk shrew,
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Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
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signals as it receives dense projections from the NTS and AP and has outputs to amygdala and BNST among other nuclei [49–53]. The NTS distribution of cisplatin-induced Fos observed in our studies shows that the rostral NTS exhibits greater expression, relative to caudal regions. Given that dense projections to the PBN originate from the rostral NTS [47], we speculate that our findings implicate activation of NTS neurons connecting monosynaptically with the PBN. With this in mind, it may be considered that a known subpopulation of these ascending PBN projections are glutamatergic [48], consistent with our gene expression data described below. Interestingly, in rats with PBN lesions, kaolin intake (an indicator of nausea) is increased dramatically [31]. Although this finding has not been followed up systematically, it is clear that the PBN neurons and/or PBN connections may exert a gating or inhibitory effect the intake of kaolin and gastric dysfunction associated with cisplatin treatment. In this context, calcitonin gene-related peptide-expressing neurons within the external lateral PBN have recently been suggested to play a crucial role in mediating anorectic circuits [54]. What is perhaps most notably underrepresented in the literature is examination of the central neurotransmitters mediating nausea and sickness behavior rodent models. The glutamatergic nature of vagal afferents [20,21] and serotonergic facilitation of glutamate signaling [22–24] in the brainstem, point to glutamate receptor signaling as a candidate pathway contributing to the emetic effects of cisplatin. Our results show AMPA (GluA1) and NMDA (NR1 and NR2B) receptor subunit upregulation in NTS-enriched DVC tissues from cisplatin treated mice. Cisplatin also elicits release of inflammatory cytokines, including TNF-α [44], which can increase glutamate release and NMDA receptor signaling [55]. Thus, it may be that TNF-α and/or other circulating cytokines also play a role in cisplatin-induce NMDA receptor upregulation cisplatin by amplifying vagal input signals [45]. In addition to the NTS, our results also show upregulation of AMPA receptors subunits and NR2B NMDA receptor unit in the PBN. As mentioned above, the PBN plays an integrative role in the mediating cisplatin-induced activation of the neuroaxis. Along these lines, recent evidence from Palmiter and colleagues points to a major role for glutamate signaling in the NTS and the PBN in the mediation of a central anorectic circuit [33]. Intriguingly, NR2B NMDA receptor subunits within the PBN may be responsible for enhanced PBN activation and engagement of a multisite “starvation” network [32,54]. Thus, we may interpret these findings together as evidence for a common nauseogenic/ anorectic pathway of NMDA-mediated processing of anorectic and noxious signals by PBN neurons. In the forebrain, we observe increased expression of NMDA receptor subunits in the amygdala as well as the NR2B subunit in the BNST. Similarly, cisplatin has been shown to induce c-Fos expression in the amygdala of the rat and musk shrew [12,18]. Thus, it is possible that activation of NMDA receptors within the amygdala and BNST represent stimulation of a nausea circuit. Lithium chloride, a potent inducer of visceral illness, has also been shown to stimulate neurons in the lateral PBN that project to the amygdala [32]. Future research should determine whether this circuit is also involved in mediation of cisplatin-induced anorexia/emesis. Finally, glutamate activity in the amygdala is linked to the transmission and learning related to visceral information in the formation of a conditioned taste aversion, a commonly used proxy for nausea in the rodent [56]. Taken together, the amygdala and BNST are both physiologically and anatomically connected to brainstem nuclei to potentially mediate neuroendocrine signals involved in feeding and nausea via glutamatergic circuits linked to the PBN. 5. Conclusions In summary, the transmission of cisplatin-induced nauseogenic and emetic information across the neuroaxis of mice extends to multiple sites and neurotransmitters in a broad network of activation. These current data suggest similar neural pathways mediating cisplatin effects
