*ENDOCANNABINOID SYSTEM AND HUMAN REPRODUCTION

THE ENDOCANNABINOID SYSTEM AND HUMAN REPRODUCTION

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INTRODUCTION

Humans have used Cannabis sativa (marijuana) for at least 12,000 years, but researchers have only recently described an endogenous cannabinoid system. Our body’s own endocannabinoid system modulates an array of critical physiological and psychological functions, including human reproduction. Endocannabinoids are widely distributed throughout the body.

Investigations into the biological basis of the multiple effects of cannabis have led to important scientific breakthroughs in recent years. Exogenous cannabinoids, those consumed from sources external to the body, have been shown to significantly alter neuroendocrine output.   Knowledge of this fact led to emergent knowledge of endogenous cannabinoids (produced by our body) as being important signaling molecules in the neuroendocrine control of homeostatic and reproductive functions, including the stress response, energy metabolism and gonadal regulation.  While we share components of an endocannabinoid system with many other organisms, ours is complex and governs critical aspects of our existence, beginning with our conception.

The endocannabinoid system is our first attachment system.   Proper functioning of our mother’s endocannabinoid system was critical to our being alive in the first place, and proper functioning or our own endocannabinoid system is critical to our continued survival.  This system, which exists in the central nervous system and brain, as well as in peripheral organ systems, is comprised of two cannabinoid receptors (CB1 and CB2), endogenous ligands called ‘endocannabinoids‘ and the specific enzymes that metabolize them, as well as transporter molecules.

The endocannabinoid system is present in mammalian reproductive organs including the testis, epididymis, prostate, ovary, uterus, sperm, all stages of the preimplantation fertilized egg, embryo and placenta, as well as in prostatic and mammary carcinomas.  This system is also present in human seminal plasma, mid-cycle oviductal fluid, follicular fluid, amniotic fluid, breast milk, and fluids from malignant ovarian cysts.  Endocannabinoid system signaling via cannabinoid receptors regulates proliferation of human mammary and prostatic carcinomas.

This system modulates follicular maturation of the egg, ovulation, normal and pathological ovarian function, placental and fetal physiology, lactation, infant physiology, and behavior.  Its presence in human reproductive fluids helps regulate multiple physiological and pathological processes in the reproductive system, and implies that exogenous cannabinoids delivered by marijuana smoke might impact these processes.

The endocannabinoid system is in operation from the earliest stages of embryonic development as it governs sperm fertility, fertilization of the egg, and implantation on the uterine wall.  It is through the essential influence and operation of this system that gestation can be successfully accomplished, as well.  It fulfills a multitude of vital roles that allows a forming organism to complete distinct pre- and postnatal development stages:

1.  Fertility of the sperm

2.  Fertilization of the egg

3.  Implantation of the embryo on the uterine wall

4.  Prenatal brain and central nervous system development

5.  Protection of brain cells including protection of the developing brain from trauma-induced neuron cell loss

4. Initiation of postnatal suckling in the newborn (working in the neonatal brain and critical for survival).

5. In addition, subtle but definite deficiencies have been described in memory, motor and addictive behaviors and in higher cognitive (‘executive’) function in the human offspring as result of prenatal exposure to marijuana.  Therefore, the endocanabinoid-CB1 receptor system is suspected to play a role in the development of structures which control these functions

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The most used ‘recreational’ cannabinoid drug, marijuana [active psychoactive component (-)delta9tetrahydrocannabinol [(-)delta9THC], has always been identified as harmful because of its negative effect on male and female reproduction. Male and female marijuana smokers show impaired fertility, owing to defective signaling pathways, aberrant hormonal regulation, or wrong timing during embryo implantation.

There is an interplay between our endocannabinoid system, sex hormones and cytokines which is a critical array involved in the control of human reproduction.  Cytokines are a category of signaling molecules that are used extensively in cellular communication. They are proteins, peptides, or glycoproteins. The term cytokine encompasses a large and diverse family of polypeptide regulators that are produced widely throughout the body by cells of diverse embryological origin.

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Signaling by the endocannabinoid system is significant in multiple reproductive events

Spermatogenesis:  The process by which male spermatogonia develop into mature spermatozoa, the mature male gametes .  Spermatogenesis is the male version of gametogenesis. In mammals it occurs in the male testes and epididymis.   Spermatogenesis is highly dependent upon optimal conditions for the process to occur correctly, and is essential for sexual reproduction.

Sertoli cell survival:  Because its main function is to nurture the developing sperm cells through the stages of spermatogenesis, the Sertoli cell has also been called the “mother” or “nurse” cell. Sertoli cells also act as white blood cell phagocytes, consuming the residual cytoplasm, the part of a cell that is enclosed within the plasma membrane, during spermatogenesis.

Placentation:  The formation, type and structure, and arrangement of the placenta. The function of placentation is to transfers nutrients from maternal tissue to a growing embryo.

