Identification of substances which regulate activity of corticotropin-releasing factor-producing neurons in the paraventricular nucleus of the hypothalamus

The stress response is a physiological system for adapting to various internal and external stimuli. Corticotropin-releasing factor-producing neurons in the paraventricular nucleus of the hypothalamus (PVN-CRF neurons) are known to play an important role in the stress response as initiators of the hypothalamic–pituitary–adrenal axis. However, the mechanism by which activity of PVN-CRF neurons is regulated by other neurons and bioactive substances remains unclear. Here, we developed a screening method using calcium imaging to identify how physiological substances directly affect the activity of PVN-CRF neurons. We used acute brain slices expressing a genetically encoded calcium indicator in PVN-CRF neurons using CRF-Cre recombinase mice and an adeno-associated viral vector under Cre control. PVN-CRF neurons were divided into ventral and dorsal portions. Bath application of candidate substances revealed 12 substances that increased and 3 that decreased intracellular calcium concentrations. Among these substances, angiotensin II and histamine mainly increased calcium in the ventral portion of the PVN-CRF neurons via AT1 and H1 receptors, respectively. Conversely, carbachol mainly increased calcium in the dorsal portion of the PVN-CRF neurons via both nicotinic and muscarinic acetylcholine receptors. Our method provides a precise and reliable means of evaluating the effect of a substance on PVN-CRF neuronal activity.

associated viral vector (AAV: AAV9-CMV-FLEX-YC) which expresses YC in the presence of Cre recombinase was bilaterally injected into the PVN in CRF-iCre mice 21 (Fig. 1a). YC expression in PVN-CRF neurons was confirmed in bigenic mice, CRF-iCre;Ai14 mice in which Cre expressing CRF neurons exclusively express the red fluorescent protein tdTomato 22 (tdTomato/YC = 86.9 ± 1.2%; YC/tdTomato = 43.6 ± 2.8%; n = 7 animals; Fig. 1b-e [v&d PVN]). We found strong tdTomato expression in various brain regions, such as the PVN, amygdala and hippocampus, where CRF neurons are already known to be distributed 23 . In the PVN, tdTomato-expressing neurons were found distributed in two regions, as previously reported 24 . tdTomato fluorescence appeared to be high in the ventral portion and low in the dorsal portion of the PVN (Fig. 1b,c). To distinguish the calcium response in the ventral and dorsal portions, we defined a mediolateral line through the dorsal edge of the third ventricle as the boundary between the ventral and dorsal portions of the PVN (vPVN and dPVN, respectively; Fig. 1c). In the vPVN and dPVN, tdTomato expression in YC-expressing cells was 93.3 ± 0.9% and 72.7 ± 4.1%, respectively (Fig. 1d), while YC expression in tdTomato-expressing cells was 39.9 ± 3.3% and 54.3 ± 3.9%, respectively (Fig. 1e). There was a greater proportion of YC-expressing cells in the vPVN than dPVN (vPVN, 65.4 ± 3.6%; dPVN, 34.6 ± 3.6%; n = 7 animals; Fig. 1f).
YC is composed of yellow and cyan fluorescent proteins (YFP and CFP, respectively) fused to a calcium binding domain. When calcium concentrations increase, YFP fluorescence will increase and CFP fluorescence will decrease, and thus the YFP/CFP (Y/C) ratio will increase. To examine the relationship between the YC signal and neuronal activity, we performed simultaneous electrophysiological recordings and calcium imaging in acute brain slices. Positive pulse currents were injected into single cells expressing YC through a recording pipette to evoke action potentials. Calcium imaging in the same neurons showed that the Y/C ratio was increased in a firing frequency-dependent manner. Pulses of 1, 2, 5 and 10 Hz current injection induced firing of 0.95 ± 0.03, 1.81 ± 0.12, 4.88 ± 0.10 and 9.69 ± 0.22 Hz, respectively (Fig. 1g), and induced increases of 5.1 ± 1.0, 9.5 ± 1.4, 20.8 ± 3.5 and 31.7 ± 4.3 ΔR/R 0 (%), respectively (n = 8 cells, Fig. 1h-j). Thus, these results confirmed that functional YC was exclusively expressed in PVN-CRF neurons and that an increase in the Y/C ratio was correlated with neuronal activity.
Screening of substances that directly affect activity of PVN-CRF neurons. Next, we performed a screen of bioactive substances to identify those which may affect the activity of PVN-CRF neurons. We prepared acute brain slices from mice expressing YC in their PVN-CRF neurons. Brain slices were mounted in a chamber on a fluorescence microscope and were perfused with artificial cerebrospinal fluid (aCSF). To suppress the indirect effects from synaptic inputs of other neurons, the voltage-gated sodium channel blocker, tetrodotoxin (TTX, 1 μM), was added to the perfused aCSF. We applied candidate substances one by one to the perfused aCSF for 2 min and monitored Y/C ratios during and after application of each substance. We confirmed that aCSF application itself did not affect the Y/C ratio, while the excitatory neurotransmitter glutamate (100 μM) increased the Y/C ratio and the inhibitory neurotransmitter GABA (100 μM) decreased the ratio (Fig. 2a-e). We then screened 63 bioactive substances, including 6 amines, 3 amino acids, 1 choline, 2 lipids, 2 nucleic acids, and 49 peptides (n = 1935 total cells from 26 animals; Table 1, Supplementary Tables S1 and S3). We defined substances which increased or decreased the Y/C ratio from the Z-score, as described in the methods section, to conservatively detect clear calcium changes based on the mean Z-scores observed after application of glutamate and GABA. Results showed that the Y/C ratio was increased by 12 substances: histamine (HA), glutamate, serotonin, carbachol (CCh), angiotensin II (AngII), noradrenaline (NA), dopamine, sulfated cholecystokinin octapeptide (CCK8S), thyrotropin releasing hormone, neuromedin C, cholecystokinin tetrapeptide (CCK4) and tyramine. Conversely, the Y/C ratio was decreased by 3 substances: GABA, nociceptin and glycine (Fig. 2f). Representative calcium signals of the responsive substances are shown in Supplementary Fig. S1, except for glutamate, GABA, HA, CCh and AngII, which are shown in Figs. 2c,d, 4b, 5b and 3b, respectively. Among the responsive substances, CCK8S, CCK4 and tyramine had not been reported in previous studies. We also identified 3 substances, www.nature.com/scientificreports/  www.nature.com/scientificreports/ www.nature.com/scientificreports/ AngII, HA and CCh, that showed different types of calcium responses between the dPVN and vPVN. Therefore, we further examined the effect of these 3 substances using selective antagonists.

