Skip to main content

Assessing the treatment of cannabidiolic acid methyl ester: a stable synthetic analogue of cannabidiolic acid on c-Fos and NeuN expression in the hypothalamus of rats

Abstract

Background

Cannabidiol (CBD), the non-psychotropic compound from Cannabis sativa, shows positive results on controlling several health disturbances; however, comparable data regarding additional chemical from C. sativa, such as cannabidiolic acid (CBDA), is scarce due to its instability. To address this limitation, a stable CBDA analogue, CBDA methyl ester (HU-580), was synthetized and showed CBDA-like effects. Recently, we described that HU-580 increased wakefulness and wake-related neurochemicals.

Objective

To extend the comprehension of HU-580´s properties on waking, the c-Fos and NeuN expression in a wake-linked brain area, the hypothalamus was evaluated.

Methods

c-Fos and NeuN expression in hypothalamic sections were analyzed after the injections of HU-580 (0.1 or 100 μg/kg, i.p.).

Results

Systemic administrations of HU-580 increased c-Fos and neuronal nuclei (NeuN) expression in hypothalamic nuclei, including the dorsomedial hypothalamic nucleus dorsal part, dorsomedial hypothalamic nucleus compact part, and dorsomedial hypothalamic nucleus ventral part.

Conclusion

HU-580 increased c-Fos and NeuN immunoreactivity in hypothalamus nuclei suggesting that this drug might modulate the sleep–wake cycle by engaging the hypothalamus.

Background

Several pieces of evidence have suggested that the non-psychotropic molecule derived from Cannabis sativa, cannabidiol (CBD), exerts positive therapeutic pharmacological properties for the management of several health disturbances, including epilepsy, pain, anxiety, among many others (Fraguas-Sánchez and Torres-Suárez, 2018; Friedman and Wongvravit, 2018; Millar, et al. 2019; Premoli, et al. 2019; Pretzsch, et al. 2019). However, only limited experimental data is available concerning the effects of another molecule from C. sativa, cannabidiolic acid (CBDA). The lack of evidence of this cannabinoid lies in its chemical instability (Citti, et al. 2018; Mechoulam and Hanus, 2002). Hence, to tackle this problem, our group has synthetized a stable CBDA analogue named CBDA methyl ester of HU-580, which produces certain CBDA-like effects more potently than CBDA. These pharmacological properties of HU-580 include the management of anxiety and depression in experimental models (Hen-Shoval, et al. 2018; Pertwee, et al. 2018). In addition, HU-580 modulates the sleep–wake cycle by increasing wakefulness as well as wake-related neurochemicals such as dopamine, serotonin, adenosine, and acetylcholine (Murillo-Rodríguez et al. 2020). Despite these fascinating results, the mechanism of action activated by HU-580 for modulation of the sleep–wake cycle is unknown. Therefore, to provide further evidence of the neurobiological effects of HU-580 on sleep control, we evaluated whether administrations of this chemical might induce changes on the expression of neural markers, such as c-Fos and neuronal nuclei (NeuN), in the hypothalamus, a brain region that has been linked to the regulation of wakefulness (Aston-Jones et al. 2001; Chen, et al. 2018; Saper et al. 2005; Sapin et al. 2010).

Methods

Ethics

All experimental procedures were performed in accordance with the Research and Ethics Committees of our Institution and met the guidelines of Mexican Standards Related to Use and Management of Laboratory Animals (DOF. NOM-062-Z00-1999), fulfilling the ARRIVE guidelines in accordance with the U.K. Animals (Scientific Procedures; Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments) as well as the National Institute of Health (NIH publication No. 80–23, revised 1996 and Guide for the Care and Use of Laboratory Animals, 8th edition, 2011).

Animals

Male Wistar rats (N = 15; 250–300 g) were singly housed in transparent acrylic cages (48 × 20 × 27 cm) with standard bedding material, chow pellets (Purina Rat Chow, México), and tap water ad libitum. Experimental conditions included housing all rats at 12-h light/dark cycle (lights on at 07:00 h; 200 lx), controlled temperature (22 ± 1 °C), and relative humidity (60 ± 10%). All efforts were made to minimize animal suffering and using the minimal number of animals required to produce reliable results.

Chemicals

HU-580 was synthetized by our group as previously described and prepared in a vehicle (VEH) solution (Hen-Shoval, et al. 2018; Pertwee, et al. 2018). Paraformaldehyde, phosphate-buffered saline (PBS), sucrose, glycerol, dimethyl sulfoxide (DMSO), solvents, and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or elsewhere. Reagents for immunohistochemical studies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), Millipore (Billerica, MA, USA), and Vector Laboratories (Burlingame, CA, USA).