Fertilization:  Or conception, is the fusion of gametes to produce a new organism. In animals, the process involves a sperm fusing with an ovum.

Preimplantation embryo development:   The preimplantation uterus contains the highest level of the endocannabinoid, anandamide (AEA), yet discovered in a mammalian tissue. Various aspects of endocannabinoid signaling in preimplantation embryo development and activation, and uterine differentiation during the preimplantation embryo-uterine dialog must happen before a pregnancy can continue.  One of the places that this endocannabinoid Anandamide (AEA) is synthesized is in the female reproductive tracts.  It acts on cannabinoid receptors expressed on the cell surface of the embryo to regulate the preimplantation embryo development and implantation.  Levels of uterine anandamide and blastocyst CB1 cannabinoid receptors are coordinately regulated to synchronize preimplantation development and uterine receptivity for implantation in mice.

Mammalian fertility absolutely depends on synchronized development of the blastocyst to the stage when it is competent to implant, and the uterus to the stage when it is receptive to implantation. There is a molecular basis for the reciprocal interaction between the embryo and the uterus.  Cannabinoid receptor, CB1, that is expressed at high levels on the surface of the trophectoderm (newly differentiated cells from the egg) and anandamide (AEA), an endocannabinoid ligand found to be produced at higher levels by the uterus before implantation and then down-regulated at the time of implantation, play a critical role in preimplantation events.

Anandamide (AEA), within a very narrow range, regulates blastocyst function and implantation by differentially modulating signaling activity via CB1 receptors.  This delicate signaling process renders the blastocyst competent for implantation in the receptive uterus.  In contrast, anandamide at a higher concentration inhibits blastocyst competency for implantation.  Besides uncovering a potentially important regulatory mechanism for synchronizing blastocyst and uterine competency to implantation, this observation has high clinical relevance, because elevated levels of anandamide induce spontaneous pregnancy loss in women.

The development of the embryo is called embryogenesis. The blastocyst is the structure formed in early embryogenesis, after the formation of the blastocoel, but before implantation.  The human blastocyst comprises 70-100 cells and is preceded by a zygote, the fertilized egg cell.  It possesses an inner cell mass, or embryoblast, which subsequently forms the placenta proper, and an outer layer of cells, or trophoblast which later forms the embryo.

Anandamide (AEA) of the endocannabinoid system appears to be an important neuro-cytokine mediator synchronizing the embryo-endometrial development as it is prepared for timed implantation on the uterine wall.   The development of the embryo into the blastocyst and transport of the embryo across the fallopian tubes is under the influence of this system.   Embryo retention in the Fallopian tube (FT) is thought to lead to ectopic pregnancy (EP), a considerable cause of morbidity.  Research is showing a possible genetic link to alterations in the endocannabinoid CB1 receptor that causes changes in the way the endocannabinoid system usually moderates the embryo’s transition to its implantation site as one of the causes of this complication.

There is a differential concentration-dependent effect of cannabinoids on the trophoblast, [trophoblast  — the layer of extraembryonic ectoderm that chiefly nourishes the embryo — mediate the implantation of the embryo into the endometrium, but they are never incorporated into the mother’s body or the fetus] with an observed inhibition of differentiation at higher doses.

Implantation:  The significance of cannabinoid/endocannabinoid signaling in early pregnancy includes endocannabinoid signaling that directs periimplantation events.  Recent research has shown that cannabinoid effects are differentially executed depending on the embryonic stage and cannabinoid levels in the environment.  Since uterine anandamide levels are lowest at the sites of implantation and highest at the interimplantation sites, the new findings imply that site-specific levels of anandamide and/or other endogenous ligands in the uterus may regulate implantation spatially by promoting trophoblast differentiation at the sites of blastocyst implantation.

If conditions, including the condition of the endocannabinoid system, are good 5-7 days after fertilization the blastula, or blastosphere, adheres, or attaches to the wall of the uterus.  Endocannabinoids signal the blastocyst to tell it exactly where on the uterine wall to attach itself.  There is only a specific period of time during which implantation is possible, called the “implantation window”.  If implantation does not occur during this window, it signifies that something is wrong. There is massive communication at this stage between the blastocyst and the endometrium, which is the inner membrane of the uterus. The blastocyst signals to the endometrium to adapt further to its presence.

Disturbed regulation of anandamide (AEA) signaling via the CB1 receptor may be associated with pregnancy failure. AEA could lower the quality of blastocysts by inducing apoptosis and inhibiting cell proliferation, thus making them incompetent for implantation.  Preimplantation embryo development to the blastocyst stage and uterine differentiation to the receptive state are prerequisites for embryo implantation.   Endocannabinoid signaling is critical to these early pregnancy events.   Anandamide (AEA) (N-arachidonoylethanolamine) and 2-AG (2-arachidonoylglycerol) are two major endocannabinoids that bind to and activate G-protein coupled cannabinoid receptors CB1 and CB2.

