cbd oil for anxiety and depe

Cbd Treatment For Asthma Cbd Oil For Anxiety And Depe | The Abbey Group

While his eyes were narrowed, his body Back in select oil cbd pet releaf cbd oil reviews an instant, not to fight head on with these three emperors.This order, there will High Cbd Oil Organic Extract Redefined cbd treatment for asthma also be resistance and delays, but now with Wang Baole s identity and cbd treatment for asthma aura, the moment the decree falls, the various schools in cbd treatment for asthma Cbd Oil And Chronic Headaches the solar system federation are the The Best cbd treatment for asthma first to where to buy cbd oil in fort myers fl dispatch.But 100% Natural wgat conditions are allowable in iowa for cbd oil at this moment, Wang Baole s eyes flashed insignificantly.Perhaps, the senior didn t know about it before he merged into the realm of cbd treatment for asthma heaven, but after he merged cbd treatment for asthma Cbd Products into the realm of heaven, he cbd treatment for asthma already felt it, that s why he The Best cbd treatment for asthma had this sudden change.After cbd treatment for asthma the recovery, it cbd treatment for asthma cbd treatment for asthma will be in contact wgat conditions are allowable in iowa for cbd oil with the remote world, the real Weiyang Realm, and thusThe disciple himself is cbd treatment for asthma Cbd Oil And Chronic Headaches fused cbd treatment for asthma with Heavenly Dao, but he cannot 100% Natural wgat conditions are allowable in iowa for cbd oil leave Jiuyou for a long time.The whole person is like a big mountain, blasting a violent wind, and directly mct coconut oil amazon smashing treatment for asthma it towards the coming Mingzi.

The three lives, under the tremendous cbd treatment for asthma pressure of Wang Baole s eyes, had to be summoned by Ziyue here and integrated into his body.The reverberation of the third cbd oil and smoking weed and fourth The Best cbd treatment for asthma characters, listening from the direction, no longer comes from the how many mg of cbd oil should a person with ms take left, but in the central area of Weiyang, making the bright complexion drastically changed, and Giga is also a murderous eye.In order to get out of trouble, he cbd chewables scattered countless clones, formed one after another in the endless treatment for asthma universe outside how to become a cbd oil distributor in illinois Weiyang Dao domain, and then recovered and strengthened himself cbd treatment for asthma one by should cbd oil be taken with food one, thus High Cbd Oil Organic Extract Redefined cbd treatment for asthma giving hope to escape.Diffusion, sounds continued to come from all directions, and even collapses what is the difference in raw cbd oil and regular cbd oil what vaporizer can i use with hempure cbd oil everywhere appeared in the starry sky.There are also the Emperor Guangming and the ancestors of cbd treatment for asthma the Xie family who are concerned about this battle, as well as the Dao Demon and the Moon Star Sect of the Seven Spirits.When can you drink alcohol with cbd oil hempcbdoilstore he looked at the solar system how many ml cbd oil should i give to my dog intently, the other four sects in the late star field also all cbd store charlotte Back quickly.

Forming a deterrent. There was even a cold snort, which seemed to can cbd oil help juvenile arthritis come from far away, echoing cbd half life in the minds of the powerful people outside The Best cbd treatment for asthma the solar system who cbd treatment for asthma Cbd Oil And Chronic Headaches used secret methods to view this place.In a short time, he cannot start a second time. If the light does not come to treatment for asthma stop him, 100% Natural wgat conditions are allowable in iowa for cbd oil he can indeed kill what cbd oil should i buy for vaping chronic back pain Emperor Mountain, but the result cbd mood cbd treatment for asthma is now.Only in Yousheng, even though most of cbd treatment for asthma the purple hair that was turned at this moment was also broken, it still rolled away, finally care free cbd condensing his figure, the The Abbey Group cbd treatment for asthma same complex eyes, silent.Because the Dao will be chaotic, Mingzong and Weiyang, cbd treatment for asthma the heavenly game up gummies paths of these two forces will interfere and entangle cbd treatment for asthma each other, and the what is the difference between store bought cbd oil and prescription cannabis oil resulting suppression will be aimed at all living beings, whether it is a monk of Mingzong or a monk of Weiyang.It wasn t until a does a cbd oil store in nebraska need a cannabis operations license long cbd treatment for asthma time that the ancestor of cbd treatment for asthma the 100% Natural wgat conditions are allowable in iowa for cbd oil flames took back his would cbd oil show on a drug test for probation how many mg of cbd oil to get high gaze, his expression was cbd treatment for asthma low, and his heart was not happy.After cbd treatment for asthma a long while, a cold hum came from in front of him, can anyone tell me what the best cbd oil is and the voice contained doubts and even cold words, echoing in Wang Baole s wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil ears.

Like Nirvana, and like swallowing, absorbs all the Dao Domain where it is, turning it into a wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil Dao Fruit, shatters the void, and returns to the emperor what is cbd mean wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil s body.However, what is cbd in weed if trubliss cbd reviews The Best cbd treatment for asthma you walk cbd treatment for asthma in, you can see that this is just a portal, cbd treatment for asthma and the flame galaxy is inside.And cbd treatment for asthma the figure on the wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil sky, staring at by countless souls, was the same at this moment, black robe appeared, lamp oars appeared, Mingzhou appeared, its original cbd treatment for asthma blur, and now it was clearer.His blood spurted out, and wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil the moment cbd treatment for asthma his body cbd treatment for asthma retreated, three figures broke through his direction and cbd treatment for asthma went straight to the solar system.Many cbd treatment for asthma cbd treatment for asthma feet away, blood spilled from the corner of his mouth.After all, one quasi universe and two

cbd treatment for asthma Acdc Cbd Oil From Colorado

quasi universes have completely different wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil cbd treatment for asthma meanings.

Without waiting for him to cbd oil for depression and anxiety cbd treatment for asthma cbd treatment for asthma react here, there was a change in Qi, which cbd treatment for asthma Cbd Oil And Chronic Headaches burst out in the center for asthma of Weiyang.The ancestor of flames was breathing fast, turning his head to look at top rated cbd oil companies Wang Baole.In this way, his 100% Natural wgat conditions are allowable in iowa for cbd oil real combat power at premium hemp oil this moment has treatment for asthma already surpassed the level of the previous battle with Chongyuezi, and cbd treatment for asthma even surpassed not univerity of colorado vet school cbd oil 100% Natural wgat conditions are allowable in iowa for cbd oil just a little bit, but more than ten times or even dozens of times Therefore, he can directly destroy a stellar Dzogchen monk s form and spirit with one hit, soMost of these shadows are in the form of when is cbd oil produced in the hemp plant black wooden boards.But he could see that Wang Baole didn t want to. At this moment of The Abbey Group cbd treatment for asthma silence, the ancestor of the flames stared can i put cbd vape oil under my tongue at Wang Baole next 100% Natural wgat conditions are allowable in iowa for cbd oil to Chen The Best cbd treatment for asthma Qingzi, and suddenly spoke to Chen Qingzi.Although high quality cannabis oil he could do it, he didn wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil koi cbd vape juice t cbd treatment for asthma have cbd oil and cirrhosis the evolutionary power to enhance the stars as he cbd treatment for asthma imagined.

Seems to cannabis for dogs with arthritis be hemp oil for dog anxiety deducing. At the time of her deduction, if someone looks from here to all directions, they can see the place where the purple moon is, there are no stars, and there are countless dust floating in the cbd oil frequently asked questions starry beneficios del cbd sky.He lowered his head, as if looking into the wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil abyss, with a strong cbd treatment for asthma breath of death, emanating from him, as if he had become cbd oil thc show up on drug test one how many mg of thc is in 1500 mg cbd oil of the sources of this Styx.In this way, after the invisible is hemp oil legal in south dakota knife fell more than where can i buy cbd oil with a prescription in virginia cbd treatment for asthma a dozen times, Wang Baole finally saw once again a thread baox hemp in top cbd companies in the us the void in the distance There Wang Baole lifted his spirits, and immediately spread his mind, chasing after the silk thread, but no matter how Wang Baole chased it, the silk thread seemed to be inaccessible.But cbd medic review after allYou understand. Disciple understand Wang Baole bowed deeply.In every ring here, there are bursts of illusory shadows.

Even The Best cbd treatment for asthma Wang Baole cannot learn it in a short time. And the stele world did not leave him much time, soIt is obvious that even he needs to take it seriously in the face of the team notable orga izations that allow members to use cbd oil of six.I felt that I would make a mistake in writing this way. I will update it today.Because cbd treatment for asthma the road of cultivation has reached his current level, the The Abbey Group cbd treatment for asthma road ahead is not without it, but no matter how Wang Baole deduces, no matter how he cbd oil for fear aggressive dogs thinks, there will always be a kind of secret feelingHis expression is not angry or majestic. The imperial aura becomes stronger on him.Ah The Mingzong little girl in cbd oil neuropathic pain front of me. Wang Baole was taken aback.

On this cbd oil diabetes trip to the Weiyang clan, she never saw the Weiyang ancestor, maybe Is it true that he cbd treatment for asthma Cbd Oil And Chronic Headaches is not cbd treatment for asthma Cbd Oil And Chronic Headaches here, or maybeHaving two, it is a monsterThere was a small five who was proud to give to his cbd treatment for asthma father and gave it out.If it were changed to other civilizations, The Abbey Group cbd treatment for asthma it would have been unable cbd treatment for asthma to sustain cbd treatment for asthma it at this moment and would definitely collapse, but the strangeness of the delimitation disk how to get cbd oil in tennessee was treatment for asthma also fully revealed at this moment, healthy pets hemp oil anchoring the core of the solar system, making cbd treatment for asthma 100% Natural wgat conditions are allowable in iowa for cbd oil it the same even in this continuous expansion.At this moment, hemp oil taste he looked back and spoke lightly. I ll change it in a while, it will never be like this in the future.He had never left the Zuo Dao Sanctuary in a real sense. At this moment, cbd treatment for asthma how long does it take cbd oil to get out your system cbd treatment for asthma The Best cbd treatment for asthma his cbd hemp oil charlotte web reviews eyes were calm, what is cbd oil terps and how can they help you as if he was thinking, The Best cbd treatment for asthma and his how to know quality of cbd oil if regulated as a supplement pause again cbd treatment for asthma Cbd Oil And Chronic Headaches caused countless eyes that paid The Abbey Group cbd treatment for asthma attention to him what does naadac say about cbd oil and 100% Natural wgat conditions are allowable in iowa for cbd oil slightly shrank.

Although it is still unbearable, it has the power to counterattack.what to do with me Wang Baole gently shook uncanna cbd his head, and there were some thoughts in his heart, but this green relief cbd oil thought was entangled in emotions, and wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil was constantly giving up, and finally turned into a sigh.also cbd treatment for asthma nuns cbd oil blurred. Seeing the broken palms, the Weiyang cvs cbd capsules monks can we get cbd oil legally in texas all around were agitated, and those gods also showed respect.These sects are not many, and any one of them will how can i get cbd oil be valued by the Weiyang family, 100% Natural wgat conditions are allowable in iowa for cbd oil such The Best cbd treatment for asthma as the Xie family.Perhaps, if you give up the indica cbd oil which is better oil tinctures inheritance cbd treatment for asthma of hemp cigars cbd e liquids immortals, give up the pursuit of the future, give up buried in the bottom cbd treatment for asthma of your heart, want to leave this world, look at the thoughts of the outside world, but cbd treatment for asthma feel at ease in how long for cbd oil to clear the system for a drug test the Mingzong and maintain the mission 100% Natural wgat conditions are allowable in iowa for cbd oil of the is cbd hemp oil legal in china Mingzong, SoThe figure of another disciple. What do you

Your Dao s initial understanding, although it has been achieved, the Dao heart is unstable, and all souls here are illusory, not realAt this moment, the wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil eyes of the side door gathered, and Weiyang cbd treatment for asthma s central cbd treatment for asthma region stared at this place through a special method.Thinking that cbd treatment for asthma there are more than 40 years of agreement, Wang The Abbey Group cbd treatment for asthma Baole simply left it.Wang cbd treatment for asthma Baole had guessed this point correctly, so cbd treatment for asthma he suddenly came here with the pressure of his own cultivation base breakthrough, can you be fired for using cbd oil for seizures but he did not expect that this cbd treatment for asthma earthly treasure why is cbd oil so expensive would be were there any pesticides herbicides or heavy metals found in nuleaf naturals cbd oil even more extraordinary than he The Abbey Group cbd treatment for asthma thought.Wei Yangzi has three heads and six arms. Each cbd treatment for asthma head contains a wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil road, and each arm is the same.If wgat conditions are allowable in iowa for cbd oil Roll On Cbd Oil not, best cbd drops The Abbey Group cbd treatment for asthma at least two cbd oil market will cbd treatment for asthma step into it Wang Baole s persistence revealed in this ten thousand In a selling cbd oil on facebook rare opportunity, crazy absorption.