in mice compared to other well-studied species, which are likely mediated by central upregulation of AMPA and NMDA receptors. In future, the use of mouse models will be advantageous to study nausea and sickness behavior on cancerous backgrounds, in combination with emetogenic treatments, to provide a greater understanding of chemotherapy-induced anorexia and nausea/vomiting. Acknowledgments We wish to thank Amber L. Alhadeff for a critical reading of the manuscript. This manuscript is based on work presented during the 2013 Annual Meeting of the Society for the Study of Ingestive Behavior, July 30 - August 3, 2013. Grants This work was supported by: The McCabe Fund, The Biobehavioral Research Center of the University of Pennsylvania School of Nursing, the University of Pennsylvania Research Foundation, and the Center for Undergraduate Research at The University of Pennsylvania. References [1] Percie du Sert N, Rudd JA, Apfel CC, Andrews PL. Cisplatin-induced emesis: systematic review and meta-analysis of the ferret model and the effects of 5-HT(3) receptor antagonists. Cancer Chemother Pharmacol 2011;67:667–86. [2] Andrews PL, Horn CC. Signals for nausea and emesis: implications for models of upper gastrointestinal diseases. Auton Neurosci 2006;125:100–15. [3] Florczyk AP, Schurig JE, Bradner WT. Cisplatin-induced emesis in the Ferret: a new animal model. Cancer Treat Rep 1982;66:187–9. [4] Gylys JA, Doran KM, Buyniski JP. Antagonism of cisplatin induced emesis in the dog. Res Commun Chem Pathol Pharmacol 1979;23:61–8. [5] Matsuki N, Ueno S, Kaji T, Ishihara A, Wang CH, Saito H. Emesis induced by cancer chemotherapeutic agents in the Suncus murinus: a new experimental model. Jpn J Pharmacol 1988;48:303–6. [6] McCarthy LE, Borison HL. Antiemetic activity of N-methyllevonantradol and nabilone in cisplatin-treated cats. J Clin Pharmacol 1981;21:30S–7S. [7] Szelenyi I, Herold H, Gothert M. Emesis induced in domestic pigs: a new experimental tool for detection of antiemetic drugs and for evaluation of emetogenic potential of new anticancer agents. J Pharmacol Toxicol Methods 1994;32:109–16. [8] Horn CC, Kimball BA, Wang H, Kaus J, Dienel S, Nagy A, et al. Why can't rodents vomit? A comparative behavioral, anatomical, and physiological study. PLoS One 2013;8:e60537. [9] Horn CC. The medical implications of gastrointestinal vagal afferent pathways in nausea and vomiting. Curr Pharm Des 2013. [10] Schwartz GJ, Zeltser LM. Functional organization of neuronal and humoral signals regulating feeding behavior. Annu Rev Nutr 2013;33:1–21. [11] De Jonghe BC, Horn CC. Chemotherapy-induced pica and anorexia are reduced by common hepatic branch vagotomy in the rat. Am J Physiol Regul Integr Comp Physiol 2008;294:R756–65. [12] Horn CC, Ciucci M, Chaudhury A. Brain Fos expression during 48 h after cisplatin treatment: neural pathways for acute and delayed visceral sickness. Auton Neurosci 2007;132:44–51. [13] Vera G, Chiarlone A, Martin MI, Abalo R. Altered feeding behaviour induced by longterm cisplatin in rats. Auton Neurosci 2006;126–127:81–92. [14] Malik NM, Moore GB, Smith G, Liu YL, Sanger GJ, Andrews PL. Behavioural and hypothalamic molecular effects of the anti-cancer agent cisplatin in the rat: a model of chemotherapy-related malaise? Pharmacol Biochem Behav 2006;83:9–20. [15] Liu YL, Malik N, Sanger GJ, Friedman MI, Andrews PL. Pica — a model of nausea? Species differences in response to cisplatin. Physiol Behav 2005;85:271–7. [16] Yamamoto K, Matsunaga S, Matsui M, Takeda N, Yamatodani A. Pica in mice as a new model for the study of emesis. Methods Find Exp Clin Pharmacol 2002;24:135–8. [17] Rudd JA, Andrews PLR. In: Hesketh PJ, editor. Management of nausea and vomiting in cancer and cancer treatment. Jones and Bartlett; 2005. p. 15–65. [18] De Jonghe BC, Horn CC. Chemotherapy agent cisplatin induces 48-h Fos expression in the brain of a vomiting species, the house musk shrew (Suncus murinus). Am J Physiol Regul Integr Comp Physiol 2009;296:R902–11. [19] Horn CC. Brain Fos expression induced by the chemotherapy agent cisplatin in the rat is partially dependent on an intact abdominal vagus. Auton Neurosci 2009;148:76–82. [20] McCann MJ, Rogers RC. Functional and chemical anatomy of a gastric vago-vagal reflex. In: Tache Y, Wingate DL, Burks TF, editors. Innervation of the gut: pathophysiological implications. Boca Raton, FL: CRC; 1994. p. 81–92. [21] Sykes RM, Spyer KM, Izzo PN. Demonstration of glutamate immunoreactivity in vagal sensory afferents in the nucleus tractus solitarius of the rat. Brain Res 1997;762:1–11. [22] Jeggo RD, Kellett DO, Wang Y, Ramage AG, Jordan D. The role of central 5-HT3 receptors in vagal reflex inputs to neurones in the nucleus tractus solitarius of anaesthetized rats. J Physiol 2005;566:939–53. [23] Funahashi M, Mitoh Y, Matsuo R. Activation of presynaptic 5-HT3 receptors facilitates glutamatergic synaptic inputs to area postrema neurons in rat brain slices. Methods Find Exp Clin Pharmacol 2004;26:615–22.