Experimental design

The rats were assigned randomly to one of two treatment conditions: vehicle (1 mL/i.p.; n = 5) or HU-580 (0.1 or 100 μg/kg/1 mL; i.p.; n = 5; each dose). To avoid circadian influences on the expression of c-Fos or NeuN, all systemic administrations were given 1 h after the beginning of the lights-on period. In addition, we used a single-blind study in which members of the laboratory that applied the administrations were not aware about the code of the treatments.

Brain tissue collection

One hour after the treatments were applied, rats were sacrificed by a lethal dose of pentobarbital (150 mg/kg; i.p.) and perfused intracardially with sodium chloride (0.9%) followed by paraformaldehyde (4.0%; Sigma-Aldrich, St. Louis, MO, USA) in PBS (0.1 M, pH 7.1) as previously described (Macías-Triana et al. 2020). Staff members of the laboratory blinded to the code of rats developed the perfusion in all rats. Later, the brains were removed, post-fixed in the same fixative solution overnight at 4 °C and then equilibrated following previous procedures by sucrose immersion (10, 20, or 30% sucrose/0.1 M PBS during 24 h each concentration or until tissue sinks). After complete equilibration by infiltration of the sucrose, the brains were cut in coronal Sects. (20 μm thickness) and collected in 1:5 serial order using a Portable Bench-top Cryostat (Leica CM1100. Leica Microsystems GmbH. Wetzlar, Germany; Macías-Triana et al. 2020). To avoid experimental bias, member of the laboratory blinded to the code of rats cut the brains. Due to the hypothalamus has been linked with wakefulness control (Heiss et al. 2018; Latifi et al. 2018; Naganuma et al. 2019), this brain area was chosen for the immunohistochemical study. The identification of the hypothalamic nuclei, including the dorsomedial hypothalamic nucleus dorsal part (DMD), dorsomedial hypothalamic nucleus compact part (DMC), and dorsomedial hypothalamic nucleus ventral part (DMV) was done by the aid of the Rat Brain Atlas which included coordinates from − 2.28 to − 3.48 mm (from Bregma according the Rat Brain Atlas (Paxinos and Watson, 2005). Once collected, the sections were stored in cryoprotective solution (glycerol [20%] and DMSO [2%] in sodium phosphate [0.1 M]) at – 20 °C (Thermo Fisher Scientific Revco, Waltham, MA, USA). The whole brain collection procedures were developed as previously reported (de-la-Cruz et al. 2018).

c-Fos and NeuN immunohistochemical analysis

Since the immediate early gene c-fos (Chung, 2015; Kovács, 2008) and NeuN (Duan et al. 2016; Gusel'nikova and Korzhevskiy, 2015) have long been known as molecular markers of neuronal activity, then the expression of these proteins was addressed in DMD, DMC, and DMV in control and HU-treated animals. In detail, slides from control and HU-580 groups (0.1 or 100 μg/kg; i.p.) were prepared for c-Fos and NeuN immunohistochemical analysis using standardized procedures as previously described (Ni et al. 2020; Plaisier et al. 2020). Serial coronal cryostat sections of the DMD, DMC, and DMV were processed for c-Fos and NeuN immunoreactivity, imaged, and quantified as described previously (de-la-Cruz et al. 2018). The slides were washed 3 times in phosphate-buffered saline (PBS; 0.1 M, pH 7.3) and later to inactivate the endogenous peroxidase, the sections were incubated in periodic acid (0.28%) during 1 min at room temperature, with hydrogen peroxide (H2O2; 3%) and methanol (10%) in PBS (0.1 M) for 20 min at room temperature. Next, the slides were washed 3 times in PBS (0.1 M, pH 7.3) and blocked with donkey or goat serum (10%) diluted in PBS (containing 0.2% Triton X-100. Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the slides were incubated with the corresponding primary antibody at 4 °C (Goat anti-c-fos 1:100; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) or mouse anti-NeuN (1:500; Millipore. Billerica, MA, USA) overnight. On the next day, the slides were again washed 3 times in PBS (0.1 M, pH 7.3) and incubated for 2 h at room temperature with the respective biotinylated secondary antibody (1:250 dilution, goat anti-mouse IgG; Sigma-Aldrich, St. Louis, MO, USA. Donkey anti-rabbit IgG; Vector Laboratories. Burlingame, CA, USA). Upon the application of the secondary antibody, the slides were washed another 3 times in PBS (0.1 M, pH 7.3) and incubated with the peroxidase complex (1:2000. Sigma-Aldrich, St Louis, MO, USA) for 1 h in a dark room. Lastly, following 3 washes in PBS, the immunoreactivity was revealed by exposing the sections to diaminobenzidine (0.05%; Sigma-Aldrich. St Louis, MO, USA) and H2O2 (0.03%) in PBS. The reaction was stopped using PBS and slides were then washed several times in PBS again. Once immunoreactivity was achieved, all slides were mounted onto chrome alum gelatin-coated slides, dehydrated through graded alcohols, cleared in xylene and cover slipped with histology slide mounting medium (DPX Mountant, Sigma-Aldrich, St Louis, MO, USA). To confirm the reproducibility of the immunohistochemical experiments, batches containing approximately the same number of slides from the experimental groups were stained using the same primary antibody simultaneously whereas the negative controls included slides analyzed under an identical immunohistochemical procedure with the exception that 1% bovine serum albumin in PBS was substituted for the primary antibody. One observer blind to the experimental codes of slides developed the c-Fos and NeuN immunohistochemistry.

