The role of central amygdaloid nucleus in regulating the nongenomic effect of aldosterone on sodium intake in the nucleus tractus solitary

Abstract Objective The central nucleus of the amygdala (CeA) has dense downward fiber projections towards the nucleus tractus solitary (NTS) and can modulate the activity of NTS taste neurons. However, whether CeA affects the nongenomic role of aldosterone (ALD) in regulating sodium intake at the NTS level remains unclear. Methods First, 40 adult male Sprague Dawley rats were divided into five groups, referring to different concentrations of ALD, to observe the sodium intake pattern compared with the vehicle (n = 8). ALD, the mineralocorticoid receptor antagonist spironolactone (SPI), and ALD + SPI were injected into the NTS. Then, the rats were divided into four groups (n = 16): bilateral/unilateral CeA electrolytic lesions, bilateral/unilateral CeA sham lesions. After recovery, one stainless steel 23‐gauge cannula with two tubes was implanted into the rat NTS, and all rats underwent a recovery period of 7 days. Then, each group was divided into two subgroups that received aldosterone or control solution injection, and the cumulative intake of 0.3 mol/L NaCl solution was recorded within 30 min. Results Bilateral CeA lesion eliminated the increased 0.3 mol/L NaCl intake induced by aldosterone microinjected into the NTS (CeA lesion: 0.3 ± 0.04 ml/30 min vs. sham lesion: 1.3 ± 0.3 ml/30 min). Unilateral CeA lesion reduced the increased NaCl intake induced by aldosterone microinjected into the NTS compared with the control group (p < .05) in the first 15 min but not in 15–30 min (p > .05). In sham lesion rats, aldosterone (5 ng/0.1 μl) still induced a significant increase in NaCl intake (aldosterone: 1.3 ± 0.3 ml/30 min vs. control: 0.25 ± 0.02 ml/30 min) (p < .05). Conclusion The results verified that the complete CeA may play an important role in aldosterone to regulate the nongenomic effect on rapid sodium intake.


INTRODUCTION
Sodium appetite is a behavioral response initiated by a salty taste and is the result of multiple types of afferent information modulated and integrated through different levels of neuroendocrine systems. The renin-angiotensin-aldosterone system plays an essential role in the regulation of sodium appetite in which aldosterone regulates sodium excretion in the kidney. It is also involved in the management of salty perception and sodium intake through the peripheral mechanism of regulation of sodium channels in the tongue epithelium and the influence of sodium-sensitive neurons in the central nervous system. Formenti et al. reported that sodium intake was significantly increased in a dose-dependent manner following long-term and chronic treatment using aldosterone in the fourth ventricle of rats (Formenti et al., 2013), observations supported by the research of Koneru et al., who found that this phenomenon could be inhibited by shRNA interference of mineralocorticoid receptors in the solitary nucleus (Koneru et al., 2014). Thus, aldosterone has been demonstrated to have far-reaching effects on the intake of sodium in the solitary nucleus (NTS). In addition to classical genomic effects, aldosterone may also operate diffusely and rapidly in a nongenomic manner in vivo (Williams, 2013). Aldosterone can bind to mineralocorticoid receptors (MRs) in the cytoplasm to form an MR-aldosterone complex, and then the complex is translocated to the nucleus, thereby exerting the regulation of renal water reabsorption through genomic effects. Notably, the first effect usually occurs 30-60 min after aldosterone release/intake. Aldosterone can also function through nongenomic effects. Nongenomic effects can increase second messengers of membrane signaling transduction pathways and thereby may exert a rapid effect at the membrane level (within minutes) without transcription or protein synthesis. Recently, our laboratory reported that injection of different doses of aldosterone into the solitary nucleus induced fast sodium intake in rats (Qiao et al., 2016).
In another respect, the central amygdaloid nucleus (CeA) represents a vital subnucleus in the amygdaloid nucleus and participates in the regulation of multiple physiological functions. For example, the CeA has complex neural links via projections with other regions of the brain (Fadok et al., 2018;Smith et al., 2016;Wang et al., 2018).

Materials and instruments
Aldosterone was purchased from Sigma and dissolved in a solution of DMSO in H 2 O (at a volume ratio of 1:100). The same concentration of DMSO was used in the control group as a vehicle. A 0.1 μl volume of drugs was injected into the bilateral NTS. A final concentration of 5 ng/0.1 μl aldosterone was used for microinjection, as previously reported (Brailoiu et al., 2013;Formenti et al., 2013;Francis et al., 2001). The following instruments were used in this study: a 1-μl microinjector (Hamilton), a stereotaxic apparatus (SN-2N, Narishige), a concentric electrode (CEA 200, MicroProbes), a lesion-making device (53500, Ugo Basile), metabolism/feeding-drinking cages, part of a feeding-drinking-activity analyzer (41800111213, UGO), and a small animal anesthetic machine (RWD).

