# CANNABINOID THERAPEUTICS: WHAT'S HOT

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# CANNABINOID THERAPEUTICS: WHAT'S HOT

Topic Editors: Fabricio A. Pamplona, Entourage Phytolab, Brazil Carsten T. Wotjak, Max Planck Institute of Psychiatry (MPI), Germany Mark Ware, McGill University, Canada

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Citation: Pamplona, F. A., Wotjak, C. T., Ware, M., eds. (2019). Cannabinoid Therapeutics: What's Hot. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-419-4

# Table of Contents

#### *06 The Grass Might Be Greener: Medical Marijuana Patients Exhibit Altered Brain Activity and Improved Executive Function After 3 Months of Treatment*

Staci A. Gruber, Kelly A. Sagar, Mary K. Dahlgren, Atilla Gonenc Rosemary T. Smith, Ashley M. Lambros, Korine B. Cabrera and Scott E. Lukas

*19 Cannabinoid Receptor Type 1 Expression in the Developing Avian Retina: Morphological and Functional Correlation With the Dopaminergic System*

Luzia da Silva Sampaio, Regina C. C. Kubrusly, Yolanda P. Colli, Priscila P. Trindade, Victor T. Ribeiro-Resende, Marcelo Einicker-Lamas, Roberto Paes-de-Carvalho, Patricia F. Gardino, Fernando G. de Mello and Ricardo A. De Melo Reis

*29 Suppression of Cisplatin-Induced Vomiting by* Cannabis sativa *in Pigeons: Neurochemical Evidences*

Ihsan Ullah, Fazal Subhan, Javaid Alam, Muhammad Shahid and Muhammad Ayaz

*39 Endocannabinoid System and Migraine Pain: An Update* Rosaria Greco, Chiara Demartini, Anna M. Zanaboni, Daniele Piomelli and Cristina Tassorelli

#### *46 Emerging Role of (Endo)Cannabinoids in Migraine*

Pinja Leimuranta, Leonard Khiroug and Rashid Giniatullin

*53 Cannabidiol as a Promising Strategy to Treat and Prevent Movement Disorders?*

Fernanda F. Peres, Alvaro C. Lima, Jaime E. C. Hallak, José A. Crippa, Regina H. Silva and Vanessa C. Abílio

#### *65 Cannabigerol Action at Cannabinoid CB1 and CB2 Receptors and at CB1 –CB2 Heteroreceptor Complexes*

Gemma Navarro, Katia Varani, Irene Reyes-Resina,

Verónica Sánchez de Medina, Rafael Rivas-Santisteban, Carolina Sánchez-Carnerero Callado, Fabrizio Vincenzi, Salvatore Casano, Carlos Ferreiro-Vera, Enric I. Canela, Pier Andrea Borea, Xavier Nadal and Rafael Franco

*79 Chemical Proteomic Analysis of Serine Hydrolase Activity in Niemann-Pick Type C Mouse Brain*

Eva J. van Rooden, Annelot C. M. van Esbroeck, Marc P. Baggelaar, Hui Deng, Bogdan I. Florea, André R. A. Marques, Roelof Ottenhoff, Rolf G. Boot, Herman S. Overkleeft, Johannes M. F. G. Aerts and Mario van der Stelt

*88 Long-Term Stress and Concomitant Marijuana Smoke Exposure Affect Physiology, Behavior and Adult Hippocampal Neurogenesis* Kitti Rusznák, Kata Csekő, Zsófia Varga, Dávid Csabai, Ágnes Bóna,

Mátyás Mayer, Zsolt Kozma, Zsuzsanna Helyes and Boldizsár Czéh

*108 Cannabidiol as a Therapeutic Alternative for Post-traumatic Stress Disorder: From Bench Research to Confirmation in Human Trials* Rafael M. Bitencourt and Reinaldo N. Takahashi

Uchini S. Kosgodage, Rhys Mould, Aine B. Henley, Alistair V. Nunn, Geoffrey W. Guy, E. L. Thomas, Jameel M. Inal, Jimmy D. Bell and Sigrun Lange

Sumana Ghosh, Sandeep Sheth, Kelly Sheehan, Debashree Mukherjea, Asmita Dhukhwa, Vikrant Borse, Leonard P. Rybak and Vickram Ramkumar

Daniel Moreira-Silva, Daniel C. Carrettiero, Adriele S. A. Oliveira, Samanta Rodrigues, Joyce dos Santos-Lopes, Paula M. Canas, Rodrigo A. Cunha, Maria C. Almeida and Tatiana L. Ferreira

Libor Uttl, Ewa Szczurowska, Kateřina Hájková, Rachel R. Horsley, Kristýna Štefková, Tomáš Hložek, Klára Šíchová, Marie Balíková, Martin Kuchař, Vincenzo Micale and Tomáš Páleníček

*223 The Endocannabinoid System and Oligodendrocytes in Health and Disease*

Alexander A. Ilyasov, Carolanne E. Milligan, Emily P. Pharr and Allyn C. Howlett

*233 Case Report: Clinical Outcome and Image Response of Two Patients With Secondary High-Grade Glioma Treated With Chemoradiation, PCV, and Cannabidiol*

Paula B. Dall'Stella, Marcos F. L. Docema, Marcos V. C. Maldaun, Olavo Feher and Carmen L. P. Lancellotti

*240 Acute Cannabinoids Produce Robust Anxiety-Like and Locomotor Effects in Mice, but Long-Term Consequences are Age- and Sex-Dependent* Chelsea R. Kasten, Yanping Zhang and Stephen L. Boehm II

Paulo Fleury-Teixeira, Fabio Viegas Caixeta, Leandro Cruz Ramires da Silva, Joaquim Pereira Brasil-Neto and Renato Malcher-Lopes

# The Grass Might Be Greener: Medical Marijuana Patients Exhibit Altered Brain Activity and Improved Executive Function after 3 Months of Treatment

Staci A. Gruber1,2,3 \*, Kelly A. Sagar1,2,3, Mary K. Dahlgren1,2,4, Atilla Gonenc1,2,3 , Rosemary T. Smith1,2, Ashley M. Lambros1,2, Korine B. Cabrera1,2 and Scott E. Lukas3,5

1 Cognitive and Clinical Neuroimaging Core, McLean Imaging Center, McLean Hospital, Belmont, MA, United States, 2 Marijuana Investigations for Neuroscientific Discovery Program, McLean Imaging Center, McLean Hospital, Belmont, MA, United States, 3 Department of Psychiatry, Harvard Medical School, Boston, MA, United States, 4 Department of Psychology, Tufts University, Medford, MA, United States, 5 Behavioral Psychopharmacology Research Laboratory, McLean Imaging

#### Edited by:

Fabricio A. Pamplona, Entourage Phytolab, Brazil

#### Reviewed by:

Tiago Bortolini, Instituto D'Or de Pesquisa e Ensino (IDOR), Brazil Eugene A. Kiyatkin, National Institute on Drug Abuse (NIH), United States Tiago Arruda Sanchez, Universidade Federal do Rio de Janeiro, Brazil

> \*Correspondence: Staci A. Gruber<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology

Received: 07 September 2017 Accepted: 22 December 2017 Published: 17 January 2018

#### Citation:

Gruber SA, Sagar KA, Dahlgren MK, Gonenc A, Smith RT, Lambros AM, Cabrera KB and Lukas SE (2018) The Grass Might Be Greener: Medical Marijuana Patients Exhibit Altered Brain Activity and Improved Executive Function after 3 Months of Treatment. Front. Pharmacol. 8:983. doi: 10.3389/fphar.2017.00983 Center, McLean Hospital, Belmont, MA, United States

The vast majority of states have enacted full or partial medical marijuana (MMJ) programs, causing the number of patients seeking certification for MMJ use to increase dramatically in recent years. Despite increased use of MMJ across the nation, no studies thus far have examined the specific impact of MMJ on cognitive function and related brain activation. In the present study, MMJ patients seeking treatment for a variety of documented medical conditions were assessed prior to initiating MMJ treatment and after 3 months of treatment as part of a larger longitudinal study. In order to examine the effect of MMJ treatment on task-related brain activation, MMJ patients completed the Multi-Source Interference Test (MSIT) while undergoing functional magnetic resonance imaging (fMRI). We also collected data regarding conventional medication use, clinical state, and health-related measures at each visit. Following 3 months of treatment, MMJ patients demonstrated improved task performance accompanied by changes in brain activation patterns within the cingulate cortex and frontal regions. Interestingly, after MMJ treatment, brain activation patterns appeared more similar to those exhibited by healthy controls from previous studies than at pre-treatment, suggestive of a potential normalization of brain function relative to baseline. These findings suggest that MMJ use may result in different effects relative to recreational marijuana (MJ) use, as recreational consumers have been shown to exhibit decrements in task performance accompanied by altered brain activation. Moreover, patients in the current study also reported improvements in clinical state and health-related measures as well as notable decreases in prescription medication use, particularly opioids and benzodiapezines after 3 months of treatment. Further research is needed to clarify the specific neurobiologic impact, clinical efficacy, and unique effects of MMJ for a range of indications and how it compares to recreational MJ use.

Keywords: medical marijuana, cannabis, neuroimaging, fMRI, cognition, executive function, MSIT

# INTRODUCTION

fphar-08-00983 January 12, 2018 Time: 13:29 # 2

Currently, 30 states and the District of Columbia have medical marijuana (MMJ) programs or pending MMJ legislation, an additional 16 states have passed laws to allow limited access to MMJ, and an estimated 2.6 million individuals in the United States are certified for MMJ use (Procon.org). Since societal attitudes toward marijuana (MJ) have generally warmed, an increasing number of individuals are turning to MMJ to help treat a variety of medical conditions, as patients often do not achieve full symptom alleviation with conventional medications and experience unwanted side effects. Data gathered from several US surveys of MMJ patients indicate that the most common indications for MMJ use included pain-related concerns (i.e., chronic pain, headaches), psychiatric disorders (i.e., anxiety, depression), and insomnia (Nunberg et al., 2011; Reinarman et al., 2011; Bonn-Miller et al., 2014; Park and Wu, 2017). Although considerable research efforts have clarified the impact of recreational MJ use, particularly among adolescent and young adult populations, to date, there is a paucity of research focused on examining the impact of MMJ use on neurobiologic measures, including brain function and structure. As the number of MMJ patients continues to grow, research efforts designed to understand potential changes associated with MMJ use are critically important.

 While these deficits have been observed in adult MJ users (Nader and Sanchez, 2017), they are most salient among MJ-using adolescents (Lisdahl et al., 2014) who are in the midst of critical neurodevelopment (Giedd et al., 1999). Furthermore, these decrements have also been linked to alterations in brain structure and function. Although the directionality of structural alterations appears to be dependent on the brain region under investigation (Batalla et al., 2013), studies show that gray and white matter alterations are associated with increased executive dysfunction (Medina et al., 2009, 2010; Churchwell et al., 2010; Clark et al., 2012; Price et al., 2015). In addition, functional magnetic resonance imaging (fMRI) studies have reported altered activation patterns within the prefrontal cortex as well as orbitofrontal, cingulate, and subcortical/limbic regions of recreational MJ users compared to non-using control subjects during tasks of executive function (Lisdahl et al., 2014, for review). Further, similar to studies of cognitive performance and brain structure, fMRI studies have revealed that earlier onset of MJ use is related to altered patterns of brain activation during tasks requiring cognitive control and inhibition (Tapert et al., 2007; Gruber et al., 2012a; Sagar et al., 2015).

Although many have posited that MMJ use would be associated with similar deficits, preliminary studies have suggested that this may not be the case. In our own recent pilot investigation (Gruber et al., 2016), the only study to date to examine the impact of whole plant-derived MMJ products on cognitive performance, we found that MMJ patients did not demonstrate decrements in performance on measures of executive function following 3 months of MMJ treatment. In fact, patients generally demonstrated improved performance on a number of measures, particularly those assessing executive function. Improvements were also noted on several measures of quality of life, sleep, and depression relative to pre-MMJ treatment levels. Differences between recreational and MMJ users may be related to a variety of factors, including age of onset of MJ use, duration, magnitude and frequency of use, and choice of actual cannabis products used. Although products used by recreational MJ consumers and MMJ patients are derived from the same plant species, they are generally utilized for different purposes (i.e., to get high/alter one's current state of being vs. symptom alleviation). Accordingly, recreational and medical users often seek different MJ products with various constituent compositions based on the desired effect. Recreational MJ users often seek products high in 19 -tetrahydrocannabinol (THC), the main psychoactive constituent of the cannabis plant, and while medical patients may also choose products with high THC levels they often seek products high in other potentially therapeutic cannabinoids. Research has begun to focus on the beneficial effects of cannabidiol (CBD), the primary non-intoxicating constituent of MJ, which has been touted for its antipsychotic, anxiolytic, anti-seizure, and anti-inflammatory properties (Rong et al., 2017). While studies from recreational MJ users have reported a relationship between higher levels of THC and poorer cognitive performance (Ramaekers et al., 2006; Kowal et al., 2015) the acute administration of CBD prior to THC has been shown to improve cognitive function (Morgan et al., 2010; Englund et al., 2013), underscoring the need for further study. Moreover, Yücel et al. (2016) recently found that although MJ users exposed to THC exhibit alterations in hippocampal volume and neurochemistry, those who utilized CBD-containing products did not demonstrate differences relative to healthy controls. Similarly, a recent review of the effects of THC and CBD on neuroanatomy concluded that MJ users are prone to brain alterations in regions with high cannabinoid receptor density, and although THC exacerbates these alterations, CBD appears to protect against these deleterious changes (Lorenzetti et al., 2016). In addition, several researchers have administered pure THC or CBD to healthy control participants to investigate the impact of these constituents on brain activation patterns using fMRI. In general, studies suggest that THC and CBD have opposite effects on cognition-related brain activation (Bhattacharyya et al., 2010, 2015; Winton-Brown et al., 2011). This may be related to the fact that THC is a CB1 agonist with strong binding affinity for CB1 receptors, while CBD appears to exert effects through more indirect mechanisms, which include additional receptor types (Zuardi, 2008; Ashton and Moore, 2011). Despite preliminary work investigating the acute effects of pure THC and CBD on neural networks associated with cognitive domains impacted by MJ use, to our knowledge, no studies thus far have examined the impact of treatment with whole-plant-derived MMJ products on brain activation patterns.