Imaging and image analysis of c-Fos- and NeuN-positive neurons

A Rat Brain Atlas (Paxinos and Watson, 2005) was used as a reference to identify the c-Fos and NeuN labeled neurons in DMD, DMC, and DMV. Immunoreactivity was visualized with an Axio Imager Microscope (A2m, Carl Zeiss AG, Oberkochen, Germany) with an attached microscope camera (AxioCam, Carl Zeiss AG, Oberkochen, Germany). The images were acquired using a computerized image analysis system ZEN (Blue Edition, Carl Zeiss AG, Oberkochen, Germany). A laboratory staff member blinded to the code of all slides, counted the c-Fos- and NeuN-positive immunostaining as previously reported (de-la-Cruz et al. 2018).

Statistical analysis

Using StatView software (version 5.0.0, SAS Institute, USA), data were analyzed by one-way analysis of variance (ANOVA) applied with multiple comparisons using the Scheffé’s post hoc analysis. Differences between groups were considered statistically significant at values of P < 0.05. Results are expressed as mean ± S.E.M. For investigating the relationship among the HU-580 doses and c-Fos and NeuN expression, Pearson’s correlation coefficient (r) was used (StatView; version 5.0.0, SAS Institute, USA). The strength of association between these variables was established if r ≥ 0.6 and P < 0.05. In addition, linear regression analysis (R2) was used to test if the dosage of HU-580 (0.1 or 100 μg/kg; i.p.) significantly would predict the increase the number of positive c-Fos and NeuN neurons. Significant statistical values for R2 were determined within the range of 0–1 and P < 0.05.

Results

Expression of c-Fos and NeuN immunoreactivity in the hypothalamic nuclei in response to HU-580

To test whether HU-580 promoted changes in c-Fos and NeuN expression, we analyzed the immunohistochemical staining in the hypothalamic nuclei including the DMD, DMC, and DMV. Figure 1A displays a representative illustration depicting the location of the relative density of c-Fos and NeuN immunoreactivity in the targeted areas. As shown for c-Fos analysis, and compared to control (Fig. 1B), systemic injections of HU-580 (0.1 or 100 μg/kg; i.p.; Fig. 1C, D, respectively) increased c-Fos expression in DMD, DMC, and DMV. Moreover, compared to the control group (Fig. 1E), similar findings were observed in NeuN expression in rats treated with HU-580 (0.1 or 100 μg/kg; i.p.; Fig. 1F, G, respectively).

Fig. 1
figure1

The schematic illustration from the rat brain atlas (Paxinos and Watson, 2005) showing the hypothalamus section taken for the immunohistochemical studies (Panel A). A representative illustration depicting the location of the relative density of c-Fos and NeuN expression in the hypothalamus. Drawing obtained from Paxinos and Watson's Atlas (2005)

Number of c-Fos-positive neurons in the hypothalamic nuclei in response to HU-580

HU-treated (0.1 or 100 μg/kg; i.p.) rats showed a significant increase in the number of Fos-positive neurons in the hypothalamic nuclei as compared to control group (F(2, 12) = 24.738; P < 0.0001; Fig. 2A). Further post hoc analysis showed significant differences between experimental treatments (Scheffé’s post hoc test: control vs. HU-580 (0.1 μg/kg), P < 0.01; control vs. HU-580 (100 μg/kg), P < 0.0001; HU-580 (0.1ug/Kg) vs. HU-580 (100 μg/kg), P < 0.01).

Fig. 2
figure2

The number of c-Fos-positive neurons in the hypothalamic nuclei in response to HU-580. A The significant increase in c-Fos expression in hypothalamus nuclei from HU-treated rats ((0.1 or 100 μg/kg; i.p.) as compared to controls (F(2, 12) = 24–738; P < 0.0001). The post hoc analysis showed significant differences among the experimental data (Scheffé’s post hoc test: control vs. HU-580 (0.1 μg/kg, P < 0.01; control vs. HU-580 (100 μg/kg), P < 0.0001; HU-580 (0.1 μg/kg) vs. HU-580 (100 μg/kg), P < 0.01). B The Pearson’s correlation coefficient analysis with a significant and positive relationship between the used doses of HU-580 (0.1 or 100 μg/kg; i.p.) and the Fos immunoreactivity (r = 0.6, P < 0.0002). Finally, the linear regression analysis showed that administrations of different doses of HU-580 predicted the enhancement in the number of Fos expression in the hypothalamic nuclei (R2 = 0.6, P < 0.0005; B)

