Long-Term Stress and Concomitant Marijuana Smoke Exposure Affect Physiology, Behavior and Adult Hippocampal Neurogenesis, Kitti Rusznák et al., 2018

Long-Term Stress and Concomitant Marijuana Smoke Exposure Affect Physiology, Behavior and Adult Hippocampal Neurogenesis

Kitti Rusznák, Kata Csekö, Zsófia Varga, Dávid Csabai, Ágnes Bóna, Mátyás Mayer, Zsolt Kozma, Zsuzsanna Helyes and Boldizsár Czéh

Frontiers in Pharmacology, 2018, 9, 786

doi: 10.3389/fphar.2018.00786. eCollection 2018




Marijuana is a widely used recreational drug with increasing legalization worldwide for medical purposes. Most experimental studies use either synthetic or plant-derived cannabinoids to investigate the effect of cannabinoids on anxiety and cognitive functions. The aim of this study was to mimic real life situations where young people smoke cannabis regularly to relax from everyday stress. Therefore, we exposed young adult male NMRI mice to daily stress and concomitant marijuana smoke for 2 months and investigated the consequences on physiology, behavior and adult hippocampal neurogenesis. Animals were restrained for 6-h/day for 5-days a week. During the stress, mice were exposed to cannabis smoke for 2  30 min/day. We burned 2 “joints” (2  0.8 g marijuana) per occasion in a whole body smoking chamber. Cannabinoid content of the smoke and urine samples was measured by HPLC and SFC-MS/MS. Body weight gain was recorded daily and we did unrestrained, whole body plethysmography to investigate pulmonary functions. The cognitive performance of the animals was evaluated by the novel object recognition and Y maze tests. Anxietyrelated spontaneous locomotor activity and self-grooming were assessed in the open field test (OFT). Adult neurogenesis was quantified post mortem in the hippocampal dentate gyrus. The proliferative activity of the precursor cells was detected by the use of the exogenous marker 5-bromo-20-deoxyuridine. Treatment effects on maturing neurons were studied by the examination of doublecortin-positive neurons. Both stress and cannabis exposure significantly reduced body weight gain. Cannabis smoke had no effect on pulmonary functions, but stress delayed the maturation of several lung functions. Neither stress, nor cannabis smoke affected the cognitive functioning of the animals. Results of the OFT revealed that cannabis had a mild anxiolytic effect and markedly increased self-grooming behavior. Stress blocked cell proliferation in the dentate gyrus, but cannabis had no effect on this parameter. Marijuana smoke however had a pronounced impact on doublecortin-positive neurons influencing their number, morphology and migration.

In summary, we report here that long-term stress in combination with cannabis smoke exposure can alter several health-related measures, but the present experimental design could not reveal any interaction between these two treatment factors except for body weight gain.

Keywords : body weight, BrdU, Cannabis sativa, chronic stress, cognitive function, doublecortin, hippocampus, self-grooming



Marijuana is the most widely used illicit drug, as about 2.5% of the world population consume cannabis regularly (UNODC, 2017). Cannabis is increasingly legalized worldwide for medical and recreational purposes (Carliner et al., 2017; EMCDDA, 2017; Webster, 2018), which results in an increasing consumption while the long-term consequences on health are not well understood (e.g., Volkow et al., 2014, 2016; Levine et al., 2017). Thus, it is important to investigate the outcomes of prolonged use.