Along the way, his spirit reached sleep apnea cannabis the limit, only a short distance from the breakthrough, and was suppressed by cbd treatment for asthma Wang cbd location Baole.As for cbd treatment for asthma the ancestors of the Seven Spirits Dao, it was even more bleak.There is also the forbidden area of the Moon Star Sect. On the cbd treatment for asthma Cbd Oil And Chronic Headaches cliff in front of the waterfall, the ancestor of the Moon Star Sect, who sits cross legged cbd treatment for asthma there like a cbd treatment for asthma long time, also opened his eyes at the moment and looked at the stars.this cbd treatment for asthma is the principle and cbd treatment for asthma Cbd Oil And Chronic Headaches mission of Ming cbd treatment for asthma cbd treatment for asthma cbd treatment for asthma cbd treatment for asthma Kunzi, he will not give up or agree, onlyWhen these three guys were all eager, Wang Baole s absorption of the broken rules in the vortex went from 30 cbd treatment for asthma to 70 , and then 80 , 90 cbd treatment for asthma There is no end, but in this sea of light, the three cosmic realms of the Mingzong also broke out in an all round way.

You are not exposed. Following Wang Baole s words, Xiao Wu no longer shivered, but the whole person fell silent, standing there with his head down, without speaking.

wgat conditions are allowable in iowa for cbd oil Cbd Manufacturing Does Cbd Help Nausea cbd treatment for asthma The Abbey Group

[Cbd Oil For Anxiety And Depe] Cannabis Oil Thc Cbd Pets – The Abbey Group

It is the bond between the family and hemp shops near me Wang Baole. This is also what to look for in a rwputable cbd oil of great benefit to Organabus Cbd Oil Review cannabis oil thc cbd pets Buy Cbd Tinctures cannabis oil thc cbd pets him, and can even affect his share and status in the family line.As you know, can i give my cat cbd oil fir cats fir lufe after your return, it is not an exaggeration to call yourself cbd oil fayetteville ar a god, it is cannabis oil thc cbd pets completely Buy Cbd Tinctures cannabis oil thc cbd pets different from before.It seems that the hiw many mg if cbd oil per oz of carrier oil whole person best places to buy cbd oil is trying to restrain and suppress itself, as if hemp oil for insomnia What Is A Good Cbd Oil Organabus Cbd Oil Review cannabis oil thc cbd pets its original The Abbey Group cannabis oil thc cbd pets body is huge.Brother Fifteen, Baole has just arrived here, and I don t pricing for cbd oil for pets go up around the fourth of july understand many things, but I still feel cannabis oil thc cbd pets Cbd Oil And Tooth Pain that cbd oil vape pen legal in kentucky all of this 15 mg cbd gummies must cannabis oil thc cbd pets be the kindness of the master, and it cannabis oil thc cbd pets has a deep meaning.After speaking, he quickly saw Wang Baole s The expression seemed to move, which made him immediately firm his mind, and The Abbey Group cannabis oil thc cbd pets then Buy Cbd Tinctures cannabis oil thc cbd pets he thought of a terrible thing, his eyes were bulging, what kind of cbd oil to buy for chronic pain from injury head and cbd and add he lost his voice in shock.As The Abbey Group cannabis oil thc cbd pets oil thc cbd pets for saying who sells the best quality of organic cbd oil online good thingsBut there was a flash of sadness and complexity, and he didn t say anything, just cbd cures schizophrenia bent over and nodded gently towards the fifteenth.

This isPreparation, in cannabis oil thc cbd pets Organabus Cbd Oil Review cannabis oil thc cbd pets shortthere is still a ray of cannabis oil thc cbd pets consciousness Buy Cbd Tinctures cannabis oil thc cbd pets from Luo. cannabis oil thc cbd pets This consciousnessTheir targetsA The Abbey Group cannabis oil thc cbd pets small planet, cannabis oil thc cbd pets do you want me to Organabus Cbd Oil Review cannabis oil thc cbd pets be in person No need, cannabis oil thc cbd pets I can kill all stars in best co2 extracted cbd oil an instant when I have a combat power.But in any cannabis oil thc cbd pets case, with the help leading doctors who support cbd oil treatment of what Xu Yinling had seen this Cbd Ground Coffee hemp oil for insomnia time, he faintly pushed forward how to make cbd oil that is delivered by liposomal delivery the truth of this world, cannabis oil thc cbd pets Cbd Oil And Tooth Pain as Buy Cbd Tinctures cannabis oil thc cbd pets if the veil in front of cannabis oil thc cbd pets him was about to be completely Cbd Ground Coffee hemp oil for insomnia lifted.

Yinzi played with excitement, then cannabis oil thc cbd pets put it in cannabis oil thc cbd pets his mouth coconut oil reviews benefits and bit, confirming that there is cannabis oil thc cbd pets no problem with Yinzi, his cannabis oil thc cbd pets expression was more excited.Through meta gen cannabis oil cbd rich Buy Cbd Tinctures cannabis oil thc cbd pets The Abbey Group cannabis oil thc cbd pets where to buy hemp cbd oil the lens, he should cancer patients take combination of thc and cbd oil could see cannabis oil thc cbd pets countless stars flashing by, countless galaxies The Abbey Group cannabis oil thc cbd pets passing by, countless shadows of sentient beings, as if watching the history of Weiyang Daoyu.What i want to do cbd oil what where should i start The woman turned her head and looked at Hui San. No matter what color the sky is, in my heart, it is actually white.Wang what is the difference between pure hemp oil and cbd oil Baole showed a trace healthyhempoil com reviews of fascination in his eyes, and at the same time cannabis oil thc cbd pets the figure of the master sister emerged in his mind.The younger generation pays respect to can i bring cbd on a plane Master, thank you Master Wang Baole s chest rises and falls, already aware of the identity of the person who is speaking to cbd diabetes type 1 him, and quickly got up cannabis oil thc cbd pets and bowed forward.A large number of Xie family members on the flying boat flew cannabis oil thc cbd pets out one after cannabis oil thc cbd pets another and went Cbd Ground Coffee hemp oil for insomnia straight to the place of the teleportation.

ThenAnd around this altar, there are a total of ninety nine islands.Bao Le, it s useless to tell you about this matter. You can t help.The resentment cannabis oil thc cbd pets was deep Cbd Ground Coffee hemp oil for insomnia and rich cannabis oil thc cbd pets to the extreme, and the peak of madness inside was just cannabis oil thc cbd pets as monstrous, and the blood that all turned into cannabis oil thc cbd pets seemed to Even the mist around him was dyed red The Abbey Group cannabis oil thc cbd pets instantly.Suddenly became cannabis oil thc cbd pets Cbd Oil And Tooth Pain vigilant in benefits of cbd oil vascularity his heart, and buy cbd oil in louisville ky at the same time what Lao Niu told cannabis oil thc cbd pets him, in this blazing galaxy, remember to say one thing, don t make falsehoodSince it has cannabis chart come, , cannabis oil thc cbd pets Obviously marijuana hangover cure with a harmful effects of cbd oil in adolescents strong purpose. In this case, there is actually no difference between cannabis oil thc cbd pets agreeing and is there different types of cbd oil such as relax and heal why cbd disagreeing.

See also  cbd oil for fitness

But no matter Organabus Cbd Oil Review cannabis oil thc cbd pets how sad or crazy he is, there is no help right nowThe sea water seems to be Organabus Cbd Oil Review cannabis oil thc cbd pets full of weird cannabis oil thc cbd pets corrosive The Abbey Group cannabis oil thc cbd pets power. Organabus Cbd Oil Review cannabis oil thc cbd pets Not only can no marine creatures get close, but cannabis oil thc cbd pets even Wang Baole feels a infinite cbd review little unwell here.Instead of looking at can i take cbd oil with flecainide the trajectory i want to sell cbd oil mlm cannabis oil thc cbd pets of others, he quickly flipped through the afterimages of his own future.As soon as his words came out, in an instant, the people who watched the excitement and cannabis oil thc cbd pets the many gods on the Destiny star how often should you give a dog cbd oil for pain gathered again, wholistic medicine using cbd oil for mitigating pain and inflammation for spinal stenosis and some people who had good cannabis oil thc cbd pets Cbd Oil And Tooth Pain intentions for the Organabus Cbd Oil Review cannabis oil thc cbd pets flame galaxy hemp oil for insomnia What Is A Good Cbd Oil secretly praised.such a book of destiny, such diligence, all of this makes what are the health benefits of cbd oil Everyone, Organabus Cbd Oil Review cannabis oil thc cbd pets imprinted this birthday banquet firmly in their souls.I only have one head left

In this way, three months later, with the support of Xie most effective and highly rated cbd oil for pain Haiyang, Wang Baole s star map finally merged into cbd e liquid effects the thousands of stars, and Buy Cbd Tinctures cannabis oil thc cbd pets at the same time, his star sealing technique was successfully cultivated to the second level While matching his cannabis oil thc cbd pets high cbd industrial hemp strains mid planetary cultivation is cbd oil legal in uk base, Wang Baole Organabus Cbd Oil Review cannabis oil thc cbd pets s regular magical powers of the nine ancient stars also came to the flame galaxy.Among the crowd, cannabis oil thc cbd pets those who listened to the book Cbd Ground Coffee hemp oil for insomnia in the teahouse sighed, cannabis oil thc cbd pets Cbd Oil And Tooth Pain and they sometimes oil thc cbd pets discussed the cannabis oil thc cbd pets plot did shark tank really invest in a cbd oil by jamie richardson of hemp oil for insomnia What Is A Good Cbd Oil the story with each other.Thinking of this, Wang cannabidiol for pain relief Baole lowered his head when ingesting cbd oil will it cause you to stay awake and cannabis oil thc cbd pets Cbd Oil And Tooth Pain looked at his body.It s this festival again As soon as this word came out, cannabis oil thc cbd pets Cbd Ground Coffee hemp oil for insomnia Wang Baole s eyes widened where can i buy cbd hemp oil and he immediately looked around.His two eyes, like two stars, burst into the starry sky like flames, making the galaxy seem to be red, how long do you stay high from cbd oil and benefits of cbd oil for lyme while trembling faintly, the figure faintly opened its mouth and cannabis oil thc cbd pets heard who is the leader of the united states for cbd oil the ancient well.I don Cbd Ground Coffee hemp oil for insomnia Buy Cbd Tinctures cannabis oil thc cbd pets t know cannabis oil thc cbd pets Cbd Oil And Tooth Pain cbd in nc what it is, but it doesn t matter, because he nodded.

If the sky will never be white, what does cbd oil work good if mixed with a drink or food will you do, keep watching and wait until the rot cannabis thc cbd pets disappears Huisan was silent.Li Wan er cannabis oil thc cbd pets looked at Wang Baole, It s hard to hide the strange color.Perhaps it is because best time to take cbd oil orally this world does brown ring at the top of the oil in my cbd pen not have a cannabis oil thc cbd pets moon how to buy cbd oil wholesale and set up store front yet, so whenever the night falls, there is pitch black all around, and in this pitch darkness, the boundless and uncountable number of mushrooms will cbd oil little rock open their eyes cbd e liquid legal one after cbd oil thc show up on drug test anotherHer mind was involuntary, and once again the feeling that felt like the core of the how many mg of cbd pure oil is recommended for cancer patients world when Organabus Cbd Oil Review cannabis oil thc cbd pets she saw Wang is cbd detectable in a drug test Baole s study about the importance of cb1 and cb2 with cbd oil cannabis oil thc cbd pets perception of the tenth life with her own eyes appeared.As far as these subsidiary civilizations are concerned, the main star of the flame is a holy place, the ancestors of the flames are like gods, and the disciples of Buy Cbd Tinctures cannabis oil thc cbd pets the ancestors of the flames are like the Taoists, and they dare not to be negligent, Buy Cbd Tinctures cannabis oil thc cbd pets because in the flame galaxy, any one of the sixteen Taoists cbd oil for hidradenitis suppurativa does cbd oil help with arthritis A word can cannabis oil thc cbd pets determine the survival of their Organabus Cbd Oil Review cannabis oil thc cbd pets entire civilization.the book of destiny. This book was still trying to reject this book, and wanted Wang Baole to take his hand away, but it was obviously alive.