Our next result, from the Pearson’s correlation coefficient analysis, showed a significant and positive relationship between the tested doses of HU-580 (0.1 or 100 μg/kg; i.p.) and the Fos immunoreactivity (r = 0.6, P < 0.0002; Fig. 2B). Current findings suggest a significant dose-dependent interaction between HU-580 and c-Fos expression in hypothalamic nuclei. In regard to the linear regression analysis, we fund that HU-580 (0.1 or 100 μg/kg; i.p.) significantly would predict the enhancements on quantitative Fos expression. Thus, administrations of different doses of HU-580 predicted the increase in the number of Fos immunoreactivity in hypothalamic nuclei (R2 = 0.6, P < 0.0005; Fig. 2B). We conclude that as higher doses of HU-580 were administered, higher Fos expression was found in hypothalamic nuclei.

Number of NeuN-positive neurons in the hypothalamic nuclei in response to HU-580

In regard to the effects of HU-580 on NeuN expression, we found a significant increase in this molecular marker in rats that received a systemic injections of HU-580 (0.1 or 100 μg/kg) compared to control group (F(2, 12) = 11.334; P < 0.001; Fig. 3A). The Scheffé’s post hoc test displayed significant differences among the experimental trials for NeuN immunoexpression in the hypothalamic nuclei (control vs. HU-580 (0.1 ug/kg), P = 0.2; control vs. HU-580 (100 μg/kg), P < 0.001; HU-580 (0.1 μg/kg) vs. HU-580 (100 μg/kg), P < 0.04). Regarding the Pearson’s correlation coefficient analysis among the doses of HU-580 (0.1 or 100 μg/kg; i.p.) and the NeuN expression, a significant and positive relationship between these experimental variables was found (r = 0.5, P < 0.0008; Fig. 3B). Therefore, data suggest that significant interactions among the different doses of HU-580 and NeuN activity in hypothalamic nuclei were present. In addition, the linear regression analysis indicated that HU-580 (0.1 or 100 μg/kg; i.p.) produced a significantly dose-related increase in quantitative NeuN neuronal expression in hypothalamic nuclei (R2 = 0.5, P < 0.001; Fig. 3C). We conclude that higher doses of HU-580 promote higher NeuN expression in hypothalamic nuclei.

Fig. 3
figure3

The number of NeuN positive neurons in the hypothalamus in response to HU-580. Systemic injections of the highest dose of HU-580 (100μg/Kg) increased the number of NeuN positive immunoreactive neurons in the hypothalamus (Panel A; F(2, 12)= 11.334; P< 0.001). The Scheffé´s post hoc test displayed significant differences between the experimental groups for NeuN expression in the hypothalamus (Control vs. HU-580 (0.1μg/Kg), P= 0.2; Control vs. HU-580 (100μg/Kg), P< 0.001; HU-580 (0.1μg/Kg) vs. HU-580 (100μg/Kg), P< 0.04). The Pearson’s correlation coefficient analysis among the doses of HU-580 (0.1 or 100μg/Kg; i.p.) and the NeuN expression showed a significant and positive relationship between these experimental variables (r= 0.5, P< 0.0008; Panel B) whereas the linear regression analysis indicated that HU-580 (0.1 or 100μg/Kg; i.p.) produced a significantly increase in quantitative NeuN neuronal expression in the hypothalamus (R2= 0.5, P< 0.001)

Discussion

Limited research has revealed the pharmacological properties of cannabidiolic acid (CBDA), a constituent of Cannabis sativa. However, CBDA is rather unstable (Chou et al. 2003; Citti et al. 2018; Crombie and Crombie, 1977), suggesting that its chemical instability proved difficult and need further studies. To address this issue, a stable analogue of CBDA named CBDA methyl ester (HU-580) was recently synthesized showing greater potency than CBDA at, for example, producing apparent anxiolytic and antidepressant effects in vivo (Hen-Shoval, et al. 2018; Pertwee, et al. 2018). To gain knowledge regarding the pharmacological profile of HU-580 on neurobiological functions, we have published that systemic injections of this compound induced wake-promoting effects accompanied by enhancements in wake-related neurochemicals such as dopamine, adenosine, and acetylcholine (Murillo-Rodríguez et al. 2020). These fascinating findings prompted a need to identify the putative neuroanatomical substrate involved in HU-580-induced sleep modulation. Thus, here we have demonstrated that systemic injections of HU-580 (0.1 or 100 μg/kg; i.p.) promoted neuronal activation as determined by c-Fos and NeuN immunohistochemical assays. Under our conditions, HU-580 enhanced c-Fos and NeuN expression in hypothalamic nuclei comprising dorsomedial hypothalamic nucleus dorsal part (DMD), dorsomedial hypothalamic nucleus compact part (DMC), and dorsomedial hypothalamic nucleus ventral part (DMV). The data we have obtained suggest that HU-580 might exert wake-promoting effects via the engagement of neuronal activity located in DMD, DMC, and DMV. Even though the mechanism of action of HU-580 underlying its regulation of wakefulness has not been discovered yet, we would like to draw the following hypothetical frame: HU-580 seems to induce neuronal activity evaluated by c-Fos and NeuN immunoreactivity in hypothalamic nuclei which has been suggested as modulator of wakefulness (Aston-Jones et al. 2001; Chen, et al. 2018; Saper et al. 2005; Sapin et al. 2010). Further studies support our hypothetical frame in regard the likely engagement of hypothalamic nuclei in the wake-promoting effects of HU-580 since current evidence shows that prolonged wakefulness induces an increase in c-Fos expression (Azeez et al. 2018).

