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Exposure to the cannabinoid agonist WIN 55, 212–2 in adolescent rats causes sleep alterations that persist until adulthood
European Journal of Pharmacology 874 (2020) 172911
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Full length article
Exposure to the cannabinoid agonist WIN 55, 212–2 in adolescent rats causes sleep alterations that persist until adulthood
T
Lorena Macías-Trianaa,b, Karen Romero-Corderoa,b, Agnes Tatum-Kuria,b, Alba Vera-Barróna,b, Diana Millán-Aldacoc, Gloria Arankowsky-Sandovald, Daniele Piomellie,f,g, Eric Murillo-Rodrígueza,b,∗ a
Laboratorio de Neurociencias Moleculares e Integrativas, Escuela de Medicina, División Ciencias de la Salud, Universidad Anáhuac Mayab, Mérida, Yucatán, Mexico Intercontinental Neuroscience Research Group, Mexico c Depto. de Neurociencia Cognitiva, División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México, Mexico d Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico e Department of Anatomy and Neurobiology, University of California, Irvine, Irvine, CA, USA f Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA g Department of Biological Chemistry, University of California, Irvine, Irvine, CA, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Adolescence Cannabinoids Sleep Rapid eye movement sleep WIN 55,212–2
Cannabis and, to a lesser extent, synthetic cannabinoids are used during adolescence, a period in which multiple brain areas are still undergoing development. Among such areas is the hypothalamus, which is implicated in the control of sleep-wake cycle. In the present report, we show that exposing adolescent rats to the cannabinoid receptor agonist WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg, i.p) for 14 days during adolescence (i.e., from post-natal day 30–44) resulted in significant sleep disturbances when the animals became adult (post-natal day 80). These included decreased wakefulness and enhanced rapid eye movement sleep. Furthermore, we found that labeling for NeuN, a marker of postmitotic neurons, was significantly increased the dorsomedial hypothalamic nucleus of rats treated with WIN 55, 212–2. The results suggest that excessive cannabinoid receptor activation during adolescence can persistently influence sleep patterns and neuronal activity later in life.
1. Introduction The endocannabinoid system is a neuromodulatory signaling complex that comprises cannabinoid receptors, their endogenous ligands, and proteins involved in the formation, transport and degradation of such ligands (Lu and Mackie, 2016). Investigation of the presence and distribution of the CB1 cannabinoid receptor in the rodent and human central nervous system has implicated this signaling system in multiple aspects of the development process (Erdozain et al., 2015; Harkany et al., 2008; Yi et al., 2016). Chronic use of cannabis during adolescence has been associated with a variety of neural and behavioral abnormalities, some of which may persist until adulthood (Chadwick et al., 2013; Meier et al., 2012; Renard et al., 2014; Rubino and Parolaro, 2016). Moreover, this vulnerability might involve brain areas linked to the control of key homeostatic functions, including as the sleep-wake cycle (Houston et al., 2014; Chen and Baram, 2015). Available data regarding the
impact of cannabinoid receptor stimulation and sleep alterations derive from studies conducted in adult animals and with either acute or subchronic treatment designs (Murillo-Rodríguez et al., 1998, 2001; 2003, 2013; 2016; Altman et al., 2019; Angarita et al., 2016; Furer et al., 2018; Goonawardena et al., 2011, 2015; Pava et al., 2014; ProspéroGarcía et al., 2016). By contrast, the persistent effects of prolonged cannabinoid receptor activation during adolescence on sleep, if any, are unknown. Amongst the elements involved in the plasticity of the brain during the development is the activity of neuronal nuclei (NeuN) protein which has been also been associated to assess the likely functional state of neurons in normal or pathological conditions (Gusel'nikova and Korzhevskiy, 2015; Duan et al., 2016). However, it is unknown whether prolonged cannabinoid receptor activation during adolescence contribute to induce effects on NeuN expression. In the present study, we report that daily injections of the cannabinoid agonist WIN-55, 212–2 in 30–44 day-old rats (i.e., during adolescence) caused significant
∗ Corresponding author. Laboratorio de Neurociencias Moleculares e Integrativas, Escuela de Medicina, División Ciencias de la Salud, Universidad Anáhuac Mayab, Carretera Mérida-Progreso Km. 15.5, A.P. 96 Cordemex, C.P. 97310, Mérida, Yucatán, Mexico. E-mail address: [email protected] (E. Murillo-Rodríguez).
https://doi.org/10.1016/j.ejphar.2020.172911 Received 31 July 2019; Received in revised form 10 December 2019; Accepted 7 January 2020 Available online 08 February 2020 0014-2999/ © 2020 Elsevier B.V. All rights reserved.
European Journal of Pharmacology 874 (2020) 172911
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attached onto the skull with dental cement. After surgery, the rats were transferred to individual cages with ad libitum access to food and water. The EEG/EMG electrodes surgery and habituation procedures were carried out as previously reported (Murillo-Rodríguez et al., 2019). The EEG/EMG electrodes remained in the skull of rats during 14 days. From the 14 days that rats hold the EEG/EMG electrodes, the first 7 days postsurgery belonged to the habitation/post-surgery recovery time whereas the remaining 7 days were considered for data analysis. However, the final sleep-wake cycle data analyses were taken from the second day once finishing the habitation/post-surgery recovery time. To eliminate experimental biases, the EEG/EMG electrode surgeries were conducted under blinded conditions.
alterations in the sleep-wake cycle when the animals reached adulthood. Additionally, we show that rats treated with WIN-55, 212-2 during adolescence display abnormally high levels of activity of the neuronal nuclear protein (NeuN), in the dorsomedial nucleus of the hypothalamus, a region involved in the control of the sleep-wake cycle (Aston-Jones et al., 2001; Arrigoni et al., 2018). 2. Materials and methods 2.1. Ethics approval The experimental procedures used in this study were approved by the Research and Ethics Committees of our Institution. All animal experiments met the ARRIVE guidelines and the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/ 63/EU for animal experiments as well as National Institute of Health (NIH publication No. 80–23, revised 1996) and Mexican Standards Related to Use and Management of Laboratory Animals (DOF. NOM062-Z00-1999). We made all possible efforts to minimize animal suffering and to reduce the number animals included in the study. All mandatory laboratory health and safety procedures were complied with in the course of conducting the experimental work reported.