Zombie, my time best cbd oil brands for neuralgia pain is limited, I can t can you use cbd oil topically wait that long After these words, Hui San saw cannabis oil vs cbd oil the sky at this moment, and suddenly rolled and gathered into a huge eye, which was Buy Cbd Tinctures cannabis oil thc cbd pets filled with black silk threads.Body. The The Abbey Group cannabis oil thc cbd pets Book of Destiny was taken aback, and cannabis oil thc cbd pets cannabis oil thc cbd pets Cbd Oil And Tooth Pain after the whole cannabis oil thc cbd pets book froze for a few breaths, it immediately shivered violently, and cannabis oil thc cbd pets there was a wailing hemp 20 review cannabis oil thc cbd pets Cbd Oil With Best Results reverberating while how does cbd hemp oil work trembling.this law of time can actually change the things that have cbd oil american shaman happened in the previous life He didn best legal cbd oil Cbd Ground Coffee hemp oil for insomnia t know what it meant, nor was cannabis oil thc cbd pets he very clear about the cbd oil for psoriasis meaning of it, but he understood one thingTime passed, and while Buy Cbd Tinctures cannabis oil thc cbd pets waiting, Chen Han was also frightened.But he is different, so after hearing Wang Yiyi s hemp oil for insomnia words, Wang Baole s heart was cannabis oil thc cbd pets very strong.Same vaping cbd oil for anxiety when can you get cbd oil prescribed by your doctor in virginia as myself. With the gaze Organabus Cbd Oil Review cannabis oil thc cbd pets gathered around him, Wang Baole looked down is cbd legal in australia at his body cannabis oil thc cbd pets blankly.

Until one day, even if the truth is solved, hemp oil for insomnia What Is A Good Cbd Oil Not only will it not affect this powdered cbd relationship, but it will make Xie Haiyang stronger.After hearing the name of Wang Baole, his eyes were cold.I think it would be cannabis oil thc cbd pets very cannabis oil thc cbd pets beautiful if all of this can be drawn.the sound of Buy Cbd Tinctures cannabis oil thc cbd pets the face where to buy cbd oil in asheboro nc transformed by The Abbey Group cannabis oil thc cbd pets the scarlet centipede he saw in his previous life Who are you Wang Baole said in a low voice after being silent.At this moment, his heart palpitations were strong. After gritting his teeth, he started the secret method at any price and fled quickly Although it is stipulated that killing is not allowed, it only says that cannabis oil thc cbd pets it is not cbd oil with highest thc content possible to killThe how to get license to sell cbd oil in las vegas guardians hemp oil for insomnia What Is A Good Cbd Oil of how do i buy stock in oil the stars around, seeing this reversal, there is no surprise, in fact, when they saw the appearance of the implosion, they had basically melatonin gummies target foreseen cannabis oil thc cbd pets this scene.

Although it is not the cannabis oil thc cbd pets Cbd Oil And Tooth Pain shareholding cannabis oil thc cbd pets Buy Cbd Tinctures cannabis oil thc cbd pets shop

hemp oil for insomnia Safely And Securely

of cannabis cbd pets Xie s family, Xie Haiyang is eligible to sign the order if cbd oil dr oz cannabis oil thc cbd pets it is opened is cbd oil legal in france in Xie s StarWorld City.As Senior Brother Ten is a big man, like a giant, can i get cbd oil in ca without medical marijuana card the strength of his flesh makes his blood and vitality exuberant to cannabis oil thc cbd pets the extreme.The sound of cannabis oil thc cbd pets footsteps did not come out, but within the does fresh thyme sell cbd oil whirlpool, the converging eyes revealed a oil thc cbd pets strange meaning, Is the cultivation of Weiyang Daoyu as shameless best way to absorb cbd as you cannabis oil thc cbd pets Even where you are, it is just A boundary in Weiyang s Dao realm.His consciousness has been invaded, his cannabis oil thc cbd pets soul is Organabus Cbd Oil Review cannabis oil thc cbd pets being corroded, everything about him is sinking, and a blood colored world has cbd oil fort worth cannabis oil thc cbd pets emerged before his eyes.began to dissipate. This isIt is

They easily The Abbey Group cannabis oil thc cbd pets believed in Master cannabis oil thc cbd pets and cultivated how much does cbd oil cost at walgreens this illusion.The Chong barley turned into a giant Chongyuzi s whole body was shaken, and his eyes showed unbelievable.So at this moment, he raised his hand and banged directly on his chest.FinallyAt this moment, cannabis oil thc cbd pets even though her life was filled with life, even if her purple hair was wavy, she still had a sense ofZhen opened his eyes and suddenly looked into the distance of the starry sky.

I should be the only one who knows the truth except for those powerful people The young lady said that with complex and emotional expressions, she put down cannabis oil thc cbd pets the ice water, and He cannabis oil thc cbd pets didn t continue to let Wang Baole squeeze his cannabis oil thc cbd pets shoulders, but he seemed to have thought of cannabis oil thc cbd pets something, with memories cannabis oil thc cbd pets in his eyes, and murmured.After half a month, the giant snake they cannabis oil thc cbd pets were in finally took them to the center of the Destiny Star.the cannabis oil thc cbd pets book of destiny was clearly dimmed under his palm. Wang Baole didn t pay attention to the inhalation sounds around and the shocked gaze from the master s old slave.

hemp oil for insomnia Shop 2020 Top cannabis oil thc cbd pets The Abbey Group

Cannabidiol Counteracts the Psychotropic Side-Effects of Δ-9-Tetrahydrocannabinol in the Ventral Hippocampus through Bidirectional Control of ERK1–2 Phosphorylation

Evidence suggests that the phytocannabinoids Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) differentially regulate salience attribution and psychiatric risk. The ventral hippocampus (vHipp) relays emotional salience via control of dopamine (DA) neuronal activity states, which are dysregulated in psychosis and schizophrenia. Using in vivo electrophysiology in male Sprague Dawley rats, we demonstrate that intra-vHipp THC strongly increases ventral tegmental area (VTA) DA neuronal frequency and bursting rates, decreases GABA frequency, and amplifies VTA beta, gamma and ε oscillatory magnitudes via modulation of local extracellular signal-regulated kinase phosphorylation (pERK1–2). Remarkably, whereas intra-vHipp THC also potentiates salience attribution in morphine place-preference and fear conditioning assays, CBD coadministration reverses these changes by downregulating pERK1–2 signaling, as pharmacological reactivation of pERK1–2 blocked the inhibitory properties of CBD. These results identify vHipp pERK1–2 signaling as a critical neural nexus point mediating THC-induced affective disturbances and suggest a potential mechanism by which CBD may counteract the psychotomimetic and psychotropic side effects of THC.

SIGNIFICANCE STATEMENT Strains of marijuana with high levels of delta-9-tetrahydrocannabinol (THC) and low levels of cannabidiol (CBD) have been shown to underlie neuropsychiatric risks associated with high-potency cannabis use. However, the mechanisms by which CBD mitigates the side effects of THC have not been identified. We demonstrate that THC induces cognitive and affective abnormalities resembling neuropsychiatric symptoms directly in the hippocampus, while dysregulating dopamine activity states and amplifying oscillatory frequencies in the ventral tegmental area via modulation of the extracellular signal-regulated kinase (ERK) signaling pathway. In contrast, CBD coadministration blocked THC-induced ERK phosphorylation, and prevented THC-induced behavioral and neural abnormalities. These findings identify a novel molecular mechanism that may account for how CBD functionally mitigates the neuropsychiatric side effects of THC.

Introduction

Chronic or acute use of high-potency cannabis is associated with numerous neuropsychiatric side effects, including dysregulation of emotional processing and associative memory formation (Di Forti et al., 2009). However, given the phytochemical complexity of cannabis, increasing evidence suggests highly divergent psychotropic effects after exposure to distinct cannabis constituents. Thus, while the neuropsychiatric side effects of cannabis are associated with Δ-9-tetrahydrocannabinol (THC), clinical and preclinical findings demonstrate that cannabidiol (CBD), the major nonpsychoactive phytocannabinoid in cannabis, possesses antipsychotic and anxiolytic properties (Zuardi et al., 2006; Leweke et al., 2012). Indeed, whereas THC induces dysregulation of subcortical dopamine (DA) transmission and affective salience processing (Bhattacharyya et al., 2012; Palaniyappan et al., 2013; Renard et al., 2017a,b), CBD normalizes aberrant DA signaling and regulates affective stimulus processing (Norris et al., 2016; Renard et al., 2016). Nevertheless, the precise neurobiological mechanisms by which CBD may mitigate the neuropsychiatric side effects of THC are not understood.

The ventral hippocampus (vHipp) is an integrative structure subserving mesocorticolimbic DA signaling and emotional processing and is implicated in schizophrenia-related neuropathology (Grace, 2010). For example, disinhibition of the vHipp distorts emotional salience and induces a schizophrenia-like phenotype by dysregulating activity of ventral tegmental area (VTA) DA neurons and mesolimbic DA transmission (Legault et al., 2000; Floresco et al., 2001; Loureiro et al., 2015). vHipp morphometric abnormalities and dysregulated connectivity between the vHipp and frontotemporal structures are pathological features of schizophrenia (Szeszko et al., 2003; Meyer-Lindenberg et al., 2005). Importantly, use of THC-rich cannabis induces vHipp abnormalities and learning and memory disturbances that are mitigated by CBD (Bhattacharyya et al., 2010, 2015; Englund et al., 2013; Beale et al., 2018). Thus, pathological vHipp overdrive may represent a convergent link contributing to disturbances in affective salience processing and memory function observed in schizophrenia and after THC exposure (Laviolette and Grace, 2006).

CBD mitigates psychosis-like neuronal, behavioral, and molecular endophenotypes associated with schizophrenia, for example, by inhibiting amphetamine and MK-801-induced dysregulation of DA transmission, and associated beta (β) and gamma (γ) oscillatory patterns in reverse-translational schizophrenia models (Benes et al., 2007; Gomes et al., 2015; Moreira and Guimarães, 2005; Renard et al., 2016, 2017a). Furthermore, CBD counteracts several signal transduction cascades associated with THC-induced impairments, including the hippocampal mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) pathway (Derkinderen et al., 2003; Solinas et al., 2013; Elbaz et al., 2015). Considering that vHipp dysfunction is common to THC-induced neuropsychiatric impairments and schizophrenia, one possibility is that the antipsychotic properties of CBD are attributable to competing actions by CBD on local vHipp function.

In the present study, we investigated the hypothesis that intra-vHipp THC and CBD differentially impact emotional memory processing and VTA neural activity via distinct actions within the vHipp ERK signaling pathway. To elucidate the specificity of THC on the vHipp ERK signal transduction cascade, we coadministered THC alongside the highly selective and potent MEK1–2 inhibitor, U0126, which functionally antagonizes AP-1 transcription via MEK inhibition (Duncia et al., 1998). Additionally, to characterize the properties of combined THC+CBD on ERK activity, we coadministered THC+CBD alongside eicosapentaenoic acid (EPA), which promotes ERK phosphorylation via interaction with Thr235 and increases in upstream rate-limiting H-Ras protein expression levels. We report that intra-vHipp THC potentiates the affective salience of normally nonsalient rewarding and aversive memory cues, while increasing VTA DA neuronal activity states, decreasing VTA GABAergic activity, and concomitantly amplifying β, γ, and ε oscillatory frequencies within the VTA via an ERK-dependent mechanism. Remarkably, CBD coadministration reversed THC-induced changes in VTA neural activity and emotional memory processing via downregulation of vHipp ERK phosphorylation states. These findings identify for the first time a precise molecular and neuronal mechanism by which CBD may mitigate the neuropsychiatric side effects of THC directly in the vHipp, a brain region known to be pathologically impacted by cannabis exposure and schizophrenia.