Angiotensin II increased [Ca 2+ ] i in the ventral portion of PVN-CRF neurons. AngII is a bioactive
peptide known to be involved in the regulation of fluid homeostasis 25 . AngII is also reported to be produced in the brain from angiotensinogen via angiotensin I by successive digestion by renin and angiotensin converting enzyme 26 . AngII functions to induce water intake in the central nervous system 27 . Application of AngII (100 nM) induced a large and long-lasting increase in the Y/C ratio in PVN-CRF neurons. This response was larger in vPVN-CRF neurons compared to dPVN-CRF neurons (Fig. 3a,b). The glutamate-induced increase in the Y/C ratio in both the vPVN and dPVN of the same slice preparation confirmed that neurons from both portions were healthy (Fig. 3c). To identify the receptors implicated in the AngII-induced [Ca 2+ ] i increase, we perfused a type-1 AngII (AT 1 ) receptor-selective antagonist, losartan (10 μM), for 10 min prior to AngII application. This resulted in complete inhibition of the AngII-induced increase in the Y/C ratio ( Fig. 3d-f, Supplementary  Fig. S2a, Supplementary Tables S2 and S4). Antagonist application alone did not cause a clear change in the Y/C ratio ( Supplementary Fig. S3). This is consistent with a previous report that PVN-CRF neurons express type-1a AngII (AT 1a ) receptors 28 , and that the AT 1a receptor is known to couple with the Gq family of G proteins 29 . Thus, our results suggest that AngII increases [Ca 2+ ] i in vPVN-CRF neurons via the AT 1 receptor.