2.6. Analysis of sleep recordings
WIN 55, 212–2 (PubChem CID: 5311501) was purchased from Sigma-Aldrich (St. Louis, MO. USA) and it was dissolved in a vehicle (VEH) composed of polyethylene glycol (PubChem CID: 174)/saline (5:95 v/v), as previously reported (Schoch et al., 2018). Additional reagents, chemicals and materials were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Immediately after surgeries, the six-pin plastic plug was connected to a 6-channel slip-ring commutator through a 50 cm shielded cable (Plastics One, Roanoke, VA, United States), allowing free moving of animals around the cage. From the slip-ring system an additional shielded cable was connected to the amplifier (Model M15LT 15A54; Grass Instruments. Quincy, MA. USA) to filter the EEG signal at 70 Hz (low-pass filter) and 0.3 Hz (high-pass filter). The EEG signals were continuously sampled at 128Hz by using a 100-bit analog-to-digital converter board (NI PCI-6033E Multifunction I/O Board and NI-DAQ Software, SCB-100 Shielded Connector Block. National Instruments. Austin, TX. USA). The EEG/EMG signals were visually scored in 12s epochs with the aid of the sleep-scoring software (ICELUS). The sleepwake cycle was characterized as follows: Wakefulness (W) consisted in desynchronization in the EEG as well as high EMG activity whereas high-amplitude slow waves with a low EMG tone relative to waking were the definition traces for slow wave sleep (SWS). Lastly, rapid eye movement sleep (REMS) was identified by regular theta activity across the EEG accompanied with low EMG activity. The EEG/EMG electrodes remained in the rat skull during 14 days. Thus, from the 14 days that rats hold the EEG/EMG electrodes, the first 7 days post-surgery belonged to the habitation/post-surgery recovery time whereas the remaining 7 days were considered for data analysis. However, the final sleep-wake cycle data analyses were taken from the second day once finishing the habitation/post-surgery recovery time. The sleep data were scored following previous procedures (Murillo-Rodríguez et al., 2019). To avoid bias, one observer blind to the experiment scored the sleep recordings.
2.4. Pharmacological administrations
2.7. Sleep deprivation
Animals (PND 30) were divided randomly into two experimental groups, and were given 14 daily injections of either vehicle (1 ml, intraperitoneally [i.p.]; n = 5) or WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg/ 1 ml, ip; n = 5 per dose). All treatments were given at 07:00 h. One observer blind to the experimental trials administered treatment.
The total sleep deprivation (TSD) procedure was carried our as previously described (Murillo-Rodríguez et al., 2017). Rats treated with vehicle (n = 5) or WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg; n = 5 per group) were kept awake for 6 h during the lights-on period (from 07:00–13:00h) by making loud noises, knocking the outer walls of the cages, or placing novel objects into the cages. The procedure was performed by a researcher blinded to treatment.
2.2. Animals A total of 20 male Wistar rats (N = 20; 250–300g; Vivarium from Centro de Investigaciones Regionales Dr. Hideyo Noguchi, Universidad Autónoma de Yucatán. Mérida, Yucatán. México) delivered on postnatal day (PND) 30 were singly housed in a climate-controlled room (humidity: 60 ± 10%; ambient temperature: 21 ± 1 °C) on a 12h light-dark cycle (lights-on: 07:00–19:00h; 200 lux) during all experiments. All rats had free access to Purina Rat Chow (Purina. México) and tap water. 2.3. Chemicals
2.5. Implanting EEG/EMG electrodes for sleep recording Adult animals (PND80) were anesthetized with a mixture of acepromazine (0.75 mg/kg), xylazine (2.5 mg/kg) and ketamine (22 mg/ kg) and were transferred to a stereotaxic frame (David Kopf Instruments. Tujunga, CA. USA) to implant electroencephalogram (EEG) and electromyogram (EMG) electrodes. The procedure included trepanation for the implantation of two stainless-steel screw electrodes placed 2 mm on either side of the sagittal sinus and 3 mm anterior to bregma (frontal cortex). Two additional screws were located 3 mm on either side of the sagittal sinus and 6 mm behind bregma (occipital cortex). Two EMG wire electrodes were inserted into the dorsal neck muscles. The surgical wounds were treated with antibiotics and anesthetics. Once implanted, the EEG/EMG electrode wires were connected into a six-pin plastic plug (Plastics One. Roanoke, VA, USA) and
2.8. Immunohistochemical studies At the end of the sleep-recording studies, rats (vehicle [n = 5] and WIN-treated animals [WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg; n = 5 each dose])) were intracardially perfused (de-la-Cruz et al., 2018). Brain coronal sections were obtained (20 μm thickness) and collected in 1:5 serial order using a Portable Bench-top Cryostat (Leica CM1100. Leica Microsystems GmbH. Wetzlar, Germany). Because of its neurobiological link with the sleep-wake cycle, hypothalamus was selected for analysis of NeuN expression (Ono and Yamanaka, 2017; Arrigoni et al., 2018). The identification of hypothalamus included slides from coordinates −0.12 to −3.48 mm (from bregma according the Rat Brain Atlas [Paxinos and Watson, 2005]). Next, standardized procedures for 2
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immunohistochemically detection of NeuN were used in collected samples (de-la-Cruz et al., 2018). The reproducibility of the immunohistochemical procedure was confirmed by repeating the whole procedure with additional batches containing approximately the same number of sections from all experimental groups with the same primary antibody. Sections under an identical immunostaining analysis with the exception that 1% bovine serum albumin in PBS substituted for the primary antibody were used as negative control. To minimize experimental bias, a researcher blinded to treatment performed the immunostaining experiments.
2.9. NeuN immunohistochemistry A similar number of sections from selected brain area from both hemispheres for each rat, for all experimental groups were counted for NeuN immunohistochemical expression. Slides were photographed with bright field, dark field microscopy (10× objective lens) with the aid of computer image analysis software (Microscope Zeiss Imager A.2. Carl Zeiss Microscopy, Oberkochen,Germany), a digital camera (AxioCam ICc1. Carl Zeiss) and the image analysis computer software (ZEN, 2012; blue edition. Carl Zeiss). The analysis of images comprised NeuN expression in sections of the hypothalamus (coordinates from bregma: 0.12 to −3.48 mm, according the Rat Brain Atlas [Paxinos and Watson, 2005]). A researcher blinded to treatment quantified NeuN labeling in the hypothalamus (bilateral sides) as described previously (de-la-Cruz et al., 2018). Fig. 1 shows the flowchart of interventions and procedures during the experiment.
2.10. Statistical analysis Results are shown as mean ± standard error of the mean. Statistical differences were determined by one-way ANOVA followed by Scheffé's post hoc test for multiple comparisons among the experimental groups. All statistical analyses were performed using StatView (version 5.0.0, SAS Institute, United States). No power analysis was performed.
3. Results 3.1. Effects of adolescence exposure to WIN 55,212–2 on total sleep time in adulthood
Fig. 1. Schematic representation of the different interventions and procedures during the experiment.
