Ken Mackie

Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system.

Immunohistochemical distribution of cannabinoid receptors in the adult rat brain was studied using specific purified antibodies against the amino-terminus of the CB1 receptor. Our results generally agree well with the previous studies using CB1 receptor autoradiography and messenger RNA in situ hybridization. However, because of its greater resolution, immunohistochemistry allowed identification of particular neuronal cells and fibers that possess cannabinoid receptors. CB1-like immunoreactivity was found in axons, cell bodies and dendrites, where it appeared as puncta in somata and processes. Both intensely and moderately or lightly stained neurons were observed. The intensely stained neurons were dispersed and only occur in cortical structures including hippocampal formation and olfactory bulb. Moderately or lightly stained neurons were found in caudate-putamen and amygdala. In the hippocampal formation only intensely stained neurons were observed. The cell bodies of pyramidal neurons in CA1 and CA3 fields appeared to be unstained but surrounded by a dense plexus of immunoreactive fibers. The granule cells in the dentate area were also immunonegative. Many intensely stained neurons were located at the base of the granule cell layer. CB1-like immunoreactive neurons and fibers were also found in the somatosensory, cingulate, perirhinal, entorhinal and piriform cortices, in claustrum, amygdaloid nuclei, nucleus accumbens and septum. Beaded immunoreactive fibers were detected in periaqueductal gray, nucleus tractus solitarius, spinal trigeminal tract and nucleus, dorsal horn and lamina X of the spinal cord. A triangular cap-like mass of immunoreactivity was found to surround the basal part of the Purkinje cell body in the cerebellum. Only small, lightly stained cells were found in the molecular layer in the cerebellum close to the Purkinje cell layer. The CB1 receptor is widely distributed in the forebrain and has a more restricted distribution in the hindbrain and the spinal cord. It appears to be expressed on cell bodies, dendrites and axons. According to the location and morphology, many, but not all, CB1-like immunoreactive neurons appear to be GABAergic. Therefore, cannabinoids and cannabinoid receptors may play a role in modulating GABAergic neurons.

International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid Receptors and Their Ligands: Beyond CB1 and CB2

There are at least two types of cannabinoid receptors (CB1 and CB2). Ligands activating these G protein-coupled receptors (GPCRs) include the phytocannabinoid Δ9-tetrahydrocannabinol, numerous synthetic compounds, and endogenous compounds known as endocannabinoids. Cannabinoid receptor antagonists have also been developed. Some of these ligands activate or block one type of cannabinoid receptor more potently than the other type. This review summarizes current data indicating the extent to which cannabinoid receptor ligands undergo orthosteric or allosteric interactions with non-CB1, non-CB2 established GPCRs, deorphanized receptors such as GPR55, ligand-gated ion channels, transient receptor potential (TRP) channels, and other ion channels or peroxisome proliferator-activated nuclear receptors. From these data, it is clear that some ligands that interact similarly with CB1 and/or CB2 receptors are likely to display significantly different pharmacological profiles. The review also lists some criteria that any novel “CB3” cannabinoid receptor or channel should fulfil and concludes that these criteria are not currently met by any non-CB1, non-CB2 pharmacological receptor or channel. However, it does identify certain pharmacological targets that should be investigated further as potential CB3 receptors or channels. These include TRP vanilloid 1, which possibly functions as an ionotropic cannabinoid receptor under physiological and/or pathological conditions, and some deorphanized GPCRs. Also discussed are 1) the ability of CB1 receptors to form heteromeric complexes with certain other GPCRs, 2) phylogenetic relationships that exist between CB1/CB2 receptors and other GPCRs, 3) evidence for the existence of several as-yet-uncharacterized non-CB1, non-CB2 cannabinoid receptors; and 4) current cannabinoid receptor nomenclature.