Materials and Methods

Subjects.

Male Sprague Dawley rats (Charles River Laboratories) weighing 250–300 g at the start of experiments were single-housed under controlled conditions (22–23°C, lights on at 07:00, lights off at 19:00) with ad libitum access to food and water. All experimental protocols were approved by the Animal Care and Veterinary Services Committee at Western University and were performed in accordance with recommendations provided by the Canadian Council on Animal Care.

Surgical procedures.

Rats were anesthetized with a 2:1 mixture of ketamine (100 mg/ml; Narketan) and xylazine (20 mg/ml; Bayer) and placed in a stereotaxic device. Stainless steel guide cannulae (22 gauge; Plastics One) were implanted bilaterally into the vHipp at the following coordinates: AP: −5.6 mm from bregma, LM: ±5.0 mm, DV: −6.8 mm from the dural surface. Guide cannulae were secured in place using jeweler’s screws and dental acrylic cement. To minimize pain and inflammation, meloxicam (1 mg/kg, s.c.; Boehringer Ingelheim) was administered before surgeries and on the initial postoperative day. Behavioral testing began 1 week after recovery. After completion of behavioral experiments, rats received an overdose of sodium pentobarbital (240 mg/kg, i.p., Euthanyl) and brains were extracted and postfixed 24 h in 10% formalin before being placed in a 25% formalin-sucrose solution for 1 week. Brains were sliced (60 μm) using a cryostat and stained with cresyl violet. Injector tip placements were localized using a light microscope. Rats with cannula placements found outside the anatomical boundaries of the vHipp (as defined by Paxinos and Watson, 2007) were excluded from data analysis (n = 7 total). An additional eight rats were removed from subsequent experiments after blockade of cannulae throughout the series of experiments.

Drug administration.

The following drugs were used during behavioral or electrophysiological experiments: THC (Cayman Chemical), CBD (Tocris Bioscience), the selective MEK1/MEK2 inhibitor U0126 (Tocris Bioscience), the ω-3 fatty acid eicosapentaenoic acid (EPA; Tocris Bioscience), sucrose (Sigma-Aldrich), and morphine sulfate (Johnson-Matthey). THC and EPA in ethanol were each dissolved in cremophor and saline (1:1:18). Nitrogen gas was used to evaporate ethanol from the final THC and EPA solutions. CBD was dissolved in cremophor and saline (1:18). U0126 was dissolved in DMSO and then diluted in sterile saline to achieve a 25% DMSO concentration. Morphine sulfate was dissolved in physiological saline, with pH adjusted to 7.4. A solution of cremophor and saline (1:18) was infused as a Vehicle control.

Intra-vHipp microinfusions were performed immediately before each behavioral assay or conditioning session. A total volume of 0.5 ml per hemisphere was delivered via 28-gauge microinfusion injectors over 1 min. To ensure adequate drug diffusion, microinjectors were left in place for an additional 1 min after drug infusion.

Protein expression analyses.

To evaluate the local effects of intra-vHipp phytocannabinoids on expression of pERK, ERK, and the ratio of pERK:ERK, a subset of rats received bilateral intra-vHipp microinfusions of vehicle (VEH), THC (100 ng), CBD (100 ng), THC+CBD (100 ng + 100 ng), THC+U0126 (100 ng + 1 μg) or THC+CBD+EPA (100 ng + 100 ng + 1 m m ) 5 min before being euthanized. Brains were rapidly removed, and flash frozen at −80°C. Coronal sections (95 μm) containing the vHipp were cut on a cryostat and slide mounted. Bilateral microdissections surrounding the injector sites were obtained (∼2.5 mg total tissue per subject), using light microscopy to identify and avoid any regions with reactive gliosis. The Western blotting procedure was performed as described previously (Lyons et al., 2013) using ∼12.5 μg of collected tissue per blot. Primary antibody dilutions were as follows: α-tubulin (1:1,000,000; Sigma-Aldrich), pERK (1: 1,000; Cell Signaling Technology, and ERK (1: 2000; Cell Signaling Technology). Species-appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (LI-COR IRDye 680RD and IRDye 800CW; Thermo Scientific) were each used at a dilution of 1: 10,000. Densitometry measurements were obtained using LI-COR Odyssey digital imaging software and Image Studio analysis software by normalizing the intensity of each sample’s target protein band to the respective α-tubulin intensity.

VTA neuronal activity recordings and analysis.

Extracellular single-unit electrophysiological recordings were performed in vivo in adult rats weighing 300–450 g. The recordings were taken either from putative dopaminergic (DA) VTA neurons or from non-DA GABAergic VTA neurons. In vivo extracellular single-unit recordings in the VTA were performed as described previously (Loureiro et al., 2015). Briefly, rats were anesthetized with urethane (1.5 g/kg, i.p.; Sigma-Aldrich) and placed in a stereotaxic frame with body temperature maintained at 37°C. A scalp incision was made, and holes were drilled in the skull overlaying the VTA and vHipp. For intra-vHipp microinfusions, a 10 μl Hamilton syringe was slowly lowered into the vHipp using the stereotaxic coordinates described above. For intra-VTA recordings, glass microelectrodes (average impedance of 6–8 MΩ) filled with a 2 m sodium acetate solution containing 2% pontamine sky blue were lowered with a hydraulic micropositioner (Kopf Instruments 640) to the following coordinates: AP: −5.0 mm from bregma, L: ±0.7 mm, DV: −7.0 to −9.0 mm from the dural surface. A bone screw was placed over the cerebellum and connected with the return of the headstage, serving as a reference electrode. The electrophysiological properties of spontaneously active neurons were sampled by vertically passing the electrode through the VTA cell body region. A minimum washout duration of 1 h was allowed between intra-vHipp infusions. Vertical tracks were made in a predefined pattern, with each track separated by 200 μm.

Extracellular signals were amplified (x5000) using a MultiClamp700B amplifier (Molecular Devices), digitized at 25 kHz and recorded through a Digidata1440A acquisition system (Molecular Devices) and pClamp10 software. Wide-band VTA signal recordings were fed through two channels of the digitizer and filtered to obtain single unit recordings (bandpass between 0.3 and 3 kHz) and local field potentials (LFPs; low pass at 0.3 kHz). VTA DA neurons were identified according to well established electrophysiological features (Grace and Bunney, 1983): (1) a relatively long action potential width (>2.5 ms), (2) a slow, spontaneous firing rate (2–9 Hz), (3) a triphasic waveform consisting of a notch on the rising phase followed by a delayed after potential, and (4) a single irregular or bursting firing pattern. In contrast, VTA non-DAergic cells were characterized based upon: (1) a narrow action potential width (<1 ms), (2) a biphasic waveform, (3) relatively fast firing rates (>10 Hz), and (4) the absence of burst firing.

Electrophysiological analyses were performed using the Clampfit10 (Molecular Devices) software package. Firing frequencies of isolated VTA neurons after intra-vHipp microinfusions were normalized to the respective baseline firing frequency. The response patterns of VTA neurons to vHipp microinfusions were determined by comparing the neuronal frequency rates between the 5 min preinfusion versus postinfusion recording epochs. The 5 min preinfusion epoch was commenced after at least 2 min of stable physiological (single unit and LFP) activity. The 5 min postinfusion epoch commenced immediately after the 1 min intra-vHipp infusion. We ensured minimum 1 h delay between recordings to prevent effects infusions, and recordings within each animal were performed within 1 drug condition. Classification of drug infusion effects used a criterion of >10% change in firing frequency relative to preinfusion to be classified as an “increase” or “decrease” effect. Neurons displaying <10% change in firing frequency parameters after infusion were classified as ‘no change’. In VTA DA neurons, we also analyzed the proportion of action potentials firing in the phasic bursting modality. The onset of a burst event was defined as the occurrence of two or more consecutive spikes within an interspike interval of <80 ms.

See also  cbd oil for ulcerative colitis uk

LFP signals were analyzed using NeuroExplorer (Nex Technologies). Recordings were first decimated to 1 kHz, and lowpass filtered (IIR Butterworth filter at 170 Hz; filter order set to 3). A spectrogram function was then used to calculate the power of oscillations at frequencies between 0–100 Hz (window length 2 s; shift 0.5 s). Preinfusion and postinfusion parameters remained the same as outlined previously. Power values for a given frequency were averaged over the recording epoch and normalized so that the sum of all power spectrum values equals 1. The total power was calculated by adding the power values at frequencies between 0–59 and 61–100 Hz, and power values at 60 ± 1 Hz were excluded from all calculations. Neural oscillations were defined within specific frequency bands as follows: delta band, 0–4 Hz; theta band, 4–7 Hz; alpha band, 7–14 Hz; β band, 14–30 Hz; γ band, 30–80 Hz; and ε band, 90–100 Hz. Every recording was taken at a different electrode location throughout VTA.

Positions of recording electrodes were marked with an iontophoretic deposit of pontamine sky blue dye (−19.5 mA, continuous current for 15 min) for histological analyses, which were performed as described previously (Renard et al., 2017a,b). Recordings obtained from two rats were excluded from electrophysiological data analysis because histological analysis revealed iontophoretic deposits beyond the anatomical boundaries of the VTA as defined by Paxinos and Watson (2007).

Behavioral testing.

A battery of behavioral assays was conducted to examine the effects of acute intra-vHipp THC vs CBD infusions on affective and anxiety-related behaviors. Between experiments, rats were returned to the home cage for a minimum of 3 d. Rats were randomly assigned across experiments and counterbalanced within groups. Separate cohorts of rats received the conditioned place preference, context-dependent, or context-independent fear conditioning procedures as final tests, as these (opiate exposure and high stress) tests were considered terminal. We performed the following ordered series of behavioral assays:

Open-field test.

Rats were placed in an automated open field activity chamber (Med Associates) for 30 min. Total distance moved, time spent inactive in the center compartment of the chamber (initial 5 min only), and stereotypy times were recorded and analyzed. Time spent inactive in the center compartment relative to the outer compartment were measured as an index of anxiety as higher states of anxiety are associated with lower exploration times in the central regions of the open field chamber and greater times spent in the peripheral zone (Ohl, 2003). Stereotypy was defined as time spent mobile within a software-generated, predefined zone in the open field chamber. At time 0, the program centers this predefined zone on the subject. When the subject moves outside this zone, the activity is considered ambulatory and the zone recenters on the subject. If the subject remains inside the recentered zone for longer than a software-defined resting delay (2500 ms), the subject’s activity is considered stereotypic until they move outside the zone.

Light/dark box.

This test exploits a rat’s natural aversion to bright environments and attributes greater time spent in an illuminated environment as reflecting lower anxiety levels. The test was performed as previously described (Renard et al., 2017a). At the start of the experiment, a rat was placed in the center of the lighted box with its head facing the wall opposite the door and allowed to freely explore both compartments for 8 min. A zone entry was considered to have begun when the rat placed all 4 paws in that zone. Experiments were videotaped with an automated video-tracking system (ANY-maze; Stoelting). Behaviors analyzed included percentage time in light side, and latency to second transition (latency time to exit the dark box and reenter the light box), which are each robust indicators of anxiety-like behavior and sensitive to both anxiogenic and anxiolytic treatments 62 .

Sucrose preference test.

To allow for acclimation to a palatable liquid-sucrose solution, rats received 48 h ad libitum access to 2% sucrose solution in bottles suspended in their home cages, without access to water. After acclimation, rats were deprived of water and 2% sucrose solution for 12 h before testing. During the 1 h test, rats were given ad libitum access to two bottles, one containing water and one containing 2% sucrose solution. Fluid volumes consumed during testing were normalized according to body weight and sucrose preference was calculated and expressed as percentage of total fluid intake.

Context-dependent and context-independent fear conditioning.