Adult rats (PND 80) that had received daily injections of the cannabinoid agonist WIN 55,212–2 (0.1, 0.3 or 1.0 mg/kg, i.p) from PND 30 to PND 44 displayed significant changes in W, SWS, and REMS. Adolescence exposure to the drug at the doses of 0.1 and 0.3 mg/kg was associated with decreased W (F(3, 13) = 24.586; P < 0.0001) and SWS (F(3, 13) = 31.264; P < 0.0001) as well as enhanced REMS (F(3, 13) = 10.208; P < 0.001; Fig. 2,A). At 1 mg/kg, WIN 55, 212–2 treatment caused effects opposite to those observed at the lower doses (Fig. 2, A). During the lights-on period (07:00–19:00h), rats that had been treated with low doses of WIN 55, 212–2 (0.1, 0.3 mg/kg) showed increased W (F(3, 13) = 10.622; P < 0.0008), decreased SWS (F(3, 13) = 14.512; P < 0.0002) as well as prolonged REMS (F(3, 13) = 4.05; P < 0.03; Fig. 2, B). Animals treated with the highest drug dose (1 mg/ kg) displayed increased W, decreased SWS and no change in REMS. During the lights-off period (19:00–07:00h; Fig. 2,C), rats treated with WIN 55,212-2 showed (i) a decrease in W (F(3, 13) = 24.256; P < 0.0001), which was especially marked at the doses of 0.1 and 0.3 mg/kg; (ii) various changes in SWS (increase at 0.1 and 1 mg/kg; decrease at 0.3 mg/kg) (F(3, 13) = 8.497; P < 0.002); and (iii) inverted-U changes in REMS (F(3, 13) = 55.595; P < 0.0001).
3.2. Effects of adolescence exposure to WIN 55,212–2 on the sleep-wake cycle in adulthood A 24h time-course of W, SWS and REMS in rats treated with vehicle or WIN 55, 212–2 is illustrated in Fig. 3. Adolescence exposure to the cannabinoid agonist was associated with a striking insomnia-like phenotype in adults. Alertness was increased (Fig. 3, A) while SWS (Fig. 3, B) and REMS (Fig. 3, C) were decreased during the lights-on period. Opposite changes were observed during the lights-off phase. Time points for W, SWS and REMS showed statistical differences among experimental groups (P < 0.05). Remarkably, animals treated with WIN 55, 212-2 displayed arrhythmic activity in W, SWS and REMS. 3.3. Effects of sleep deprivation In the TSD experiment, control rats experienced the expected rebound changes in SWS and REMS (Fig. 4, A). Such changes were markedly suppressed in animals that had been treated with WIN 55, 212-2 during adolescence: both SWS (F(3, 13) = 52.253; P < 0.0001) and REMS (F(3, 13) = 75.2586; P < 0.0001) were substantially reduced. A time-course analysis of sleep rebound period for SWS and 3
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Fig. 2. Effects on total time (24h of sleep recordings) of wakefulness (W; (F(3, = 24.586; P < 0.0001)), slow wave sleep (SWS; (F(3, 13) = 31.264; P < 0.0001)), and rapid eye movement sleep (REMS; (F(3, 13) = 10.208; P < 0.001)) in adult rats (PND80) that received at PND30 and during 2 weeks, a daily injection of WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg, i.p.; Panel A). Effects on lights-on period (07:00–19:00h) showed statistical changes in W (F(3, 13) = 10.622; P < 0.0008), SWS (F(3, 13) = 14.512; P < 0.0002), and REMS (F(3, 13) = 4.05; P < 0.03; Panel B). In addition, statistical differences were found during the lights-off period (19:00–07:00h) for W(F(3, 13) = 24.256; P < 0.0001), SWS (F(3, 13) = 8.497; P < 0.002), and REMS (F(3, 13) = 55.595; P < 0.0001; * vs. control, # vs. WIN-0.1, & vs. WIN-0.3; Panel C). 13)
Fig. 3. Hourly time (24h) of W (Panel A), SWS (Panel B), and REMS (Panel C) in adult rats (PND80) that received at PND30 and during 2 weeks, a daily injection of either vehicle (control; 1 ml, intraperitoneally [i.p.]) or WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg, i.p. * vs. control, P < 0.05; # vs. WIN-0.1, P < 0.05; & vs. WIN-0.3; P < 0.05). The white horizontal bar represents the lights-on period whereas the black horizontal bar indicates the lights-off phase.
REMS is shown in Fig. 4C and D, respectively. 3.4. Effects on NeuN expression in hypothalamus Finally, we evaluated whether subchronic exposure to WIN 55, 2122 might induce neuronal changes in the dorsomedial hypothalamic nucleus an important regulatory center of circadian activity. Compared to controls (Fig. 5, A), rats that had received WIN 55, 212-2 during adolescence showed dose-dependent increases in immunoreactive NeuN levels (F(3, 13) = 645.91; P < 0.0001; Fig. 5, B-E, respectively). 4
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now reached a staggering 6.8 million (Hasin et al., 2015). As with other psychoactive drugs, cannabis use typically starts in early teenage years and progressively increases throughout adolescence (NIDA, 2015). Teenage boys and girls experiment with cannabis more than any other recreational substance and a substantial percentage of them (5.8% of 12th graders in 2013) also tried synthetic cannabis substitutes such as Spice and K2 (NIDA, 2015). Though striking, these numbers are likely to grow in the near future as risk perception of cannabis's potential consequences continues to decrease (Keyes et al., 2011; Pacek et al., 2015). Furthermore, the accelerating diffusion of medicinal cannabis-derived products – e.g. in childhood epilepsy and autism – is exposing new groups of young people to the drug. These trends are of particular concern within a social context in which changes to legislation and broadening acceptance fuel financial interests that can potentially override public health and safety concerns (Kalant, 2015; Piomelli, 2015). Brain networks controlling human cognition and affect are still actively developing during the teenage years (Spear, 2000; Steinberg, 2005). The plasticity of these structures makes them particularly sensitive to the chronic effects of cannabis (Lubman et al., 2015; Schneider, 2008). Indeed, there is an overall consensus across epidemiological surveys that adolescence-onset use of the drug is associated with impairments in cognition and affective functioning that continue into adulthood even after use of the drug has stopped (Schweinsburg et al., 2008). For example, a prospective study of 1037 individuals followed from birth to age 38 years (the ‘Dunedin cohort’) found a significant association between prolonged cannabis exposure in adolescence and cognitive decline later in life (Meier et al., 2012). Increased risk of developing neuropsychiatric disorders –including addiction, depression and schizophrenia– has also been documented (Chadwick et al., 2013; Renard et al., 2014). In agreement with these epidemiological findings, a growing number of animal studies indicate that adolescent treatment with the main psychoactive component of cannabis, Δ9-tetrahydrocannabinol, or one of its synthetic mimics causes long-term impairments in sociality and memory, increased reward seeking, and dysregulated affect (for review, see Rubino and Parolaro, 2016; Schneider, 2008). In the present study, we investigated the long-term effects of adolescent exposure to the synthetic cannabinoid agonist WIN 55, 212–2 on adult sleep patterns. Our results suggest that continued stimulation of cannabinoid receptors during adolescence (PND30-44) causes alterations in the sleep-wake cycle that persist into adulthood (PND70). Interestingly, rats treated with WIN 55, 212-2 developed an insomnialike profile during the lights-on period, whereas an opposite profile was observed in the lights-off phase. In addition, the sleep rebound period after sleep deprivation was markedly suppressed in rats treated with WIN 55, 212–2, whose sleep-wake cycle also displayed an arrhythmic profile. It is worth mentioning in this context that sleep abnormalities have been associated with cannabis consumption, though they have not been linked to adolescent use (Angarita et al., 2016; Furer et al., 2018; Altman et al., 2019). In parallel with changes in sleep patterns, we also observed a marked elevation in NeuN expression in the dorsomedial hypothalamus of adult rats that had received WIN 55, 212-2 during adolescence. Overstimulation of cannabinoid receptors might be directly or indirectly responsible for this effect. Consistent with this possibility, Xapelli et al. (2013) showed that CB1 activation produced proliferation and differentiation of stem/progenitor cell cultures into NeuN-positive neurons. Alternatively, the sleep disruption described here might be responsible for the changes in NeuN expression. Future experiments will need to address this question. The dorsomedial hypothalamus plays a key role in sleep regulation (Aston-Jones et al., 2001; Chou et al., 2003; Tyree et al., 2018). However, since this brain region is not only involved in sleep modulation (Seoane-Collazo et al., 2015; Krause and Ingraham, 2017; Abuzzahab et al., 2019), the neuronal substrates that respond to treatment with
Fig. 4. Total sleep time of the sleep rebound for SWS and REMS (Panel A) after total sleep deprivation (6h) in adult rats (PND80) that received during adolescence a daily injection of either vehicle (control; 1 ml, intraperitoneally [i.p.]) or WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg, i.p). WIN-treated animals showed hourly changes in the sleep rebound after total sleep deprivation (6h) for SWS (F(3, 13) = 52.253; P < 0.0001; Panel B), and REMS (F(3, 13) = 75.2586; P < 0.0001; Panel C; * vs. control, # vs. WIN-0.1, & vs. WIN0.3).