Endocannabinoid activation at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity

Endogenous cannabinoids acting at CB(1) receptors stimulate appetite, and CB(1) antagonists show promise in the treatment of obesity. CB(1) (-/-) mice are resistant to diet-induced obesity even though their caloric intake is similar to that of wild-type mice, suggesting that endocannabinoids also regulate fat metabolism. Here, we investigated the possible role of endocannabinoids in the regulation of hepatic lipogenesis. Activation of CB(1) in mice increases the hepatic gene expression of the lipogenic transcription factor SREBP-1c and its targets acetyl-CoA carboxylase-1 and fatty acid synthase (FAS). Treatment with a CB(1) agonist also increases de novo fatty acid synthesis in the liver or in isolated hepatocytes, which express CB(1). High-fat diet increases hepatic levels of the endocannabinoid anandamide (arachidonoyl ethanolamide), CB(1) density, and basal rates of fatty acid synthesis, and the latter is reduced by CB(1) blockade. In the hypothalamus, where FAS inhibitors elicit anorexia, SREBP-1c and FAS expression are similarly affected by CB(1) ligands. We conclude that anandamide acting at hepatic CB(1) contributes to diet-induced obesity and that the FAS pathway may be a common molecular target for central appetitive and peripheral metabolic regulation.

Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations

Using a new antibody developed against the C‐terminus of the cannabinoid receptor (CB1), the immunostaining in the hippocampus revealed additional axon terminals relative to the pattern reported previously with an N‐terminus antibody. Due to a greater sensitivity of this antibody, a large proportion of boutons in the dendritic layers displaying symmetrical (GABAergic) synapses were also strongly immunoreactive for CB1 receptors, as were axon terminals of perisomatic inhibitory cells containing cholecystokinin. Asymmetrical (glutamatergic) synapses, however, were always negative for CB1. To investigate the effect of presynaptic CB1 receptor activation on hippocampal inhibition, we recorded inhibitory postsynaptic currents (IPSCs) from principal cells. Bath application of CB1 receptor agonists (WIN55,212‐2 and CP55,940) suppressed IPSCs evoked by local electrical stimulation, which could be prevented or reversed by the CB1 receptor antagonist SR141716A. Action potential‐driven IPSCs, evoked by pharmacological stimulation of a subset of interneurons, were also decreased by CB1 receptor activation. We also examined the effects of CB1 receptor agonists on Ca2+‐independent miniature IPSCs (mIPSC). Both agonists were without significant effect on the frequency or amplitude of mIPSCs. Synchronous gamma oscillations induced by kainic acid in the CA3 region of hippocampal slices were reversibly reduced in amplitude by the CB1 receptor agonist CP 55,940, which is consistent with an action on IPSCs. We used CB1–/– knock‐out mice to confirm the specificity of the antibody and of the agonist (WIN55,212‐2) action. We conclude that activation of presynaptic CB1 receptors decreases Ca2+‐dependent GABA release, and thereby reduces the power of hippocampal network oscillations.

The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus

Balanced control of neuronal activity is central in maintaining function and viability of neuronal circuits. The endocannabinoid system tightly controls neuronal excitability. Here, we show that endocannabinoids directly target hippocampal glutamatergic neurons to provide protection against acute epileptiform seizures in mice. Functional CB1 cannabinoid receptors are present on glutamatergic terminals of the hippocampal formation, colocalizing with vesicular glutamate transporter 1 (VGluT1). Conditional deletion of the CB1 gene either in cortical glutamatergic neurons or in forebrain GABAergic neurons, as well as virally induced deletion of the CB1 gene in the hippocampus, demonstrate that the presence of CB1 receptors in glutamatergic hippocampal neurons is both necessary and sufficient to provide substantial endogenous protection against kainic acid (KA)-induced seizures. The direct endocannabinoid-mediated control of hippocampal glutamatergic neurotransmission may constitute a promising therapeutic target for the treatment of disorders associated with excessive excitatory neuronal activity.

Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.

Molecular composition of the endocannabinoid system at glutamatergic synapses.

Endocannabinoids play central roles in retrograde signaling at a wide variety of synapses throughout the CNS. Although several molecular components of the endocannabinoid system have been identified recently, their precise location and contribution to retrograde synaptic signaling is essentially unknown. Here we show, by using two independent riboprobes, that principal cell populations of the hippocampus express high levels of diacylglycerol lipase α (DGL-α), the enzyme involved in generation of the endocannabinoid 2-arachidonoyl-glycerol (2-AG). Immunostaining with two independent antibodies against DGL-α revealed that this lipase was concentrated in heads of dendritic spines throughout the hippocampal formation. Furthermore, quantification of high-resolution immunoelectron microscopic data showed that this enzyme was highly compartmentalized into a wide perisynaptic annulus around the postsynaptic density of axospinous contacts but did not occur intrasynaptically. On the opposite side of the synapse, the axon terminals forming these excitatory contacts were found to be equipped with presynaptic CB1 cannabinoid receptors. This precise anatomical positioning suggests that 2-AG produced by DGL-α on spine heads may be involved in retrograde synaptic signaling at glutamatergic synapses, whereas CB1 receptors located on the afferent terminals are in an ideal position to bind 2-AG and thereby adjust presynaptic glutamate release as a function of postsynaptic activity. We propose that this molecular composition of the endocannabinoid system may be a general feature of most glutamatergic synapses throughout the brain and may contribute to homosynaptic plasticity of excitatory synapses and to heterosynaptic plasticity between excitatory and inhibitory contacts.