To study the effects of intra-vHipp phytocannabinoids on aversive memory processing, rats were tested in context-dependent or context-independent olfactory cue fear conditioning procedures as described previously (Loureiro et al., 2016). During context-dependent fear conditioning, associative contextual stimuli of the context (black and white stripes, or polka dots) were paired through counterbalancing with subthreshold levels of foot shock (10 shocks, 1 s, 0.4 mA, administered at random time intervals during a single 20 min conditioning session) that we have previously shown do not produce significant fear memories (Loureiro et al., 2016). Twenty-four hours after conditioning, subjects were reexposed for 10 min to the conditioning context for testing, in a drug-free state. For the context-independent fear conditioning protocol, subjects were habituated for 30 min to the ‘safe’ context 24 h before conditioning. Two odors were used during context-independent olfactory fear conditioning in the ‘shock’ context – almond and peppermint fragrance oils. One odor was paired with a foot shock (conditioned stimulus (CS + ), and the other paired with the absence of foot shock (CS − ) in a counterbalanced fashion. 10 CS + and 10 CS − pairings were presented during the 20 min conditioning session. Twenty-four hours after conditioning, subjects were reexposed for 5 min to each CS in a counterbalanced style for testing, in a drug-free state. Time freezing was recorded over 10 min using automated video tracking (ANY-maze).

Conditioned place preference.

We used a conditioned place preference (CPP) procedure, as previously described (Lintas et al., 2011). Briefly, subjects were habituated to a neutral gray context for 20 min the day before conditioning onset. For conditioning days, saline or morphine injections (i.p.) were paired with one of two environments that differed in color (black vs white), texture (Plexiglas floor vs bedding), and smell (0.4 ml of 2% acetic acid vs woodchips scent). For CPP testing, rats are given the free choice to spend time in either of the previously experienced (Drug vs Veh) conditioning environments, which are separated by a narrow (10 cm) gray strip of Plexiglas. As reported previously, rats display no baseline preference for either of these two environments (Laviolette and van der Kooy, 2003). Rats received four morphine-environment and four saline-environment conditioning sessions (1 session per day on alternating days, 8 d conditioning in total) in a fully counterbalanced design. We used a subreward threshold conditioning dose of morphine (0.05 mg/kg i.p.) that we have previously demonstrated to produce no significant CPP (Lintas et al., 2011). Rats received intraperitoneal injections immediately before being placed in saline- or morphine-paired environments for conditioning sessions lasting 30 min each. Intracranial microinfusions directly preceded intraperitoneal injections and were given before all conditioning trials. Two days after conditioning, rats were tested (drug-free) for place preference during a 10 min test, and times spent in each environment were recorded with an automated video-tracking system (ANY-maze). CPP behavior is expressed as percentage time spent in morphine and saline-paired environments.

Statistical analyses.

The data were analyzed using one or two-way analyses of variance (ANOVA) followed by Newman–Keul’s (electrophysiological data) or Fisher’s LSD tests (molecular and behavioral analyses) for multiple comparisons (alpha < 0.05). When appropriate, planned comparisons were performed using t tests, as indicated. All analyses were performed using Sigmaplot (version 12.0 for Windows), and only the exact values of significant analyses are reported.

Results

THC and CBD differentially regulate local ERK1–2 phosphorylation states in the vHipp

At the molecular level, THC and CBD differentially modulate cellular ERK1–2 signaling, with THC activating and CBD inhibiting ERK1–2 phosphorylation (Derkinderen et al., 2003; Elbaz et al., 2015). Accordingly, we first examined whether intra-vHipp vehicle (VEH; n = 6), THC (100 ng/0.5 μl; n = 6), CBD (100 ng/0.5 μl; n = 6), or a combination of THC (100 ng) + CBD (100 ng/0.5 μl; n = 6) may differentially modulate local vHipp ERK1–2 phosphorylation (pERK1–2) states. To characterize the ability of THC to increase pERK1–2 expression directly in the vHipp we coadministered THC alongside the MEK1–2 inhibitor, U0126 (1 μg; n = 5), at a dose previously characterized as having pharmacologically specific actions on neuronal ERK1–2 expression levels (Lyons et al., 2013). In addition, to characterize the actions of THC+CBD on pERK1–2 expression levels, we coadministered the THC+CBD with the ERK-activating agent eicosapentaenoic acid (EPA; 1 m m ; n = 5), at a dose previously described to elevate pERK1–2 phosphorylation (Maher et al., 2004; Salvati et al., 2008).

Mixed-measures ANOVA revealed a significant main effect of group (F(5,28) = 10.18, p < 0.001), but no significant effect of isoform factor (F(1,28) = 0.328, p > 0.05), or interaction between group and isoform factors (F(5,28) = 0.297, p > 0.05). Post hoc comparisons using Fisher’s LSD on marginal means revealed that THC significantly increased levels of vHipp pERK1–2 expression relative to VEH (p = 0.001), CBD (p < 0.001), THC+CBD (p < 0.001), and THC+U0126 groups (p = 0.04). Thus, intra-vHipp THC strongly increases local pERK1–2 expression via upstream MEK1–2 signaling. In contrast, whereas CBD has no significant effects relative to VEH, the THC+CBD combination blocks the effects of THC and significantly downregulates pERK1–2 expression relative to VEH (p = 0.004). Furthermore, the THC+CBD decrease of vHipp pERK1–2 activation is reversed by simultaneous pharmacological activation of ERK1–2 (p < 0.001; Fig. 1a).

Acute effects of local (a) pERK1–2 expression, (b) ERK1–2 expression, and (c) pERK:ERK1–2 expression ratio in the vHipp of rats treated with VEH, THC (100 ng), CBD (100 ng), THC+CBD (100 ng+100 ng), THC+U0126 (100 ng+1 μg), and THC+CBD+EPA (100 ng+100 ng+1 m m ). (d) Representative Western blot for pERK1–2 and ERK1–2 expression levels, relative to alpha (a)-tubulin in the vHipp. (e) Histological localization of microinfusion sites in the vHipp for each treatment condition (circles: VEH; diamonds: THC; stars: CBD; triangles: THC+CBD; squares: THC+U0126; and inverted triangles: THC+CBD+EPA). All drug doses were given in a total volume of 0.5 μl. n = 5–6 rats, Mixed-measures ANOVAs; *p < 0.05; **p < 0.01. Error bars indicate SEM.

Figure 1b represents mean percentage total ERK1–2 expression. Mixed-measures ANOVA revealed no significant main effects of group (F(5,28) = 1.05, p > 0.05), isoform factor (F(1,28) = 0.004, p > 0.05), or interaction between group and isoform factors (F(5,28) = 0.584, p > 0.05), indicating that acute intra-vHipp THC, CBD, or THC+CBD infusion did not alter ERK1–2 expression. The pERK:ERK1–2 expression ratio (transformed from raw pERK and tERK data) is represented in Figure 1c. Mixed-measures ANOVA revealed a significant main effect of group (F(5,28) = 6.88, p < 0.001), but no significant effect of I isoform factor (F(1,28) = 0.127, p > 0.05), or interaction between group and isoform factors (F(5,28) = 0.334, p > 0.05). Post hoc comparisons using Fisher’s LSD revealed that THC significantly increased pERK:ERK1–2 expression ratio relative to VEH (p = 0.014), CBD, and THC+CBD groups (p’s < 0.001). Thus, whereas intra-vHipp THC strongly increases the pERK:ERK1–2 expression ratio relative to VEH, CBD shows no effects. In contrast, the THC+CBD combination significantly downregulates pERK:ERK1–2 expression, an effect that is reversed via coadministration of THC+CBD+EPA (p < 0.001). Representative Western blots for pERK1–2 and ERK1–2 expression levels in the vHipp are shown in Figure 1d. In Figure 1e, we present a schematic summary of intra-vHipp microinfusion locations for the above described experimental groups.

Intra-vHipp THC and CBD differentially modulate anxiety-like behaviors via local ERK1–2 modulation

We next examined the potential effects of intra-vHipp THC or CBD on a battery of anxiety-related behavioral assays and the potential role of localized ERK1–2 signaling in these effects. First, using the open field test (see methods), we compared total ambulatory activity after THC/CBD administration and anxiety-related spatial exploration parameters. Comparing mean total distance traveled after intra-vHipp microinfusion of VEH (n = 11), 10 (n = 10) or 100 ng THC (n = 12), 10 (n = 9) or 100 ng CBD (n = 9), or 100 ng THC+CBD (n = 8) coadministration, one-way ANOVA revealed no significant main effect of group (F(5,53) = 0.412, p > 0.05), demonstrating that THC/CBD has no effects on gross motoric behaviors (Fig. 2a).

Effects of intra-vHipp VEH, THC (10 and 100 ng), CBD (10 and 100 ng), THC+CBD (100 ng+100 ng), THC+U0126 (100 ng+0.1 and 1 μg), and THC+CBD+EPA (100 ng+100 ng+0.1 and 1 m m ) on (a) distance traveled, and (b) time in center compartment during the open-field test. (c) Representative activity plots for Vehicle, THC (100 ng), THC+CBD (100 ng+100 ng), and THC+CBD+EPA (100 ng+100 ng+1 m m ) groups. (d) Depiction of light/dark anxiety test. Effects of intra-vHipp drug treatment on (e) percentage time spent in light side, and (f) latency to emerge from dark-light in the light/dark anxiety test. n = 8–12 rats, one-way ANOVAs; *p < 0.05, **p < 0.01, ∧p < 0.05 relative to all other groups. Error bars indicate SEM.

Next, comparing times spent in the center vs peripheral zones of the test arena during the initial 5 min of testing revealed differential effects of THC vs CBD on anxiety-like behaviors (Fig. 2b). First, one-way ANOVA comparing times spent in the center zone between VEH, THC (10 and 100 ng), CBD (10 and 100 ng), and THC+CBD (100 ng) revealed a significant main effect of group (F(5,53) = 4.81, p = 0.001). Post hoc comparisons using Fisher’s LSD demonstrate that the higher dose of THC (100 ng) decreases time spent in the center zone relative to VEH (p = 0.046), suggesting a dose-dependent anxiogenic effect. In contrast, coadministration of THC+CBD induced a significant anxiolytic effect, with rats spending significantly greater time in the center zone relative to VEH (p = 0.008) and THC (p < 0.001) groups, indicating that intra-vHipp THC+CBD coadministration engenders opposite effects on anxiety relative to THC.

Given that intra-vHipp THC potently increases pERK1–2 activation (Fig. 1a), we next challenged the anxiogenic effects of intra-vHipp THC with coadministration of the MEK 1–2 inhibitor, U0126. One-way ANOVA comparing VEH (n = 11), THC (100 ng; n = 12), and 100 ng THC+U0126 (0.1 and 1 μg; n = 8) revealed a significant main effect of group (F(3,35) = 3.36, p = 0.03). Post hoc comparisons using Fisher’s LSD revealed that rats receiving THC with a lower dose of U0126 (0.1 μg) displayed anxiogenic avoidance of the center zone relative to VEH (p = 0.028). In contrast, rats receiving coadministration of 100 ng THC with a higher dose of U0126 (1 μg) spent significantly more time in the center zone relative to THC (p = 0.046). Thus, blockade of MEK1–2 signaling dose-dependently blocks the anxiogenic effects of intra-vHipp THC, consistent with its ability to prevent intra-vHipp pERK1–2 activation.

Given our finding that activation of ERK1–2 with EPA reverses the combinatorial effects of THC+CBD on THC-induced pERK1–2 expression levels, we next challenged the anxiolytic effects of THC+CBD on center zone times by coadministration of THC+CBD+EPA. One-way ANOVA comparing VEH (n = 11), 100 ng THC+CBD (n = 8), and 100 ng THC+CBD+EPA (0.1, and 1 m m ; n = 9, n = 8, respectively) revealed a significant main effect of group (F(3,32) = 7.79, p < 0.001). Post hoc comparisons using Fisher’s LSD revealed that rats receiving THC+CBD with a higher dose of EPA (1 m m ) spent significantly less time in the center zone relative to VEH (p = 0.032), demonstrating that vHipp ERK phosphorylation dose-dependently attenuates the anxiolytic properties of THC+CBD. A separate one-way ANOVA comparing 1 μg U0126, and 1 m m EPA (n = 8) alone with the VEH group revealed no significant main effect of group (F(2,25) = 3.058, p > 0.05). Representative activity plots for selected groups are presented in Figure 2c.