A physiological tone of anandamide is critical to preimplantation events in mice, since either silencing or amplification of anandamide signaling causes retarded development and oviductal retention of embryos via CB1, leading to deferred implantation and compromised pregnancy outcome. Region- and stage-specific uterine expression of anandamide and 2-AG, creates endocannabinoid gradients conducive to implantation.  Aberrant functioning of these pathways impacting uterine anandamide and/or 2-AG levels would compromise pregnancy outcome.

Endocannabinoids such as anandamide can have adverse effects on pregnancy and embryonic development.  It is essential that their presence be adequately regulated by a good-working endocannabinoid system.  The activity of the degradative enzyme anandamide hydrolase may therefore be crucial for prevention of excessive concentrations of anandamide in the uterus, and thus prevention of pregnancy failure or female infertility.  Decreased anandamide hydrolase activity and expression in peripheral lymphocytes is an early (<8 weeks of gestation) marker of spontaneous abortion, and may prove useful as a diagnostic tool for large-scale, routine monitoring of gestation.

postimplantation embryonic growth:  About eight weeks after fertilization an embryo is then called a fetus.

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Labor and birthing are also affected by the endocannabinoid system.  The CB1 receptor regulates labor by interacting with the corticotrophin-releasing hormone-driven endocrine axis.  Abnormalities of this receptor system contribute to pre-term births.

Lactation is also influenced by the endocannabinoid system.  Endocannabinoids are present in breast milk where they help stimulate postnatal appetite.   The presence of the endocannabinoid 2-arachidonoyl glycerol and CB1 receptors have been detected in human milk which gives this system a critical role in oral motor control of suckling during neonatal development – probably through an effect on tongue muscles.

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Anandamide, also known as N-arachidonoylethanolamine or AEA, is an endogenous cannabinoid neurotransmitter found in animal and human organs. It is a lipid-signal molecule that naturally works with this system’s receptors.  AEA is released through enzyme actions from membrane phospholipids when neurons are stimulated.  Retrograde AEA signals from depolarized postsynaptic neurons inhibit neurotransmitter release at synapses.

Anandamide’s effects can be either central, in the brain, or peripheral, in other parts of the body. These distinct effects are mediated primarily by CB1 cannabinoid receptors in the central nervous system, and CB2 cannabinoid receptors (mainly involved in functions of the immune system) in the periphery.

Anandamide is produced in the ovary where it is under hormonal control and plays a role in:

Folliculogenesis:  Is the maturation of the ovarian follicle, a densely-packed shell of somatic cells that contains an immature oocyte. Folliculogenesis describes the progression of a number of small primordial follicles into large preovulatory follicles that enter the menstrual cycle.  Contrary to male spermatogenesis which can last indefinitely, folliculogenesis ends when the remaining follicles in the ovaries are incapable of responding to the hormonal cues that previously recruited some follicles to mature. This depletion in follicle supply signals the beginning of the menopause.  In a larger perspective, the whole folliculogenesis from primordial to preovulatory follicle is located in the stage of meiosis one of ootidogenesis in oogenesis.  The embryonic development doesn’t differ from the male one, but follows the common path before gametogenesis.   For each primary oocyte that undergoes meiosis, only one functional oocyte is produced. The other two or three cells produced are called polar bodies. Polar bodies have no function and eventually deteriorate.  The primary oocyte turns into a secondary oocyte in mature ovarian follicles. Unlike the sperm, the egg is arrested in the secondary stage of meiosis until fertilization.  Upon fertilization by sperm, the secondary oocyte continues the second part of meiosis and becomes a zygote.

Preovulatory follicle maturation:  Primordial follicles are indiscernible to the naked eye. However, they develop to primary, secondary and finally mature vesicular follicles.  In humans, oocytes are established in the ovary before birth and may lie dormant awaiting initiation for up to 50 years.  Ovarian follicle is the basic unit of female reproductive biology and is composed of roughly spherical aggregations of cells found in the ovary. They contain a single oocyte (aka ovum or egg). These structures are periodically initiated to grow and develop, culminating in ovulation of usually a single competent oocyte.

Oocyte maturity:  Female eggs are not mature until the proceed through a maturation process.  An oocyte (pronounced oh’a-site), ovocyte, or rarely ocyte, is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is part of the ovary development. The germ cells produce a primordial germ cell (PGC) which becomes an oogonium which marks the start of mitosis. After mitosis stops (due to actions of retinoic acid and the mesenephros) meiosis starts. This stage the oogonia is now an Oocyte.

OvulationOvulation is the process in the females menstrual cycle by which a mature ovarian follicle ruptures and discharges an ovum (also known as an oocyte, female gamete, or casually, an egg) that participates in reproduction.