Histamine increases [Ca 2+ ] i in the ventral portion of PVN-CRF neurons.
HA is a monoamine released from histaminergic neurons as a neurotransmitter in the brain 30 . Histaminergic neurons are known to be involved in various physiological functions 31 , such as the maintenance of wakefulness 32 . Application of HA  (Fig. 4a,b), in contrast to glutamate which induced an increase in the Y/C ratio in both the dPVN and vPVN within the same slice preparation (Fig. 4c). To identify receptors that may play a role in the [Ca 2+ ] i increase in vPVN-CRF neurons, a histamine H 1 (H 1 ) receptor-selective antagonist, pyrilamine (1 μM), was perfused for 10 min prior to HA application. This resulted in the complete inhibition of the HA-induced increase in the Y/C ratio by co-application of pyrilamine ( Fig. 4d-

Carbachol increases [Ca 2+ ] i in the dorsal portion of PVN-CRF neurons.
CCh is an agonist of both nicotinic and muscarinic acetylcholine receptors (nACh and mACh, respectively). Acetylcholine (ACh) is derived from choline and produced in cholinergic neurons, and is known to be involved in various physiological functions 35 such as memory, learning and wakefulness 36 . Application of CCh (100 μM) induced a larger increase in the Y/C ratio in the dPVN than vPVN (Fig. 5a,b), while glutamate induced an increase in the Y/C ratio in both the dPVN and vPVN within the same slice preparation (Fig. 5c). Since this increase in the Y/C ratio can be induced via nACh receptors and/or mACh receptors, we tried to discriminate between the receptors implicated in the [Ca 2+ ] i increase. First, we perfused the nACh receptor-selective antagonist, hexamethonium (100 μM), for  Tables 2 and  4). Next, to determine the involvement of the mACh receptor, a mACh receptor-selective antagonist, atropine (100 nM), was tested. Atropine partially diminished the CCh-induced increase in the Y/C ratio (75.1 ± 2.6%, n = 48 cells; Fig. 5f, Supplementary Fig. S2c, Supplementary Tables 2 and 4). Antagonist application alone did not result in a clear change in the Y/C ratio ( Supplementary Fig. S3). It has been reported that microinjection of ACh increases Crh mRNA expression in the PVN 37 . It is possible that this increase in mRNA expression could be explained by the direct effect of ACh on PVN-CRF neurons. Thus, our results suggest that CCh increases [Ca 2+ ] i in the dPVN via both nACh receptors and mACh receptors.