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
R.A. Holland et al. / Physiology & Behavior xxx (2014) xxx–xxx [24] Wang Y, Ramage AG, Jordan D. Presynaptic 5-HT3 receptors evoke an excitatory response in dorsal vagal preganglionic neurones in anaesthetized rats. J Physiol 1998;509(Pt 3):683–94. [25] De Jonghe BC, Hayes MR, Zimmer DJ, Kanoski SE, Grill HJ, Bence KK. Food intake reductions and increases in energetic responses by hindbrain leptin and melanotan II are enhanced in mice with POMC-specific PTP1B deficiency. Am J Physiol Endocrinol Metab 2012;303:E644–51. [26] Liu YL, Malik NM, Sanger GJ, Andrews PL. Ghrelin alleviates cancer chemotherapyassociated dyspepsia in rodents. Cancer Chemother Pharmacol 2006;58:326–33. [27] Hayes MR, Skibicka KP, Bence KK, Grill HJ. Dorsal hindbrain 5′-adenosine monophosphate-activated protein kinase as an intracellular mediator of energy balance. Endocrinology 2009;150:2175–82. [28] Hayes MR, Skibicka KP, Leichner TM, Guarnieri DJ, DiLeone RJ, Bence KK, et al. Endogenous leptin signaling in the caudal nucleus tractus solitarius and area postrema is required for energy balance regulation. Cell Metab 2010;11:77–83. [29] Paxinos G, Franklin KBJ. Paxinos and Franklin's the mouse brain in stereotaxic coordinates. 4th ed. Amsterdam: Boston: Elsevier/Academic Press; 2013. [30] Bence KK, Delibegovic M, Xue B, Gorgun CZ, Hotamisligil GS, Neel BG, et al. Neuronal PTP1B regulates body weight, adiposity and leptin action. Nat Med 2006;12:917–24. [31] Horn CC, De Jonghe BC, Matyas K, Norgren R. Chemotherapy-induced kaolin intake is increased by lesion of the lateral parabrachial nucleus of the rat. Am J Physiol Regul Integr Comp Physiol 2009;297:R1375–82. [32] Wu Q, Zheng R, Srisai D, McKnight GS, Palmiter RD. NR2B subunit of the NMDA glutamate receptor regulates appetite in the parabrachial nucleus. Proc Natl Acad Sci U S A 2013;110:14765–70. [33] Wu Q, Clark MS, Palmiter RD. Deciphering a neuronal circuit that mediates appetite. Nature 2012;483:594–7. [34] Wang FB, Powley TL. Vagal innervation of intestines: afferent pathways mapped with new en bloc horseradish peroxidase adaptation. Cell Tissue Res 2007;329:221–30. [35] Horn CC, Richardson EJ, Andrews PL, Friedman MI. Differential effects on gastrointestinal and hepatic vagal afferent fibers in the rat by the anti-cancer agent cisplatin. Auton Neurosci 2004;115:74–81. [36] Phillips RJ, Baronowsky EA, Powley TL. Afferent innervation of gastrointestinal tract smooth muscle by the hepatic branch of the vagus. J Comp Neurol 1997;384:248–70. [37] Fukui H, Yamamoto M, Sato S. Vagal afferent fibers and peripheral 5-HT3 receptors mediate cisplatin-induced emesis in dogs. Jpn J Pharmacol 1992;59:221–6. [38] Andrews PL, Davis CJ, Bingham S, Davidson HI, Hawthorn J, Maskell L. The abdominal visceral innervation and the emetic reflex: pathways, pharmacology, and plasticity. Can J Physiol Pharmacol 1990;68:325–45. [39] Hesketh PJ. Chemotherapy-induced nausea and vomiting. N Engl J Med 2008;358:2482–94. [40] Reynolds DJ, Barber NA, Grahame-Smith DG, Leslie RA. Cisplatin-evoked induction of c-fos protein in the brainstem of the ferret: the effect of cervical vagotomy and the anti-emetic 5-HT3 receptor antagonist granisetron (BRL 43694). Brain Res 1991;565:231–6.