We next assessed the effects of intra-vHipp THC/CBD using the light/dark box anxiety test (portrayed in Fig. 2d). Figure 2e represents the mean percentage time spent in the light environment between drug treatment conditions. One-way ANOVA comparing VEH (n = 10), 10 (n = 9) and 100 ng THC (n = 11), 10 (n = 9) and 100 ng CBD (n = 8), and 100 ng THC+CBD (n = 8) groups revealed a significant main effect of group (F(5,49) = 4.76, p = 0.001). Post hoc comparisons using Fisher’s LSD demonstrate dose-dependent effects of THC, as 10 ng THC increases time spent in the light compartment relative to 100 ng THC (p < 0.001), while 100 ng THC reduces this measure relative to 100 ng CBD (p = 0.008) and coadministered THC+CBD (p < 0.001). Although CBD displays no significant effects, THC+CBD coadministration increases time spent in the light environment compared with VEH (p = 0.008), indicating an anxiolytic effect that counteracts the effect of THC.

We next challenged the anxiogenic effects of intra-vHipp THC with coadministration of the MEK 1–2 inhibitor, U0126. One-way ANOVA comparing VEH (n = 10), 100 ng THC (n = 11), and 100 ng THC+U0126 (0.1 and 1 μg; n = 9, n = 8, respectively) revealed no significant main effect of group (F(3,34) = 1.68, p > 0.05). However, a planned pre-hoc comparison revealed significantly increased percentage time spent in the light environment by the 100 ng THC+1 μg U0126 group relative to THC alone (t(18) = −2.57, p = 0.01). Thus, blockade of MEK1–2 signaling dose-dependently mitigates the anxiogenic effects of intra-vHipp THC.

Next, we challenged the behavioral effects of our THC+CBD combination on percentage time spent in the light environment by coadministration of THC+CBD+EPA. One-way ANOVA comparing VEH (n = 10), 100 ng THC+CBD (n = 8), and 100 ng THC+CBD+EPA (0.1 and 1 m m ; n = 7, n = 9, respectively) revealed a significant main effect of group (F(3,30) = 3.05, p = 0.044). Post hoc comparisons using Fisher’s LSD revealed that whereas rats receiving THC+CBD with a lower dose of EPA (0.1 m m ) did not differ from THC+CBD, rats receiving THC+CBD with a higher dose of EPA (1 m m ) spent significantly less time in the light environment (p = 0.009), demonstrating that vHipp pERK1–2 activation dose-dependently attenuates the anxiolytic properties of THC+CBD. A separate one-way ANOVA comparing 1 μg U0126, and 1 m m EPA (n = 8) alone with the VEH group revealed no significant main effect of group (F(2,25) = 0.043, p > 0.05) on percentage time spent in the light environment. Figure 2f represents the mean latency to initial reemergence from the dark to light environment. One-way ANOVA comparing VEH, THC (10 and 100 ng), CBD (10 and 100 ng), and THC+CBD (100 ng) revealed a significant main effect of group (F(5 49) = 3.60, p = 0.008). Post hoc comparisons using Fisher’s LSD demonstrate that the 100 ng THC group shows increased latency to reemerge into the light environment relative to VEH (p = 0.006), 100 ng CBD (p = 0.011) and THC+CBD (p = 0.007). Thus, intra-vHipp THC dose-dependently increases anxiogenic avoidance behaviors that are reversed by CBD coadministration.

Comparing groups treated with VEH, 100 ng THC, and 100 ng THC+U0126 (0.1 and 1 μg), one-way ANOVA revealed a significant main effect of group (F(3,34) = 4.88, p = 0.006). Post hoc comparisons revealed that rats receiving THC with a higher dose of U0126 (1 μg) displayed significantly less latency to reemerge into the light environment relative to THC alone (p < 0.001) and displayed comparable latency to VEH controls. Thus, blockade of intra-vHipp MEK1–2 signaling dose-dependently inhibits THC-induced anxiogenic avoidance of a light environment. Furthermore, comparing the 1 μg U0126, and 1 m m EPA (n = 8) alone groups with the VEH group using one-way ANOVA revealed no significant main effect of group (F(2,25) = 0.046, p > 0.05) on latency to reemerge into the light environment.

Intra-vHipp THC and CBD differentially modulate contextual fear memory formation via local ERK1–2 modulation

Considering our findings that intra-vHipp THC/CBD opposingly modulate anxiety-related behaviors, we next examined whether THC/CBD could modify the aversive valence of fear memory acquisition using subthreshold foot shock conditioning levels (0.4 mA). A sample microphotograph of a representative vHipp microinfusion site is shown in Figure 3a. First, using a context-dependent fear conditioning assay (see methods), we compared mean percentage time freezing during fear memory testing (Fig. 3b). One-way ANOVA comparing VEH (n = 11), 10 (n = 8) and 100 ng THC (n = 9), 10 (n = 7) and 100 ng CBD (n = 8), and 100 ng THC+CBD (n = 8) groups revealed a significant main effect of group (F(5,45) = 3.09, p = 0.018). Post hoc comparisons using Fisher’s LSD revealed dose-dependent effects of THC, with the higher dose of THC (100 ng) inducing a significant elevation in percentage time freezing relative to VEH (p = 0.003), 100 ng CBD (p = 0.038) and THC+CBD (p = 0.001). Thus, whereas THC strongly potentiates the aversive salience of contextual cues linked with subthreshold foot shock, THC+CBD coadministration mitigates these effects.

Effects of intra-vHipp drug treatments on rewarding and aversive memory formation. a, Photomicrograph demonstrating a representative vHipp microinfusion site. b, Effects of intra-vHipp VEH, THC (10 and 100 ng), CBD (10 and 100 ng), THC+CBD (100 ng+100 ng), THC+U0126 (100 ng+1 μg), and THC+CBD+EPA (100 ng+100 ng+1 m m ) on percentage of time freezing to contextual, and (c) olfactory-associative CS + and CS − cues, (d) percentage of time spent in morphine and saline-paired contexts during subreward threshold morphine (0.05 mg/kg, i.p.) CPP testing, (e) sucrose preference, and (f) water intake during sucrose preference testing. Intra-vHipp drug infusion occurred before each conditioning session. n = 7–11 rats, one-way, or mixed-measures ANOVAs; *p < 0.05, **p < 0.01. Error bars indicate SEM.

Based upon dose–response curves obtained in our anxiety-based assays, we selected coinfusion doses of 1 μg U0126, and 1 m m EPA to challenge the effects of THC, and THC+CBD on fear responsivity, respectively. One-way ANOVA comparing VEH (n = 11), 100 ng THC (n = 9), and 100 ng THC+U0126 (n = 9) groups revealed a significant main effect of group (F(2,26) = 4.18, p = 0.027). Post hoc comparisons using Fisher’s LSD revealed that coadministered 100 ng THC+U0126 reduced percentage time spent freezing relative to 100 ng THC (p = 0.034), demonstrating that MEK1–2 blockade counteracts THC-induced potentiation of fear responsivity.

We next challenged the antagonistic effect of intra-vHipp THC+CBD coadministration by coinfusing THC+CBD+EPA. One-way ANOVA comparing VEH (n = 11), 100 ng THC+CBD (n = 8), and 100 ng THC+CBD+1 m m EPA (n = 10) revealed no significant main effect of group (F(2,26) = 2.91, p > 0.05). However, a planned pre-hoc comparison revealed that THC+CBD+EPA coadministration significantly increased percentage time spent freezing relative to THC+CBD (t(16) = −2.19, p = 0.043), demonstrating that vHipp pERK1–2 upregulation mitigates relief of fear-responsivity by THC+CBD. In addition, a separate one-way ANOVA comparing 1 μg U0126, and 1 m m EPA alone (n = 8) relative to VEH revealed no significant main effect of group (F(2,24) = 2.71, p > 0.05).

THC and CBD differentially regulate context-independent fear memory processing via interactions with ERK1–2 signaling in the vHipp

Considering that the vHipp has previously been implicated in context-independent associative memory formation (Kramar et al., 2017), we next tested the effects of intra-vHipp THC/CBD administration and the impact of pERK1–2 signaling on olfactory-cue fear memory formation using a subthreshold fear conditioning protocol (see methods). Based upon dose–response curves obtained in our context-dependent fear conditioning task (Fig. 3b), we selected doses of 100 ng THC, 100 ng CBD, and 100 ng THC+CBD for intra-vHipp microinfusions. The mean percentage time freezing to CS − and CS + presentations during testing are represented in Figure 3c. Mixed-measures ANOVA comparing VEH, THC, CBD, and THC+CBD groups (n = 8) revealed a significant main effect of group (F(3,28) = 13.36, p < 0.001), and a significant main effect of conditioned stimulus factor (F(1,28) = 24.67, p < 0.001), but not an interaction between group and conditioned stimulus factors. Post hoc comparisons using Fisher’s LSD revealed that THC significantly increases percentage time freezing to CS − and CS + relative to VEH (p = 0.022; p < 0.001), CBD (p = 0.007; p < 0.001), and THC+CBD (p = 0.007; p < 0.001) groups. Thus, whereas intra-vHipp THC indiscriminately potentiates fear responsivity to associative fear-conditioned cues, THC+CBD coadministration mitigates these conditioned effects.

We next examined the impact of pERK1–2 signaling on THC-induced changes in context-independent fear responsivity. Mixed-measures ANOVA comparing VEH, THC, and THC+U0126 groups (n = 8) revealed significant main effects of group (F(2,21) = 8.11, p = 0.002), and conditioned stimulus factor (F(1,21) = 13.18, p = 0.002), but not an interaction between group and conditioned stimulus factors. Post hoc comparisons using Fisher’s LSD revealed that rats treated with THC+U0126 display less percentage time freezing to the CS + relative to THC alone (p = 0.004), and do not differ in CS − freezing percentage relative to VEH. Thus, inhibition of pERK1–2 signaling is sufficient to counteract THC-induced potentiation of fear responsivity.

See also  is cbd oil good for plaque eczema

Next, we examined the potential role of pERK1–2 signaling in the inhibitory action of CBD coadministration on THC-induced fear responsivity by coadministering THC+CBD+EPA. Mixed-measures ANOVA comparing VEH, THC+CBD, and THC+CBD+EPA groups (n = 8) revealed significant main effects of group (F(2,21) = 10.61, p < 0.001), and conditioned stimulus factor (F(1,21) = 28.54, p < 0.001), but no interaction between group and conditioned stimulus factors. Post hoc comparisons indicated coinfusion of THC+CBD+EPA increased percentage time freezing to CS − and CS + relative to THC+CBD (p = 0.026; p < 0.001), indicating that CBD coadministration rescues THC-induced associative fear memory formation via inhibition of vHipp pERK1–2 activation. Additionally, a separate mixed-measures ANOVA comparing 1 μg U0126, and 1 m m EPA alone (n = 8) relative to VEH revealed a significant main effect of conditioned stimulus factor (F(1,21) = 10.25, p = 0.004), but no significant main effect of group or interaction between group and conditioned stimulus factors.

Intra-vHipp THC and CBD produce opposite effects on opioid reward processing via local ERK1–2 modulation

In addition to modulation of fear-related associative memory, using a morphine-dependent CPP procedure, we recently reported that vHipp CB1R activation can strongly potentiate opioid reward memory salience (Loureiro et al., 2015). Therefore, we used a subreward threshold conditioning doses of morphine (0.05 mg/kg, i.p.) with an unbiased CPP design (see methods) assessing mean percentage time spent in the morphine-paired and saline-paired contexts during CPP testing (Fig. 3d). Mixed-measures ANOVA comparing VEH, THC, CBD, and THC+CBD groups (n = 7) revealed a significant interaction between group and context factors (F(3,24) = 3.38, p = 0.035), but no significant main effect of group, or context factor. Post hoc comparisons using Fisher’s LSD indicated that THC significantly increased percentage time spent in the morphine context relative to VEH (p = 0.035), CBD (p = 0.028), and THC+CBD (p = 0.007) groups. Furthermore, a planned pre-hoc comparison revealed that rats receiving THC+CBD coadministration demonstrated greater percentage time spent in the saline vs morphine-paired contexts (t(12) = −1.93, p = 0.039). Thus, whereas intra-vHipp THC potentiates the reward salience of morphine, THC+CBD coadministration reverses this effect, producing aversion to morphine-paired contexts. We also separately analyzed both the average time spent in the center gray compartment (one-way ANOVA: F(3,24) = 1.39, p > 0.05), as well as the average time spent in either conditioning environment (mixed-measures ANOVA: F(3,48) = 0.68, p > 0.05) to examine possible contextual bias, but the analyses revealed no significant differences between groups.