In order to investigate whether pilot observations of improved executive function (Gruber et al., 2016) persist with larger sample sizes and to determine whether these changes co-occur with altered brain activation patterns, MMJ patients from an ongoing longitudinal study underwent fMRI while completing the Multi-Source Interference Test (MSIT). The MSIT is a robust measure of cognitive interference, a core facet of executive functioning, which is related to attentional control and inhibitory processing and requires actively shifting attention by inhibiting automatic responses (Lezak et al., 2004). This task reliably activates frontal brain regions associated with executive functioning, particularly the cingulo-frontal-parietal (CFP) network (Bush and Shin, 2006). Given our previous findings (Gruber et al., 2016), we hypothesized that following 3 months of treatment, MMJ patients would demonstrate improved task performance, and that this improvement would coincide with changes in brain activation patterns measured by fMRI. We have previously utilized the MSIT to better characterize patterns of cingulate and frontal brain activation within clinical and non-clinical cohorts (Gruber et al., 2012a, 2017), and although no studies thus far have examined MMJ patients using neuroimaging techniques, we hypothesized that improved MSIT task performance would be associated with increased activation in these regions following 3 months of MMJ treatment. We also posited that these changes would occur in the context of improved mood and quality of life ratings.

#### MATERIALS AND METHODS

#### Participants

To date, of 45 consented participants, 41 MMJ patients were enrolled and data from 22 patients' pre-treatment (Visit 1) and 3-month check-in visits (Visit 2) were available for analyses. In addition, patients who were free of MRI contraindications also completed neuroimaging procedures (n = 15). In order to qualify for study entry, patients had to be over the age of 18, and have an estimated IQ of 75 or higher as assessed by the Wechsler Abbreviated Scale of Intelligence (WASI; Wechsler, 1999). In order to minimize the effects of previous MJ exposure on study findings, patients were required to be MJ naïve or be abstinent from MJ use for at least 2 years for their pre-treatment visit. Patients were also required to be certified for MMJ use, or describe a plan to use industrial hemp derived products (which do not currently require certification). All subjects received payment for each study visit, and those who completed MRI procedures were compensated additionally in accordance with Partners IRBapproved protocol procedures.

#### Study Design

Prior to participation, all study procedures were explained, and each participant was required to provide written informed consent in accordance with the Declaration of Helsinki. This document and all study procedures were approved by the Partners Institutional Review Board. Eligible participants were enrolled in a larger longitudinal study designed to assess the impact of MMJ on cognition and brain function over the course of 12–24 months. Patients completed all assessments and imaging prior to initiation of MMJ treatment and again after 3 months of treatment.

As part of a larger neuroimaging protocol, participants completed the MSIT (Bush et al., 2003; Bush and Shin, 2006) with concurrent fMRI scanning using identical task parameters as reported in our previous studies of recreational MJ users, patients with bipolar disorder, and healthy controls (Gruber et al., 2012a, 2017). Using aspects from well-established measures of cognitive interference (e.g., Stroop, Simon, and Flanker tasks), the MSIT incorporates both spatial and flanker types of interference to measure cognitive control (Bush et al., 2003; Bush and Shin, 2006). During the task, three-digit stimuli sets comprised of the numbers 0, 1, 2, or 3 are presented briefly on a screen. Each set contains two identical distractor numbers and a target number that differs from the distractors. Using a button box, participants report the identity of the target number that differs from the two distractor numbers during two conditions: during the Control condition, distractor numbers are always zeros, and the identity of the target number always corresponds to its position on the button box (i.e., 100, 020, 003). During the Interference condition, patients are required to inhibit a prepotent response in favor of a less automatic response (i.e., indicate the identity of the target number rather its position). Distractor numbers are always numbers other than 0, and the identity of the target number is always incongruent with its position on the button box (e.g., 211, 232, 331, etc.). Performance is measured by reaction time and percent accuracy, which can be further subdivided by error type. Omission errors occur when no response is given and are typically reflective of slower or overloaded cognitive processing while commission errors, or incorrect responses, generally indicate difficulty inhibiting inappropriate responses. The entire task is comprised of four blocks of control trials alternating with four blocks of interference trials; the task begins and ends with a fixation period (30 s), making the total run time 6 min and 36 s (see **Figure 1** for graphic representation of the task design).

Patients also completed a battery of self-report rating scales. Briefly, these included the Profile of Mood States (POMS), Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI), Pittsburgh Sleep Quality Index (PSQI), Barratt Impulsiveness Scale (BIS-11), and the Short Form-36 Health Survey (SF-36), a measure of functional health and quality of life. During each study visit, participants also provided information regarding dose, frequency, and duration of use for all conventional medications, which were categorized into different classes, including opioids, antidepressants, mood stabilizers, and benzodiazepines. Percent change data was calculated to assess potential changes in medication use from pre- to post-3 months of MMJ treatment.

After completing pre-treatment assessments, patients began MMJ treatment at their discretion. Although patients selected their own products and determined their own treatment regimens, we collected detailed data about MMJ use patterns and products. Between study visits, patients submitted biweekly diaries documenting MMJ use and were contacted by phone on

a monthly basis to acquire information regarding MMJ product type, frequency, magnitude, and modes of use using a modified timeline follow-back procedure (TLFB; Sobell et al., 1998). Following a minimum of 3 months of regular MMJ treatment, patients returned for their first of several check in visits (Visit 2) where they repeated all study measures. In addition, participants were asked to provide a sample of their most frequently used MMJ product(s) to an outside laboratory (ProVerde Laboratories, Inc.) for cannabinoid constituent profiling. These analyses, which quantified the levels of 10 major cannabinoids including THC and CBD, will be used to identify the unique effects of specific cannabinoids in future analyses.

#### Statistical Analyses

Descriptive statistics were calculated for demographic and MMJ use variables. Repeated-measure analyses of variance (ANOVAs) were used to assess changes in clinical state from Visit 1 to Visit 2. The assumption of homogeneity of variance was confirmed using Levene's F; however, Shapiro–Wilk tests indicated that data for the MSIT were not normally distributed. Accordingly, nonparametric, repeated-measures Wilcoxon Signed Rank Tests were used to assess changes from Visit 1 to Visit 2 for MSIT data. It is of note, however, that the non-parametric tests resulted in similar findings as the ANOVAs; all significant results remained. For the MSIT analyses, alpha was set at 0.05 for the response time and percent accuracy variables. In cases where percent accuracy differed significantly between Visits 1 and 2, comparisons of the two different error types (omission, commission) utilized a Bonferroni correction for multiple comparisons (α/2 = 0.025).

#### fMRI Methods and Analyses

All imaging was performed on a Siemens Trio whole body 3T MRI scanner (Siemens Corporation, Erlangen, Germany) using a 12-channel phased array head coil. For the MSIT, 40 contiguous coronal slices were acquired from each participant, ensuring whole brain coverage (5 mm thick, 0 mm skip), and images were collected with TR = 3000, using a single shot, gradient pulse echo sequence (TE = 30 ms, flip angle = 90, with a 20 cm field of view and a 64 × 64 acquisition matrix; in plane resolution 3.125 mm × 3.125 mm × 3.125 mm). A total of 132 images per slice were collected.

fMRI images were analyzed using SPM8 (version 4667, Wellcome Department of Imaging Neuroscience, University College, London, United Kingdom) software package running in MATLAB (version R2010b, MathWorks, Natick, MA, United States). First, blood-oxygen-level dependent (BOLD) fMRI data were corrected for slice timing and for motion in SPM8 using a two-step intra-run realignment algorithm that uses the mean image created after the first realignment as a reference. A criterion of 3 mm of head motion in any direction was used as an exclusionary criterion. The realigned images were then normalized to an EPI template in Montreal Neurological Institute stereotactic space using DARTEL. Normalized images were resampled into 3 mm3 voxels and then spatially smoothed using an isotropic Gaussian kernel with 6 mm full width at half maximum. Global scaling was not used, high-pass temporal filtering with a cut-off of 168 s was applied, and serial autocorrelations were modeled with an AR(1) model in SPM8. Using a general linear model, statistical parametric images were calculated individually for each subject showing Interference > Control. These images were subsequently entered into second level model, subjected to a voxel-wise contrast and t-test to assess statistical significance. In addition to the realignment during the preprocessing, effects of motion were further corrected by removing motion related components from the data by including the calculated motion parameters from the realignment as regressors in the GLM (e.g., six nuisance regressors corresponding to three directions of translation and three axes of rotation). Regions of interest (ROI) masks were created using the Wake Forest University Pickatlas utility (Maldjian et al., 2003) and included cingulate and frontal regions. Specifically, the cingulate ROI was comprised of both bilateral anterior and mid cingulate regions (22,302 voxels) while the frontal ROI was comprised of bilateral superior frontal, middle frontal and inferior frontal gyri (6,857 voxels; see Supplementary Figure 1). These regions were selected as the cingulate (CC) and frontal cortices are associated with inhibitory processing and are reliably activated during the completion of the MSIT (Bush and Shin, 2006; Gruber et al., 2012a, 2017). Contrast analyses consisted of the subtraction of one map from

the other; for example, the cingulate activity of Visit 1 was subtracted from cingulate activity of Visit 2 to determine which areas showed increased activity over the course of treatment. As in previous studies (Heckers et al., 2004; Harrison et al., 2007; Yucel et al., 2007; Shin et al., 2011; Harding et al., 2012), the fixation point was not included in planned contrast analyses. The statistical threshold was set at p < 0.05 for cluster level family-wise-error (FWE), p < 0.001 for voxel level FWE with a minimum cluster extent k = 15 contiguous voxels in accordance with previously published manuscripts that have utilized the MSIT and have used a k-value of 15 (Gruber et al., 2012a) or lower (Harding et al., 2012; Bush et al., 2013). In addition to utilizing previously published k-values, we also conducted Monte Carlo simulations (Ward, 2000) to determine a more rigorous cluster extent for p < 0.001 which yielded k = 91. As two ROIs were used for analyses, data was also corrected for multiple comparisons, generating a new statistical threshold (p < 0.0005). One patient was excluded from MSIT analyses as they requested early termination of scanning procedures.

### RESULTS

#### Demographics and MMJ Use

All patients (11 male, 11 female) were between the ages of 28–74 (M = 50.64, SD = 13.15) who reported seeking MMJ treatment for a variety of conditions including pain (n = 13), anxiety/PTSD (n = 10), sleep (n = 10), mood (n = 8), and "other" conditions (n = 8), which included gastrointestinal issues, difficulty with attention, and additional indications not specified by the state of Massachusetts. Patients in the current sample were generally welleducated; all had earned a high school diploma, many completed advanced education (M = 15.91 years, SD = 1.97), and all were of at least average intelligence as measured by the WASI (M = 117.23, SD = 7.63). Upon initiation of MMJ treatment, all patients reported at least weekly use, which ranged from 1.5 times per week to multiple times per day. As noted in **Table 1**, patients reported using MMJ products an average of 5.34 days per week and 1.83 times per day for an overall average of 10.26 total episodes of MMJ use per week. Patients also indicated various routes of administration, including smoking and vaporizing flower, as well as use of oil and concentrates (vaporized and oral administration), tinctures, edibles, and topicals.

#### MSIT Behavioral Performance

Relative to pre-treatment, patients demonstrated improved MSIT performance following 3 months of MMJ treatment (**Table 2**). During the Control condition, patients exhibited improved performance, marked by fewer omission errors; however, qualitative analyses revealed that patients approached near perfect levels of performance pre-and post-treatment for this condition. During the Interference condition, patients performed notably better at Visit 2, demonstrating significantly fewer omission and a trend for fewer commission errors, and thus significantly improved percent accuracy. In addition, MMJ patients also demonstrated faster response times during Visit 2, relative to Visit 1, across both Control and Interference trials.

TABLE 1 | Demographics and MMJ use.

aWASI, Wechsler Abbreviated Scale of Intelligence; b reflects average use from the start of regular treatment through Visit 2.