No such differences were noted in other hypothalamic areas (data not shown). 4. Discussion The prevalence of cannabis use in the adult population of the US has climbed from 4.1% in 2001–2002 to 9.5% in 2012–2013, while the total number of Americans diagnosed with cannabis use disorder has 5
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Fig. 5. Immunohistochemical expression of NeuN in hypothalamus of adult rats that received during adolescence a daily injection of either vehicle (control; 1 ml, intraperitoneally [i.p.], Panel A) or WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg, i.p.; Panels B–D). A dose-dependent effect was found in WIN-treated rats by displaying an enhancement in the number of NeuN expression (F(3, 13) = 645.91; P < 0.0001; Panel E; * vs. control; # vs. WIN-0.1; & vs. WIN0.3). Abbreviations: 3V, third ventricle. Scale bar: 100 μm.
opens a new avenue for research in the cannabinoid field for future studies that should address these concerns. In summary, prolonged exposure to the cannabinoid agonist WIN 55, 212-2 during adolescence disrupts sleep patterns and changes NeuN expression in adulthood. Together, these findings suggest that prolonged hyperactivation of cannabinoid receptors during adolescence can result in persistent alteration in the sleep-wake cycle, which persist into adulthood.
WIN, 55, 212-2 should be investigated in the future. The dorsomedial hypothalamus includes a heterogeneous mixture of diverse neurons that serve integrative and regulatory functions in sleep as well as in feeding, stress and other functions. The hypocretin/orexin neurons are among the best studied to date (Schiappa et al., 2018; Wang et al., 2018; Arrigoni et al., 2018; Burdakov, 2019). It would be interesting therefore to determine whether the neuronal population that responded to adolescent WIN 55, 212–2 exposure expresses hypocretin/orexins, as expected from the role played by this peptide in sleep rhythms (Nixon et al., 2015; Alakuijala et al., 2016; Li et al., 2017; Leung and Mourrain, 2018; Mäkelä et al., 2018; Spiegelhalder et al., 2019). Whether the sleep changes induced by WIN 55, 212–2 administration during adolescence might be related to disruptions in hypocretin activity is an issue that deserves further investigation. Similarly, it will be important to examine whether alterations in sleep-wake cycle rhythm caused by WIN 55, 212-2 might be related to changes in retinal morphology, biochemical disturbances in melatonin or disturbances in “clock” genes expression. Our study has several limitations. We found changes in NeuN expression in the ventromedial hypothalamic nucleus after WIN 55, 212–2 administration. Although this brain region has been linked to the control of the sleep-wake cycle (Ono and Yamanaka, 2017; Arrigoni et al., 2018; Boes et al., 2018), it remains to be elucidated whether other brain areas might also be involved. It is worth noting that NeuN may not show a uniform profile of expression within a specific brain region (Hight et al., 2010), so a limitation of our study is that NeuN might be detected in a non-uniform fashion within the hypothalamus or other brain areas. Moreover, it is difficult to determine if the alterations found in the sleep-wake cycle may be cause or effect of the changes observed in NeuN activity. In line with this, previous reports have demonstrated neuroanatomical changes in adolescent brain areas, including the hippocampus, after cannabis use (Koenders et al., 2017; Blest-Hopley et al., 2018, 2019; Kaag et al., 2018; Orr et al., 2019). Finally, one additional limitation of the present work is that it did not evaluate the effects of cannabis extracts or their psychotropic constituent, Δ9-tetrahydrocannabinol. This would provide additional translational value to our study, and will be evaluated in future studies. Importantly, the sleep-wake cycle in animals treated with WIN 55, 212-2 during adolescence displayed an arrhythmic profile. It is worth mentioning that sleep abnormalities have been associated with cannabis consumption (Angarita et al., 2016; Furer et al., 2018; Altman et al., 2019). Whether the alterations in the rhythm of the sleep-wake cycle are related to changes in the retinal morphology, biochemical disturbances in melatonin or disturbances in “clock” gene expression,
Credit author statement Lorena Macías-Triana: Methodology, formal analyses, review & editing the draft of the Manuscript; Karen Romero-Cordero: Methodology, formal analyses, review & editing the draft of the Manuscript; Agnes Tatum-Kuri: Methodology, formal analyses, review & editing the draft of the Manuscript; Alba Vera-Barrón: Methodology, formal analyses, review & editing the draft of the Manuscript; Diana Millán-Aldaco: Methodology (IHC studies), review & editing the draft of the Manuscript; Gloria Arankowsky-Sandoval: Methodology, provided equipment, review & editing the draft of the Manuscript; Daniele Piomelli: Conceptualization, Methodology, formal analyses, review & editing the draft of the Manuscript; Eric Murillo-Rodríguez: Conceived, designed and supervised the project, analyzed data and wrote the Manuscript. All authors approved the final version of the Manuscript. Declaration of competing interest None. Acknowledgements This work was supported by The University of California Institute for Mexico and the United States (UC MEXUS) and Consejo Nacional de Ciencia y Tecnología (CONACyT; México) under Grant CN-17-19 and Escuela de Medicina, Universidad Anáhuac Mayab (Mérida, Yucatán. México) under Grant PresInvEMR2017 given to E. M.-R. and the NIDA Center of Excellence ‘Impact of Cannabinoids Across the Lifespan’ (DA044118, given to DP). The authors thank Pedro Aquino for his technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejphar.2020.172911. 6
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Full length article
Exposure to the cannabinoid agonist WIN 55, 212–2 in adolescent rats causes sleep alterations that persist until adulthood
T
Lorena Macías-Trianaa,b, Karen Romero-Corderoa,b, Agnes Tatum-Kuria,b, Alba Vera-Barróna,b, Diana Millán-Aldacoc, Gloria Arankowsky-Sandovald, Daniele Piomellie,f,g, Eric Murillo-Rodrígueza,b,∗ a
Laboratorio de Neurociencias Moleculares e Integrativas, Escuela de Medicina, División Ciencias de la Salud, Universidad Anáhuac Mayab, Mérida, Yucatán, Mexico Intercontinental Neuroscience Research Group, Mexico c Depto. de Neurociencia Cognitiva, División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México, Mexico d Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico e Department of Anatomy and Neurobiology, University of California, Irvine, Irvine, CA, USA f Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA g Department of Biological Chemistry, University of California, Irvine, Irvine, CA, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Adolescence Cannabinoids Sleep Rapid eye movement sleep WIN 55,212–2
Cannabis and, to a lesser extent, synthetic cannabinoids are used during adolescence, a period in which multiple brain areas are still undergoing development. Among such areas is the hypothalamus, which is implicated in the control of sleep-wake cycle. In the present report, we show that exposing adolescent rats to the cannabinoid receptor agonist WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg, i.p) for 14 days during adolescence (i.e., from post-natal day 30–44) resulted in significant sleep disturbances when the animals became adult (post-natal day 80). These included decreased wakefulness and enhanced rapid eye movement sleep. Furthermore, we found that labeling for NeuN, a marker of postmitotic neurons, was significantly increased the dorsomedial hypothalamic nucleus of rats treated with WIN 55, 212–2. The results suggest that excessive cannabinoid receptor activation during adolescence can persistently influence sleep patterns and neuronal activity later in life.