Discussion
In this study, we found 15 candidate physiological substances affecting activity of PVN-CRF neurons (Fig. 6). These substances are possibly implicated in various physiological functions through activation or inhibition of PVN-CRF neurons. For example, activity of PVN-CRF neurons is increased or decreased by negative or positive valence, respectively 2 . Specifically, optogenetic activation or inhibition of PVN-CRF neurons induced or reduced stress-related behavior, respectively 2, 38 . This kind of behavior-inducible neuronal activity can be mediated by biophysiological substances. For example, AngII is thought to be involved in the stress response via the PVN 39 .  www.nature.com/scientificreports/ The expression of AT 1 receptors is reported to be increased in the PVN following stress 40 . Furthermore, the AT 1a receptor in the PVN was shown to be essential for the expression of anxiety behavior 41 . However, these studies did not reveal the direct involvement of PVN-CRF neurons in these responses since they did not directly manipulate PVN-CRF neurons. A recent study identified that the AT 1a receptor is exclusively expressed in PVN-CRF neurons, and showed that AngII is implicated in the stress response via PVN-CRF neurons 28 . Although our screening system identified many novel substances which affect the activity of PVN-CRF neurons, it might not have detected all substances that can affect PVN-CRF neurons. For example, glucagonlike peptide-1 (GLP-1) expressing neurons are reported to innervate PVN-CRF neurons, as observed by electron microscopy 42 . This effect on PVN-CRF neurons was confirmed by electrophysiology combined with optogenetics 43 . GLP-1 was shown to directly increase [Ca 2+ ] i in isolated PVN-CRF neurons 44 . In our screening system, a small number of PVN-CRF neurons showed an increase in [Ca 2+ ] i in response to GLP-1; however, we classified GLP-1 as "not-responsive" since most of the neurons did not show a sufficient response (Fig. 1f). This difference can be attributed to the fact that we used simple criteria to define "responsive" substances; we used just a single threshold and the majority of the neural population to determine if there was an "increasing" or "decreasing" effect on [Ca 2+ ] i . Although a previous study showed that the Y 1 receptor 45 and EP 4 receptor 46 are expressed in PVN-CRF neurons, our screening system did not show clear changes in [Ca 2+ ] i by application of ligands for these receptors, i.e. neuropeptide Y and prostaglandin E 2 . These results suggest that substances classified as "not-responsive" in our screen might represent false negatives since [Ca 2+ ] i is just one of the indicators of neural activity.
Furthermore, this study did not address the more detailed mechanisms that regulate PVN-CRF neuronal activity. For example, in our experiments we added TTX to suppress multi-synaptic modulation 47 of PVN-CRF neurons. Therefore, it is possible that substances classified as not-responsive might affect the activity of PVN-CRF neurons in an indirect manner via their effects on other neurons. The possible existence of TTX-resistant sodium channels (Na v 1.5, Na v 1.8 and Na v 1.9) 48 might also be taken into consideration. Among these channels, Na v 1.8 is reported to be expressed in the PVN; however, its sodium channel current (I Na ) accounts for less than 2% of the total I Na 49 . In addition, we sequentially screened multiple substances; however, GPCR itself can be modulated by specific substances via some mechanisms, such as desensitization/resensitization 50 and heterodimerization 51,52 . As shown in Supplementary Table S3, we applied each substance in different orders in multiple brain slices from different animals to minimize potential effects from the precedingly applied substances. Recently, sexual dimorphism has been reported in the function of an NMDA receptor subunit in CRF-producing neurons 53,54 . It is thus possible that this type of sexual dimorphism could exist for other receptors. Since we randomly used both male and female animals in this study, we could not examine potential sexual dimorphism. However, the methods we adopted in the present study are flexible and can also be applied to evaluating the complex regulatory mechanisms of PVN-CRF activity by selecting the appropriate conditions such as the absence of TTX, presence of other antagonists, combinations of substances or evaluation in either sex.
Our screening system showed that dPVN-CRF neurons and vPVN-CRF neurons have different features in their response to various substances. For example, AngII and HA increased [Ca 2+ ] i mainly in the vPVN-CRF neurons, which makes a contrast to the observation that CCh increased [Ca 2+ ] i mainly in the dPVN-CRF neurons. The major proportion of the vPVN-CRF neurons have been well-recognized to project to the median eminence and regulate the HPA-axis, whereas part of the dPVN-CRF neurons are known to project to pre-autonomic neurons in the lower brain stem, and possibly to the spinal cord, and they may be involved in the regulation of autonomic output from the spinal preganglionic neurons 11 . Therefore, we assume that the differences in responses between the vPVN-CRF neurons and dPVN-CRF neurons could be related to the differences in expression of respective receptors among CRF neuronal subclasses, which are involved in different physiological functions. It was reported that α7 subunit-containing nicotinic acetylcholine (α7nACh) receptors are expressed in magnocellular neurons and not in parvocellular neurons 55 , but it is not clear whether α7nACh receptors are expressed in a particular subclass of CRF neurons in the dPVN, which needs to be examined further. On the other hand, AngII induced larger increases in [Ca 2+ ] i in some vPVN-CRF neurons compared to the responses in other neurons in the same anatomical location (Fig. 3b). This makes a contrast to the more uniform [Ca 2+ ] i increase when it was induced by glutamate (Fig. 3c). These results imply the potential existence of subpopulations among the vPVN-CRF neurons. It is necessary to confirm distribution of receptor expression more in detail to understand the mechanism underlying the present results.
It is not clear whether the changes in calcium signals, observed in the present study, are the consequences of electrophysiological firing of the PVN-CRF neurons. Calcium signals can be affected not only by voltage-gated calcium channels sensitive to changes in membrane potential, but also by additional machinery, including other calcium pumps and transporters, as well as the transfer of Ca 2+ among cytosol and other organelles such as in mitochondria and the endoplasmic reticulum 56 . For example, NA is reported to increase the levels of phosphoextracellular signal-regulated kinases 1/2 (ERK1/2) in PVN-CRF neurons via the α 1 adrenoreceptor 57 , while ERK1/2 can be regulated by intracellular calcium signaling to induce changes in gene expression 58 . It is also reported that NA activates phospholipase C via the α 1 adrenoreceptor, and can subsequently induce an increase in [Ca 2+ ] i through both the release of intracellular Ca 2+ stores as well as extracellular Ca 2+ influx in isolated PVN neurons 59 . It is possible that the changes in [Ca 2+ ] i observed in the present study might be implicated in such intracellular calcium signaling, independent of electrophysiological changes.
NA is known to act as an important regulator of PVN-CRF neurons 60 . However, the mechanism by which it regulates PVN-CRF neuronal activity may not be uniform, and the presence of multiple underlying mechanisms has been proposed. For example, low concentrations of NA increased firing of neurons in the PVN, whereas high concentrations of NA decreased firing of them 61 . NA has also been shown to indirectly increase the frequency and amplitude of excitatory post-synaptic current in parvocellular neurons in the PVN via glutamatergic interneurons expressing α 1 adrenoreceptors; while, conversely, NA can also directly hyperpolarize parvocellular Scientific RepoRtS | (2020) 10:13639 | https://doi.org/10.1038/s41598-020-70481-5 www.nature.com/scientificreports/ neurons via the β adrenoreceptors 62 . Furthermore, NA is known to increase [Ca 2+ ] i in isolated PVN neurons via the α 1 receptors 59 . In our study, although the mean Z-score of the NA-induced [Ca 2+ ] i increase was smaller than that of the other 5 substances (Fig. 2f), the [Ca 2+ ] i signal transiently dropped in the middle of an increment, also suggesting the presence of complex regulatory mechanism ( Supplementary Fig. S1b). More detailed experiments are required to examine the effect of NA on PVN-CRF neurons. In summary, our method for calcium imaging provided a precise and reliable way to compare the effect of various substances on the activity of PVN-CRF neurons. The present study will contribute to the understanding of the mechanisms of stress responses, and to the development of future studies aimed at elucidating the actual physiological function of the calcium-modulating effects of the substances identified in this screen.