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ESTROGEN AND THE ENDOCANNABINOID SYSTEM

As an example of how systems operating within the human body are interconnected and related – as well as an example of how they affect us —  it is now known that the fatty acid amide hydrolase (FAAH), which is the enzyme that degrades the endocannabinoid anandamide (AEA), has been shown to be regulated by estrogen.  Researchers have examined the anxiolytic (relieves symptoms of anxiety) and antidepressant effects of estrogen because of its involvement with the endocannabinoid system.  Their collective research findings indicate that estrogen may elicit changes in emotional behavior through an endocannabinoid mechanism.  This finding led them to further examine how FAAH might represent a therapeutic target for anxiety and depression in women.

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Cannabinoids inhibit glutamatergic and GABAergic synaptic input onto neurons of the hypothalamic arcuate nucleus (ARC).  Estrogen modulates this process.  GABA is the chief inhibitory neurotransmitter in the mammalian central nervous system. It plays an important role in regulating neuronal excitability throughout the nervous system. In humans, GABA is also directly responsible for the regulation of muscle tone.

Hypothalamic arcuate nucleus (ARC) neurons, including POMC neurons, receive glutamatergic and GABAergic synaptic inputs that are presynaptically inhibited by cannabinoids, and differentially modulated by estrogen. These opposing effects of estrogen on the cannabinoid regulation of amino acid neurotransmission excite POMC neurons, and lend additional insight into the mechanisms underlying estrogen-induced anorexia and negative feedback of the reproductive axis.

Pro-opiomelanocortin (POMC) is a precursor polypeptide.  This gene encodes a polypeptide hormone precursor that encodes proteins synthesized mainly in cells of the anterior pituitary.  In other tissues POMC gives rise to peptides with roles in pain and energy homeostasis, melanocyte stimulation, and immune modulation

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AN EXAMPLE OF ENDOCANNABINOID-CONTROLLED REPRODUCTIVE RHYTHYM AND TIMING

Gonadotropin-releasing hormone (GnRH), also known as Luteinizing-hormone releasing hormone (LHRH), is a key reproductive neurohormone discovered in the brain in 1977.  GNRH secretion and action in the pituitary gland is regulated by our internal cannabinoid system.

GNRH activity is very low during childhood and is activated at puberty. During the reproductive years, pulse activity is critical for successful reproductive function as controlled by sensitive feedback loops.  At the pituitary, GNRH stimulates the synthesis and secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). These processes are controlled by the size and frequency of GNRH pulses, as well as by feedback from androgens and estrogens. Low frequency GNRH pulses lead to FSH release, whereas high frequency GNRH pulses stimulate LH release.

GNRH secretion is pulsatile in all vertebrates, and is necessary for correct reproductive function. There are differences in GNRH secretion between females and males:  In males, GNRH is secreted in pulses at a constant frequency, but in females the frequency of the pulses varies during the menstrual cycle with a large surge of GNRH occurring just before ovulation.  This single hormone, GNRH1, signaled by the endocannabinoid system, controls a complex process of follicular growth, ovulation, and corpus luteum maintenance in the female, and spermatogenesis in the male.

Exogenous cannabinoids substances consumed from outside the body, interfere with the body’s natural processes because they exert negative effects on reproduction by reducing LH secretion and suppressing gonadal function.  These effects are due primarily to hypothalamic dysfunction.  Hypothalamic GnRH neurons produce and secrete at least two different endocannabinoids and possess CB1 and CB2 receptors that are coupled to proteins whose activation leads to inhibition of GnRH secretion.

Endocannabinoids, our own naturally occurring endogenous lipid-signaling molecules, are intended to bind to CB receptors on neighboring cells, and perhaps GnRH neurons, to exert feedback control over GnRH function. This network appears to serve as a novel mechanism for regulating GnRH secretion where critically important reproductive functions as diverse as the onset of puberty, timing of ovulation, duration of lactational infertility, and initiation/persistence of menopause are affected.

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Our own endocannabinoid system, particularly anandamide (AEA), has an important role not only in regulating female fertility, but also in controlling sperm function and male fertility.  Sperm cells have a complete and efficient endocannabinoid system.  They contain and express cannabinoid receptors for neurotransmitters that are also affected by psychoactive drugs. Activation of these receptors controls, at different time-points, sperm functions required for fertilization.   Anandamide (AEA) signaling regulates sperm functions required for fertilization in human reproductive tracts, a fact that implies that the smoking of marijuana can impact these processes.

Anandamide (AEA) signaling directly affects sperm functions required for fertilization and provides additional evidence for common signaling processes in neurons and sperm. It is now known that AEA is present in human seminal plasma, midcycle oviductal fluid, and follicular fluid.  Sperm are sequentially exposed to these reproductive fluids as they move from the vagina to the site of fertilization in the oviduct. 