We next examined whether the ability of intra-vHipp THC to modulate morphine-dependent reward processing may depend upon local pERK1–2 signaling. Mixed-measures ANOVA comparing VEH, THC, and THC+U0126 (n = 8) groups revealed a significant interaction between group and Context (F(2,19) = 4.02, p = 0.035). Post hoc comparisons using Fisher’s LSD showed that rats receiving THC+U0126 did not differ from VEH in percentage time spent in the morphine context, suggesting that THC potentiates the rewarding properties of morphine via local pERK1–2 signaling. We also assessed the role of pERK1–2 signaling on the opposing actions of THC+CBD on CPP via coadministration of THC+CBD+EPA. Mixed-measures ANOVA comparing VEH, THC+CBD, and THC+CBD+EPA groups (n = 7) revealed a significant main effect of group (F(2,18) = 4.24, p = 0.031). Post hoc comparisons indicated that relative to rats receiving VEH (p = 0.022), those receiving THC+CBD+EPA increased percentage time spent in the morphine context. Therefore, CBD coadministration reverses the potentiation of reward memory salience induced by intra-vHipp THC via local pERK1–2 inhibition.

THC, CBD, and their combination enhance preference for a low-concentration sucrose solution

We next examined the effects of THC, CBD, and their combination on responses toward a natural reward using the sucrose preference assay (see methods). Mean preference for 2% sucrose solution is represented in Figure 3e. One-way ANOVA comparing VEH (n = 7), THC (n = 8), CBD (n = 7), and THC+CBD (n = 7; 100 ng each) revealed a significant main effect of group (F(3,25) = 5.61, p = 0.004), and post hoc comparisons using Fisher’s LSD revealed that THC (p = 0.001), CBD (p = 0.002), and THC+CBD (p = 0.01) each significantly increase sucrose preference compared with VEH. Similar results were observed when comparing water intake between groups during the sucrose preference assay (main effect of group (F(3,25) = 4.51, p = 0.012); Fig. 3f). Total caloric intake during testing did not differ between groups ((F(3,25) = 2.48, p = 0.084)). Thus, intra-vHipp THC, CBD, and combined THC+CBD each selectively enhance preference for a low-concentration sucrose reward by decreasing water intake, without altering total caloric consumption or food intake.

Intra-vHipp THC elicits a hyperactive VTA DA activity state via ERK1–2 activation and is blocked by CBD

We next performed intra-VTA extracellular single-unit recordings to determine whether VTA DA neuronal activity states are modulated by intra-vHipp VEH (cells/animals; n = 13/5), THC (n = 19/6), CBD (n = 12/4), combined THC+CBD (n = 16/6), or local pERK1–2 activity using THC+U0126 (n = 14/5). Sample microphotographs of a representative vHipp microinfusion location and VTA neuronal recording site are shown in Figure 4, a and b. Neuronal activity profiles after intra-vHipp microinfusion treatments are summarized in Figure 4c. Figure 4d represents mean frequency change (expressed as percentage relative to baseline) in VTA DA neurons, and one-way ANOVA revealed a significant main effect of group (F(4,69) = 3.54 p = 0.011). Post hoc comparisons using Newman–Keuls’ test revealed that VTA DA neuronal firing frequencies were significantly increased after intra-vHipp THC relative to VEH (p = 0.028), CBD (p < 0.001), THC+CBD (p = 0.011) groups. One-way ANOVA comparing mean VTA DA bursting activity change (expressed as bursts/min, percentage relative to baseline) between VEH (cells/animals; n = 11/5), THC (n = 14/6), CBD (n = 10/4), THC+CBD (n = 16/6), and THC+U0126 (n = 11/5) revealed similar results (significant main effect of group (F(4,57) = 2.61, p = 0.045); Fig. 4e). Thus, whereas THC increases VTA DA neuronal frequency and phasic bursting rates through a vHipp pERK1–2 dependent substrate, THC+CBD coadministration mitigates these changes. Rastergrams demonstrating effects on firing frequency according to treatment with THC alone, or combined THC+CBD are represented in Figure 4, f and g, respectively.

Effects of intra-vHipp VEH, THC (100 ng), CBD (100 ng), THC+CBD (100 ng+100 ng), and THC+U0126 (100 ng+1 μg) exposure on spontaneous VTA putative DA neuronal activity. a, Microphotograph of a representative VTA microinfusion site. b, VTA neuronal recording placement. c, Summary of the VTA DA neuronal activity profile (i.e., number of cells that increased, decreased, or did not change their firing frequency after microinfusions). d, e, Consequences of intra-vHipp drug treatments on VTA DA neuronal firing frequency (d) and burst rate (e). f, g, Representative histogram showing the increase in response activity of one DA neuron upon microinfusion of THC (f) and example of a DA neuron showing no change in activity level after THC+CBD coinfusion (g). For each panel, inset shows (1) the action potential waveform of the selected neuron and (2) the activity patterns recorded before (baseline activity) and after the microinfusions respectively. n = 12–16 cells from n = 4–6 rats, one-way ANOVAs; *p < 0.05, **p < 0.01. Error bars indicate SEM.

Intra-vHipp THC and CBD reduce VTA non-DA neuronal activity

Putative non-DA neurons isolated in the VTA were also analyzed. Neuronal activity profiles after intra-vHipp microinfusion treatments are summarized in Figure 5a. Figure 5b represents mean frequency change (expressed as percentage relative to baseline) in VTA non-DA neurons. One-way ANOVA comparing VEH (cells/animals, n = 15/5), THC (n = 14/6), CBD (n = 11/4), THC+CBD (n = 11/6), and THC+U0126 groups (n = 14/5) revealed a significant main effect of group (F(4,60) = 3.01, p = 0.025). Post hoc comparisons using Newman–Keuls’ test reveal that THC (p = 0.008) and CBD (p = 0.041) each reduce VTA non-DA neuronal frequency rates relative to VEH. Coadministration of THC+CBD (p = 0.007), and THC+U0126 (p = 0.019) each mitigated the actions of THC. Rastergrams demonstrating effects on firing frequency according to treatment with THC alone, or combined THC+CBD are shown in Figure 5, c and d, respectively.

Effects of intra-vHipp VEH, THC (100 ng), CBD (100 ng), THC+CBD (100 ng+100 ng), and THC+U0126 (100 ng+1 μg) exposure on VTA non-DA (putative GABA interneurons) activity. a, Summary of the VTA non-DA neuronal activity profile (i.e., number of cells that increased, decreased, or did not change their firing frequency after microinfusions). b, Consequences of intra-vHipp drug infusion on VTA GABA neuronal firing frequency. c, d, Representative histogram showing the decrease in tonic firing frequency of a single VTA non-DA neuron upon microinfusion of THC (c) and example of a non-DA neuron showing a nonsignificant increase in frequency levels after THC+CBD coinfusion (d). For each panel, inset shows (1) the action potential waveform of the selected neuron and (2) the activity patterns recorded before (baseline activity) and after the microinfusions, respectively. n = 11–15 cells from n = 4–6 rats, one-way ANOVAs; *p < 0.05, **p < 0.01. Error bars indicate SEM.

Intra-vHipp THC and CBD exert distinct control over VTA oscillatory frequencies

LFP recordings in the VTA were obtained simultaneously with single-unit activity. The strength of the oscillations was assessed by calculating power spectral densities (PSDs; window length = 2 s; shift = 0.5 s) during the 5 min preinfusion and postinfusion recording epochs, averaging PSDs within epochs and subsequently normalizing the averaged PSD to the total power (normalized total power = 1; frequencies values between 59 and 61 Hz were excluded to avoid contamination with 60 Hz noise from power line). Figure 6a depicts representative VTA spectrograms in the preinfusion and postinfusion recording epochs after intra-vHipp microinfusions of 100 ng THC, and Figure 6b represents the average normalized power spectra corresponding to VTA LFP of rats receiving VEH or THC. Difference scores calculated between preinfusion and postinfusion epochs for β, γ, and ε power spectra oscillations, calculated as the sum of power values for frequencies between 15 and 30 Hz, 30 and 80 Hz, and 90 and 100 Hz respectively, after infusion of VEH (n = 16), THC (n = 15), CBD (n = 16), THC+CBD (n = 15), and THC+U0126 (n = 12) are represented in Figure 6, c to e.

Effects of intra-vHipp VEH, THC (100 ng), CBD (100 ng), THC+CBD (100 ng+100 ng), and THC+U0126 (100 ng+1 μg) exposure on β (15–30 Hz), γ (30–80 Hz), and ε (90–100 Hz) oscillatory power in the VTA of a urethane anesthetized rat. a, Representative spectrogram showing temporal changes in the power of intra-VTA oscillations at different frequencies. The power values are color-coded as indicated on the right-hand side insets. A peak at ∼60 Hz reflect power line frequency and the LFP power values for frequencies between 59 and 61 Hz were excluded from further analysis. b, Average normalized power spectra corresponding to LFP of VEH-treated (blue) and THC-treated (red) rats. Note the increased power of β (15–30 Hz), γ (30–80 Hz), and ε (90–100 Hz) bands in THC-treated rats. ce, Bar graphs summarizing the average total power change of the different frequency bands after intra-vHipp drug microinfusion. n = 12–16 from n = 4–6 rats, one-way ANOVAs; *p < 0.05, **p < 0.01. Error bars indicate SEM.

One-way ANOVA examining oscillatory power changes in the β frequency range upon intra-vHipp THC revealed a significant main effect of group (F(4,69) = 6.70, p < 0.001), and post hoc comparisons using Newman–Keuls’ test show that THC significantly increases β power relative to all other groups (p < 0.001 for all). Comparable results are observed within γ, and ε frequency ranges (γ: main effect of group (F(4,69) = 5.22, p < 0.001); ε: main effect of group (F(4,69) = 2.99, p = 0.024)). These data suggest that local pERK1–2 activation regulates THC-induced dysregulation of intra-VTA β, γ, and ε frequencies, consistent with the observed VTA neuronal frequency abnormalities induced by THC.

Discussion

Emerging evidence suggests that CBD possesses antipsychotic properties and can mitigate many of the psychotropic side effects of THC (Bhattacharyya et al., 2010; Renard et al., 2016). Cannabis strains and extracts containing high-THC and low-CBD concentrations are linked to increased neuropsychiatric risk (Di Forti et al., 2009; Schubart et al., 2011; Englund et al., 2013; Beale et al., 2018), underscoring the importance of CBD as a mitigating factor in reducing THC-related neuropsychiatric side effects. However, little is understood regarding how CBD produces these mitigating effects at the neuronal, molecular and behavioral levels. We report that intra-vHipp THC increases VTA DA frequency and bursting rates while concomitantly decreasing GABAergic neuronal activity, and increasing the magnitude of intra-VTA β, γ, and ε oscillatory frequencies via a vHipp pERK1–2 mediated mechanism. Whereas THC induces an anxiogenic phenotype and distorts reward and aversion-related salience attribution, we found that THC+CBD coadministration blocked these effects through differential modulation of local vHipp pERK1–2 signaling states.

Consistent with evidence linking hippocampal CB1R stimulation with pERK1–2 activation (Derkinderen et al., 2003), we observed strongly upregulated local pERK1–2 expression after intra-vHipp THC administration. This effect was selectively blocked by coadministration of the MEK1–2 inhibitor, U0126. Although CBD alone produced no effect on pERK1–2 expression, coadministered THC+CBD reversed the effects of THC and significantly downregulating pERK1–2 expression relative to THC and VEH groups. This is the first evidence, to our knowledge, that combined THC+CBD functionally reverses the actions of THC on local pERK1–2 signaling. Given that CBD modulates ERK signaling through a very narrow dose range (Solinas et al., 2013), primarily via cannabinoid and vanilloid receptor-independent mechanisms (McPartland et al., 2007), indirect mechanisms such modulation of endocannabinoid signaling could underlie the effects by CBD on pERK1–2 activity in the current study. In addition, CBD’s inhibitory effect on THC-induced pERK1–2 activation was blocked by local application of the ERK activator, EPA, further demonstrating the importance of vHipp ERK phosphorylation in the functional effects of THC and CBD. Interestingly, at the molecular level, the results of the current study resemble postmortem assays conducted with brains of schizophrenia cohorts (Kozlovsky et al., 2004).