Electrolytic CeA lesions
Rats were anaesthetized with isoflurane (5% induction, 2% maintenance at 1 L/min) and then fixed in the stereotaxic apparatus, ensuring that the skull was level between the bregma and the lambda. After disinfection with alcohol, the fur and skin on the skull were removed, and the calvarium was exposed. The location of the CeA was ascertained based on a brain map of rats (2.5 mm caudal to bregma, 4.2 mm from the midline, and at a depth of 7.5 mm from the cortical surface). A microdrill was used to create a hole at the CeA to expose the dura mater.
After removal of the fragments and dura mater, concentric electrodes were implanted bilaterally. Electrolytic lesions were created bilaterally at a constant current (400 μA for 25 s) at stereotaxic points above each side. A clip attached to the tail was used as an indifferent electrode.
Sham lesion rats underwent the same surgical procedure and had an electrode placed at the same coordinates but without any current being passed. A lesion was created on a single side of the CeA by electrically stimulating only the left electrode, while the right electrode was disconnected from the output. All other steps of the surgery remained the same. Rats were allowed to recover for 3-5 days following creation of the lesion prior to the subsequent experiments being performed.

Implantation of cannulae in the solitary nucleus
Implantation of cannulae in the solitary nucleus was performed in rats 3-5 days after creation of electrolytic lesions in the CeA. Briefly, rats were anaesthetized and fixed in the stereotaxic apparatus, ensuring that the skull was level between the bregma and the lambda. After disinfection with alcohol, the fur and skin on the skull were removed, and the calvarium was exposed. The stereotaxic coordinates of the NTS were 13.9 mm caudal to the bregma, 0.5 mm lateral to the midline, and 7.8 mm below the surface of the skull. Each cannula was fixed with three screws and dental acrylic resin, and an obturator (30 gauge) was inserted. A prophylactic dose of penicillin was administered intramuscularly when the surgery was completed. All rats were allowed to recover for at least 7 days in metabolic cages following surgery, and a two-bottle choice test was conducted synchronously during the recovery period. The metabolic cage is a component of the analyzer and measures food and water intake and monitors activity.

Microinjection
Drugs were microinjected into the NTS using a 1-μl Hamilton syringe connected by PE-10 polyethylene tubing to the 30-gauge injection cannula (1 mm longer than the guide cannula). The injection volume into the NTS was 0.1 μl, with injection conducted over a 1-min period followed by an additional 1 min with the injection cannula remaining in place. The injection cannula was then replaced with the obturator. Prior to the experiment officially beginning, rats were injected three times to ensure that they became accustomed to the injection process.

Behavioral tests
Rats were allowed to recover for 3 days following creation of electrolytic lesions and for 7 days after implantation of cannulae in the solitary nucleus prior to the start of the following experiments.
Two-bottle choice test training was conducted during the recovery.
1. Two-bottle choice test: After the operation, the rats were placed in a metabolic cage for two-bottle selection of distilled water and 0.3 mol/L NaCl for experimental training, freely eating and ingesting distilled water and 0.3 mol/L NaCl every day, once the intake of 0.3 mol/L NaCl was stabilized (usually 4-5 days), and then different drugs could be administered for experiments.

Histological analysis
Immediately after the experiments, 2% Pontamine Sky Blue solution (0.2 μl) was injected into the NTS. The rats were then deeply anaesthetized with a high dose of chloral hydrate and perfused transcardially with PBS followed by 10% buffered formalin. In each rat, the brain was removed, fixed, frozen-sectioned (40 μm thick) in the coronal plane, and analyzed under a light microscope to confirm the injection sites in the NTS, in accordance with the atlas of Paxinos and Watson. Coronal brain sections were cut at 40 μm thick and then Nissl stained to confirm the locations of lesions in the amygdala via microscopic inspection.

Data analysis
Statistical analysis was performed using a Statistical Program for Social Sciences statistical software (SPSS 18.0). All data are presented as means ± standard error of the mean (SEM) and were analyzed into account Fitts's assumption, sodium intake was analyzed as cumulative and noncumulative data, and since there were no differences in the outcome of the hypothesis testing, such findings were presented as cumulative data as it is more usual.

Histology of localization analysis
A total of 167 rats were used in this experiment; among those, 82 rats were used to verify the effect of ALD/SPI on sodium intake, and the other 70 rats were used to determine whether CeA can affect the nongenomic effect of ALD on sodium intake. Histologic localization indicated that among the 82 rats, the cannulae were placed accurately in 73 rats, and the majority of the injection sites were located in the middle of the solitary nucleus ( Figure 1). The data gathered from these rats were used to analyze the effects of drug injection in the solitary nucleus regarding sodium intake. Data relating to rats that had histologically inaccurate localization were not significant compared with the vehicle treatment on sodium intake. Thus, the data were not included in the statistical analysis.

Intake of 0.3 mol/L NaCl in rats with bilateral aldosterone injection in the solitary nucleus after electrolytic lesions in the CeA
The results showed an increase in sodium intake with increasing ALD concentration compared with vehicle, and the time course curves were significantly different between treatments [F(4, 35) = 45.76, p < .05)].
There was a peak in the first 5 min, which could be seen as an obvious rapid increase, as shown in Figure 3a. Compared with SPI group, the SPI + ALD group still stimulated sodium intake, which represented that SPI could not effectively block this rapid increase in sodium intake (Figure 3b), and this result indicated that this rapid regulation of ALD toward sodium intake may be a nongenomic effect. there was a significant difference in sodium intake in the first 15 min, with a difference that was not significant over the subsequent 15 min (p > .05). Compared with the unilateral lesion + ALD group, the sham lesion + ALD group showed a significant difference in sodium intake in the second 15 min (p < .05), as shown in Figure 5.