#### MSIT fMRI Data

Interestingly, in addition to improved task performance, MMJ patients exhibited notable changes in brain activation patterns in terms of both magnitude and location from Visit 1 to Visit 2. Results are provided in **Table 3** which includes data for both the a priori threshold of k = 15 and also indicates which values survived the new threshold of k = 91 determined by the Monte Carlo simulations. After initiating MMJ treatment, patients generally exhibited increased activation within both the cingulate and frontal ROIs. Specifically, within the CC ROI, single-sample analyses revealed no significant activation at Visit 1, yet at Visit 2 patients exhibited focal activation within the midcingulate cortex (k = 165). Within-subjects contrast analyses between Visit 1 and Visit 2 revealed no significant activation differences for Visit 1 > Visit 2, but the Visit 2 > Visit 1 contrast indicated activation differences within the right anterior cingulate (k = 43). Within the frontal ROI, single-sample analyses revealed activation at Visit 1 within the left superior (k = 65) and the right inferior frontal gyrus (k = 19), and at Visit 2, within the right inferior (k = 575), left middle frontal gyrus (k = 217), and the left precentral gyrus (k = 19). Within-subjects contrast analyses between Visit 1 and Visit 2 yielded no significant activation differences for the Visit 1 > Visit 2 contrast; however, the Visit 2 > Visit 1 contrast revealed significant activation differences within the right middle gyrus (k = 88) and superior frontal gyrus (k = 25). See **Figure 2**.

#### Clinical Ratings and Conventional Medication Use

Following 3 months of MMJ treatment, patients reported some improvement on measures of mood and quality of life (**Table 4**). Across all rating scales, no significant worsening

of clinical state or quality of life was observed. Moreover, consistent with a previous report (Gruber et al., 2016), patients reported significant improvements on measures of depression (BDI), impulsivity (BIS-11), sleep (PSQI), and quality of life (SF-36). Specifically, on the SF-36, patients indicated significantly improved energy/fatigue and fewer role limitations due to physical health, which reflects how often patients' physical health affects their work and other life

TABLE 2 | Repeated measures Wilcoxon signed rank tests assessing Multi-Source Interference Test (MSIT) performance at pre-treatment and after 3 months of MMJ use (post-treatment).

df = 1,21; acorrected for multiple comparisons using Bonferroni method; ∗ results significant at p ≤ 0.05 when α = 0.05 or, for Bonferroni corrected analyses, at p ≤ 0.025 when α = 0.025; ˆresults trending toward significance at p ≤ 0.10 when α = 0.05 or, for Bonferroni corrected analyses, at p ≤ 0.05 when α = 0.025.

TABLE 3 | Multi-Source Interference Task (Interference-Control condition): activation local maxima within cingulate cortex (CC) and frontal cortex regions of interest (ROIs).

The statistical threshold was initially set at p < 0.05 for cluster level family-wise-error (FWE), and p < 0.0005 for voxel level FWE (corrected for multiple comparisons) with a minimum cluster extent k = 15 contiguous voxels. Bolded results indicate values that survived the Monte Carlo simulation minimum cluster extent (k = 91).

activities. A trend also emerged suggesting improved social functioning.

In addition to improvements in clinical state and quality of life, following 3 months of MMJ treatment, patients reported reductions in their use of conventional pharmaceutical products across several drug classes. Specifically, patients taking opioids reported a 47.69% reduction in use and those prescribed benzodiazepines reported a 46.91% reduction in use. Antidepressant use decreased by 22.35% while the use of mood stabilizers decreased by 28.57% between Visit 1 and Visit 2.

### DISCUSSION

Following 3 months of MMJ treatment, patients exhibited improved task performance and related alterations in frontal brain activation patterns during the completion of the MSIT, a measure of executive function and cognitive control, relative to pre-MMJ treatment. Within the cingulate cortex (CC), patients did not exhibit any significant pre-treatment activation during the Interference condition of the MSIT; however, after 3 months of treatment, robust activation was noted within this region. In fact, the magnitude of activation significantly increased over the course of treatment such that post-treatment activation patterns appeared more similar to that of healthy controls observed in previous studies (Bush and Shin, 2006; Gruber et al., 2012a). Activation within the frontal ROI was also notably increased following 3 months of MMJ treatment relative to pre-MMJ treatment. Taken together, these changes may be reflective of a potential "normalization" of brain function following 3 months of MMJ use.

Further, changes in brain activation patterns were observed in the context of improved task performance and self-reported improvements in mood and quality of life as well as reduced sleep disturbance and lower motor impulsivity, consistent with previously published preliminary data (Gruber et al., 2016). It is possible that improvements in symptomatology (i.e., relief of symptoms, improved mood/sleep) are directly related to observed improvements in cognitive function and alterations in brain activation. Patients in the present study most commonly endorsed pain and anxiety as their reasons for MMJ certification; both of these conditions have previously been associated with reduced cognitive performance (Moriarty et al., 2011; Vytal et al., 2013). Symptom improvement may therefore result in improved cognitive performance, and subsequently impact patterns of brain activation during completion of these tasks.

In addition to reduced symptomatology resulting in improved cognitive performance, it is also possible that several other factors may have also contributed to the observed changes. Patients reported notable decreases in their use of opioids, benzodiazepines, antidepressants, and mood stabilizers, and it is possible that reductions in conventional medications influenced changes in brain activation patterns.  In particular, Bell et al. (2005) reported that use of mood stabilizers is related to hypoactivation of the CFP network, a key region implicated in cognitive interference processing. While we are not aware of fMRI research focused on the effects of short-term prescriptive doses of opioids in humans, one study examining opioid dependence reported that normalization of frontal brain activation patterns was related to days since last drug use (Bunce et al., 2015). Reduction or cessation of use of these medications may therefore alter patterns of brain activation. Accordingly, future studies are needed to disentangle the effects

TABLE 4 | Mood and health ratings at pre-treatment and after 3 months of MMJ use (post-treatment).

Clinical rating scales (POMS, BDI, BAI), lower scores reflect lower levels of clinical symptoms; BIS-11, lower scores indicate lower levels of self-reported impulsivity; PSQI, lower scores reflect improved sleep quality; SF-36, higher scores indicate higher quality of life. adf = 1,21; bdf = 1,18; ∗bolded results are significant at p ≤ 0.05; ˆ italicized results approach significance at p ≤ 0.10.

of MMJ treatment and conventional medication use on brain activation patterns, and may benefit from limiting clinical samples to only those on a single specific class of conventional medication.

Although findings from this study indicate improvements in cognitive task performance and more normalized patterns of brain activation after 3 months of MMJ treatment, previous studies, exclusively focused on recreational MJ users, have reported decrements in cognitive performance and accompanying atypical neural alterations. A recent review highlighting neuroimaging findings in recreational MJ users found evidence for altered frontal neural function during completion of executive function tasks (Weinstein et al., 2016), a finding observed in our own previous research (e.g., Gruber and Yurgelun-Todd, 2005; Gruber et al., 2012a; Sagar et al., 2015). A number of critical factors may account for the differences between our current findings in MMJ patients relative to findings from recreational MJ consumers. The majority of studies of recreational MJ use have included adolescent and young adult populations. Overwhelmingly, studies have demonstrated that early/adolescent onset of recreational MJ use is related to poorer task performance and changes in brain structure and function (Lisdahl et al., 2013, 2014; Jacobus and Tapert, 2014; Levine et al., 2017). Given that participants in the current sample are adults (Mean age = 50.64) who are well-beyond the critical stages of neurodevelopment (Giedd et al., 1999), they are likely less vulnerable to the adverse neural effects of THC. Interestingly, recent preclinical evidence indicates that THC may have the potential to improve cognition in older individuals (Bilkei-Gorzo et al., 2017). Mature and old mice administered low doses of THC demonstrated a reversal of age-related cognitive decline, hypothesized to be related to upregulation of the aging endocannabinoid system via increased signaling secondary to low dose THC exposure. Moreover,

the same exposure resulted in cognitive decrements among young mice. Additional research is needed to more fully understand the mechanisms underlying these improvements and to examine the impact of cannabis and cannabinoids in older adult populations as well as the effects of low doses of THC, as these factors likely influence the impact of MJ use.

It is also important to consider patterns of MJ use, including frequency and duration of use, in order to understand potential reasons for the different outcomes among recreational users and medical patients. In the current study, all patients reported using MMJ at least weekly; on average, they reported using 5 days per week and 1–2 times per day. Traditionally, studies of recreational users have examined chronic, heavy use; although criteria for "heavy use" can vary across investigations, most studies have required participants to use MJ at least 1–4 days per week (Tait et al., 2011; Gruber et al., 2012a; Macher and Earleywine, 2012; Dougherty et al., 2013; Cousijn et al., 2014), similar to the frequency of use among the current sample of medical patients. Given these similarities, it is unlikely that differences between recreational and MMJ patients are solely attributable to frequency of use. Additionally, studies of recreational MJ users typically include consumers with a longer duration of MJ use relative to the current sample of MMJ patients, and differences in cumulative exposure should also be considered. For this reason, our ongoing study is designed to examine MMJ users after increasingly longer periods of use to explore the impact of longer durations of MMJ use on cognitive function.

Further, MMJ patients and recreational MJ users also typically differ in terms of the products they use. Recreational MJ products are often prized for high THC levels, and the goal of the recreational consumer is to change their current state of being or to 'get high.' MMJ patients seek symptom alleviation and tend to choose products with rich and varied cannabinoid profiles including constituents other than THC, which may also impact clinical state, cognitive processing and other domains. For example, CBD, which has been touted for its clinical benefits (Rong et al., 2017), has demonstrated efficacy in mitigating the negative cognitive effects of THC (Yücel et al., 2016) and appears to exert opposite effects on task-related brain activation relative to THC (Colizzi and Bhattacharyya, 2017). In addition, although there is a paucity of research in this area, some studies have examined the direct impact of acute CBD administration on cognitive performance. Englund et al. (2013) reported that administration of CBD prior to the administration of THC resulted in better episodic memory relative to placebo pre-treatment in healthy controls. Morgan et al. (2010) examined verbal memory performance in current recreational MJ users and found that those using products without CBD (confirmed by hair sample analysis) performed more poorly on verbal memory measures than those with detectable levels of CBD. While no studies have examined the impact of whole plant-derived MMJ products or assessed the long-term impact of MMJ treatment, some studies have utilized fMRI techniques to examine the acute effects of

individual cannabinoids. Borgwardt et al. (2008) studied the acute impact of THC, CBD, and placebo on executive function in healthy controls using a Go/No go task. Although the authors did not report any performance differences between cannabinoids or placebo, fMRI data demonstrated that THC reduced activation in frontal and anterior cingulate regions, while CBD reduced activation in temporal and insular regions relative to placebo. In addition, Bhattacharyya et al. (2010) found that intravenous administration of THC and CBD resulted in opposite effects on brain activation patterns across multiple regions during the completion of memory, inhibitory function, and affective measures. Given these findings, data from the present study may reflect the direct neurobiologic effects of cannabinoids, as increased endocannabinoid signaling is associated with improved cognition (Egerton et al., 2006), reduced stress response, emotional regulation, and increased endogenous reward signaling (Hill and McEwen, 2010; Befort, 2015), and as previously noted, specific alterations in brain activation patterns (Borgwardt et al., 2008; Bhattacharyya et al., 2010). Results may also reflect the indirect impact of whole plant-derived products, which include an array of cannabinoid constituents, and may exert downstream affects on multiple receptor types and neural systems (i.e., pain and reward circuitry). While THC and CBD are generally the most abundant cannabinoids in patients' products, and as noted, several studies have begun to explore their impact on cognition and brain activation patterns, a number of other cannabinoids including cannabigerol (CBG), cannabinol (CBN), cannabichromene (CBC), and tetrahydrocannabidivarin (THCV), are often present in MMJ products, and may have moderated or indirectly affected the impact typically associated with THC exposure (Englund et al., 2016). Further research is clearly indicated for assessing the specific impact of individual cannabinoids on cognitive and clinical variables in patients using cannabis for medical purposes.

#### Limitations and Future Directions

Despite the compelling nature of the study findings, several limitations must be noted. First, the current investigation is designed as an observational, longitudinal pre–post study in which patients choose their own products and treatment regimen. The ability to assess the impact of whole plant-derived cannabis-based products is more ecologically valid than studies involving synthetic or non-plant derived products; however, the current legal landscape prohibits the use of dispensarybased products within a clinical trial model and allows only the use of products supplied by the National Institute on Drug Abuse (NIDA). While NIDA's drug supply program has expanded their portfolio of MJ products available for research, their supply does not currently include the range and scope of products (i.e., product type, potency, constituent profiles, etc.) that patients are seeking and obtaining through dispensaries and caregivers across the nation. Accordingly, as a clinical trial model could not be utilized, the present study collected comprehensive data on product source, selection, dose, frequency, and mode of use. Further, as previously noted, patients also provided a sample of their most frequently

used MMJ products for cannabinoid constituent profiling. Interestingly, 13 of the 22 patients (59%) in the current study were identified as taking products high in CBD which may have contributed to study findings given previous data highlighting its clinical benefits (McGuire et al., 2017; Rong et al., 2017). As initial laboratory analyses revealed a range of cannabinoid constituents from patients' products, additional analyses will be conducted to examine the impact of specific cannabinoids and their relationship with cognitive and clinical variables. Further, future studies will assess potential differences between patients who choose products high in THC compared to those using products high in CBD, as these data may provide critical information regarding efficacy of individual constituents and combinations of constituents for specific indications and conditions to better inform selection of MMJ products.