1. Introduction The endocannabinoid system is a neuromodulatory signaling complex that comprises cannabinoid receptors, their endogenous ligands, and proteins involved in the formation, transport and degradation of such ligands (Lu and Mackie, 2016). Investigation of the presence and distribution of the CB1 cannabinoid receptor in the rodent and human central nervous system has implicated this signaling system in multiple aspects of the development process (Erdozain et al., 2015; Harkany et al., 2008; Yi et al., 2016). Chronic use of cannabis during adolescence has been associated with a variety of neural and behavioral abnormalities, some of which may persist until adulthood (Chadwick et al., 2013; Meier et al., 2012; Renard et al., 2014; Rubino and Parolaro, 2016). Moreover, this vulnerability might involve brain areas linked to the control of key homeostatic functions, including as the sleep-wake cycle (Houston et al., 2014; Chen and Baram, 2015). Available data regarding the
impact of cannabinoid receptor stimulation and sleep alterations derive from studies conducted in adult animals and with either acute or subchronic treatment designs (Murillo-Rodríguez et al., 1998, 2001; 2003, 2013; 2016; Altman et al., 2019; Angarita et al., 2016; Furer et al., 2018; Goonawardena et al., 2011, 2015; Pava et al., 2014; ProspéroGarcía et al., 2016). By contrast, the persistent effects of prolonged cannabinoid receptor activation during adolescence on sleep, if any, are unknown. Amongst the elements involved in the plasticity of the brain during the development is the activity of neuronal nuclei (NeuN) protein which has been also been associated to assess the likely functional state of neurons in normal or pathological conditions (Gusel'nikova and Korzhevskiy, 2015; Duan et al., 2016). However, it is unknown whether prolonged cannabinoid receptor activation during adolescence contribute to induce effects on NeuN expression. In the present study, we report that daily injections of the cannabinoid agonist WIN-55, 212–2 in 30–44 day-old rats (i.e., during adolescence) caused significant
∗ Corresponding author. Laboratorio de Neurociencias Moleculares e Integrativas, Escuela de Medicina, División Ciencias de la Salud, Universidad Anáhuac Mayab, Carretera Mérida-Progreso Km. 15.5, A.P. 96 Cordemex, C.P. 97310, Mérida, Yucatán, Mexico. E-mail address: [email protected] (E. Murillo-Rodríguez).
https://doi.org/10.1016/j.ejphar.2020.172911 Received 31 July 2019; Received in revised form 10 December 2019; Accepted 7 January 2020 Available online 08 February 2020 0014-2999/ © 2020 Elsevier B.V. All rights reserved.
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attached onto the skull with dental cement. After surgery, the rats were transferred to individual cages with ad libitum access to food and water. The EEG/EMG electrodes surgery and habituation procedures were carried out as previously reported (Murillo-Rodríguez et al., 2019). The EEG/EMG electrodes remained in the skull of rats during 14 days. From the 14 days that rats hold the EEG/EMG electrodes, the first 7 days postsurgery belonged to the habitation/post-surgery recovery time whereas the remaining 7 days were considered for data analysis. However, the final sleep-wake cycle data analyses were taken from the second day once finishing the habitation/post-surgery recovery time. To eliminate experimental biases, the EEG/EMG electrode surgeries were conducted under blinded conditions.
alterations in the sleep-wake cycle when the animals reached adulthood. Additionally, we show that rats treated with WIN-55, 212-2 during adolescence display abnormally high levels of activity of the neuronal nuclear protein (NeuN), in the dorsomedial nucleus of the hypothalamus, a region involved in the control of the sleep-wake cycle (Aston-Jones et al., 2001; Arrigoni et al., 2018). 2. Materials and methods 2.1. Ethics approval The experimental procedures used in this study were approved by the Research and Ethics Committees of our Institution. All animal experiments met the ARRIVE guidelines and the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/ 63/EU for animal experiments as well as National Institute of Health (NIH publication No. 80–23, revised 1996) and Mexican Standards Related to Use and Management of Laboratory Animals (DOF. NOM062-Z00-1999). We made all possible efforts to minimize animal suffering and to reduce the number animals included in the study. All mandatory laboratory health and safety procedures were complied with in the course of conducting the experimental work reported.