Animals.
All experiments were carried out in accordance with Nagoya University Regulations on Animal Care and Use in Research. All experiments were approved by the Institutional Animal Care and Use Committees of the Research Institute of Environmental Medicine, Nagoya University, Japan (approval #19232 and #19268). All efforts were made to reduce the number of animals used and to minimize the pain and suffering of animals. Adult (> 4 weeks old) CRF-iCre (Crh tm2(icre)Ksak ) mice 21 and mice generated by crossing with Ai14 (B6;129S6-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J) mice were used in this study. Animals were maintained on a 12-h light-dark cycle under ad libitum feeding and drinking conditions. Room temperature was maintained at 23 ± 2 °C.
Buffers. The

Adeno-associated virus (AAV).
AAV9-CMV-FLEX-YC-Nano50-WPRE (1.0 × 10 13 copies/mL) was generated according to the protocol described in the supplementary methods. Briefly, pHelper, pAAV-RC (serotype 9) and pAAV-CMV-FLEX-YC-Nano50-WPRE plasmids were transfected into AAV-293 cells (Agilent Technologies) using the calcium phosphate method. Three days after transfection, cells were collected and suspended in PBS and AAVs were purified by ultracentrifugation. The final virus solvent was a mixture of PBS and OptiPrep (Alere Technologies AS), where the ratio of the solution depended on the AAV titer.

Virus vector injection.
Mice were anesthetized with 1-2% isoflurane and fixed on a stereotaxic frame.
The scalps were opened and the skull above the injection site was drilled. A glass pipette (GC150-10; Harvard Apparatus) made with a puller (P-97, Sutter Instrument) was used for injection of AAV. In the bilateral PVN (in mm, AP − 0.5 from the bregma, ML 0.5 from the midline, DV − 4.2 from the brain surface), 600 μL/site of AAV solution was injected by air pressure pulses regulated by a Pneumatic Picopump (World Precision Instruments) with a pulse generator (SEN-7103, Nihon Kohden). The injected animals were used for subsequent experiments at least 3 weeks after injection. Fixed brain slices. Mice were deeply anesthetized with isoflurane and perfused with 25 mL chilled saline followed by 25 mL chilled 10% formalin. After decapitation, each skull was carefully removed and brains were placed into chilled 10% formalin for post-fixation overnight. After post-fixation, brains were placed into 30% sucrose containing PBS for cryoprotection for at least 48 h. After cryoprotection, brains were placed into O.C.T. compound (Sakura Finetek Japan) and frozen at − 80 °C for 20 min then placed into a − 20 °C cryostat (CM3050 S; Leica Biosystems). Embedded brains were fixed on a stage using O.C.T. compound and sliced at a thickness of 40 μm.
Cell counts. Every 1 in 4 cryo-sectioned brain slices were used for cell counts. Slices were mounted on slide glasses and were visualized and imaged using a confocal microscope (LSM710, Carl Zeiss). Images of YFP and tdTomato fluorescence were obtained and the number of cells positive for each signal was counted manually. Acute brain slices. Animals were anesthetized with isoflurane and decapitated. The brains were immediately removed from the head and incubated in ice-cold cutting solution. Brains were sliced at a thickness of 250 μm using a vibratome (VT1200S, Leica). The slices were incubated in aCSF at 35ºC for 1 h then at room temperature for at least 1 h.
Substances for screening. The compounds used for screening are listed in Table 1 Electrophysiological recordings. A glass pipette was made from a glass capillary (GC150-10, Harvard Apparatus) using a puller (P-1000, Sutter Instrument) to have a pipette resistance of 4-10 MΩ. KCl-based pipette solution was used as the internal solution. For patch-clamp recordings, an amplifier (Axopatch 200B, Molecular Devices) and a digitizer (Axon Digidata 1550A, Molecular Devices) were used. After identifying a cell expressing YC, the cell was contacted and ruptured with a glass pipette and maintained in a whole-cell current clamp mode. Negative current was injected to suppress spontaneous firing. Once the resting membrane potential was stable for > 30 s, command current (300 pA, 5 ms) was injected with a specific frequency (1, 2, 5 and 10 Hz) for 10 s sequentially with a gap of more than 1 min between each frequency. Data was acquired using software (Clampex 10.7, Molecular Devices).