The current study utilized a pre–post, within-subjects design in which all patients are MJ naïve at Visit 1 and are followed over the course of 12–24 months in order to clarify the impact of prolonged duration of exposure to MMJ treatment. As results from the current study represent only data from baseline and patients' first check-in visit after 3 months of MMJ treatment, data must be considered preliminary. Additional analyses, which are planned for the future, are needed to understand the impact of MMJ over longer treatment periods. Further, as a result of the pre–post study design, repeated administration of cognitive measures was required, and thus practice effects cannot be completely ruled out. Although no studies to date have specifically examined practice effects for the MSIT, given the lengthy duration of time between visits (at least 3 months) and the computerized nature of the task, it is highly unlikely that practice effects would persist. In addition, given the longitudinal nature of the design, each subject's baseline assessment serves as their own control from which to assess change after initiation of MMJ treatment. It could, however, prove beneficial for future investigations to also recruit a control group of patients who report similar symptoms (i.e., pain, insomnia, anxiety, etc.) but who do not choose to utilize MMJ. Comparing outcomes of MMJ patients and "treatment as usual" patients over time could strengthen findings if MMJ patients display more positive outcomes relative to those who do not use MMJ but suffer from similar symptoms or conditions.

In addition, statistical thresholds for fMRI analyses were set in accordance with previous investigations (Shin et al., 2011; Gruber et al., 2012a; Harding et al., 2012) in order to aid in the interpretation of findings. While more stringent thresholds were also applied and are noted within the results, it is important to recognize that some findings did not survive the more rigorous thresholds, a common issue in fMRI studies with limited sample sizes.

In order to gain a more thorough understanding of how MMJ impacts cognitive functioning, it will be important for future studies to replicate current study findings using other measures of executive function and to examine additional cognitive domains. As executive functioning has been shown to be impacted by recreational MJ use (for review, Crean et al., 2011), this domain was targeted for the current investigation; however, it is crucial to examine additional cognitive variables, including verbal memory, which has also been shown to be sensitive to MJ use (for review, Solowij and Battisti, 2008; Broyd et al., 2016).

Finally, this study included MMJ patients using products for a variety of indications, which resulted in a varied clinical sample. While this approach provides a broad assessment of the impact of MMJ, it is likely that individual conditions and symptoms will have unique patterns of responses associated with MMJ treatment. A number of medical and psychiatric conditions have been shown to negatively impact cognitive processing; accordingly, future studies may derive additional power by limiting inclusion to patients with a single condition or indication (i.e., patients using MMJ exclusively for pain) or including only patients taking medications from a single drug class.

#### CONCLUSION

To our knowledge, this study represents the first neuroimaging investigation of patients using marijuana for medical purposes. Following 3 months of MMJ treatment, brain activation patterns appear more similar to those exhibited by healthy controls from previous studies than at pre-treatment. This finding provides strong evidence that MMJ treatment may normalize brain activity. Importantly, these changes were accompanied by improved task performance as well as positive changes in ratings of clinical state, impulsivity, sleep, and quality of life. Further, patients reported notable decreases in their use of conventional medications, including opioids. In light of the national opioid epidemic, these data clearly underscore the need to expand and extend this study to determine if a reduction in opioid use persists with continued MMJ treatment. Results from the current study raise the possibility that the observed improvements in cognition and related changes in functional activation patterns may be related to direct and/or indirect effects of cannabinoids, specifically within an adult population beyond the stages of critical neuromaturation. Patients utilizing MMJ appear to use products with different cannabinoid profiles (i.e., high CBD) relative to recreational users, which is also likely to impact cognitive function. Observed changes may also be related to secondary or more indirect effects, including the reduction of clinical symptoms, improved sleep, and decreased use of conventional medications. Additional studies using both observational and clinical trial models to examine the impact of actual MMJ products used by patients are needed to clarify the underlying neural mechanisms associated with clinical and behavioral changes that accompany MMJ treatment.

#### AUTHOR CONTRIBUTIONS

SG conceptualized and designed the current study in consultation with SL. KS assisted SG in manuscript preparation with

additional help provided by the remaining authors. SG, KS, RS, AL, and KC recruited the patients, carried out the study procedures and administered the neuropsychological and clinical assessments. MD completed the statistical analyses and AG completed the neuroimaging analyses.

#### FUNDING

This project was funded by generous private donations to the Marijuana Investigations for Neuroscientific Discovery (MIND) Program at McLean Hospital.

#### REFERENCES

#### ACKNOWLEDGMENTS

The authors thank Integr8, Canna Care, and Dr. David Rideout at MMJ Physician Practice for their assistance in facilitating patient recruitment.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2017.00983/full#supplementary-material

systems over time in treatment in prescription opioid-dependent patients. J. Addict. Med. 9, 53–60. doi: 10.1097/ADM.0000000000000087

brain structure, and function. Front. Psychiatry 4:53. doi: 10.3389/fpsyt.2013. 00053

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Gruber, Sagar, Dahlgren, Gonenc, Smith, Lambros, Cabrera and Lukas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cannabinoid Receptor Type 1 Expression in the Developing Avian Retina: Morphological and Functional Correlation With the Dopaminergic System

Luzia da Silva Sampaio1 , Regina C. C. Kubrusly 2 , Yolanda P. Colli 1 , Priscila P. Trindade1 , Victor T. Ribeiro-Resende1 , Marcelo Einicker-Lamas 3 , Roberto Paes-de-Carvalho4 , Patricia F. Gardino1 , Fernando G. de Mello1 and Ricardo A. De Melo Reis 1 \*

1Laboratório de Neuroquímica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2Laboratório de Neurofarmacologia, Instituto Biomédico, Universidade Federal Fluminense, Niterói, Brazil, 3Laboratório de Biomembranas, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 4Laboratório de Neurobiologia Celular, Programa de Neurociências, Universidade Federal Fluminense, Niterói, Brazil

#### Edited by:

Fabricio A. Pamplona, Entourage Phytolab, Brazil

#### Reviewed by:

Victoria P. Connaughton, American University, United States Eric Murillo-Rodriguez, Anahuac Mayab University, Mexico

> \*Correspondence: Ricardo A. De Melo Reis<EMAIL_ADDRESS>
Received: 15 December 2017 Accepted: 19 February 2018 Published: 12 March 2018

#### Citation:

da Silva Sampaio L, Kubrusly RCC, Colli YP, Trindade PP, Ribeiro-Resende VT, Einicker-Lamas M, Paes-de-Carvalho R, Gardino PF, de Mello FG and De Melo Reis RA (2018) Cannabinoid Receptor Type 1 Expression in the Developing Avian Retina: Morphological and Functional Correlation With the Dopaminergic System. Front. Cell. Neurosci. 12:58. doi: 10.3389/fncel.2018.00058 The avian retina has been used as a model to study signaling by different neuro- and gliotransmitters. It is unclear how dopaminergic and cannabinoid systems are related in the retina. Here we studied the expression of type 1 and 2 cannabinoid receptors (CB1 and CB2), as well as monoacylglycerol lipase (MAGL), the enzyme that degrades 2-arachidonoylglycerol (2-AG), during retina development. Our data show that CB1 receptor is highly expressed from embryonic day 5 (E5) until post hatched day 7 (PE7), decreasing its levels throughout development. CB1 is densely found in the ganglion cell layer (GCL) and inner plexiform layer (IPL). CB2 receptor was also found from E5 until PE7 with a decrease in its contents from E9 afterwards. CB2 was mainly present in the lamination of the IPL at PE7. MAGL is expressed in all retinal layers, mainly in the IPL and OPL from E9 to PE7 retina. CB1 and CB2 were found both in neurons and glia cells, but MAGL was only expressed in Müller glia. Older retinas (PE7) show CB1 positive cells mainly in the INL and co-expression of CB1 and tyrosine hydroxylase (TH) are shown in a few cells when both systems are mature. CB1 co-localized with TH and was heavily associated to D1 receptor labeling in primary cell cultures. Finally, cyclic AMP (cAMP) was activated by the selective D1 agonist SKF38393, and inhibited when cultures were treated with WIN55, 212–2 (WIN) in a CB1 dependent manner. The results suggest a correlation between the endocannabinoid and dopaminergic systems (DSs) during the avian retina development. Activation of CB1 limits cAMP accumulation via D1 receptor activation and may influence embryological parameters during avian retina differentiation.

Keywords: cannabinoid, retina, development, dopamine, cAMP

# INTRODUCTION

The retina is a unique tissue located in the posterior part of the eye involved with light transduction in visual information. The vertebrate retina, easily accessible to experimental manipulation, formed by seven major cell types, reviewed in Masland (2012), is extensively used as an experimental model to investigate cell interactions. Neurons and glia cells interact bidirectionally, and the avian retina has been used in the last 40 years, as a model to study the role of neuro-glia interactions in cell migration and development (Reis et al., 2007; de Melo Reis et al., 2008).

Dopamine is the main catecholamine found in a subtype of retinal amacrine cells located in the inner nuclear layer (INL; Reis et al., 2007). Emergence of amacrine cells begins at early stages (around embryonic day 3, E3) and is concluded at E9 (Prada et al., 1991; Calaza Kda and Gardino, 2010). Tyrosine hydroxylase (TH) expression is only found around E12, while D1/D5 type dopaminergic receptors coupled to cyclic AMP (cAMP) production are fully functional on E7 (de Mello, 1978) as compared to the end of the proliferation period (Gardino et al., 1993). Indeed, analysis of chick retina sections show TH positive dopaminergic amacrine cells located in the dorsal retina at E13/E14, and beginning to be defined at E16, with a rising arborization complexity until hatching, in a way that at E18/2 days post hatching dopaminergic cells are uniformly distributed throughout the retina (Gardino et al., 1993). In addition, specific dopaminergic amacrine cell subpopulation shows a more restrict neurogenesis period than the general amacrine cell population. However, dopaminergic system (DS) development only becomes complete in the post-embryonic phase, when TH expressing in amacrine cells in the INL are found in a highly connected wiring pattern with retinal ganglion cells, located in the ganglion cell layer (GCL), exerting a major inhibitory input. TH expression is modulated and dependent on several extrinsic and intrinsic factors that determine the dopaminergic phenotype, such as the cAMP signaling cascade activated by PACAP (Reis et al., 2007; Fleming et al., 2013). However, a clear picture of dopaminergic amacrine cell development is still not completely understood.

The endocannabinoid system (ECS) is present in almost all classes of vertebrates (Cottone et al., 2013). The modulation of cannabinoid activity is mediated by selective type-1 and type-2 cannabinoid receptors (CB1 and CB2), which are members of the G protein-coupled receptor (GPCR) family that inhibit adenylate cyclase leading to a decrease in cAMP levels. Specific endogenous endocannabinoids agonists drive these receptors. These bioactive lipids, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are synthesized and degraded by a limited group of enzymes presented in different cell types (Howlett, 2002). One of these enzymes, monoacylglycerol lipase (MAGL), is a serine hydrolase involved in the metabolism of 2-AG in the brain (Chanda et al., 2010).

In the CNS, CB1 are found mainly in presynaptic neurons, functioning as a regulatory mechanism for the release of both excitatory and inhibitory neurotransmitters (Katona and Freund, 2008).

The ECS is expressed at very early stages of the avian retinal development, as evidenced by CB1 expression in neurons (Leonelli et al., 2005). Recently, CB2 has been described in Müller glia in mammals (Bouskila et al., 2013). Straiker et al. (1999) showed for the first time that CB1 is expressed in the avian retina in two synaptic retinal layers, the inner plexiform and outer plexiform layers (IPL and OPL, respectively). CB1 expression was also reported in some amacrine and ganglion cell bodies and axons (Straiker et al., 1999). We recently showed that CB1 and CB2 receptors are found in both neurons and glial avian retinal cells in culture. These receptors regulate avian retina signaling (GABA release, calcium mobilization and cAMP levels) at critical embryonic stages during synapse formation (Kubrusly et al., 2018).

Dopaminergic neurons are modulated by ECS in different areas of the CNS. CB1 and endocannabinoids are abundant in the DS, acting to modulate dopamine release. It has been reported that selective and non-selective CB1 agonists decrease the dopamine release in the guinea-pig retina (Schlicker et al., 1996). However, the relationship between dopaminergic and ECSs during retinal development is still unclear. Therefore, we characterized the expression of CB1 and CB2 receptor, and also MAGL, during the development of avian retina as well as the possible functional correlation between the dopaminergic and ECSs in this process.

# MATERIALS AND METHODS

All materials used were of analytical grade. Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12), fetal calf serum (FCS) and gentamycin were obtained from Gibco (USA).

### Animals

All experiments involving animals were approved by and carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Federal University of Rio de Janeiro (permit number IBCCF-035), and experimental procedures were carried out in accordance with the guidelines of the Brazilian Society of Neuroscience and Behavior (SBNeC). Fertilized White Leghorn eggs (Gallus gallus) were obtained from a local hatchery and kept in an appropriate incubator under 12 h light and 12 h dark cycles until the day of use. The eyes from chick embryos ranging from 5, 7, 9 and 14 days of incubation to 7 days post-hatching stage were used in this study. We started with embryonic day 5 (E5) since amacrine cells begin to differentiate early (E3; Calaza Kda and Gardino, 2010), CB1 receptors are found around E5 (Leonelli et al., 2005), and the first dopaminergic marker is described around E6 (dopamine transporter, DAT, Kubrusly et al., 2003). D1 receptors are found around E7 (de Mello, 1978), and amacrine neurons end their migration process (Gardino et al., 1993). A peak on retinal synaptogenesis is found around E14 (Calaza Kda and Gardino, 2010). And at P7, DS in the retina is fully differentiated (Gardino et al., 1993).

# Preparation of Tissue and Retinal Radial Sections

After decapitation, the embryos and chicks eyes were enucleated and fixed for 2 h in 4% paraformaldehyde in phosphate buffer 0.16 M, pH 7.4 (PB); after these procedures, the eyes were washed in PB three times. In 24 h, the eyes were cryoprotected in sucrose 15% and 30%, embedded in medium optimal cutting temperature (O.C.T. compound, Sakura Finetek, Torrance, CA, USA), frozen and then cut perpendicularly to the vitreal surface on a Leica CM3050 S cryostat (12 µm).