Ejaculated mammalian sperm require several hours exposure to secretions in female reproductive tracts, or incubation in appropriate culture medium in vitro, before acquiring the capacity to fertilize eggs.

In addition, research on animals is suggesting that unfertilized eggs have enzymes required to release anandamide (AEA) from membrane phospholipids.  This process might be activated by the fertilizing sperm so that the release of anandamide (AEA) may then react with the endocannabinoid receptors in nearby sperm to block them from entering the egg after it has already been fertilized   This prevents what is known as polyspermy from occurring.

Cannabinoids (the main active components of marijuana) have been shown to exert different adverse effects on male reproduction both in vertebrates and invertebrates.  They exert negative effects on hypothalamic-hypophyseal reproductive hormone secretion and testicular endocrine and exocrine functions.   In research with rats a connection has been found between the cannabinoid CB1 receptor, oxytocin and penile erection.  The presence of anandamide (AEA) at the cannabinoid CB1 receptor creates a relaxation response because it blocks oxytocinergic neurons that mediate penile erection.

A large amount of experimental data have clearly shown that cannabinoids negatively influence important sperm functions, including motility, capacitation and acrosome reaction, three fundamental processes necessary for oocyte penetration.  These inhibitory effects, which lead to the inhibition of sperm fertilizing ability, are mediated by the direct action of cannabinoids on sperm through the activation of the cannabinoid receptor subtype CNR1  that has been shown to be expressed in mature sperm.

The consumption of externally supplied cannabinoids is known to affect sperm motility by interrupting the ability of sperm to move properly toward an egg.  It is known that insulin is an autocrine factor that affects this capacity through involvement of the endocannabinoid system which is involved in the signaling cascade that induces the process of controlling sperm energy homeostasis.

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CANNABIS USE DURING PREGNANCY

The consumption by women of cannabis derivatives during pregnancy and/or lactation affects the development of their offspring.  Like other psychoactive drugs, cannabinoids including those that contain the psychoactive ingredients of marijuana, can cross the placental barrier and can be secreted into the maternal milk.

Cannabis is widely abused by women at reproductive age and during pregnancy.  Animal studies show a particular vulnerability of the developing brain to prenatal chronic cannabinoid administration, and show that prenatal exposure to cannabinoids can induce a long-term alteration of endocannabinoid system in brain areas involved in learning-memory, motor activity and emotional behavior.  Subtle but definite deficiencies have been described in memory, motor and addictive behaviors and in higher cognitive (‘executive’) function in the human offspring as result of prenatal exposure to marijuana.

Endocannabinoid CB1 receptor system (not the CB2 receptor) may play a role in the development of structures which control these functions, including the nigrostriatal pathway and the prefrontal cortex.  Cannabinoids are also known to affect the expression of key genes for neural developmental leading to neurotransmitter and behavioral disturbances particularly in males.

Manipulating the endocannabinoid system by pre- and/or postnatal administration of cannabinoids or maternal marijuana consumption has significant, yet subtle effects on the offspring.  Alterations in the dopamine, GABA and endocannabinoid systems have been reported in offspring, while enhanced drug seeking behaviour and impaired executive (prefrontal cortical) function have also been observed.  The relatively mild nature of the disruptive effects of prenatal cannabinoids may be understood in the framework of the intricate timing requirements and frequently biphasic effects of the (endo)cannabinoids.

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Research studies have also been done to explore the influence of prenatal cannabinoid exposure on the gene expression of a key protein for brain development, the neural adhesion molecule L1.  Activation of cannabinoid receptors increases the levels of the full-length L1 and alters those of some active proteolytic fragments of this protein whose generation has been associated with specific steps in the process of neuritic elongation in cultured neurons.

L1 plays an important role in processes of cell proliferation and migration, neuritic elongation and guidance, and synaptogenesis in the developing brain. Results confirmed that the levels of L1 transcripts were significantly increased after prenatal delta9-THC exposure in several regions such as the fimbria, stria terminalis, stria medullaris and corpus callosum, which share the properties of being white matter regions and containing, exclusively during development, an abundant population of cannabinoid CB1 receptors, which are also the major targets for the action of plant-derived cannabinoids.

L1-mRNA levels were also increased in grey matter structures such as the septum nuclei and the habenula, but remained unchanged in most of the grey matter structures analyzed (cerebral cortex, basolateral amygdaloid nucleus, hippocampus, thalamic and hypothalamic nuclei, basal ganglia and subventricular zones) and also in a few white matter structures (fornix and fasciculus retroflexus).

An important aspect of these observations is that the increase in L1-mRNA levels reached statistical significance only in the case of delta9-THC-exposed males but not in the case of delta9-THC-exposed females where only trends or no effects were detected.  This supports previous evidence on a sexual dimorphism, with greater effects in male fetuses, for the action of cannabinoids in the developing brain.