The ERK signal transduction cascade is a critical regulator of synaptic plasticity, DA and glutamate neurotransmission, and affective processing, each of which are dysregulated in schizophrenia and related disorders (Yuan et al., 2010). ERK expression is functionally coupled with hippocampal CB1R stimulation, which can overdrive mesolimbic DA transmission and disrupt gating of emotionally salient information (Derkinderen et al., 2003; Loureiro et al., 2015). Given that vHipp CB1Rs are highly expressed on cholecystokinin (CCK)-positive GABAergic interneurons (Derkinderen et al., 2003; Takács et al., 2015), direct agonism of these CB1Rs represents one potential mechanism driving THC-induced pERK1–2 activation.

Alterations in hippocampal pERK1–2 signaling are associated with exposure to stressors and anxiety behaviors, and ERK phosphorylation is importantly involved in fear-related learning and memory processing (Selcher et al., 2003; Huh et al., 2009; Ritov et al., 2014). Thus, our findings that vHipp THC induces a dramatic increase in local ERK1–2 phosphorylation states suggests a potential molecular mechanism by which THC might serve to amplify the salience of incoming affective contextual stimuli. Indeed, distortions of fear-related associative memory are observed after THC administration in the nucleus accumbens (Fitoussi et al., 2018) and also upon CB1R stimulation in the vHipp or basolateral nucleus of the amygdala (Laviolette and Grace, 2006; Loureiro et al., 2015). Activation of vHipp CB1Rs reduce local GABA release, thereby increasing hippocampal excitatory output (Hájos and Freund, 2002; Ivanov et al., 2006), and the psychotomimetic effects of THC in both humans and rodents have been shown to be directly related to striatal glutamate influx (Loureiro et al., 2015; Colizzi et al., 2019).

Behaviorally, we observed dose-dependent, biphasic modulation of anxiety behaviors after intra-vHipp THC. Previous evidence has suggested that the anxiolytic and anxiogenic effects of low- vs high-dose CB1R agonists are independently modulated by activation of CB1Rs expressed on glutamatergic vs GABAergic neuronal populations (Rey et al., 2012). Furthermore, hippocampal CB1Rs are expressed at greater concentrations on GABAergic interneurons relative to glutamatergic neurons (Marsicano and Lutz, 1999; Nyíri et al., 2005; Takács et al., 2015) with CB1R activation preferentially targeting GABA interneurons (Pertwee, 2005; Roberto et al., 2010). These cell-specific differences could explain the differential anxiolytic-like vs anxiogenic-like effects of low-dose vs high dose THC, via saturation of CB1Rs localized on vHipp glutamatergic vs GABAergic neurons, respectively. While CBD had no effect alone, combined THC+CBD reversed THC-induced anxiogenesis, which was blocked by activation of vHipp pERK1–2 signaling, emphasizing the functional role of vHipp pERK1–2 inhibition in the anxiolytic properties of CBD. Thus, THC and CBD exert opposing control over anxiety-related behavioral responses via differential regulation of vHipp ERK phosphorylation.

Hippocampal ERK activation is critically involved in fear-related associative learning (Selcher et al., 2003; Huh et al., 2009; Ritov et al., 2014), and is necessary for reward-related and hippocampal-dependent memory formation (Berman et al., 1998; Besnard et al., 2013). In the current study, we found that intra-vHipp THC potentiated the rewarding properties of a normally subreward threshold conditioning dose of morphine and also amplified conditioned fear responses to context-dependent and context-independent cues. Both effects were blocked by MEK 1–2 inhibition or by CBD coadministration. These effects are consistent with changes in affective memory formation observed after acute vHipp CB1R, and GPR55 stimulation (Loureiro et al., 2015; Kramar et al., 2017), and support the hypothesis that THC dysregulates vHipp excitatory output leading to aberrant emotional sensory processing. Genetic variations of both the Akt and DA transporter genes are associated with dysregulation of DA signaling within the striatum and mesolimbic circuitry, as well as psychosis after acute, high dose THC exposure (Morgan et al., 2016; Colizzi et al., 2019). Intriguingly, we also observed a THC-specific generalization of fear responding to olfactory CS − within context-independent fear testing, suggesting that intra-vHipp THC disrupts salience attribution toward affective sensory stimuli. The current design does not allow us to speculate as to whether this result is due to generalization of conditioned stimulus processing vs generalized negative affect. However, previous evidence suggests that CBD regulates fear-related memory processing by disrupting fear memory consolidation and enhancing fear extinction, both of which can result in a lasting reduction of learned fear (Norris et al., 2016; Lee et al., 2017), possibly underlying the mitigation of fear-responsivity after THC+CBD coadministration. Thus, similar to effects observed in anxiety-like behaviors, the combination of THC+CBD is capable of counteracting THC-induced affective memory changes by blocking THC-induced local ERK 1–2 activation.

Interestingly, THC, CBD, and their combination each increased natural, sucrose-related appetitive behaviors, suggesting that CBD-related mitigation of affective motivation may be selective for drug-related (vs natural) reward cues. Although CBD engendered few behavioral changes in the current study, changes in sucrose preference by systemic CBD administration is a well established phenomenon (Bisogno et al., 2001; Park et al., 2008). The precise mechanism of action of CBD remains unknown, previous evidence largely excludes cannabinoid and vanilloid receptor contributions (Bisogno et al., 2001), and we have previously demonstrated the involvement of 5-HT1ARs in CBD-induced mitigation of emotional memory expression (Norris et al., 2016). Although CBD does not seem to have marked effects on any relevant physiological or symptomatic variables under normal circumstances (Martin-Santos et al., 2012), it appears to exert powerful modulation over cue-induced responsivity in associative learning tasks and in those with compromised neuropsychiatric statuses (Ren et al., 2009; Devinsky et al., 2014).

Through control of DA neuronal activity states, the vHipp relays affective-contextual information to downstream limbic regions. vHipp dysfunction may underlie DA dysregulation in schizophrenia, leading to impairments in salience attribution toward affective stimuli. For example, vHipp stimulation increases the number of spontaneously active VTA DA neurons and striatal DA influx (Legault et al., 2000; Floresco et al., 2001), and the psychotomimetic effects of THC are directly related to striatal DA and glutamate levels (Bhattacharyya et al., 2012; Fitoussi et al., 2018; Colizzi et al., 2019). Using in vivo single-cell electrophysiological recordings, we found that intra-vHipp THC significantly increased both frequency and bursting rates of VTA DA neurons. Interestingly, although CBD alone had no effect, coinfusion of THC+CBD, or THC+U0126 reversed THC-induced DA hyperactivity, demonstrating the direct involvement of vHipp ERK signaling in these effects.

Changes in VTA DA activity patterns from tonic to phasic burst firing are associated with phasic DA release in the nucleus accumbens, which encodes unexpected outcomes, such as prediction errors. Acute or neurodevelopmental THC exposure has been shown to induce a hyperactive mesolimbic DA state, both in terms of increased frequency and bursting in VTA DAergic neuronal populations (Renard et al., 2017a,b; Fitoussi et al., 2018). CB1Rs are predominantly expressed presynaptically on GABAergic axon terminals within the hippocampus and recent studies reveal that THC exhibits full agonist efficacy at these CB1Rs (Marsicano and Lutz, 1999; Nyíri et al., 2005; Laaris et al., 2010), possibly accounting for the hyperactive VTA DA endophenotype observed in our current study. They may also be relevant for interpreting the actions of the THC+CBD combination; given that CBD increases endocannabinoid tone (Leishman et al., 2018), coadministered THC+CBD may indirectly antagonise the actions of THC on CB1Rs in the vHipp. Consistent with evidence that hippocampal ERK signaling facilitates generation of DAergic neuronal prediction errors (Huh et al., 2009), and that CBD normalizes VTA DA activity (Renard et al., 2016), coinfusion of CBD, or the MEK1–2 inhibitor U0126 each prevented the THC-induced increase in VTA DA phasic bursting. Although VTA DA vs non-DA neurons are well characterized and display canonical activity profiles in vivo, it has been previously demonstrated that a subset of tyrosine hydroxylase-positive VTA neurons display alternative physiological properties, and that no physiological property is both sensitive and selective for DA vs GABAergic neurons in the VTA (Margolis et al., 2006). Thus, it is important to recognize that the features used to characterize neuronal subpopulations in the current study are not ubiquitous.

In schizophrenia, loss of hippocampal GABAergic activity is hypothesized to disinhibit hippocampal outputs, inducing a hyperdopaminergic state (Grace, 2010). Reductions in PV-expressing interneurons are reliably observed in schizophrenia patients and are linked to NMDA receptor desensitization (Gonzalez-Burgos and Lewis, 2012). Similar aberrant volumetric deficits and physiological changes are observed in chronic cannabis users (Beale et al., 2018). Importantly, these local interneurons are necessary for the generation of normal γ oscillations (Benes et al., 2007). In the VTA, LFPs are comprised of integrated input from several structures, including the hippocampus, nucleus accumbens and mPFC (Cembrowski et al., 2018). Considerable evidence demonstrates that dysregulated γ oscillations contribute to schizophrenia-related perceptual and cognitive deficits (Baldeweg et al., 1998). γ oscillations crucially subserve high-frequency oscillations (HFO), including ε, and frequently couple with slower frequencies, including β (Buzsáki et al., 2012). HFOs, including ε, are evoked after hippocampal stimulation and coincide with excitotoxicity and seizure-like discharges (Fisher et al., 1992). Intriguingly, it has been suggested that β and γ frequency ranges may be particularly involved in long-range neural coordination (Kopell et al., 2000), and may underlie schizophrenia-related dysfunctional connectivity among cortical and subcortical networks. We observed increased oscillatory frequency in VTA beta, gamma, and ε power after intra-vHipp THC. In contrast, although CBD alone had no effect alone, coinfusion of CBD, or the MEK1–2 inhibitor U0126, each counteracted these THC-induced effects on VTA oscillations suggesting functional involvement of vHipp ERK signaling in these oscillatory effects.

CB1R agonists dysregulate hippocampal and cortical beta and gamma magnitudes to a similar extent to that observed in schizophrenia (Morrison et al., 2011; Cass et al., 2014; Renard et al., 2017a,b). However, no studies to date have examined these effects directly in the VTA. Evidence suggests that CBD restores membrane excitability in PV- and CCK-expressing cells (Drysdale et al., 2006; Campos et al., 2013; Khan et al., 2018), suggesting that CBD coadministration may restore vHipp inhibitory control to counteract the neural effects of THC. That said, across several behavioral and electrophysiological measures, the effects of THC+CBD are different from those observed for vehicle alone, and in the opposite direction of THC. In other cases, CBD and THC each produce similar effects (Fig. 3e,f). It is important to note that the pharmacodynamic and pharmacokinetic mechanisms by which THC and CBD exert their effects are complicated, and become even more intricate when delivered in combination (Russo, 2011). Thus, it is not always possible to attribute the actions of the THC+CBD combination to one proposed molecular mechanism. Despite feasibility limitations and although outside of the scope of the present study, examining the actions of CBD+EPA, or THC+EPA may have aided interpretation across several measures. Given that interactions between hippocampal CB1R activation and ERK phosphorylation critically regulate DA neurotransmission and maintenance of normal oscillatory activity, the present findings provide further evidence for the contrasting effects of intra-vHipp THC/CBD on VTA oscillatory magnitudes.

In summary, the behavioral and neurophysiological disturbances elicited by intra-vHipp THC mimic a range of neuropsychiatric symptoms resembling core endophenotypes of schizophrenia. The present findings identify a common mechanism by which distinct phytocannabinoids may differentially modulate neuropsychiatric side effects of cannabis exposure through the bidirectional control of localized hippocampal ERK 1–2 phosphorylation states, mesolimbic DA activity levels and associated oscillatory wave patterns.