Limitations of the study

Indeed, we recognize several limitations of our findings as follows: (i) the c-Fos study lacks the characterization of certain neuronal types. Moreover, despite that Fos shows a fast and transient induction curve in activated neurons (Kim, et al. 2019) and the half-life of this protein is ~ 40–60 min (Kovács, 2008; Stancovski et al. 1995), the activity of Fos is not strictly correlated with neuronal activity (Cirelli and Tononi, 2000; Ito et al. 2005); (ii) some additional neuronal populations might be involved in HU-580’s effects. For instance, DMD sends rostral afferents to the ventrolateral preoptic nucleus (Deurveilher et al. 2002; Lu et al. 2001), a region in which lesions cause insomnia (Gvilia, 2010; Lüthi, 2019; Peyron, et al. 1998). Therefore, it is likely the engagement of the ventrolateral preoptic nucleus in HU-580’s effects; (iii) to advance the current comprehension of the mechanism underlying the effects of HU-580 on c-Fos and NeuN expression, it will be necessary to determine the identity of the responding neurons to HU-580 and to understand how the drug activates these neurons. Since the hypothalamic nuclei also projects to the lateral hypothalamic area which many neurons contain the wake-promoting neuropeptide hypocretin also known as orexin (Arrigoni et al. 2019; Backholer et al. 2009; Chen et al. 2018; Eyigor, et al. 2012; Nollet et al. 2011; Ono and Yamanaka, 2017; Peyron and Kilduff, 2017; Sapin et al. 2010; Sakurai, et al. 1998; Tyree et al. 2018; Wang, et al. 2018), it is highly possible that neurons reacting to HU-580 might be hypocretinergic; (iv) whether HU-580 modulates neurons located in hypothalamic nuclei will require further study by using alternative experimental approaches such as electrophysiological recordings, double-staining, or optogenetic procedures. The study, in its present form, is very limited in scope; however, it provides, for the very first time, that HU-580 exerts effects on c-Fos and NeuN expression in hypothalamus.

Conclusions

The new pharmacological data we have now obtained suggest that HU-580 can enhance the expression of c-Fos and NeuN activity in hypothalamus, a brain area related to the regulation of wakefulness. The results obtained in this investigation allow to conclude that HU-580 might engage hypothalamic nuclei activity in rats for regulation of wakefulness. Indeed, further studies are still required to determine the mechanism of action that underlies the sleep–wake cycle effects of HU-580.

Availability of data and materials

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

Abbreviations

ANOVA:

Analysis of variance

CBD:

Cannabidiol

CBDA:

Cannabidiolic acid

DMC:

Dorsomedial hypothalamic nucleus compact part

DMD:

Dorsomedial hypothalamic nucleus dorsal part

DMOS:

Dimethyl sulfoxide

DMV:

Dorsomedial hypothalamic nucleus ventral part

HU-580:

CBDA methyl ester

PBS:

Phosphate-buffered saline

r :

Pearson’s correlation coefficient

R 2 :

Linear regression analysis

VEH:

Vehicle

References

  1. Arrigoni E, Chee MJS, Fuller PM. To eat or to sleep: that is a lateral hypothalamic question. Neuropharmacol. 2019;154:34–49. https://doi.org/10.1016/j.neuropharm.2018.11.017.

    CAS  Article  Google Scholar 

  2. Aston-Jones G, Chen S, Zhu Y, Oshinsky ML. A neural circuit for circadian regulation of arousal. Nat Neurosci. 2001;4:732–8. https://doi.org/10.1038/89522.

    CAS  Article  PubMed  Google Scholar 

  3. Azeez IA, Del Gallo F, Cristino L, Bentivoglio M. Daily fluctuation of orexin neuron activity and wiring: the challenge of “chronoconnectivity.” Front Pharmacol. 2018;9:1061. https://doi.org/10.3389/fphar.2018.01061.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Backholer K, Smith J, Clarke IJ. Melanocortins may stimulate reproduction by activating orexin neurons in the dorsomedial hypothalamus and kisspeptin neurons in the preoptic area of the ewe. Endocrinol. 2009;150:5488–97. https://doi.org/10.1210/en.2009-0604.