#### Mixed Retinal Cells in Culture

Primary cell cultures of chick retinal cells at stage E8 (8-days embryo) were prepared as described (Paes de Carvalho and de Mello, 1982). Briefly, retinas were dissected on a Ca2+ and Mg2+-free salt balanced medium (CMF) and cleared of the pigmented epithelium. The retinas were dissociated using TrypLEr (Thermo Fisher Scientific) and incubated at 37◦C for 10 min. After brief centrifugation, the pellet was suspended in 1 ml of DMEM/F-12 medium supplemented with 10% FCS and mechanically dissociated with a Pasteur pipette. Cells were plated on 13 mm coverslips (Marienbad, German; 1.6 × 106 cells), or 33.7 mm plates (TPP, Trasadingen, Switzerland; 20 × 106 cells), previously coated with Poly-L-Lysine. The cultures were maintained in an incubator at 37◦C under 5% CO2 atmosphere for 6 days (E8C6 cells in culture). E8C6 on 13 mm coverslips were fixed for 15 min in 4% paraformaldehyde in PBS.

#### Immunofluorescence

Retina sections and primary cell cultures of retinal cells were washed three times with PBS and incubated for 30 min with 0.15% Triton X-100 and 3% FCS in PBS. This buffer was removed and the primary antibodies of interest added in an overnight incubation at 4◦C. The sections and cultures were washed with PBS and incubated with fluorochrome-conjugated secondary antibody for 2 h at room temperature protecting from the light. After rinsing with PBS, the retinas and cultures were mounted using a PBS containing 40% glycerol and DAPI 0.04 µg/ml solution and were examined in a Zeiss Axio Imager 2 Fluorescence Microscope with Apotome and/or confocal microscope (LSM 510 Meta, Zeiss, Germany).

For the immunofluorescence results, specific antibodies were used according to the recommendation of each manufacturer. For primary antibodies, anti-CB1 (anti-cannabinoid receptor 1 produced in goat—SAB2500190 from Sigma-Aldrich), dilution of 1:100; anti-CB2 (CB2 antibody (M-15) produced in goat—sc-10076 from Santa Cruz Biotechnology) dilution of 1:100; anti-MAGL (anti-MAGL produced in rabbit—ab152002 from abcam) dilution of 1:100; anti-TH (antityrosine hydroxylase produced in rabbit—AB152 from EMD Millipore) dilution of 1:100; anti-MAP2 (anti-MAP2 produced in mouse—ab11267 from abcam) dilution of 1:400; anti-2M6 (monoclonal anti-2M6 (kindly provided by Dr. B. Schlosshauer; Max-Planck-Institute, Tübingen, Germany, Schlosshauer et al., 1991)), dilution of 1:400; anti-D1 (anti-dopamine D1R produced in rabbit—NBP2–16213 from Novusbio) dilution of 1:100.

For secondary antibodies, Alexa Fluorr 488 donkey anti-goat (A11055 produced from Thermo Fisher Scientific); Alexa Fluorr 488 donkey anti-rabbit (A21206 produced from Thermo Fisher Scientific); Alexa Fluorr 594 donkey anti-rabbit (A21207 produced from Thermo Fisher Scientific); Alexa Fluorr 594 donkey anti-mouse (A21203 produced from Thermo Fisher Scientific), all were used at the 1:500 dilution.

#### Western Blotting

Fresh retinas from embryos and chicks, and E8C6 cells culture were washed twice with PBS, homogenized with a lysis buffer (EDTA 10 mM, HEPES-Tris 50 mM, Sucrose 1 M, trypsin inhibitor 0.15 mg/ml) and the total protein concentration was quantified (Lowry et al., 1951). Total proteins were separated by polyacrylamide gel electrophoresis (10% SDS-PAGE) and transferred to nitrocellulose membranes (Laemmli, 1970). After 1 h in blocking buffer (5% low fat dried milk in Tris-buffered saline—TBS), the immunodetection was performed by incubating the membrane with a specific primary antibody overnight at 4◦C. The membranes were washed with TBS and incubated with secondary horseradish peroxidase (HRP)-conjugated antibody for 2 h at room temperature. The proteins of interest were finally detected using LuminataTM Forte (Merck Millipore) and ChemiDoc MP system (Bio-Rad).

For the western blotting results, we used specific antibodies, according to the recommendation of each manufacturer. For primary antibodies, anti-CB1 (anti-cannabinoid receptor CB1 (1–77) produced in rabbit–209550 from Calbiochem) was used at a dilution of 1:1000; anti-CB2 (anti-CNR2 antibody produced in mouse—WH0001269M1 from Sigma-Aldrich) was used at a dilution of 1:1000; anti-TH (anti-tyrosine hydroxylase produced in rabbit—AB152 from EMD Millipore) was used at a dilution of 1:500; anti-D1 (anti-dopamine D1R produced in rabbit—NBP2–16213 from Novusbio) was used at a dilution of 1:500; anti-DARP32 (anti-DARPP-32 antibody [EP720Y] produced in rabbit—ab40801 from abcam) was used at a dilution of 1:500; anti-Nurr-1 (anti-Nurr-1 antibody produced in rabbit—ab93332 from abcam) was used at a dilution of 1:500; anti-ERK1/2 (anti p44/42 MAPK (Erk1/2) antibody produced in mouse–4696 from cell signaling) was used at a dilution of 1:1000; anti-β actin (anti-β-actin clone AC-15 produced in mouse—A5441 from Sigma-Aldrich) was used at a dilution of 1:5000. For secondary antibodies, HRP-conjugated goat anti-mouse (A5278 from Sigma-Aldrich) and HRP-conjugated goat anti-rabbit (A0545 from Sigma-Aldrich) were used at a dilution of 1:5000 (for the β actin protocol, we used the dilution 1:25,000). For statistical analysis, densitometry for each protein of interest was performed using Scion Image software followed by ANOVA.

#### cAMP Accumulation

Accumulation of cAMP was assayed according to the competitive binding assay as described previously (Kubrusly et al., 2005). Mixed retinal cells were washed with PBS and pre-treated with 5 × 10−4 M IBMX, an inhibitor of phosphodiesterases, for 15 min in 1 ml of DMEM/F-12 plus 20 mM HEPES pH 7.4 at 37◦C. To measure adenylyl cyclase activity, we used 5 × 10−5 M SKF38393,

a D1R agonist, for 30 min at 37◦C, or 1 × 10−7 M forskolin, a general activator for this enzyme. The treatments performed were 10−6 M AM251, a CB1 antagonist, for 15 min; 10−6 M WIN55, 212–2, a non-selective cannabinoid receptor agonist, for 30 min. The reaction was stopped by addition of 100 µl trichloroacetic acid 100% (5% final) acid followed by storage at −20◦C for 24 h. After centrifugation (1000 g for 10 min), the supernatant was passed through an ion-exchange resin column (Dowex 50) to remove the trichloroacetic acid and other nucleotides. The samples containing cAMP were then incubated in the presence of the regulatory subunit of PKA and a fixed trace amount of [3H]cAMP in 50 mM acetate buffer, pH 4.0, at 4◦C for 90 min. The reaction was interrupted by the addition of 200 mM phosphate buffer at pH 6.0. The samples were filtered through Millipore filters and the radioactivity determined in a Tri Carb 2810 TR Perkin Elmer liquid scintillation analyzer.

#### Statistical Analysis

All the results are shown as means ± SEM. Graphpad Prism5r software was used to analyze the results by one way ANOVA, followed by Tukey post-test. For all the results, p < 0.05 was taken as the significance level, and the number of experiments is described in each figure legend.

#### RESULTS

Our results confirm that CB1 receptor is highly expressed in the chick retina. Indeed, we analyzed its expression during ontogenesis from embryonic day 9 (E9) for immunofluorescence and E5 for western blotting, until post-hatched day 7 (PE7; **Figures 1A,B**). Western blotting data show a decrease, but not statistically different, in the CB1 expression during avian retinal development (**Figure 1B**). CB1 is located in almost all retinal layers from E14 to PE7, and is more densely present in the GCL and IPL (**Figure 1A**).

We also evaluated the expression of other ECS proteins, such as CB2 receptor and MAGL, the enzyme that degrades the endocannabinoid agonist 2-AG. Our data clearly show the expression of CB2 receptor throughout development, between E5 and PE7 retinas (**Figure 1D**). We also observed a decrease in CB2 protein content from E5 afterwards and that these receptors are mainly present in the lamination of the IPL at PE7 retinas (**Figure 1C**). MAGL is expressed in all retinal layers, but more clearly in the IPL and OPL from E9 to PE7 retina (**Figure 1E**).

To test our hypothesis of a link between the ECS and the DS, we asked whether the CB1 receptor is co-expressed with TH in retina dopaminergic cells. We performed western blotting for TH at different stages, confirming that TH is highly expressed at PE7 (**Figure 2A**). TH amacrine neurons express CB1 receptor in the INL and GCL cells around PE7 (**Figure 2B**). Indeed, CB1 positive cells mainly in the INL are found co-expressed with a few TH positive cells (**Figure 2B**).

Since dopaminergic neurons express CB1 receptor, we decided to investigate whether this receptor modulate dopaminergic functions in the avian retina. For this purpose, we used primary cell cultures of mixed neurons and Müller glia (**Figure 3**), as these cells express both CB1 and CB2 receptors (Kubrusly et al., 2018) and also MAGL. CB1 and CB2 are found both in neurons and glia cells, but MAGL is only expressed in Müller glia. CB1 is highly expressed in neuron bodies (**Figure 3**), and in Müller cells purified culture (**Figure 3C**).

CB1 was also found to be co-localized with TH and heavily associated with D1 receptor labeling in primary cell cultures (**Figures 4A,B**). To explore the presence of selective dopaminergic markers in mixed retinal cell cultures, we performed western blot analysis of different proteins associated with the DS. Data revealed the clear expression of the nuclear receptor related 1 protein (Nurr1), D1 receptor and dopamineand cAMP-regulated phosphoprotein 32 (Darpp32; **Figure 4C**) in addition to CB1.

Next, E8C6 cultures were exposed to SKF38393 (SKF), a selective D1 agonist, which was able to increase the cAMP levels when compared to controls. Our results show that the cAMP accumulation induced by SKF is lost when cultures were pretreated with WIN55, 212–2 (WIN), a non-selective CB agonist (**Figure 5**). When cultures were pre-treated with AM251, a CB1 selective antagonist, and then co-exposed to SKF+WIN, the SKF response was restored (**Figure 5**), indicating that the repressive effect of WIN on SKF promoted cAMP accumulation was due, mainly, to activation of CB1 receptors.

# DISCUSSION

Here we show the expression of CB1 receptors in the avian retina throughout development, being detected since an early embryonic stage (E5) up to a more mature period (PE7). Moreover, we show a decrease in CB1 protein content during development, but no significance was reached upon ANOVA. CB1 is mainly located in ganglion as well as amacrine cells, present in the GCL and in the INL, respectively. Our work confirms previous studies that have shown the presence of CBs and endocannabinoids metabolic enzymes in the retina of several species such as goldfish, rat, mouse, chick and monkey (Schwitzer et al., 2016). Some of these previous data have shown CB1 expression in ganglion, horizontal and amacrine cells, cone pedicles and rod spherules of photoreceptors (Zabouri et al., 2011a). Leonelli et al. (2005) showed for the first time that, in avian retina, CB1 emerges in E4, in ganglion cells, while at E18 CB1 is expressed in neurons located in the GCL and INL.

TH co-expression in PE7 avian retina. As shown, a discrete TH co-expression with CB1 that is mainly present in neighboring cells in the INL. ApoTome images in upper row and confocal images in lower row. Scale bar of 20 µm, all images were obtained with 40× magnification. N = 5 for each analysis.

We recently showed that CB1 and CB2 receptors are found in both retinal neurons and Müller glia of chick embryos. We found that WIN decreases cAMP production in retinal cells in basal conditions, decreases the number of glial cells that increased Ca2+ evoked by ATP, and inhibited [3H]-GABA release induced by KCl or L-Aspartate (Kubrusly et al., 2018). Now we evaluated the expression of CB1, CB2 receptor and MAGL enzyme in the developing retina. CB2 has been reported in rat, mouse and monkey but not in avian retina. CB2 has been shown to be expressed in photoreceptors, horizontal, amacrine and ganglion cells and also in fibers in the INL in adult rat retina (López et al., 2011). Interestingly, CB2 expression appears in Müller cells in the monkey retina (Bouskila et al., 2013). However, there is no information regarding CB2 expression during development of the avian retina. Our data show that CB2 is expressed beginning at E5 and is clearly observed in the lamination of the IPL between E14 and PE7 (**Figure 1C**). In early retinogenesis, up to E5, retinal cells are essentially in the neuroblastic layer made of multipotent progenitors that generate early-born cells (RGCs, horizontal, some types of

FIGURE 3 | Expression of cannabinoid markers in primary retinal cell in cultures. Expression of CB1, CB2 and MAGL in mixed cultures of avian retina cells, displaying neurons and glia prepared from 8-day old embryos (E8), as described. In (A), CB1, CB2, MAGL (all in green) and MAP2 (red) as a specific neuronal marker. In (B), expression of CB1, CB2, MAGL (green) and 2M6 (red) as a selective glial marker for Müller cells. In (C), purified culture of Müller cells show expression of CB1 (green). Scale bar of 20 µm, all images were obtained with 40× magnification in a fluorescence microscope with ApoTome software. N = 5.

amacrines and cones). Around E14, late-born progenitors give rise to bipolar, rods and Müller glia, so the precise number and proportion of cells into different layers form a fully mature retina (Martins and Pearson, 2008). Since CB1 (Xapelli et al., 2013) and CB2 (Bravo-Ferrer et al., 2017) receptors are correlated with proliferation and/or neurogenesis in the central nervous system, their presence early in the embryonic avian retina could modulate the generation of retinal cells. Moreover, activation of these receptors at early stages suggests that the retinal circuitry might be altered (morphologically) or in terms of function (CBs inhibit the release of GABA and aspartate (Kubrusly et al., 2018), and decrease cAMP levels, as shown in **Figure 5**. This could possibly reflect in changes during synapse remodeling during retinal development.

magnification in a fluorescence microscope with ApoTome software. N = 3 for each analysis.

