**1. Introduction**

Adolescence is the developmental period of transition between childhood and adulthood, on average starting at age 12 and ending at age 18 [1,2]. This period is marked by significant neuroplasticity in the prefrontal cortex and limbic regions, two brain regions involved in development of adult behavior and cognitive functions [1,3].

Cannabis use among adolescents is very high, with 9.4% of 8th graders, 23.9% of 10th graders, and 36.5% of 12th graders reporting cannabis use in the last 12 months in 2016 [4]. This event is concerning as cannabis abuse can lead to persistent cognitive impairments in learning, attention and memory [5–10]. Moreover, early cannabis use before 16 years of age increases the risk of developing psychiatric disorders, including anxiety-related symptoms [11–13]. Anxiety appears to be the most common complication arising from heavy cannabis use, with up to 20% of cannabis users experiencing anxiety [14] while the prevalence of anxiety in the general population is estimated to be around 6–17% [15].

The primary psychoactive component of cannabis is delta-9-tetrahydrocannabinol (THC). The biological effects of THC are mainly mediated by members of the G proteincoupled receptor (GPCR) family, such as cannabinoid receptors (CB1R and CB2R).

The cannabinoid receptors together with their naturally occurring ligands (anandamide and 2-arachidonoyl glycerol) and the enzymes responsible for their biosynthesis

**Citation:** Sestan-Pesa, M.; Shanabrough, M.; Horvath, T.L.; Miletta, M.C. Impaired Ghrelin Signaling Does Not Lead to Alterations of Anxiety-like Behaviors in Adult Mice Chronically Exposed to THC during Adolescence. *Biomedicines* **2023**, *11*, 144. https:// doi.org/10.3390/biomedicines11010144

Academic Editor: Wesley M. Raup-Konsavage

Received: 22 November 2022 Revised: 17 December 2022 Accepted: 4 January 2023 Published: 6 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

constitute the endocannabinoid system [16,17]. This system plays a critical role in the maturation of brain circuits during adolescence by regulating excitatory and inhibitory neurotransmission [18]. Further, CB1R expression increases dramatically in regions such as the prefrontal cortex, striatum, and hippocampus [19]. Imaging studies have shown decreased cortical thickness in the right superior prefrontal cortex (PFC), bilateral insula and bilateral superior cortices in adolescent cannabis users compared to adolescents who do not use cannabis [20], as well as a decrease in volume of the right medial orbitofrontal cortex [21] and bilateral hippocampus [22,23].

Ghrelin is a hormone mainly produced in the gu<sup>t</sup> [24]. It stimulates potent orexigenic effects through metabolic homeostatic regulatory mechanisms in the hypothalamus and by increasing food reward and motivation through mesolimbic activation [25,26]. Ghrelin mediates both peripheral and central physiological functions through the growth hormone secretagogue receptor (GHSR) [27]. Ghrelin's role in regulating mood is very complex and it has a dual role in regulating anxiety. In some cases, injecting ghrelin centrally increased anxiety-like behaviors assessed by elevated plus maze [28], while other reports sugges<sup>t</sup> the opposite effect, with ghrelin injections showing a decrease in anxiety-like behaviors as assessed by elevated plus maze [29]. This discrepancy might be related to the timing of the behavioral experiments. Another factor that contributes to modulateing ghrelin's effect on behavior is food availability, with ghrelin increasing locomotion in the absence of food [30] and decreasing locomotion in the presence of food [31]. Findings in ghrelin knockout mice also demonstrate the controversial relationship between ghrelin and anxiety. Ghrelin knockout (Ghr −/−) mice appear to be less anxious than their wild-type counterparts under non-stressed conditions, but display more anxious behavior under mild stress conditions (15 min restraint) [32]. Of note is that stress increases ghrelin and corticosterone concurrently. GHSR and ghrelin knockout mice showed decreased plasma levels of corticosterone after chronic social defeat stress and acute restraint stress, as well as increased anxiety-like behavior [32,33]. Taken together, these findings sugges<sup>t</sup> that ghrelin and GHSR are important for the ability of animals to cope with anxiety-inducing stressors. GHSR and the cannabinoid CB1R are expressed within overlapping brain regions that are crucial for feeding (hypothalamus), reward and motivation (Ventral tegmental area/VTA, nucleus accubens/NAC). Both systems mutually interact to a significant extent in the regulation of homeostatic as well as hedonic food intake [34–37]. Further, systemic pretreatment with the CB1R antagonist rimonabant significantly reduced intracerebroventricular ghrelin-induced NAC dopamine release and hyperlocomotion in mice [38]. Despite this knowledge, there are limited data on the mutual role of cannabis and ghrelin in promoting anxiety-like behaviors. Therefore, we aim to test the way in which GHSR −/− mice (and their wildtype counterparts) would respond to chronic THC administration during adolescence. To investigate the long-term effects of THC on behavior relating to anxiety, we exposed the animals to 10 mg of THC daily (via pulmonary route) during sexual maturation (6–8 weeks old mice), which roughly corresponds to adolescence in humans. After 14 days of THC administration, animals (male and female mice) could recover for additional 4 weeks. At 12 weeks of age, behavioral testing was performed to evaluate any long-term effects from THC administration (Figure 1A).

**Figure 1.** Experiment design and equipment for THC administration. (**A**) Experimental design, time course for THC (or vehicle) administration and behavioral testing. (**B**) Entire apparatus used to administer THC and vehicle under a chemical hood. (**C**) Tubing leading from administration box to activated charcoal trap. (**D**) Tubing leading from activated charcoal trap to the activated charcoal filter, which then leads to the vacuum line. (**E**) Open-ended tube with Volcano mouthpiece attached. (**F**) Balloon attached to the open-ended mouthpiece, sealed with parafilm.

#### **2. Materials and Methods**