    CAS  Article  Google Scholar 

  5. Chen KS, Xu M, Zhang Z, et al. A hypothalamic switch for REM and non-REM sleep. Neuron. 2018;97:1168-1176.e4. https://doi.org/10.1016/j.neuron.2018.02.005.

    CAS  Article  PubMed  Google Scholar 

  6. Chou TC, Scammell TE, Gooley JJ, Gaus SE, Saper CB, Lu J. Critical role of dorsomedial hypothalamic nucleus in a wide range of behavioral circadian rhythms. J Neurosci. 2003;23:10691–702. https://doi.org/10.1523/JNEUROSCI.23-33-10691.2003.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Chung LA. Brief introduction to the transduction of neural activity into Fos signal. Dev Reproduction. 2015;19:61–7. https://doi.org/10.12717/DR.2015.19.2.061.

    Article  Google Scholar 

  8. Cirelli C, Tononi G. On the functional significance of c-fos induction during the sleep-waking cycle. Sleep. 2000;23:453–69.

    CAS  Article  Google Scholar 

  9. Citti C, Pacchetti B, Vandelli MA, Forni F, Cannazza G. Analysis of cannabinoids in commercial hemp seed oil and decarboxylation kinetics studies of cannabidiolic acid (CBDA). J Pharm Biomed Anal. 2018;149:532–40. https://doi.org/10.1016/j.jpba.2017.11.044.

    CAS  Article  PubMed  Google Scholar 

  10. Citti C, Palazzoli F, Licata M, et al. Untargeted rat brain metabolomics after oral administration of a single high dose of cannabidiol. J Pharm Biomed Anal. 2018;161:1–11. https://doi.org/10.1016/j.jpba.2018.08.021.

    CAS  Article  PubMed  Google Scholar 

  11. Crombie L, Crombie WML. Cannabinoid acids and esters: miniaturized synthesis and chromatographic study. Phytochem. 1977;16:1413–20. https://doi.org/10.1016/S0031-9422(00)88794-4.

    CAS  Article  Google Scholar 

  12. de-la-Cruz M, Millán-Aldaco D, Soriano-Nava DM, Drucker-Colín R, Murillo- Rodríguez E. The artificial sweetener Splenda intake promotes changes in expression of c-Fos and NeuN in hypothalamus and hippocampus of rats. Brain Res. 2018;1700:181–9. https://doi.org/10.1016/j.brainres.2018.09.006.

    CAS  Article  PubMed  Google Scholar 

  13. Deurveilher S, Burns J, Semba K. Indirect projections from the suprachiasmatic nucleus to the ventrolateral preoptic nucleus: a dual tract-tracing study in rat. Eur J Neurosci. 2002;16:1195–213. https://doi.org/10.1046/j.1460-9568.2002.02196.x.

    Article  PubMed  Google Scholar 

  14. Duan W, Zhang YP, Hou Z, et al. Novel Insights into NeuN: from Neuronal Marker to Splicing Regulator. Mol Neurobiol. 2016;53:1637–47. https://doi.org/10.1007/s12035-015-9122-5.

    CAS  Article  PubMed  Google Scholar 

  15. Eyigor O, Minbay Z, Kafa IM. Chapter Eleven - Glutamate and Orexin Neurons. Vitam Horm. 2012;89:209–22. https://doi.org/10.1016/B978-0-12-394623-2.00011-1.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Fraguas-Sánchez AI, Torres-Suárez AI. Medical use of cannabinoids. Drugs. 2018;78:1665–703. https://doi.org/10.1007/s40265-018-0996-1.

    CAS  Article  PubMed  Google Scholar 

  17. Friedman LK, Wongvravit JP. Anticonvulsant and neuroprotective effects of cannabidiol during the juvenile period. J Neuropathol Exp Neurol. 2018;77:904–19. https://doi.org/10.1093/jnen/nly069.

    CAS  Article  PubMed  Google Scholar 

  18. Gusel’nikova VV, Korzhevskiy DE. NeuN as a neuronal nuclear antigen and neuron differentiation marker. Acta Naturae. 2015;7:42–7.

    CAS  Article  Google Scholar 

  19. Gvilia I. Underlying brain mechanisms that regulate sleep-wakefulness cycles. Int Rev Neurobiol. 2010;93:1–21. https://doi.org/10.1016/S0074-7742(10)93001-8.

    Article  PubMed  Google Scholar 

  20. Heiss JE, Yamanaka A, Kilduff TS. Parallel arousal pathways in the lateral hypothalamus. eNeuro. 2018;5:ENEURO.0228–18.2018. https://doi.org/10.1523/ENEURO.0228-18.2018.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Hen-Shoval D, Amar S, Shbiro L, et al. Acute oral cannabidiolic acid methyl ester reduces depression-like behavior in two genetic animal models of depression. Behav Brain Res. 2018;51:1–3. https://doi.org/10.1016/j.bbr.2018.05.027.