Cannabinoids thus seem to influence the expression of L1 in specific brain structures during the prenatal period, which, considering the role played by this protein in different events related to neural development, might explain the neurotransmitter and behavioral disturbances reported after prenatal consumption of marijuana.

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BRAIN DEVELOPMENT

“Wiring and firing neuronal networks — endocannabinoids take center stage”

The endocannabinoid system has recently been shown to play an important role in brain development.  Endocannabinoids regulate a broad range of physiological functions in the postnatal brain and are implicated in the neuropathogenesis of psychiatric and metabolic diseases.  Accumulating evidence indicates that endocannabinoid signaling also serves key functions during neurodevelopment, and is inherently involved in the control of neural progenitor proliferation, lineage segregation, and the migration and phenotypic specification of immature neurons.

The spatiotemporal expression of cannabinoid receptors and endocannabinoid-metabolizing enzymes during brain development guides major developmental processes including neurogenesis, cell differentiation, cell migration, neuronal specification and synaptogenesis.  Expression of endocannabinoid components plays an important physiological role in fine-tuning neurotransmission.

Furthermore, pharmacological experiments and transgenic animal models have shown the impact that disrupted endocannabinoid signaling has on normal brain development.   This research reveals the danger of both cannabis abuse and exposure to cannabinoid drugs during embryonic development, childhood and adolescence.

Recent advances in developmental biology define fundamental endocannabinoid-driven cellular mechanisms that also contribute to our understanding of the molecular substrates of prenatal drug, in particular cannabis, actions. There are organizing principles of endocannabinoid signaling systems in the developing cerebrum which have a sequence of decision points and underlying signaling pathways upon CB1 cannabinoid receptor activation that contribute to neuronal diversification in the developing brain. These novel principles critically affect the formation of complex neuronal networks in the brain.

Actions of cannabinoid receptors in the developing human brain have also been identified based on the detection of early appearing elements of the endogenous cannabinoid system (receptors and ligands) during the brain’s development. The “atypically” distributed location of CB1 receptors, which display a transient presence in white matter areas of the pre- and postnatal nervous system in several neural pathways of the fetal brain, suggests that this system may play a role in specific molecular events related to neural development.

Animal research indicates that the endocannabinoid-CB1 receptor (‘ECBR’) system fulfills a number of roles in the developing organism.  It regulates neural progenitor differentiation into neurones and glia and guiding axonal migration and synaptogenesis.  Cannabinoid CB1 receptors and their ligands emerge in early fetal ages during neural development and are thought to play a major role in brain development.   They are abundantly expressed in certain brain regions that play key roles in processes related to cell proliferation and migration, neuritic elongation and guidance, and synaptogenesis.

This fact supports the notion that the cannabinoid system might play a modulatory role in the regulation of these processes.   This modulatory action might be exerted, among others, through regulating the levels of several key elements in these processes, such as the L1 protein. Considering the role played by the regulating the synthesis of the cell adhesion molecule L1 in different events related to neural development, scientific observations support the occurrence of a physiological mechanism by which the cannabinoid system might regulate these processes.

This action has been observed in various white matter areas of the fetal rat forebrain.  The cannabinoid system modulates the levels of L1 in these several brain structures during specific periods of development (late gestation) and very early postnatal days, which correlates with the periods in which an atypical distribution of CB1 receptors can be found in the developing brain. However, the magnitude of the effects of cannabinoids on L1 appears to be influenced by two factors: gender and age of development.  CB1 receptors in forebrain white matter regions during fetal rat brain development also provide evidence for a role of these receptors in axonal growth and guidance.

Endocannabinoid signaling controls pyramidal cell specification and long-range axon patterning, but has only recently been identified as axon guidance cues that shape the connectivity of local GABAergic interneurons in the developing cerebrum.  Endocannabinoid functions during pyramidal cell specification and establishment of long-range axonal connections have only recently been discovered, as well.

Research shows that endocannabinoid signaling is operational in subcortical proliferative zones from embryonic day 12 in the mouse telencephalon and controls the proliferation of pyramidal cell progenitors and radial migration of immature pyramidal cells.   When layer patterning is accomplished, developing pyramidal cells rely on endocannabinoid signaling to initiate the elongation and fasciculation of their long-range axons.   Endocannabinoids are fundamental developmental cues controlling pyramidal cell development during corticogenesis.

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ENDOCANNABINOID NEUROPROTECTION

The fact that exogenous and endogenous cannabinoids have potential neuroprotective agents in a number of neurodegenerative disorders of the adult has led to new research on the neuroprotective actions the endocannabinoid system might have during prenatal brain developmental stages.  It has been found that anandamide (AEA) has neuroprotectant properties in the developing pre- and post-natal brain and can prevent damage due to traumas.  Neuroprotective effects of AEA in white matter involve increased survival of preoligodendrocytes and better preservation of myelination.