Advocates argue that marijuana is a safe and natural alternative for the treatment of a variety of medical and mental health conditions, but ambiguous data are reported in the literature on the health risks imposed by chronic cannabis use. Cannabidiol (CBD), a major constituent of Cannabis sativa and several components of the endocannabinoid system are increasingly viewed as potential ‘druggable’ targets for the treatment for anxiety-related disorders (Blessing et al., 2015; Lee et al., 2017; Patel et al., 2017). Indeed, anxiety is among the top five medical symptoms for which North Americans report using medical marijuana while its anxiolytic effectiveness is still not well documented (Turna et al., 2017). The correlation between cannabis use and cognitive enhancement or impairment is also ambiguous. While numerous clinical and preclinical data suggest a strong correlation between marijuana exposure and impaired cognition, it does not conclusively demonstrate that cannabis consumption alone is sufficient to cause these deficits in humans (Broyd et al., 2016; Curran et al., 2016; Volkow et al., 2016; Levine et al., 2017). At the same time, there are reports on positive effect of cannabis use on various cognitive and executive functions (Osborne et al., 2017; Gruber et al., 2018; Tervo-Clemmens et al., 2018).

The consequences of prolonged marijuana inhalation on respiratory health and lung cancer is also debated (Gates et al., 2014; Martinasek et al., 2016; Chatkin et al., 2017; Stone et al., 2018). Similarly, there is conflicting data on the effect of cannabis use on body weight. Cannabis is known to stimulate appetite and potentially promote weight gain in patients suffering from human immunodeficiency virus or cancer, whereas, findings of the large epidemiological studies in the general population, consistently indicate that users of marijuana tend to have lower body mass indices (Sansone and Sansone, 2014).

Adult neurogenesis in the hippocampal dentate gyrus is a unique form of neuroplasticity that has received substantial attention during the recent years. Adult-born neurons in the dentate play an essential role in normal cognitive functioning and in specific forms of learning (Denny et al., 2014; Anacker and Hen, 2017). Stress is a potent inhibitor of the proliferative activity of the precursor cells and also blocks the survival of the newly generated neurons (Gould et al., 1997; Czéh et al., 2001, 2002; Cameron and Schoenfeld, 2018). In consequence, stress-induced disruption of adult neurogenesis may play a role in the development of various psychiatric disorders, including depression, anxiety, and schizophrenia (Santarelli et al., 2003; Snyder et al., 2011; Surget et al., 2011; Kim et al., 2012; Schoenfeld and Cameron, 2015).

Cannabinoid receptors are highly expressed in the hippocampus, and recent studies suggest that facilitation of the cannabinoid signaling in the hippocampus may prevent stress-induced behavioral changes (Campos et al., 2013; Scarante et al., 2017; Fogaça et al., 2018). Numerous studies investigated the effect of various cannabinoids on adult hippocampal neurogenesis (Prenderville et al., 2015), but these studies used either plant-derived extracts or synthetic cannabinoids. The results of these studies are also inconsistent. Some studies report on positive, stimulatory effect (Jiang et al., 2005; Palazuelos et al., 2006; Marchalant et al., 2009; Wolf et al., 2010; Rivera et al., 2011; Suliman et al., 2018), while others document negative, inhibitory effect on adult neurogenesis (Realini et al., 2011; Abboussi et al., 2014; Lee et al., 2014; Steel et al., 2014), or no effect at all (Kochman et al., 2006; Ma´ckowiak et al., 2007; Steel et al., 2014).

Most of the preclinical studies use either synthetic cannabinoid agents or cannabis extracts to investigate the physiological and behavioral consequences. Typically, animals are injected with various doses of the synthetic compound in an acute or chronic (10–14 days) treatment protocol. The aim of the present study was to mimic a real life situation where cannabis is smoked by adolescents on a daily basis to ease everyday stress. We used young adult mice and subjected them to daily stress over a 2-month period and during the stress exposure the animals were also exposed to cannabis smoke. To investigate the health risks of such a treatment protocol, we examined body weight gain, pulmonary functions, emotional responses and cognitive performance, as well as cellular changes in adult hippocampal neurogenesis. The Cannabis sativa used in this experiment was obtained from the local Hungarian Police Superintendancy. To determine the amount of tetrahydrocannabinol (19-THC), cannabinol (CBN), and cannabidiol (CBD) content of our sample, we did an HPLC analysis of the marijuana smoke and metabolites of these compounds were measured in urine samples collected from the animals