    CAS  Article  Google Scholar 

  22. Ito Y, Inoue D, Kido S, Matsumoto T. c-Fos degradation by the ubiquitin-proteasome proteolytic pathway in osteoclast progenitors. Bone. 2005;37:842–9. https://doi.org/10.1016/j.bone.2005.04.030.

    CAS  Article  PubMed  Google Scholar 

  23. Kim SH, Park JY, Shin HE, et al. The influence of rapid eye movement sleep deprivation on nociceptive transmission and the duration of facial allodynia in rats: a behavioral and Fos immunohistochemical study. J Headache Pain. 2019;20:21. https://doi.org/10.1186/s10194-019-0977-0.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Kovács KJ. Measurement of immediate-early gene activation- c-fos and beyond. J Neuroendocrinol. 2008;20:665–72. https://doi.org/10.1111/j.1365-2826.2008.01734.x.

    CAS  Article  PubMed  Google Scholar 

  25. Latifi B, Adamantidis A, Bassetti C, Schmidt MH. Sleep-wake cycling and energy conservation: role of hypocretin and the lateral hypothalamus in dynamic state-dependent resource optimization. Front Neurol. 2018;9:790. https://doi.org/10.3389/fneur.2018.00790.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Lu L, Zhang YH, Chou TC, Gaus SE, Elmquist JK, Shiromani P, Saper CB. 2001 Contrasting effects of ibotenate lesions of the paraventricular nucleus and subparaventricular zone on sleep-wake cycle and temperature regulation. J Neurosci. 2001;21:4864–74. https://doi.org/10.1523/JNEUROSCI.21-13-04864.2001.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Lüthi A. Sleep: The Very Long Posited (VLPO) Synaptic pathways of arousal. Curr Biol. 2019;29:R1310–2. https://doi.org/10.1016/j.cub.2019.11.012.

    CAS  Article  PubMed  Google Scholar 

  28. Macías-Triana L, Romero-Cordero K, Tatum-Kuri A, Vera-Barrón A, Millán-Aldaco D, Arankowsky-Sandoval G, Piomelli D, Murillo-Rodríguez E. Exposure to the cannabinoid agonist WIN 55, 212–2 in adolescent rats causes sleep alterations that persist until adulthood. Eur J Pharmacol. 2020;874:172911. https://doi.org/10.1016/j.ejphar.2020.172911.

    CAS  Article  PubMed  Google Scholar 

  29. Mechoulam R, Hanus L. Cannabidiol: an overview of some chemical and pharmacological aspects. Part I: chemical aspects. Chem Phys Lipids. 2002;121:35–43. https://doi.org/10.1016/s0009-3084(02)00144-5.

    CAS  Article  PubMed  Google Scholar 

  30. Millar SA, Stone NL, Bellman ZD, Yates AS, England TJ, O’Sullivan SE. A systematic review of cannabidiol dosing in clinical populations. Brit J Clin Pharmacol. 2019;85:1888–900. https://doi.org/10.1111/bcp.14038.

    CAS  Article  Google Scholar 

  31. Murillo-Rodríguez E, Arankowsky-Sandoval G, Pertwee RG, Parker L, Mechoulam R. Sleep and neurochemical modulation by cannabidiolic acid methyl ester in rats. Brain Res Bull. 2020;155:166–73. https://doi.org/10.1016/j.brainresbull.2019.12.006.

    CAS  Article  PubMed  Google Scholar 

  32. Naganuma F, Kroeger D, Bandaru SS, Absi G, Madara JC, Vetrivelan R. Lateral hypothalamic neurotensin neurons promote arousal and hyperthermia. PLoS Biol. 2019;17:e3000172. https://doi.org/10.1371/journal.pbio.3000172

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Ni RJ, Wang J, Shu YM, Xu L, Zhou JN. Mapping of c-Fos expression in male tree shrew forebrain. Neurosci Lett. 2020;714:134603. https://doi.org/10.1016/j.neulet.2019.134603

    CAS  Article  PubMed  Google Scholar 

  34. Nollet M, Gaillard P, Minier F, Tanti A, Belzung C, Leman S. Activation of orexin neurons in dorsomedial/perifornical hypothalamus and antidepressant reversal in a rodent model of depression. Neuropharmacol. 2011;61:336–46. https://doi.org/10.1016/j.neuropharm.2011.04.022.

    CAS  Article  Google Scholar 

  35. Ono D, Yamanaka A. Hypothalamic regulation of the sleep/wake cycle. Neurosci Res. 2017;118:74–81. https://doi.org/10.1016/j.neures.2017.03.013.