Experimental evidence supports the role of endocannabinoids as a candidate therapy for excitotoxic perinatal brain lesions.  The neuroprotective effects of the endogenous cannabinoid anandamide (AEA) were found in studies that used a well-defined rodent model of neonatal excitotoxic brain lesions, showing that AEA provides dose-dependent and long-lasting protection of developing white matter and cortical plate.

The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol, are important retrograde messengers that inhibit neurotransmitter release via presynaptic CB1 receptors. In addition, cannabinoids are known to modulate the cell death/survival decision of different neural cell types, leading to different outcomes that depend on the nature of the target cell and its proliferative/differentiation status. Thus, cannabinoids protect primary neurons, astrocytes and oligodendrocytes from apoptosis, whereas transformed glial cells are prone to apoptosis by cannabinoid challenge.

Moreover, a potential role of the endocannabinoid system in neurogenesis and neural differentiation has been proposed. Recent research shows that endocannabinoids stimulate neural progenitor proliferation and inhibit hippocampal neurogenesis in normal adult brain. Cannabinoids inhibit cortical neuron differentiation and promote glial differentiation. On the other hand, experiments with differentiated neurons have shown that cannabinoids also regulate neuritogenesis, axonal growth and synaptogenesis. These new observations support findings that endocannabinoids constitute a newly identified family of lipid signaling cues responsible for the regulation of neural progenitor proliferation and differentiation, acting as instructive proliferative signals through the CB1 receptor.

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FURTHER CONSIDERATIONS ABOUT ENDOCANNABINOIDS AND NEURO DEVELOPMENT

Adult rats submitted to a single prolonged episode of maternal deprivation show behavioral alterations that resemble specific symptoms of schizophrenia. Accordingly, this experimental procedure has been proposed as an animal model of schizophrenia based on the neurodevelopmental hypothesis.  Researchers have recently reported that maternal deprivation induced sex-dependent alterations in the hippocampus of neonatal young rats.

In view of recent evidence for important implications of the cerebellum in neurodevelopmental psychiatric diseases, possible degenerative changes in the cerebellar cortex have been suggested.   In a rat study, maternal deprivation MD induced significant increases in the number of cells that indicated degenerating neurons as well as other changes, but only in males.

The two cannabinoid compounds reversed or lessened these effects. These results provide new insights regarding the psychopathological implications of the cerebellum, the role of the endocannabinoid system in neural development, and the possible neurodevelopmental basis of gender differences in schizophrenia.  It appears that cannabinoids are dysregulated in a number of psychiatric disorders and might be involved in their pathogenesis. There is now evidence that manipulation of the endocannabinoid system could be a therapeutic target for a variety of conditions

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CENTRAL NERVOUS SYSTEM (CNS) DEVELOPMENT

Emerging information describes the functions of endocannabinoid signaling during central nervous system (CNS) (of which the brain is a part) development.  In the postnatal brain, endocannabinoids act as retrograde messengers that regulate the function of many synapses.  Increasing the knowledge of basic developmental and signaling principles that are controlled by endocannabinoids will provide important insights into the molecular mechanisms that establish functional neuronal circuits in the brain.  By contrast, the understanding of endocannabinoid functions that regulate fundamental developmental processes such as cell proliferation, migration, differentiation and survival  during patterning of the CNS is just beginning to unfold.

The endocannabinoid system exerts important modulatory functions in the central nervous system (CNS), particularly the retrograde control of excitatory or inhibitory synapses, which enables this system to participate in the control of important neurobiological processes in healthy conditions.

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ENDOCANNABINOID IMPLICATIONS FOR IMMUNITY

Cell-cell communication by endocannabinoids during immune surveillance of the central nervous system

The immune system is designed to defend the organism from hazardous infection. The way by which cells of the immune system perform this function can be dangerous for the survival and function of the neuronal network in the brain. An attack of immune cells inside the brain includes the potential for severe neuronal damage or cell death and therefore impairment of central nervous system (CNS) function. To avoid such undesirable action of the immune system, the CNS harbors an impressive arsenal of cellular and molecular mechanisms enabling strict control of immune reactions–the so-called “immune privilege”.

Under inflammatory and pathological conditions, loss of control of the central nervous system (CNS) immune system results in the activation of neuronal damage cascades frequently associated with neurological disease. On the other hand, processes of neuroprotection and neurorepair after neuronal damage depend on a steady and tightly controlled immune surveillance. Accordingly, the immune system serves a highly specialized function in the CNS including negative feedback mechanisms that control immune reactions.

Recent studies have revealed that endocannabinoids participate in one of the most important ones of the brain’s negative feedback system. The central nervous system (CNS) endocannabinoid system participates crucially in neuronal cell-cell-communication and signal transduction, e.g., by modulating synaptic input and protecting neurons from excitotoxic damage. Over the last decade, it has also become evident that endocannabinoids play an important role in the communication between immune cells and in the interaction between nerve and immune system during CNS damage. Thus, therapeutic intervention in the CNS endocannabinoid system may help to restore the well-controlled and finely tuned balance of immune reactions in pathological conditions.