GPR55 is a cannabinoid receptor that increases intracellular calcium and inhibits M current.

The CB1 cannabinoid receptor mediates many of the psychoactive effects of Δ9THC, the principal active component of cannabis. However, ample evidence suggests that additional non-CB1/CB2 receptors may contribute to the behavioral, vascular, and immunological actions of Δ9THC and endogenous cannabinoids. Here, we provide further evidence that GPR55, a G protein-coupled receptor, is a cannabinoid receptor. GPR55 is highly expressed in large dorsal root ganglion neurons and, upon activation by various cannabinoids (Δ9THC, the anandamide analog methanandamide, and JWH015) increases intracellular calcium in these neurons. Examination of its signaling pathway in HEK293 cells transiently expressing GPR55 found the calcium increase to involve Gq, G12, RhoA, actin, phospholipase C, and calcium release from IP3R-gated stores. GPR55 activation also inhibits M current. These results establish GPR55 as a cannabinoid receptor with signaling distinct from CB1 and CB2.

Modulation of Ca2+ channels by G-protein beta gamma subunits.

CALCIUM ions entering cells through voltage-gated Ca2+ channels initiate rapid release of neurotransmitters and secretion of hormones. Ca2+ currents can be inhibited in many cell types by neurotransmitters acting through G proteins via a membrane-delimited pathway independently of soluble intracellular messengers1–4. Inhibition is typically caused by a positive shift in the voltage dependence and a slowing of channel activation and is relieved by strong depolarization resulting in facilitation of Ca2+currents1,4–6. This pathway regulates the activity of N-type and P/ Q-type Ca2+ channels1,2,7, which are localized in presynaptic terminals8,9 and participate in neurotransmitter release10–13. Synaptic transmission is inhibited by neurotransmitters through this mechanism1,4. G-protein a subunits confer specificity in receptor coupling1–4,14–17, but it is not known whether the Gα or Gβγ subunits are responsible for modulation of Ca2+channels. Here we report that Gβγ subunits can modulate Ca2+ channels. Transfection of Gβγ into cells expressing P/Q-type Ca2+ channels induces modulation like that caused by activation of G protein-coupled receptors, but Gα subunits do not. Similarly, injection or expression of Gβγ subunits in sympathetic ganglion neurons induces facilitation and occludes modulation of N-type channels by noradrenaline, but Gα subunits do not. In both cases, the Gγ subunit is ineffective by itself, but overexpression of exogenous Gβ subunits is sufficient to cause channel modulation.

Distribution of Cannabinoid Receptors in the Central and Peripheral Nervous System

CB1 cannabinoid receptors appear to mediate most, if not all of the psychoactive effects of delta-9-tetrahydrocannabinol and related compounds. This G protein-coupled receptor has a characteristic distribution in the nervous system: It is particularly enriched in cortex, hippocampus, amygdala, basal ganglia outflow tracts, and cerebellum--a distribution that corresponds to the most prominent behavioral effects of cannabis. In addition, this distribution helps to predict neurological and psychological maladies for which manipulation of the endocannabinoid system might be beneficial. CB1 receptors are primarily expressed on neurons, where most of the receptors are found on axons and synaptic terminals, emphasizing the important role of this receptor in modulating neurotransmission at specific synapses. While our knowledge of CB1 localization in the nervous system has advanced tremendously over the past 15 years, there is still more to learn. Particularly pressing is the need for (1) detailed anatomical studies of brain regions important in the therapeutic actions of drugs that modify the endocannabinoid system and (2) the determination of the localization of the enzymes that synthesize, degrade, and transport the endocannabinoids.