The main endocannabinoids are AEA and 2-AG, described in the retina of several vertebrates along with the main enzymes that degrade them (Schwitzer et al., 2016). MAGL, one of these enzymes, is present in rat retinal layers mainly in amacrine and Müller cells (Cécyre et al., 2014). We found that, in the avian retina, MAGL is expressed beginning at E9 and up to post-hatched PE7, where it is detected in all the five retinal layers, mostly in IPL and GCL (**Figure 1E**). Our data provide that MAGL is located primarily in the glial compartment as the enzyme is co-localized with 2M6 (**Figure 3B**, lower panel), at least in vitro. As glial cells are the last to be generated in the retina (Martins and Pearson, 2008), perhaps this explains the appearance of labeling at E9. We still cannot affirm how the developmental profile of CB1, CB2, MAGL and endocannabinoids match the interactions between different retinal neurons and Müller glia. However, data on **Figure 5** suggest an important interplay between receptors that decrease the levels of cAMP (CB1 and CB2) and those that increase cAMP levels (dopamine, adenosine or PACAP) during retina differentiation.

In addition, CB receptor activation decreases the number of glial cells that increased Ca2+ evoked by ATP, and inhibited [ 3H]-GABA release induced by KCl or L-Aspartate (Kubrusly et al., 2018). The first part of the retina that differentiates is the central part and RGCs are the first to emerge. Synaptogenesis peak in the avian retina around E14, and it is known to have

FIGURE 5 | Endocannabinoid system (ECS) modulates the function of dopamine-induced increase in cyclic AMP (cAMP) levels. E8C6 retinal mixed cell culture was subjected to cAMP quantification analysis, as described. When the selective D1 receptor agonist, SKF38393 (SKF) at 5 × 10−5 M was used, cAMP levels increased significantly. When the culture was treated with WIN after SKF stimulation, the SKF-induced increase in cAMP levels was lost. However, in cultures pretreated with AM251, a selective CB1 antagonist, cAMP levels were increased (AM251 + SKF + WIN), similar to SKF-only application. AM251 was added 15 min before SKF and WIN, which were added for 30 min, all at 37◦C. ∗∗∗< 0.001. N = 6 for each analysis.

two main axis: glutamatergic in the vertical axis (photoreceptors, bipolar and the RGCs) and GABAergic in the horizontal axis (horizontal and amacrine; Barnstable, 1993). Moreover, Müller glia is an active compartment that shapes the circuitry (de Melo Reis et al., 2008). The ECS seems to be a powerful regulator of the efficacy of retinal circuitry.

The retina presents the main neurotransmitters found in the brain such as glutamate (Connaughton, 1995), GABA (Bringmann et al., 2009) and dopamine (Reis et al., 2007). A direct action of the ECS on retinal transmitter release and in general retinal physiology has been described in different vertebrate retinas mainly in retinal bipolar cells (Straiker et al., 1999). Interestingly, dopamine and noradrenaline release is inhibited by CB1 activation in perfused guinea-pig retina (Schwitzer et al., 2016). In the developing vertebrate retina, the DS is one of the first phenotypes to appear mainly in amacrine and interplexiform cells (Reis et al., 2007). Here, we first evaluated the emergence of CB1 and TH co-expression in PE7 avian retina. We observed that CB1 is co-localized with dopaminergic neurons (**Figure 2**) present in the IPL. CB1 is also localized in neighboring cells in IPL and also GCL, suggesting a more complex role in retina signaling. It is likely that CB1 positive cells are responsive for the dopamine released by TH amacrine cells and vice versa. Bosier et al. (2007) showed an important relationship between the ECS and DS using a murine neurospheres model, where CB1 agonists promoted a reduction in TH expression.

It is known since the 70s that dopamine increases cAMP levels in the chick retina and in mixed cultures of embryonic chick retina cells (de Mello, 1978). Since it is described that CB1 expression occurs in retinal cells in response to dopaminergic stimuli (Fan and Yazulla, 2005), we investigated the correlation between ECS and the DS. Primary cell cultures of mixed neurons and Müller glia express CB1 and CB2 receptors as well as MAGL enzyme. CB1 is predominantly in cell bodies while CB2 is found mainly in neuronal processes. On the other hand, MAGL is mainly located in Müller cells.

In order to evaluate a functional correlation between CB1 and D1 receptors, a mixed neuron-glia culture was used at E8C6, a stage where most of dopaminergic proteins are expressed. The D1 receptor agonist SKF38393 promotes a significant increase in cAMP accumulation in retina cells (Castro et al., 1999). In order to test the effect of the ECS on this D1-mediated effect, we added AM251, a CB1 receptor selective antagonist, and WIN 55, 212–2 (WIN), a non-selective cannabinoid CB1/CB2 agonist. Our results show that WIN decreases cAMP accumulation induced by SKF, and this response is blocked when AM251 was used as a pretreatment. Additionally, WIN did not change cAMP accumulation promoted by forskolin (not shown), a general adenylyl cyclase activator, suggesting that WIN is selectively acting on dopaminergic receptor level. Therefore, we provide evidence here that CB1 receptor can modulate the response of D1 receptor activation through cAMP accumulation. We conclude that the endocannabinoid and the DS interact in retinal cells.

Emergence of the ECS (CB1/CB2 receptors), endocannabinoids and enzymes during embryonic retinal development might influence dopaminergic communication that seems to use two separated systems during development. One operates in early stages of retina formation, and the other, later in development. No TH is detected in the tissue between E5 and E10/12. However, E5 retinas display several cells (neuroblasts) clearly labeled for L-Dopa decarboxylase (DDC; da Silva et al., 2009). At this stage, or even before, the presumptive neuroretina is already associated with the pigmented epithelium, characterized by the presence of melanin. Melanin, as dopamine, use as precursor L-Dopa that in the epithelium comes from tyrosinase activity. Our group has shown that conditioned media from the pigmented epithelium of embryonic eyes fed to embryonic retina tissue prior to TH expression, is capable, of synthesizing dopamine (Kubrusly et al., 2003). This activates cAMP formation prior to TH expression. Therefore, based on the findings that D1 (increasing cAMP) or D2 receptors activation (decreasing cAMP levels) in embryonic avian retina can modify the dynamics of neurites outgrowth, interference with these systems via CBs might influence dopaminergic circuitry. D1 and CB1 have been shown to exhibit antagonistic effects in goldfish bipolar retinal cells, as D1 activation increases cAMP level, while CB1 (WIN) reduces the concentration of cAMP (Fan and Yazulla, 2005). Our data strengthen this point using avian retinal cells to assess the interaction between the endocannabinoid and DSs.

A recent article has revealed in a pioneer way the presence of both cannabinoid receptors (CB1R and CB2R, the synthesizing and degrading enzymes in mice, shrews and monkey retinae (Bouskila et al., 2016). These data, added to ours in chicks (and many others) suggest a strong conservation of the ECS in the retinal tissue in vertebrates. This opens a possibility to create strategies to ameliorate vision problems with cannabinoid modulators. A recent article has shown that activation of CB2 receptors with a selective agonist (JWH-133) increases inflammation in human retinal pigment epithelium, decreasing cellular viability through release of pro-inflammatory cytokines and might be a central element in avoiding vision problems (Hytti et al., 2017). Both neuronal and glial CB1 and CB2 receptors might have an important role in retinal development through modulation of intracellular pathways (Kubrusly et al., 2018). Indeed, 2-AG is found in early retinal development together with DAGLα, 2-AG degrading enzyme), while MAGL, develops later (Cécyre et al., 2014). FAAH, (AEA, degrading enzyme) emerges transiently at postnatal day 1 in ganglion and cholinergic amacrine cells (Zabouri et al., 2011b).

Moreover, we show that endocannabinoids may act on the developing CNS (here exemplified by the retina), possibly influencing developmental characteristics of CNS systems such as plasticity during synapses formation (Fleming et al., 2013). We also call attention to the possibility that cannabinoid consumption might have a dramatic effect in the developing CNS.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

LSS and RCCK: conception and design, provision of study material, collection and assembly of data, data analysis and interpretation, manuscript writing and final approval of manuscript. YPC, PPT, VTR-R, ME-L and PFG: collection and assembly of data, data analysis and interpretation. RP-C, FGM and RAMR: conception and design, provision of study material, assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript, financial support and administrative support.

#### ACKNOWLEDGMENTS

Grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Instituto Nacional de Ciência e Tecnologia de Neurociência Translacional (INCT-INNT) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) supported this work. We are indebted to the technical support of Luciano C. Ferreira and Aurizete Bizerra.

**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 da Silva Sampaio, Kubrusly, Colli, Trindade, Ribeiro-Resende, Einicker-Lamas, Paes-de-Carvalho, Gardino, de Mello and De Melo Reis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Suppression of Cisplatin-Induced Vomiting by Cannabis sativa in Pigeons: Neurochemical Evidences

Ihsan Ullah1,2 \*, Fazal Subhan2 , Javaid Alam2,3, Muhammad Shahid2 and Muhammad Ayaz4 \*

1 Department of Pharmacy, University of Swabi, Swabi, Pakistan, 2 Department of Pharmacy, University of Peshawar, Peshawar, Pakistan, 3 Drug and Herbal Research Centre, Faculty of Pharmacy, University Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 4 Department of Pharmacy, University of Malakand, Chakdara, Pakistan

Cannabis sativa (CS, family Cannabinaceae) has been reported for its anti-emetic activity against cancer chemotherapy-induced emesis in animal models and in clinics. The current study was designed to investigate CS for potential effectiveness to attenuate cisplatin-induced vomiting in healthy pigeons and to study the impact on neurotransmitters involved centrally and peripherally in the act of vomiting. Highperformance liquid chromatography system coupled with electrochemical detector was used for the quantification of neurotransmitters 5-hydroxytryptamine (5HT), dopamine (DA) and their metabolites; Di-hydroxy Phenyl Acetic acid (Dopac), Homovanillic acid (HVA), and 5-hydroxy indole acetic acid (5HIAA) centrally in specific brain areas (area postrema and brain stem) while, peripherally in small intestine. Cisplatin (7 mg/kg i.v.) induce emesis without lethality across the 24 h observation period. CS hexane fraction (CS-HexFr; 10 mg/kg) attenuated cisplatin-induced emesis ∼ 65.85% (P < 0.05); the reference anti-emetic drug, metoclopramide (MCP; 30 mg/kg), produced ∼43.90% reduction (P < 0.05). At acute time point (3rd h), CS-HexFr decreased (P < 0.001) the concentration of 5HT and 5HIAA in the area postrema, brain stem and intestine, while at 18th h (delayed time point) CS-HexFr attenuated (P < 0.001) the upsurge of 5HT caused by cisplatin in the brain stem and intestine and dopamine in the area postrema. CS-HexFr treatment alone did not alter the basal neurotransmitters and their metabolites in the brain areas and intestine except 5HIAA and HVA, which were decreased significantly. In conclusion the anti-emetic effect of CS-HexFr is mediated by antiserotonergic and anti-dopaminergic components in a blended manner at the two different time points, i.e., 3rd and 18th h in pigeons.

#### Keywords: cisplatin, emesis, Cannabis sativa, pigeon, neurotransmitters

#### INTRODUCTION

Cytotoxic agents like cisplatin and cyclophosphamide have the side effects of nausea and vomiting most feared by patients undergoing chemotherapy (Hesketh et al., 2003a). These stressful side effects often result in poor compliance and even refusal of treatment (Tanihata et al., 2000; Hesketh, 2008). The D2 receptor blocker "metoclopramide" was found to be effective against chemotherapy induced vomiting (CIV) at higher doses, where the anti-emetic effect is reported to be mediated

#### Edited by:

Fabricio A. Pamplona, Entourage Phytolab, Brazil

#### Reviewed by:

Maria Grazia Morgese, University of Foggia, Italy Daniel Souza Monteiro De Araújo, Federal Fluminense University, Brazil

#### \*Correspondence:

Ihsan Ullah<EMAIL_ADDRESS><EMAIL_ADDRESS>Muhammad Ayaz<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology

Received: 29 November 2017 Accepted: 28 February 2018 Published: 16 March 2018

#### Citation:

Ullah I, Subhan F, Alam J, Shahid M and Ayaz M (2018) Suppression of Cisplatin-Induced Vomiting by Cannabis sativa in Pigeons: Neurochemical Evidences. Front. Pharmacol. 9:231. doi: 10.3389/fphar.2018.00231

**29**

through antagonism of 5-hydroxytryptamine type 3 (5HT3) receptors (Coronas et al., 1975; Miner and Sanger, 2012). These findings led to the discovery of 5HT3 receptor antagonists (Ondansetron, etc.).