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[41] Dey D, Abad J, Ray AP, Darmani NA. Differential temporal changes in brain and gut substance P mRNA expression throughout the time-course of cisplatin-induced vomiting in the least shrew (Cryptotis parva). Brain Res 2010;1310:103–12. [42] Higa GM, Auber ML, Altaha R, Piktel D, Kurian S, Hobbs G, et al. 5-Hydroxyindoleacetic acid and substance P profiles in patients receiving emetogenic chemotherapy. J Oncol Pharm Pract 2006;12:201–9. [43] Carpenter DO, Briggs DB, Strominger N. Responses of neurons of canine area postrema to neurotransmitters and peptides. Cell Mol Neurobiol 1983;3:113–26. [44] Ramesh G, Reeves WB. TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity. J Clin Invest 2002;110:835–42. [45] Hermann GE, Rogers RC. TNFalpha: a trigger of autonomic dysfunction. Neuroscientist 2008;14:53–67. [46] Ray AP, Chebolu S, Ramirez J, Darmani NA. Ablation of least shrew central neurokinin NK1 receptors reduces GR73632-induced vomiting. Behav Neurosci 2009;123:701–6. [47] Grill HJ, Hayes MR. Hindbrain neurons as an essential hub in the neuroanatomically distributed control of energy balance. Cell Metab 2012;16:296–309. [48] Gill CF, Madden JM, Roberts BP, Evans LD, King MS. A subpopulation of neurons in the rat rostral nucleus of the solitary tract that project to the parabrachial nucleus express glutamate-like immunoreactivity. Brain Res 1999;821:251–62. [49] Jia HG, Rao ZR, Shi JW. An indirect projection from the nucleus of the solitary tract to the central nucleus of the amygdala via the parabrachial nucleus in the rat: a light and electron microscopic study. Brain Res 1994;663:181–90. [50] Krukoff TL, Harris KH, Jhamandas JH. Efferent projections from the parabrachial nucleus demonstrated with the anterograde tracer Phaseolus vulgaris leucoagglutinin. Brain Res Bull 1993;30:163–72. [51] Papas S, Ferguson AV. Electrophysiological characterization of reciprocal connections between the parabrachial nucleus and the area postrema in the rat. Brain Res Bull 1990;24:577–82. [52] Voshart K, van der Kooy D. The organization of the efferent projections of the parabrachial nucleus of the forebrain in the rat: a retrograde fluorescent doublelabeling study. Brain Res 1981;212:271–86. [53] Norgren R. Taste pathways to hypothalamus and amygdala. J Comp Neurol 1976;166:17–30. [54] Carter ME, Soden ME, Zweifel LS, Palmiter RD. Genetic identification of a neural circuit that suppresses appetite. Nature 2013;503:111–4. [55] Jara JH, Singh BB, Floden AM, Combs CK. Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK-dependent death. J Neurochem 2007;100:1407–20. [56] Miranda MI, Ferreira G, Ramirez-Lugo L, Bermudez-Rattoni F. Glutamatergic activity in the amygdala signals visceral input during taste memory formation. Proc Natl Acad Sci U S A 2002;99:11417–22.
Please cite this article as: Holland RA, et al, Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.02.038