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Endocannabinoids not only help to maintain neuroendocrine homeostasis, but also take part in immunological changes occurring during early pregnancy.  Endocannabinoids inhibit the release of leukaemia inhibitory factor (LIF) from peripheral T lymphocytes.  Research also suggests that endocannabinoids might be critical in regulating the lymphocyte-dependent cytokine network associated with human fertility and successful pregnancy.  During human gestation, the interplay between the endocannabinoid system and the hormone-cytokine array involved in the control of human pregnancy, shows that the endocannabinoids take part in the immunological adaptation occurring during early pregnancy.

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A clinical study performed on 100 healthy women showed that a low FAAH activity in lymphocytes correlates with spontaneous abortion, whereas anandamide (AEA) transporter and cannabinoid receptors in these cells remain unchanged. These results add the endocannabinoids to the hormone-cytokine array involved in the control of human pregnancy.

Physiological concentrations of progesterone stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human lymphocytes.  An inverse association has been reported between fatty acid amide hydrolase (the anandamide degrading enzyme) in human lymphocytes and miscarriage.  Stimulation of FAAH occurs through up-regulation of gene expression at the transcriptional and translational level, and is partly mediated by the Th2 cytokines.

There is potential relevance of the immunological activity of CB2 receptors at the maternal/fetal interface.  CB2 receptors are found in the uterine wall, and play an important role in pregnancy.  CB2 receptors are about 100 times as common here as CB1 receptors are in the brain, and concentrations are just as high in a developing embryo.  Anandamide (AEA) provides synchronization between the embryo and the uterus by slowing down the embryo’s development until the uterine wall is able to be implanted upon.  AEA may also take part in embryo selection, determining if an embryo will implant — and thus be viable — by whether it has the right concentrations of CB2 receptors.  AEA also prevents one embryo from implanting too close to another, as AEA levels near an implanted embryo fall sharply.

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In recent years, it has been demonstrated that high circulating levels of the endogenous cannabinoid anandamide (AEA) , resulting from low expression of its metabolizing enzyme fatty acid amide hydrolase (FAAH), may contribute to spontaneous miscarriage and poor outcome in women undergoing in vitro fertilization. The site of action of this compound, however, had not been determined until a study examined the distribution of the cannabinoid receptors, CB1 and CB2, and the endocannabinoid-metabolizing enzyme FAAH in first trimester human placenta. Researchers found that FAAH is expressed throughout the human first trimester placenta, in extravillous trophoblast columns, villous cytotrophoblasts, syncytiotrophoblasts (outer syncytial layer of the trophoblasts and actively invade the uterine wall during implantation) , and macrophages (white blood cells).

Furthermore, FAAH mRNA levels appear to be regulated during gestation, with levels peaking at 11 wk before declining again. The immune system-associated cannabinoid CB2 receptors were found to be localized only to placental macrophages.  It is likely that CB2 receptors are involved in normal immune responses during early human pregnancy.  Interestingly, the cannabinoid receptor CB1 was not identified in first trimester placenta despite having previously been shown to be present in placental tissues at term. These findings suggest that the placenta may form a barrier preventing maternal-fetal transfer of anandamide and/or modulate local levels of anandamide by regulation of FAAH expression with gestation.

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MEDICINAL USES OF CANNABINOID SUBSTANCES FOR CHILDREN

Research findings about our endocannabinoid system have potential medical and public policy ramifications related to the incidence of marijuana abuse by youth and adults in our society.   The massive amount of scientific information about the critical involvement of this system as it is interfered with by cannabis use must be considered in any consideration of the legalization of the substance, as well.  The documented reproductive effects of marijuana are among the most important concerns in the ongoing debate about medicinal use of marijuana and cannabinoids. The endocannabinoid system remains a new target for correcting in infertility in both men and women, and for improving reproductive health in humans.

Children may respond positively to medicinal applications of cannabinoids without undesirable central nervous effects.   Developmental observations have shown that CB1 receptors develop only gradually during the postnatal period, which correlates with an insensitivity to the psychoactive side effects of cannabinoid and THC treatment in the young.

Excellent clinical results have been reported in pediatric oncology and in case studies of children with severe neurological disease or brain trauma.  Cannabinoid treatment for children or young adults with cystic fibrosis is also recommended in order to achieve an improvement of their health condition including improved food intake and reduced inflammatory exacerbations.

Researchers suggest that a dysfunctional endocannabinoid CB1 receptor system may exist in infants with growth failure resulting from an inability to ingest food, which may resolve the enigma of “non-organic failure-to-thrive” (NOFTT).

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Background research articles for this essay are posted here:

*Endocannabinoids – Fertility, pregnancy, lactation, infants and children

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