Dopamine (DA), 5-hydroxytryptamine (5HT), and neuropeptide substance P are involved in emetic circuitry. The neurotransmitter 5HT (Serotonin) is primarily responsible for the initiation of the vomiting produced by cisplatin (Grunberg and Koeller, 2003). Up to 95% of 5HT is present in the enterochromaffin (EC) cells in the gastrointestinal mucosa along with substance P (Diemunsch and Grelot, 2000; Minami et al., 2003). The noxious stimulus caused by highly emetogenic chemotherapy (HEC) agents like cisplatin results in the release of 5HT (Wolff and Leander, 1997; Percie du Sert et al., 2011). The released 5HT then activates 5HT3 receptors on vagal afferents which stimulate the brain centers to initiate the vomiting response (Hesketh et al., 2003b). Furthermore, in human and animal studies, there is evidence for the increased level of the 5-HT metabolite, 5-Hydroxy Indole Acetic Acid (5HIAA, urine) (Cubeddu et al., 1995; Veyrat-Follet et al., 1997), 5HT in the intestinal mucosa (ileal segment), Tryptophan Hydroxylase (TPH, ileum), Aromatic L-amino Decarboxylase (AADC, ileum) (Endo et al., 1993) and in the brain stem (Minami, 1995), following cisplatin treatment. Furthermore, a decrease in Monoamine Oxidase (MAO, ileum) has also been reported (Endo et al., 1993). This enhancement in 5HT biosynthesis and reduction in degradation ultimately led to the upsurge of serotonin which initiates the vomiting response (Ullah, 2013).

The selective activation of D2 receptors, localized in the limbic system, hypothalamus, amygdala and in the brain stem emetic circuitry trigger the vomiting response (Le Moine and Bloch, 2004). This involvement of dopamine receptors advocates dopamine as important mediator for vomiting act. Dopaminergic agonists like apomorphine have been reported to be emetic in a variety of species including dogs (Foss et al., 1998), ferrets (Osinski et al., 2003, 2005), least shrew (Darmani et al., 1999), and humans (Schofferman, 1976). The emetic action of apomorphine and loperamide has been suggested to be mediated in the chemoreceptor trigger zone/area postrema through stimulation of dopamine receptors. Where, the ablation of area postrema abolished the vomiting response advocating the involvement of area postrema in the mediation of vomiting by apomorphine and loperamide (Miller and Leslie, 1994; Yoshikawa et al., 1996).

The identification of cannabinoid receptors resulted in the discovery of endocannabinoids (Pacher et al., 2006). Delta-9- Tetrahydrocannabinol (19 -THC) and synthetic cannabinoids exert their cannabimimetic effects via CB1 receptors (Mackie and Stella, 2006). CB1 receptors are primarily located centrally and peripherally while CB2 receptors occur mainly on immune cells (Pertwee, 2006). Furthermore, a family of nuclear hormone receptor PPAR (α, β, and γ) are also been reported to be involved in the mediation of some effects in analgesia, antiinflammatory, neuroprotection, cardiovascular, gastrointestinal, and anti-tumor properties of some cannabinoids (O'sullivan, 2016). Endocannabinoids like oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are reported to activate PPARα. Other endocannabinoids including noladin ether, virodhamine, 2-arachidonoyl-glycerol, and Anandamide are also shown to stimulate PPARα and transient receptor potential vanilloid type 1 (TRPV1) cation channel. In continuation, Synthetic cannabinoids like WIN55,212-2 activates the transcriptional activity of PPARα and PPARγ (Brown, 2007; O'sullivan, 2016; Stampanoni Bassi et al., 2017). Activation of the Endocannabinoid system, PPARγ and CB1 receptors are associated with decrease in the dopaminergic activity in the basal ganglia and levodopa induced abnormal involuntary movements (AIMs) which can be extrapolated to the anti-emetic effect of CS in the brain stem emetic center (Martinez et al., 2015; Stampanoni Bassi et al., 2017).

Cannabinoids have been shown to affect neuronal circuits that modulate nausea, vomiting, and other gastrointestinal functions. Evidence is emerging regarding the interaction of cannabinoid (CB1), serotonin (5HT3), neurokinin-1 (NK1) and dopamine receptors (D2 and D3), implicating an important role for cannabinoids in vomiting circuits. The old era of neurotransmitter understanding advocate primarily the involvement of the monoaminergic neurotransmitters especially serotonergic system, while the late phase is associated with monoaminergic system excluding the serotonergic system (Tanihata et al., 2000). The current literature provide evidences for the substantial overlapping of serotonergic, dopaminergic, and neurokininergic mechanisms for the entire time course of cisplatin-induced vomiting (Saito et al., 2003; Darmani et al., 2009; Higgins et al., 2012).

Considering the relevance of DA and 5HT in cisplatininduced vomiting, this study was designed to evaluate the participation of these monoamine neurotransmitters and their metabolites in cisplatin-induced vomiting, and to examine the impact of Cannabis sativa (CS) extract on neurotransmitters implicated in the act of vomiting in specific brain areas and intestine in pigeons. Cannabis sativa hexane fraction was selected based on our previous studies where it was proved to be anti-emetic against cisplatin-induced vomiting in pigeon model (Ullah et al., 2012).

#### MATERIALS AND METHODS

#### Animals

Pigeons of either sex (mixed breed, Department of Pharmacy, University of Peshawar, Peshawar, Pakistan) weighing between 250 and 350 g were used. They were housed in groups of eight (n = 8) at 22–26◦C under a 12 h light/dark cycle and had free access to food (locally available food; Millet + Wheat) and water before and during experimentation. All of the experimental procedures were approved by the Ethical Committee of the Department of Pharmacy, University of Peshawar (Ref. No. 5/EC/Pharm) and were in accordance with the UK Animal Scientific Procedure Act, 1986 (Ullah, 2013).

#### Drugs and Chemicals

High-performance liquid chromatography (HPLC) grade acetonitrile (99.9%), methanol (99.9%), 1-octane sulfonic acid sodium salt (>98%) (Fisher Scientific, United Kingdom), sodium

dihydrogen orthophosphate (99%) and ethylene diamine tetra acetic acid (≥99%) (EDTA) were purchased from the Merck local distributor in Peshawar, Pakistan. Noradrenaline (≥98%), DOPAC (≥98%), dopamine (≥99%), 5HIAA (≥98%), HVA (≥98%), and serotonin (≥99%), were from Acros Organics, Belgium. Cisplatin (≥99.9%) was from Korea United Pharm., Inc. (South Korea). Metoclopramide (MCP; ≥98%) was purchased in solution from GlaxoSmithKline (GSK Pakistan, Ltd.). Commercial grade n-Hexane was from Haq Chemicals Peshawar (Pakistan). The plant was collected at a farm, from Malakand Division (Khyber Pukhtoonkhwa, Pakistan) at its bloom season and was authenticated by Prof. Dr. Muhammad Ibrar, Department of Botany, University of Peshawar, a specimen was preserved in the herbarium for future reference (voucher No. 8717) (Ullah, 2013).

#### Extraction of Cannabis sativa

Leaves and flowering tops of Cannabis sativa plant were separated, shade dried, coarsely ground and then extracted as shown in the extraction scheme (**Figure 1**) (Ali et al., 2017; Ayaz et al., 2017).

# Drug Formulation

Cisplatin was dissolved in normal saline by heating up to 60◦C and then cooled up to 40–45◦C before administration (Ullah et al., 2014). Cannabis sativa n-hexane fraction (CS-HexFr) was dissolved in absolute ethanol, mixed with emulsifier and made the volume with distilled water in such a way that the final mixture consists of ethanol: emulsifier: distilled water in a ratio of 5: 5: 90 (Feigenbaum et al., 1989a; Ullah, 2013).

# Drug Administration

Cotton wool and methylated spirit were used to sterilize the skin prior to drug administration. Intravenous (Cisplatin) and

intramuscular (Treatment) administrations were done through brachial wing vein and chest muscle, respectively using Neoject 2 ml non-pyrogenic syringes with sharp painless needles (27G × 1/200 for the i.v. route, and 23G × 1 00 for the i.m. route). Immediately, after the last injection, the animals were put back in the specially designed confining/observation cages and the number of Retching plus Vomiting (R + V) and latency to first vomit were recorded for 24 h. At the end of experiment, body weight loss was calculated. Subsequently, the animals were decapitated to terminate the experiment (Ullah, 2013).

# Anti-emetic Assay

On the day of experiment, the pigeons were placed in individual cages specially designed for video observation. Cisplatin at the dose of 7 mg/kg was administered intravenously via the brachial wing vein at t = 0 (Ullah et al., 2014). The behavior of the pigeons was then recorded for 24 h. Food and water were available during the observation period and each animal was used only once. The vomiting response with or without oral expulsion was considered as one vomiting episode (Preziosi et al., 1992). The latency to first vomit and the number of vomiting episodes were recorded. A vomiting episode was considered complete when the pigeon adapted relaxed posture. Jerking episodes, which are indicative of vomiting intensity, were also recorded. In these studies, CS fraction and MCP or respective vehicles, were administered 30 min before cisplatin administration, In case of twice (BD) administration of CS the second dose was administered intramuscularly at 12th of cisplatin administration (Ullah, 2013).

## Tissue Sampling for Neurotransmitters Analysis

Two discrete parts of the brain (brain stem and area postrema) as well as the intestinal samples 5–6 cm from the pylorus (initial segment of Jejunum) were collected for the neurotransmitter analysis and the effects of CS-HexFr and MCP were investigated. The dissection of brain parts was carried out according to the atlas of Karten and Hodos (1967) and Duvernoy and Risold (2007). In brief, after decapitation of experimental animals, the dorsal surface of the skull was exposed by making an incision along the mid line and the temporal muscles were stripped off to expose skull bone. After exposing the skull, bones, and meninges were carefully removed in a way to expose the brain hemispheres and especially to make brain stem prominent from the ventral aspect. The long strip of capillaries stretching from the obex on the median line to the lateral angles of the fourth ventricle (area postrema) was dissected followed by dissection of brain stem. Jejunal samples of about 2 cm were rapidly removed and washed with ice cold saline. The collected samples were rapidly frozen on an ice plate and stored at −80◦C until analysis (Ullah, 2013).

### Determination of Neurotransmitters and Their Metabolites

Tissue samples were homogenized in cold 0.2% perchloric acid (PCA) at 5000 rpm with the help of Teflon glass homogenizer (Wise stir HS 30 E). After centrifugation

evaporator to get CS-HexFr.

(Centurion, United Kingdom) at 12000 g/min (4◦C) and filtered through a 0.45 micron filter. Neurotransmitters and their metabolites were analyzed using High-Performance Liquid Chromatography system (HPLC, Shimadzu, Japan) coupled with Electrochemical Detection (ECD, ESA Coulochem III model 5300), a pump (model LC-20AT), and an analytical column (Teknokroma 3 × 150, 3 um). The mobile phase consisted of 94 mM sodium dihydrogen orthophosphate, 40 mM Citric acid, 2.3 mM sodium 1-octane sulfonic acid, 50 uM EDTA, and 10% acetonitrile (pH 3). The flow rate was maintained at 0.6 mL/min. The standards used were noradrenaline hydrochloride (NA), 3, 4 dihydroxyphenylacetic acid (DOPAC), dopamine hydrochloride (DA), 5-hydroxyindole-3-acetic acid (5HIAA), Homovanillic acid (HVA), and serotonin (5HT). The HPLC method already reported by our laboratory (Ullah et al., 2014) was used where all the neurotransmitters and their metabolites were separated within 13 min (Ullah, 2013).

### Statistical Analysis

The differences between means were evaluated using a one way analysis of variance (ANOVA) followed by Dunnett or Tukey multiple comparison tests. P < 0.05 was considered as statistically significant. The animals which showed complete suppression of Retching Plus Vomiting (R + V) were not included in statistical analysis for latency. Data represent the mean ± SEM unless otherwise indicated.

# RESULTS

### Anti-emetic Effect of Cannabis sativa Hexane Fraction (CS-HexFr)

Cisplatin at the dose of 7 mg/kg (Ullah et al., 2014) induced reliable R + V in all the animals tested with intense vomiting occurring in the first 3 h while the treatments attenuated it **Figure 2** and **Table 1**.

In these experiments, cisplatin-induced R + V following a latency of ∼ 67 min that comprised ∼ 41 episodes. CS hexane fraction (CS-HexFr; 5, 10, and 15 mg/kg) attenuated cisplatininduced R + V in non-dose-dependent manner (**Figure 3**), showing significant reduction with 10 mg/kg once (OD) and twice (BD) dosing up to 17 ± 3.4 (58.53% protection) and

(E) Cannabis sativa hexane fraction 10 mg BD treatment; the arrow indicates second dose administration, and (F) Cannabis sativa hexane fraction 15 mg treatment.

Data represents the mean ± SEM of the total numbers of retches + vomits occurring during 1 h intervals (n = 7–8).

TABLE 1 | Effect of Cannabis sativa hexane fraction (CS-HexFr) or standard metoclopramide (MCP) on cisplatin-induced Retching plus Vomiting (R + V) and jerking during a 24 h observation period.

∗P < 0.05, ∗∗P < 0.01 as compared to cisplatin control (ANOVA followed by Tukey's post hoc test).