    CAS  Article  PubMed  Google Scholar 

  36. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. San Diego: Academic; 2005.

    Google Scholar 

  37. Pertwee RG, Rock EM, Guenther K, et al. Cannabidiolic acid methyl ester, a stable synthetic analogue of cannabidiolic acid, can produce 5-HT1A receptor-mediated suppression of nausea and anxiety in rats. Brit J Pharmacol. 2018;175:100–12. https://doi.org/10.1111/bph.14073.

    CAS  Article  Google Scholar 

  38. Peyron C, Kilduff TS. Mapping the hypocretin/orexin neuronal system: an unexpectedly productive journey. J Neurosci. 2017;37:2268–72. https://doi.org/10.1523/JNEUROSCI.1708-16.2016.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Peyron C, Tighe DK, van den Pol AN, et al. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci. 1998;18:9996–10015. https://doi.org/10.1523/JNEUROSCI.18-23-09996.1998.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Plaisier F, Hume C, Menzies J. Neural connectivity between the hypothalamic supramammillary nucleus and appetite- and motivation-related regions of the rat brain. J Neuroendocrinol. 2020;32:e12829. https://doi.org/10.1111/jne.12829

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Premoli M, Aria F, Bonini SA, et al. Cannabidiol: recent advances and new insights for neuropsychiatric disorders treatment. Life Sci. 2019;224:120–7. https://doi.org/10.1016/j.lfs.2019.03.053.

    CAS  Article  PubMed  Google Scholar 

  42. Pretzsch CM, Voinescu B, Mendez MA, et al. The effect of cannabidiol (CBD) on low-frequency activity and functional connectivity in the brain of adults with and without autism spectrum disorder (ASD). J Psychopharmacol. 2019;33:1141–8. https://doi.org/10.1177/0269881119858306.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Sakurai T, Amemiya A, Ishii M, et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell. 1998;92:1–696. https://doi.org/10.1016/s0092-8674(02)09256-5.

    Article  PubMed  Google Scholar 

  44. Saper CB, Scammell TE, Lu J. Hypothalamic regulation of sleep and circadian rhythms. Nature. 2005;437:1257–63. https://doi.org/10.1038/nature04284.

    CAS  Article  PubMed  Google Scholar 

  45. Sapin E, Bérod A, Léger L, Herman PA, Luppi PH, Peyron C. A very large number of GABAergic neurons are activated in the tuberal hypothalamus during paradoxical (REM) sleep hypersomnia. PLoS ONE. 2010;5:e11766. https://doi.org/10.1371/journal.pone.0011766

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. Stancovski I, Gonen H, Orian A, Schwartz AL, Ciechanover A. Degradation of the proto-oncogene product c-Fos by the ubiquitin proteolytic system in vivo and in vitro: identification and characterization of the conjugating enzymes. Mol Cell Biol. 1995;15:7106–16. https://doi.org/10.1128/mcb.15.12.7106.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Tyree SM, Borniger JC, de Lecea L. Hypocretin as a Hub for Arousal and Motivation. Front Neurol. 2018;9:413. https://doi.org/10.3389/fneur.2018.00413.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Wang C, Wang Q, Ji B, et al. The orexin/receptor system: molecular mechanism and therapeutic potential for neurological diseases. Front Mol Neurosci. 2018;11:220. https://doi.org/10.3389/fnmol.2018.00220.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We have no acknowledgements.

Funding

This study was supported by the Escuela de Medicina, Universidad Anáhuac Mayab (Mérida, Yucatán. México), grant number PresInvEMR2019 given to E. M.-R.

Author information

Affiliations

Authors

Contributions

E M-R conceived, designed, performed the experiments, analyzed data and wrote the paper; D M-A performed research and collected data; G A-S, TY, RGP, LP, and RM analyzed data. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Eric Murillo-Rodríguez.

Ethics declarations

Ethics approval and consent to participate

All procedures were performed in accordance with the Research and Ethics Committees of our Institution and met the guidelines of Mexican Standards Related to Use and Management of Laboratory Animals (DOF. NOM-062-Z00-1999), fulfilling the ARRIVE guidelines in accordance with the U.K. Animals (Scientific Procedures; Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments) as well as the National Institute of Health (NIH publication No. 80–23, revised 1996 and Guide for the Care and Use of Laboratory Animals, 8th edition, 2011).

Consent for publication

All authors read and approved this manuscript for publication.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Murillo-Rodríguez, E., Millán-Aldaco, D., Arankowsky-Sandoval, G. et al. Assessing the treatment of cannabidiolic acid methyl ester: a stable synthetic analogue of cannabidiolic acid on c-Fos and NeuN expression in the hypothalamus of rats. J Cannabis Res 3, 31 (2021). https://doi.org/10.1186/s42238-021-00081-1

Download citation

Keywords

  • Cannabis
  • Hypothalamus
  • Rat
  • Sleep
  • Wakefulness