2.6. Analysis of sleep recordings
WIN 55, 212–2 (PubChem CID: 5311501) was purchased from Sigma-Aldrich (St. Louis, MO. USA) and it was dissolved in a vehicle (VEH) composed of polyethylene glycol (PubChem CID: 174)/saline (5:95 v/v), as previously reported (Schoch et al., 2018). Additional reagents, chemicals and materials were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Immediately after surgeries, the six-pin plastic plug was connected to a 6-channel slip-ring commutator through a 50 cm shielded cable (Plastics One, Roanoke, VA, United States), allowing free moving of animals around the cage. From the slip-ring system an additional shielded cable was connected to the amplifier (Model M15LT 15A54; Grass Instruments. Quincy, MA. USA) to filter the EEG signal at 70 Hz (low-pass filter) and 0.3 Hz (high-pass filter). The EEG signals were continuously sampled at 128Hz by using a 100-bit analog-to-digital converter board (NI PCI-6033E Multifunction I/O Board and NI-DAQ Software, SCB-100 Shielded Connector Block. National Instruments. Austin, TX. USA). The EEG/EMG signals were visually scored in 12s epochs with the aid of the sleep-scoring software (ICELUS). The sleepwake cycle was characterized as follows: Wakefulness (W) consisted in desynchronization in the EEG as well as high EMG activity whereas high-amplitude slow waves with a low EMG tone relative to waking were the definition traces for slow wave sleep (SWS). Lastly, rapid eye movement sleep (REMS) was identified by regular theta activity across the EEG accompanied with low EMG activity. The EEG/EMG electrodes remained in the rat skull during 14 days. Thus, from the 14 days that rats hold the EEG/EMG electrodes, the first 7 days post-surgery belonged to the habitation/post-surgery recovery time whereas the remaining 7 days were considered for data analysis. However, the final sleep-wake cycle data analyses were taken from the second day once finishing the habitation/post-surgery recovery time. The sleep data were scored following previous procedures (Murillo-Rodríguez et al., 2019). To avoid bias, one observer blind to the experiment scored the sleep recordings.
2.4. Pharmacological administrations
2.7. Sleep deprivation
Animals (PND 30) were divided randomly into two experimental groups, and were given 14 daily injections of either vehicle (1 ml, intraperitoneally [i.p.]; n = 5) or WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg/ 1 ml, ip; n = 5 per dose). All treatments were given at 07:00 h. One observer blind to the experimental trials administered treatment.
The total sleep deprivation (TSD) procedure was carried our as previously described (Murillo-Rodríguez et al., 2017). Rats treated with vehicle (n = 5) or WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg; n = 5 per group) were kept awake for 6 h during the lights-on period (from 07:00–13:00h) by making loud noises, knocking the outer walls of the cages, or placing novel objects into the cages. The procedure was performed by a researcher blinded to treatment.
2.2. Animals A total of 20 male Wistar rats (N = 20; 250–300g; Vivarium from Centro de Investigaciones Regionales Dr. Hideyo Noguchi, Universidad Autónoma de Yucatán. Mérida, Yucatán. México) delivered on postnatal day (PND) 30 were singly housed in a climate-controlled room (humidity: 60 ± 10%; ambient temperature: 21 ± 1 °C) on a 12h light-dark cycle (lights-on: 07:00–19:00h; 200 lux) during all experiments. All rats had free access to Purina Rat Chow (Purina. México) and tap water. 2.3. Chemicals
2.5. Implanting EEG/EMG electrodes for sleep recording Adult animals (PND80) were anesthetized with a mixture of acepromazine (0.75 mg/kg), xylazine (2.5 mg/kg) and ketamine (22 mg/ kg) and were transferred to a stereotaxic frame (David Kopf Instruments. Tujunga, CA. USA) to implant electroencephalogram (EEG) and electromyogram (EMG) electrodes. The procedure included trepanation for the implantation of two stainless-steel screw electrodes placed 2 mm on either side of the sagittal sinus and 3 mm anterior to bregma (frontal cortex). Two additional screws were located 3 mm on either side of the sagittal sinus and 6 mm behind bregma (occipital cortex). Two EMG wire electrodes were inserted into the dorsal neck muscles. The surgical wounds were treated with antibiotics and anesthetics. Once implanted, the EEG/EMG electrode wires were connected into a six-pin plastic plug (Plastics One. Roanoke, VA, USA) and
2.8. Immunohistochemical studies At the end of the sleep-recording studies, rats (vehicle [n = 5] and WIN-treated animals [WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg; n = 5 each dose])) were intracardially perfused (de-la-Cruz et al., 2018). Brain coronal sections were obtained (20 μm thickness) and collected in 1:5 serial order using a Portable Bench-top Cryostat (Leica CM1100. Leica Microsystems GmbH. Wetzlar, Germany). Because of its neurobiological link with the sleep-wake cycle, hypothalamus was selected for analysis of NeuN expression (Ono and Yamanaka, 2017; Arrigoni et al., 2018). The identification of hypothalamus included slides from coordinates −0.12 to −3.48 mm (from bregma according the Rat Brain Atlas [Paxinos and Watson, 2005]). Next, standardized procedures for 2
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immunohistochemically detection of NeuN were used in collected samples (de-la-Cruz et al., 2018). The reproducibility of the immunohistochemical procedure was confirmed by repeating the whole procedure with additional batches containing approximately the same number of sections from all experimental groups with the same primary antibody. Sections under an identical immunostaining analysis with the exception that 1% bovine serum albumin in PBS substituted for the primary antibody were used as negative control. To minimize experimental bias, a researcher blinded to treatment performed the immunostaining experiments.
2.9. NeuN immunohistochemistry A similar number of sections from selected brain area from both hemispheres for each rat, for all experimental groups were counted for NeuN immunohistochemical expression. Slides were photographed with bright field, dark field microscopy (10× objective lens) with the aid of computer image analysis software (Microscope Zeiss Imager A.2. Carl Zeiss Microscopy, Oberkochen,Germany), a digital camera (AxioCam ICc1. Carl Zeiss) and the image analysis computer software (ZEN, 2012; blue edition. Carl Zeiss). The analysis of images comprised NeuN expression in sections of the hypothalamus (coordinates from bregma: 0.12 to −3.48 mm, according the Rat Brain Atlas [Paxinos and Watson, 2005]). A researcher blinded to treatment quantified NeuN labeling in the hypothalamus (bilateral sides) as described previously (de-la-Cruz et al., 2018). Fig. 1 shows the flowchart of interventions and procedures during the experiment.
2.10. Statistical analysis Results are shown as mean ± standard error of the mean. Statistical differences were determined by one-way ANOVA followed by Scheffé's post hoc test for multiple comparisons among the experimental groups. All statistical analyses were performed using StatView (version 5.0.0, SAS Institute, United States). No power analysis was performed.
3. Results 3.1. Effects of adolescence exposure to WIN 55,212–2 on total sleep time in adulthood
Fig. 1. Schematic representation of the different interventions and procedures during the experiment.