Substance screening.
To monitor cell autonomous effects and to suppress the effects of synaptic inputs from other neurons, the voltage-gated sodium channel blocker tetrodotoxin (1 μM) was added to the aCSF. For a single brain slice, 10 substances, at most, were screened sequentially. Each candidate substance was dissolved in aCSF. The solution was then applied for 2 min via perfusion. The time between applications was at least 5 min. When any change in calcium signal was observed, the next substance was not applied until the signal returned to baseline and was stable for an additional 5 min. As controls, for detection of baseline, increased and decreased calcium concentrations, aCSF, glutamate and GABA were applied, respectively. Each substance was examined at least twice in different orders in multiple slices from different animals (Supplementary Table S3).

Antagonist experiments.
To examine the effects of an antagonist, the antagonist was applied for at least 3 min before the onset of the subject substance application. The subject substance was diluted into the antagonist solution and applied for 2 min. The antagonist was further applied for at least 5 min after the offset of substance application. After antagonist application, normal aCSF was perfused.
Analysis. Images of YFP and CFP were motion corrected and aligned if needed using an original program based on the Scale-Invariant Feature Transform 63 written in MATLAB (R2019a, MathWorks). Regions of interest (ROIs) were drawn to surround cell bodies. ROIs that included two or more cells, or cells that disappeared before the end of an experiment were omitted. The intensity of YFP and CFP was measured using Fiji 64 , and subsequent calculations including the Y/C ratio were performed in MATLAB. For patch-clamp recordings, the raw Y/C ratio was used for analysis. The peak Y/C ratio during current injection of each stimulation was subtracted by the mean Y/C ratio during the period 30 s before current injection (R 0 ) to obtain the peak ΔR. ΔR was divided by R 0 to obtain the peak ΔR/R 0 .
For screening and antagonist experiments, the value of the Y/C ratio was corrected using the calculations described in the supplementary methods.
For screening, the mean Z-score during the 5 min after the onset of each substance application was used. Quartiles of the mean Z-score for each substance were calculated from the combined screening experiments. When the third quartile was greater than 2, the substance was defined as increasing [Ca 2+ ] i ; conversely, when the first quartile was less than − 1, the substance was defined as decreasing [Ca 2+ ] i . These definitions were established to conservatively detect a clear calcium change based on mean Z-scores observed after application of glutamate and GABA.
For antagonist experiments, the integral of the Z-score during the 5 min after onset of subject substance application was calculated for both the 1st (substance only) and 2nd (with or without antagonist) sessions (Z 1st and Z 2nd , respectively). Z 2nd was divided by Z 1st to obtain the 2nd/1st ratio for control and antagonist effects.
Statistics. All data were presented as the mean ± standard error of the mean (s.e.m.). Statistical analysis was performed using software (OriginPro, Version 2019, OriginLab). A p-value less than 0.05 was considered statistically significant. To test normality, the Shapiro-Wilk test was performed. When data did not show normality, to test differences in distribution between control and subject compounds, the Mann-Whitney U test was performed. To test population differences, a Kruskal-Wallis test followed by the Dunn's test was performed.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.