Change in intake of 0.3 mol/L NaCl in rats with electrolytic sham lesions in the CeA in which aldosterone was injected outside of the solitary nucleus
In rats with electrolytic sham lesions in the CeA, injections of 5 ng/0.1 μl aldosterone outside of the solitary nucleus (misplaced injection) failed to induce accelerated intake of 0.3 mol/L NaCl (p > .05).

DISCUSSION
The results demonstrate that electrolytic lesions of the bilateral CeA abolished the increased intake of sodium induced by aldosterone injections in both sides of the solitary nucleus, suggesting that there F I G U R E 2 Photomicrographs of coronal rat brain sections showing the typical sites of bilateral electrolytic lesions of the CeA. Scale bar = 1 mm. Abbreviations: BLA, basolateral amygdala; CeA, central nucleus of amygdala; opt, optic tract may be a facilitation mechanism in the CeA that is indispensable for aldosterone-induced rapid sodium intake in rats.
The amygdaloid nucleus belongs to the limbic system of the fore- between the CeA and a number of regions of the brain associated with sodium intake regulation, such as signals from the HSD2 neuron (Geerling et al., 2006;Resch et al., 2017) in the solitary nucleus, relayed by the parabrachial nucleus, projected upward to the lateral CeA, and then projected directly by the medial CeA to the HSD2 neuron, which may be an important neural loop where the CeA participates in the regulation of sodium intake (Gasparini et al., 2017). In addition, the CeA has a fibrous connection with the nucleus of the reward system and emits fiber projections to neuroendocrine nuclei, such as the chamber nucleus. As the CeA has a two-way fiber link with the brain stem group that transmits taste and visceral information and is closely The mechanism by which CeA regulates sodium intake has been intensively studied in previous studies. In rats with electrolytic lesions on both sides, spontaneous sodium intake decreased significantly, and sodium intake induced by subcutaneous injection of oxidative corticosterone, α 2 -adrenergic receptor antagonist, or Ang II was also reduced by creating a CeA lesion (Smith et al., 2016). Previous studies suggested that sodium appetite induced by various facilitation stimuli, such as Ang II and corticosteroids, depends on CeA facilitation mechanisms. The data in our study also demonstrate that the facilitation mechanism of CeA was indispensable for sodium intake induced by aldosterone in the solitary nucleus. However, compared with previous F I G U R E 3 Cumulative intake of 0.3 mol/L NaCl by rats that received ALD injections at different concentrations into the nucleus tractus solitary (NTS) (a). Intake of 0.3 mol/L NaCl induced by sodium depletion followed by ALD or SPI treatment (b). n = number of animals. *p < .05, when each treatment group is compared with the vehicle group. Error bars show means ± SEM. # p < .05, when the treatment group is compared with the "10 ng SPI" group reports, we found that bilateral lesions of the CeA had no significant effect on sodium intake during the experiment. The exact reason for this difference remains unclear, and it is assumed that in this study, we observed sodium intake over a short period of time (30 min) rather than over the long term. Furthermore, the majority of previous studies used 0.5 mol/L NaCl as a sodium source, which is more aversive to rats than the 0.3 mol/L NaCl used in this study.
In summary, the results of this study indicated that CeA is indispensable for aldosterone's nongenomic effects of sodium intake in the solitary nucleus of rats. Complete CeA is required for aldosterone's nonge-F I G U R E 4 Cumulative 0.3 mol/L NaCl intake by rats submitted bilateral sham or electrolytic lesions of the central nucleus of the amygdala (CeA) that received bilateral injections of aldosterone (5 ng/0.1 μl) or control into the nucleus tractus solitary (NTS). n = number of animals. Error bars show means ± SEM. *p < .05, when each treatment group is compared with the vehicle group nomic effects of rapid sodium intake. There may be a descending regulatory mechanism in the CeA in which aldosterone regulates sodium intake in the solitary nucleus.

ACKNOWLEDGMENTS
This study was supported by National Natural Science Foundation of China (Program No. 82001087) and the Fundamental Research Funds for the Central Universities (xzy012019104).

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Data that support the findings of this study and custom code used to analyze data are available from the corresponding author upon reasonable request.
F I G U R E 5 Cumulative 0.3 mol/L NaCl intake by rats submitted unilateral sham or electrolytic lesions of the central nucleus of the amygdala (CeA) that received bilateral injections of aldosterone (5 ng/0.1 μl) or control into the nucleus tractus solitary (NTS). n = number of animals. *p < .05, when each treatment group is compared with the vehicle group. Error bars show means ± SEM. # p < .05, when the treatment group is compared with the "unilateral lesion + ALD" group