Adult rats (PND 80) that had received daily injections of the cannabinoid agonist WIN 55,212–2 (0.1, 0.3 or 1.0 mg/kg, i.p) from PND 30 to PND 44 displayed significant changes in W, SWS, and REMS. Adolescence exposure to the drug at the doses of 0.1 and 0.3 mg/kg was associated with decreased W (F(3, 13) = 24.586; P < 0.0001) and SWS (F(3, 13) = 31.264; P < 0.0001) as well as enhanced REMS (F(3, 13) = 10.208; P < 0.001; Fig. 2,A). At 1 mg/kg, WIN 55, 212–2 treatment caused effects opposite to those observed at the lower doses (Fig. 2, A). During the lights-on period (07:00–19:00h), rats that had been treated with low doses of WIN 55, 212–2 (0.1, 0.3 mg/kg) showed increased W (F(3, 13) = 10.622; P < 0.0008), decreased SWS (F(3, 13) = 14.512; P < 0.0002) as well as prolonged REMS (F(3, 13) = 4.05; P < 0.03; Fig. 2, B). Animals treated with the highest drug dose (1 mg/ kg) displayed increased W, decreased SWS and no change in REMS. During the lights-off period (19:00–07:00h; Fig. 2,C), rats treated with WIN 55,212-2 showed (i) a decrease in W (F(3, 13) = 24.256; P < 0.0001), which was especially marked at the doses of 0.1 and 0.3 mg/kg; (ii) various changes in SWS (increase at 0.1 and 1 mg/kg; decrease at 0.3 mg/kg) (F(3, 13) = 8.497; P < 0.002); and (iii) inverted-U changes in REMS (F(3, 13) = 55.595; P < 0.0001).
3.2. Effects of adolescence exposure to WIN 55,212–2 on the sleep-wake cycle in adulthood A 24h time-course of W, SWS and REMS in rats treated with vehicle or WIN 55, 212–2 is illustrated in Fig. 3. Adolescence exposure to the cannabinoid agonist was associated with a striking insomnia-like phenotype in adults. Alertness was increased (Fig. 3, A) while SWS (Fig. 3, B) and REMS (Fig. 3, C) were decreased during the lights-on period. Opposite changes were observed during the lights-off phase. Time points for W, SWS and REMS showed statistical differences among experimental groups (P < 0.05). Remarkably, animals treated with WIN 55, 212-2 displayed arrhythmic activity in W, SWS and REMS. 3.3. Effects of sleep deprivation In the TSD experiment, control rats experienced the expected rebound changes in SWS and REMS (Fig. 4, A). Such changes were markedly suppressed in animals that had been treated with WIN 55, 212-2 during adolescence: both SWS (F(3, 13) = 52.253; P < 0.0001) and REMS (F(3, 13) = 75.2586; P < 0.0001) were substantially reduced. A time-course analysis of sleep rebound period for SWS and 3
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Fig. 2. Effects on total time (24h of sleep recordings) of wakefulness (W; (F(3, = 24.586; P < 0.0001)), slow wave sleep (SWS; (F(3, 13) = 31.264; P < 0.0001)), and rapid eye movement sleep (REMS; (F(3, 13) = 10.208; P < 0.001)) in adult rats (PND80) that received at PND30 and during 2 weeks, a daily injection of WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg, i.p.; Panel A). Effects on lights-on period (07:00–19:00h) showed statistical changes in W (F(3, 13) = 10.622; P < 0.0008), SWS (F(3, 13) = 14.512; P < 0.0002), and REMS (F(3, 13) = 4.05; P < 0.03; Panel B). In addition, statistical differences were found during the lights-off period (19:00–07:00h) for W(F(3, 13) = 24.256; P < 0.0001), SWS (F(3, 13) = 8.497; P < 0.002), and REMS (F(3, 13) = 55.595; P < 0.0001; * vs. control, # vs. WIN-0.1, & vs. WIN-0.3; Panel C). 13)
Fig. 3. Hourly time (24h) of W (Panel A), SWS (Panel B), and REMS (Panel C) in adult rats (PND80) that received at PND30 and during 2 weeks, a daily injection of either vehicle (control; 1 ml, intraperitoneally [i.p.]) or WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg, i.p. * vs. control, P < 0.05; # vs. WIN-0.1, P < 0.05; & vs. WIN-0.3; P < 0.05). The white horizontal bar represents the lights-on period whereas the black horizontal bar indicates the lights-off phase.
REMS is shown in Fig. 4C and D, respectively. 3.4. Effects on NeuN expression in hypothalamus Finally, we evaluated whether subchronic exposure to WIN 55, 2122 might induce neuronal changes in the dorsomedial hypothalamic nucleus an important regulatory center of circadian activity. Compared to controls (Fig. 5, A), rats that had received WIN 55, 212-2 during adolescence showed dose-dependent increases in immunoreactive NeuN levels (F(3, 13) = 645.91; P < 0.0001; Fig. 5, B-E, respectively). 4
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now reached a staggering 6.8 million (Hasin et al., 2015). As with other psychoactive drugs, cannabis use typically starts in early teenage years and progressively increases throughout adolescence (NIDA, 2015). Teenage boys and girls experiment with cannabis more than any other recreational substance and a substantial percentage of them (5.8% of 12th graders in 2013) also tried synthetic cannabis substitutes such as Spice and K2 (NIDA, 2015). Though striking, these numbers are likely to grow in the near future as risk perception of cannabis's potential consequences continues to decrease (Keyes et al., 2011; Pacek et al., 2015). Furthermore, the accelerating diffusion of medicinal cannabis-derived products – e.g. in childhood epilepsy and autism – is exposing new groups of young people to the drug. These trends are of particular concern within a social context in which changes to legislation and broadening acceptance fuel financial interests that can potentially override public health and safety concerns (Kalant, 2015; Piomelli, 2015). Brain networks controlling human cognition and affect are still actively developing during the teenage years (Spear, 2000; Steinberg, 2005). The plasticity of these structures makes them particularly sensitive to the chronic effects of cannabis (Lubman et al., 2015; Schneider, 2008). Indeed, there is an overall consensus across epidemiological surveys that adolescence-onset use of the drug is associated with impairments in cognition and affective functioning that continue into adulthood even after use of the drug has stopped (Schweinsburg et al., 2008). For example, a prospective study of 1037 individuals followed from birth to age 38 years (the ‘Dunedin cohort’) found a significant association between prolonged cannabis exposure in adolescence and cognitive decline later in life (Meier et al., 2012). Increased risk of developing neuropsychiatric disorders –including addiction, depression and schizophrenia– has also been documented (Chadwick et al., 2013; Renard et al., 2014). In agreement with these epidemiological findings, a growing number of animal studies indicate that adolescent treatment with the main psychoactive component of cannabis, Δ9-tetrahydrocannabinol, or one of its synthetic mimics causes long-term impairments in sociality and memory, increased reward seeking, and dysregulated affect (for review, see Rubino and Parolaro, 2016; Schneider, 2008). In the present study, we investigated the long-term effects of adolescent exposure to the synthetic cannabinoid agonist WIN 55, 212–2 on adult sleep patterns. Our results suggest that continued stimulation of cannabinoid receptors during adolescence (PND30-44) causes alterations in the sleep-wake cycle that persist into adulthood (PND70). Interestingly, rats treated with WIN 55, 212-2 developed an insomnialike profile during the lights-on period, whereas an opposite profile was observed in the lights-off phase. In addition, the sleep rebound period after sleep deprivation was markedly suppressed in rats treated with WIN 55, 212–2, whose sleep-wake cycle also displayed an arrhythmic profile. It is worth mentioning in this context that sleep abnormalities have been associated with cannabis consumption, though they have not been linked to adolescent use (Angarita et al., 2016; Furer et al., 2018; Altman et al., 2019). In parallel with changes in sleep patterns, we also observed a marked elevation in NeuN expression in the dorsomedial hypothalamus of adult rats that had received WIN 55, 212-2 during adolescence. Overstimulation of cannabinoid receptors might be directly or indirectly responsible for this effect. Consistent with this possibility, Xapelli et al. (2013) showed that CB1 activation produced proliferation and differentiation of stem/progenitor cell cultures into NeuN-positive neurons. Alternatively, the sleep disruption described here might be responsible for the changes in NeuN expression. Future experiments will need to address this question. The dorsomedial hypothalamus plays a key role in sleep regulation (Aston-Jones et al., 2001; Chou et al., 2003; Tyree et al., 2018). However, since this brain region is not only involved in sleep modulation (Seoane-Collazo et al., 2015; Krause and Ingraham, 2017; Abuzzahab et al., 2019), the neuronal substrates that respond to treatment with
Fig. 4. Total sleep time of the sleep rebound for SWS and REMS (Panel A) after total sleep deprivation (6h) in adult rats (PND80) that received during adolescence a daily injection of either vehicle (control; 1 ml, intraperitoneally [i.p.]) or WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg, i.p). WIN-treated animals showed hourly changes in the sleep rebound after total sleep deprivation (6h) for SWS (F(3, 13) = 52.253; P < 0.0001; Panel B), and REMS (F(3, 13) = 75.2586; P < 0.0001; Panel C; * vs. control, # vs. WIN-0.1, & vs. WIN0.3).