14.1 ± 2.9 (65.85% protection), respectively (P < 0.01; **Table 1**) during 24 h of observation period. The CS-HexFr was found to be effective as it suppressed R + V up to 16 h of observation period while standard metoclopramide provided protection up to 8 h (**Figure 4**). CS-HexFr 10 mg BD and standard MCP increased the latency to first vomit significantly (P < 0.01).

None of the treatments induced vomiting when administered alone.

# Effect of MCP or CS-HexFr on Cisplatin-Induced Jerks and Weight Loss

In the cisplatin control group, animals lost ∼15% of their starting body weight. The body weight loss in the standard MCP (30 mg/kg) treated group was 11.9 ± 1.1%, while CS-HexFr (10 mg/kg BD) reduced body weight loss up to 8.9% (P < 0.05, **Table 1**). All other treatments failed to reduce body weight loss significantly. The jerking behavior (reflects the vomiting intensity in pigeons; one vomiting episode may contain 2–80 jerks) observed in cisplatin control and standard MCP groups were 570 ± 63 and 361 ± 25, respectively, while no treatment decreased the jerking behavior up to the observation period (24 h) except CS-HexFr (10 mg/kg BD) where the jerking episodes were reduced significantly (570 ± 63 → 239 ± 59; P < 0.05, **Table 1**).

#### Effect of Standard MCP or CS-HexFr on Basal Level of Neurotransmitters and Their Metabolites in the Brain Areas and Small Intestine

The standard MCP treatment reduced the concentration of 5HIAA in the area postrema (P < 0.05) and brain stem (P < 0.001) as compared to basal level. In addition, the decrease in the concentration of HVA was also observed in the area postrema (P < 0.05, **Table 2**). As depicted in **Table 2**, treatment with CS-HexFr (10 mg/kg) had no significant effects on NA, DA and its metabolites DOPAC and HVA, 5HT and its metabolite 5HIAA in the brain areas (AP and BS) and intestine. Though, the concentration of DA at the level of AP and intestine was increased significantly (P < 0.001) as compared to basal level.

## Effect of Standard MCP or CS-HexFr on the Level of Neurotransmitters and Their Metabolites in the Brain Areas and Small Intestine at 3rd Hour After Cisplatin Administration

Cisplatin treatment significantly increased the concentration of 5-hydroxytryptamine (5HT) in the brainstem and intestine (P < 0.001; **Table 3**) as compared to basal level, while a non-significant increase was observed in the area postrema. In addition, cisplatin also caused a significant increase in the concentration of 5HIAA in the area postrema (P < 0.05), brain stem (P < 0.001), and intestine (P < 0.001). The treatment with standard MCP at the dose of 30 mg/kg failed to change the concentration of NA, DOPAC, DA, and HVA in all the brain areas (AP and BS) and intestine, but reduced the concentration of 5HT in the area postrema, brain stem, and intestine significantly (P < 0.05–0.001) as compared to cisplatin control (**Table 3**). In addition to its inhibitory effects on 5HT, MCP also decreased

and 15 mg/kg) against cisplatin-induced vomiting during a 24 h observation period; each bar represents the mean ± SEM of vomiting episodes occurring during 4 h periods (n = 5–8). Values significantly different from cisplatin control are denoted as ∗P < 0.05, 2∗P < 0.01, 3∗P < 0.001 (ANOVA followed by Tukey's post hoc test).

TABLE 2 | Effect of standard metoclopramide (MCP) or Cannabis sativa hexane fraction (CS-HexFr) on basal level of neurotransmitters (ng/mg tissue wet weight) and their metabolites in brain areas and the small intestine of pigeons.

∗P < 0.05, ∗∗∗P < 0.001 as compared to saline (ANOVA followed by Tukey's post hoc analysis; n = 6–8).

5HIAA concentration in both the brain areas (AP and BS) and intestine significantly (P < 0.01–0.001, **Table 3**). CS-HexFr (10 mg/kg) significantly reduced the 5HIAA and 5HT concentrations in the brain areas (AP and BS) and intestine (P < 0.001) while no effects were seen on the levels of NA and DOPAC. On the contrary, CS-HexFr (10 mg) treatment caused an increase in the concentration of DA in AP, BS, and intestine that was significant (P < 0.001) as compared to the cisplatin control (**Table 3**).

#### Effect of Standard MCP or CS-HexFr on the Level of Neurotransmitters and Their Metabolites in the Brain Areas and Small Intestine at 18th Hour After Cisplatin Administration

Cisplatin increased the level of DA significantly (P < 0.001) in the AP, while a non-significant trend toward increase was observed in the brain stem (**Table 4**). 5HT concentrations were also raised in the area postrema (P < 0.01), brain stem (P < 0.001) and intestine (P < 0.001), without effecting the levels of NA, DOPAC, 5HIAA, HVA (**Table 4**). Treatment with standard metoclopramide (MCP; 30 mg/kg) significantly decreased the upsurge of DA in the area postrema (P < 0.001; **Table 4**). Furthermore, a decrease in the concentration of 5HT was also observed in the area postrema (P < 0.01), brain stem and intestine (P < 0.001) and 5HIAA concentration (P < 0.01) in the area postrema as compared to cisplatin control (**Table 4**). Cannabis sativa hexane fraction (CS-HexFr) at the dose of 10 mg/kg decreased significantly (P < 0.001) the upsurge in the concentration of DA in the brain area of AP (P < 0.001) while decrease in 5HT was observed in the brain stem and intestine (P < 0.001; **Table 4**).

#### DISCUSSION

In the present study, we screened n-hexane fraction of Cannabis sativa (CS-HexFr) against cisplatin-induced retching and vomiting (R + V) in the pigeon vomiting model, where it was found to be effective to attenuate cisplatin-induced R + V.

TABLE 3 | Effect of standard metoclopramide (MCP) or Cannabis sativa hexane fraction (CS-HexFr) on neurotransmitters (ng/mg tissue wet weight) and their metabolites in brain areas and small intestine 3 h after cisplatin treatment in pigeons.

Neurotransmitter or metabolite levels significantly different from cisplatin control are denoted by ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, while values significantly different from basal levels are denoted by #P < 0.05, ###P < 0.001 (ANOVA followed by Tukey's post hoc analysis; n = 6–8).

TABLE 4 | Effect of standard metoclopramide (MCP) or Cannabis sativa hexane fraction (CS-HexFr) on neurotransmitters (ng/mg tissue wet weight) and their metabolites in brain areas and the small intestine at 18 h after cisplatin treatment in pigeons.

Neurotransmitter and metabolites values significantly different from cisplatin control are denoted by ∗∗P < 0.01, ∗∗∗P < 0.001, while values significantly different from basal level are indicated by ##P < 0.05, ###P < 0.001 (ANOVA followed by Tukey's post hoc analysis; n = 6–8).

CS-HexFr at the dose of 10 mg/kg once and twice daily dosing provided up to 58.53% (17 ± 3.4 episodes) and 65.85% (14.1 ± 2.9 episodes) protection, respectively (**Table 1**). The n-hexane extract has been reported to contain cannabis major active constituent Delta-9-tetrahydrocannabinol (19 - THC) which has been in use for the treatment of various diseases including management of CIV in clinics and the enhancement of appetite. 19 - THC is also found to have anti-inflammatory, spasmolytic, analgesic, and anti-glaucoma activity (Carlini, 2004). Furthermore, Sallan et al. (1975) have shown that the active component of CS (19 - THC) has anti-emetic effects, by its ability to stimulate presynaptic cannabinoid CB1 receptors (Darmani, 2001) and subsequent inhibition of monoamine neurotransmitters release (Darmani et al., 2003).

Metoclopramide (MCP), a clinically relevant anti-emetic with dopamine and 5-HT3 receptor antagonist properties (Al-Zubaidy and Mohammad, 2005), was used as a positive control. The dose of MCP that we selected is higher than that required to antagonize cisplatin-induced emesis in other species (Zhang et al., 2006), and was based on a previous study in the pigeon showing activity against reserpine-induced emesis (Coronas et al., 1975). The metoclopramide was selected as standard drug because of the intrinsic emetic activity of 5HT3 receptor antagonists in pigeon (unpublished data).

Cisplatin which belongs to the highly emetogenic class of cancer chemotherapeutic agents is in use for the screening of anti-emetic potential of current anti-emetic agents. Cisplatin (4–10 mg/kg) has been used by several investigators to induce vomiting in pigeons (Feigenbaum et al., 1989b; Wolff and Leander, 1995). However, Tanihata et al. (2000) used a lower dose of 4 mg/kg and longer observation periods (∼72 h). In fact, our colony of pigeons had shown a reliable vomiting response at 7 mg/kg up to 24 h of observation period (Ullah et al., 2014, 2015) and we therefore, used the dose of 7 mg/kg to induce emesis.

The current evidences about the involvement of neurotransmitters implicate the overlap of serotonergic, dopaminergic, and neurokininergic systems in the whole time course of cisplatin-induced vomiting (Darmani et al., 2009). The neurotransmitters especially 5HT (serotonin) and dopamine are considerable mediators of vomiting induced by cancer chemotherapy treatments (Johnston et al., 2014) and the detection of neurotransmitter metabolites in biological samples suggest the involvement of serotonin and dopamine in the triggering of vomiting response (Veyrat-Follet et al., 1997; Gralla et al., 1999). Furthermore, substance P also plays an important role in the mediation of the vomiting response. Since increased turnover of 5-HT, dopamine and substance P occur during both phases of vomiting in the brainstem and intestine in vomit-competent animals as well as humans (Darmani et al., 2009), further investigations are needed to investigate the effects of substance P in both the phases of vomiting. At 3rd h, the reduction in the concentration of 5HT and the metabolite 5HIAA by CS-HexFr (10 mg) in the brain areas (AP and BS) and intestine (**Table 3**) correlate well with the suppression of the vomiting response, where serotonin has been reported to be the mediator of acute vomiting response of cisplatininduced vomiting (Higgins et al., 2012; Ullah et al., 2014). Similarly, the reduction in the concentration of dopamine in the area postrema and 5HT concentration in the brain areas and intestine at 18th h of cisplatin administration (**Table 4**) further support the anti-emetic action of CS-HexFr later in the emetic episode. At 3rd h of cisplatin administration, dopamine concentration has been quantified as significantly high (P < 0.001) in the area postrema, brain stem and intestine, which is paradoxical with regard to the anti-emetic effect of CS-HexFr. The paradox can be hypothesized of (1) Switching of efficacy from agonist to antagonist of cannabis active constituent (2) Differential interaction with Gi or Gs signal transduction proteins (3) Pharmacokinetic factors, etc. (Darmani, 2010).

A number of studies suggest the involvement of CB1 receptor activation for the anti-emetic action of Cannabis sativa (19 -THC) (Darmani, 2001; Van Sickle et al., 2003) against

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various emetogenic agents. The CB1 receptors are co-localized with 5HT3 receptors in the nucleus tractus solitarius (NTS) in the brain stem and Gastrointestinal tract (GIT) (Hermann et al., 2002), where the action of THC on these receptors inhibit the release of monoamines, especially 5HT, in the least shrew model (Darmani and Johnson, 2004) and Pigeon model (present study). Our study provides further evidence for the involvement of serotonin and dopamine (and their metabolites) in the control of cisplatin-induced emesis over a 24 h period in the pigeon. Our current data in **Figure 1** (VEH/Pt) does not support the time periods as acute- and delayed-emetic phases, which indicate that the acute emetic phase is probably between first and second hour post-cisplatin injection, and there is no identifiable delayed phase since the frequency of emesis/jerks gradually declines and there is no upsurge of emesis per hour later on through 24 h observation period. The two time points, i.e., 3rd and 18th h post-cisplatin administration have been selected to find a clue for any mechanistic differences in the mediation of cisplatin-induced vomiting throughout the observation period in pigeons.

In summary, this study provides evidence for the involvement of serotonin and dopamine differentially at the two different time points in the triggering of vomiting response by cisplatin in pigeons. Furthermore, the suppression of the behavioral signs of cisplatin-induced vomiting by CS-HexFr is supported by attenuation of the cisplatin-induced 5HT upsurge at acute time point (3rd h) and dopamine and 5HT upsurge at delayed time point (18th h).

#### AUTHOR CONTRIBUTIONS

IU conceived the project, performed experimental work, data collection, analysis, literature search, and manuscript preparation. FS supervised research work, helped in study design, and drafted the final version of the manuscript. MS, JA, and MA helped in project design, behavioral experiments, performed statistical analysis, and corrected the final version of the manuscript. All authors read and approved the final manuscript for publication.

#### ACKNOWLEDGMENTS

We sincerely thank Higher Education Commission of Pakistan for sponsoring the studies. We are thankful to Korea United Pharm, Inc., Korea for donating Cisplatin active material for this study. The contribution from Dissertation of IU is also hereby acknowledged.

of dorsal vagal complex to inhibit emesis in ferrets. Am. J. Physiol. Gastrointest. Liver Physiol. 285, G566–G576. doi: 10.1152/ajpgi.00113.2003

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Ullah, Subhan, Alam, Shahid and Ayaz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Endocannabinoid System and Migraine Pain: An Update

Rosaria Greco1 \*, Chiara Demartini 1 , Anna M. Zanaboni 1,2, Daniele Piomelli 3 and Cristina Tassorelli 1,2

1 Laboratory of Neurophysiology of Integrative Autonomic Systems, Headache Science Centre, IRCCS Mondino Foundation, Pavia, Italy, 2 Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy, 3 Department of Anatomy and Neurobiology, University of California, Irvine, Irvine, CA, United States