No such differences were noted in other hypothalamic areas (data not shown). 4. Discussion The prevalence of cannabis use in the adult population of the US has climbed from 4.1% in 2001–2002 to 9.5% in 2012–2013, while the total number of Americans diagnosed with cannabis use disorder has 5
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Fig. 5. Immunohistochemical expression of NeuN in hypothalamus of adult rats that received during adolescence a daily injection of either vehicle (control; 1 ml, intraperitoneally [i.p.], Panel A) or WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg, i.p.; Panels B–D). A dose-dependent effect was found in WIN-treated rats by displaying an enhancement in the number of NeuN expression (F(3, 13) = 645.91; P < 0.0001; Panel E; * vs. control; # vs. WIN-0.1; & vs. WIN0.3). Abbreviations: 3V, third ventricle. Scale bar: 100 μm.
opens a new avenue for research in the cannabinoid field for future studies that should address these concerns. In summary, prolonged exposure to the cannabinoid agonist WIN 55, 212-2 during adolescence disrupts sleep patterns and changes NeuN expression in adulthood. Together, these findings suggest that prolonged hyperactivation of cannabinoid receptors during adolescence can result in persistent alteration in the sleep-wake cycle, which persist into adulthood.
WIN, 55, 212-2 should be investigated in the future. The dorsomedial hypothalamus includes a heterogeneous mixture of diverse neurons that serve integrative and regulatory functions in sleep as well as in feeding, stress and other functions. The hypocretin/orexin neurons are among the best studied to date (Schiappa et al., 2018; Wang et al., 2018; Arrigoni et al., 2018; Burdakov, 2019). It would be interesting therefore to determine whether the neuronal population that responded to adolescent WIN 55, 212–2 exposure expresses hypocretin/orexins, as expected from the role played by this peptide in sleep rhythms (Nixon et al., 2015; Alakuijala et al., 2016; Li et al., 2017; Leung and Mourrain, 2018; Mäkelä et al., 2018; Spiegelhalder et al., 2019). Whether the sleep changes induced by WIN 55, 212–2 administration during adolescence might be related to disruptions in hypocretin activity is an issue that deserves further investigation. Similarly, it will be important to examine whether alterations in sleep-wake cycle rhythm caused by WIN 55, 212-2 might be related to changes in retinal morphology, biochemical disturbances in melatonin or disturbances in “clock” genes expression. Our study has several limitations. We found changes in NeuN expression in the ventromedial hypothalamic nucleus after WIN 55, 212–2 administration. Although this brain region has been linked to the control of the sleep-wake cycle (Ono and Yamanaka, 2017; Arrigoni et al., 2018; Boes et al., 2018), it remains to be elucidated whether other brain areas might also be involved. It is worth noting that NeuN may not show a uniform profile of expression within a specific brain region (Hight et al., 2010), so a limitation of our study is that NeuN might be detected in a non-uniform fashion within the hypothalamus or other brain areas. Moreover, it is difficult to determine if the alterations found in the sleep-wake cycle may be cause or effect of the changes observed in NeuN activity. In line with this, previous reports have demonstrated neuroanatomical changes in adolescent brain areas, including the hippocampus, after cannabis use (Koenders et al., 2017; Blest-Hopley et al., 2018, 2019; Kaag et al., 2018; Orr et al., 2019). Finally, one additional limitation of the present work is that it did not evaluate the effects of cannabis extracts or their psychotropic constituent, Δ9-tetrahydrocannabinol. This would provide additional translational value to our study, and will be evaluated in future studies. Importantly, the sleep-wake cycle in animals treated with WIN 55, 212-2 during adolescence displayed an arrhythmic profile. It is worth mentioning that sleep abnormalities have been associated with cannabis consumption (Angarita et al., 2016; Furer et al., 2018; Altman et al., 2019). Whether the alterations in the rhythm of the sleep-wake cycle are related to changes in the retinal morphology, biochemical disturbances in melatonin or disturbances in “clock” gene expression,
Credit author statement Lorena Macías-Triana: Methodology, formal analyses, review & editing the draft of the Manuscript; Karen Romero-Cordero: Methodology, formal analyses, review & editing the draft of the Manuscript; Agnes Tatum-Kuri: Methodology, formal analyses, review & editing the draft of the Manuscript; Alba Vera-Barrón: Methodology, formal analyses, review & editing the draft of the Manuscript; Diana Millán-Aldaco: Methodology (IHC studies), review & editing the draft of the Manuscript; Gloria Arankowsky-Sandoval: Methodology, provided equipment, review & editing the draft of the Manuscript; Daniele Piomelli: Conceptualization, Methodology, formal analyses, review & editing the draft of the Manuscript; Eric Murillo-Rodríguez: Conceived, designed and supervised the project, analyzed data and wrote the Manuscript. All authors approved the final version of the Manuscript. Declaration of competing interest None. Acknowledgements This work was supported by The University of California Institute for Mexico and the United States (UC MEXUS) and Consejo Nacional de Ciencia y Tecnología (CONACyT; México) under Grant CN-17-19 and Escuela de Medicina, Universidad Anáhuac Mayab (Mérida, Yucatán. México) under Grant PresInvEMR2017 given to E. M.-R. and the NIDA Center of Excellence ‘Impact of Cannabinoids Across the Lifespan’ (DA044118, given to DP). The authors thank Pedro Aquino for his technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejphar.2020.172911. 6
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