Michaela Egertová

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.

Localisation of cannabinoid receptors in the rat brain using antibodies to the intracellular C-terminal tail of CB1

The CB1‐type cannabinoid receptor mediates physiologic effects of Δ9‐tetrahydrocannabinol, the psychoactive ingredient of the drug marijuana. In this report, the authors analyse the expression of CB1 in the rat brain by using antibodies to the C‐terminal 13 amino acids of the receptor. Western blot analysis of rat brain membranes revealed a prominent immunoreactive band with a molecular mass (≈53 kDa) consistent with that predicted for CB1 from the rat cDNA sequence. In addition, however, less intense immunoreactive bands corresponding to glycosylated (≈62 kDa) and putative N‐terminally shorter (≈45 kDa) isoforms of CB1 were detected. The distribution of CB1‐immunoreactivity in rat brain was similar to the distribution of binding sites for radiolabelled cannabinoids, with high levels of expression in the olfactory system, the hippocampal formation, the basal ganglia, the cerebellum, and the neocortex. This provides important evidence that CB1 is likely to be largely responsible for mediating effects of cannabinoids in the brain. CB1 immunoreactivity was associated with nerve fibre systems and axon terminals but was not detected in neuronal somata. This is consistent with the presynaptic inhibitory effects of cannabinoids on neurotransmitter release in the brain. Detailed immunocytochemical analysis of anatomically or functionally related regions of the brain revealed the location of CB1 receptors within identified neural circuits. Determination of the cellular and subcellular location of CB1 within known neuronal circuits of the brain provides an anatomic framework for interpretation of the neurophysiologic and behavioural effects of cannabinoids. J. Comp. Neurol. 422:159–171, 2000.

A NEW PERSPECTIVE ON CANNABINOID SIGNALLING : COMPLEMENTARY LOCALIZATION OF FATTY ACID AMIDE HYDROLASE AND THE CB1 RECEPTOR IN RAT BRAIN

CB1–type cannabinoid receptors in the brain mediate effects of the drug cannabis. Anandamide and sn–2 arachidonylglycerol (2–AG) are putative endogenous ligands for CB1 receptors, but it is not known which cells in the brain produce these molecules. Recently, an enzyme which catalyses hydrolysis of anandamide and 2–AG, known as fatty acid amide hydrolase (FAAH), was identified in mammals. Here we have analysed the distribution of FAAH in rat brain and compared its cellular localization with CB1–type cannabinoid receptors using immunocytochemistry. High concentrations of FAAH activity were detected in the cerebellum, hippocampus and neocortex, regions of the rat brain which are enriched with cannabinoid receptors. Immunocytochemical analysis of these brain regions revealed a complimentary pattern of FAAH and CB1 expression with CB1–immunoreactivity occurring in fibres surrounding FAAH–immunoreactive cell bodies and/or dendrites. In the cerebellum, FAAH was expressed in the cell bodies of Purkinje cells and CB1 was expressed in the axons of granule cells and basket cells, neurons which are presynaptic to Purkinje cells. The close correspondence in the distribution of FAAH and CB1 in rat brain and the complimentary pattern of FAAH and CB1 expression at the cellular level provides important new evidence that FAAH may participate in cannabinoid signalling mechanisms of the brain.

Cannabinoid CB1 receptor expression in rat spinal cord

While evidence implicates the endogenous cannabinoid system as a novel analgesic target at a spinal level, detailed analysis of the distribution of the cannabinoid receptor CB1 in spinal cord has not been reported. Here, immunocytochemical studies were used to characterize the CB1 receptor expression in rat spinal cord. Staining was found in the dorsolateral funiculus, the superficial dorsal horn (a double band of CB1 immunoreactivity (ir) in laminae I and II inner/III transition), and lamina X. Although CB1-ir was present in the same laminae as primary afferent nociceptor markers, there was limited colocalization at an axonal level. Interruption of both primary afferent input by dorsal root rhizotomy and descending input by rostral spinal cord hemisection produced minor changes in CB1-ir. This and colocalization of CB1-ir with interneurons expressing protein kinase C subunit γ-ir suggest that the majority of CB1 expression is on spinal interneurons. These data provide a framework and implicate novel analgesic mechanisms for spinal actions of cannabinoids at the CB1 receptor.

Localisation of cannabinoid receptor 1 in rat dorsal root ganglion using in situ hybridisation and immunohistochemistry

In this study we used in situ hybridisation and double-labelling immunohistochemistry to characterise cannabinoid receptor 1 (CB(1)) expression in rat lumbar dorsal root ganglion (DRG) neurons.Approximately 25% of DRG neurons expressed CB(1) mRNA and displayed immunoreactivity for CB(1). Sixty-nine percent to 82% of CB(1)-expressing cells were also immunoreactive for neurofilament 200, indicative of myelinated A-fibre neurons, which tend to be large- and medium-sized DRG neurons (>600 microm(2)). Approximately 10% of CB1-expressing cells also expressed transient receptor potential vanilloid family ion channel 2 (TRPV2), the noxious heat-transducing channel found in medium to large lightly myelinated Adelta-fibre DRG neurons. Seventeen percent to 26% of CB(1)-expressing cells co-stained using Isolectin B4, 9-10% for calcitonin gene-related peptide and 11-20% for transient receptor potential vanilloid family ion channel 1 (TRPV1), predominantly markers of small non-myelinated C-fibre DRG neurons (<600 microm(2)). These findings suggest that whilst a wide range of DRG neuron phenotypes express CB(1), it is predominantly associated with myelinated fibres.

MRAP and MRAP2 are bidirectional regulators of the melanocortin receptor family

The melanocortin receptor (MCR) family consists of 5 G protein-coupled receptors (MC1R–MC5R) with diverse physiologic roles. MC2R is a critical component of the hypothalamic–pituitary–adrenal axis, whereas MC3R and MC4R have an essential role in energy homeostasis. Mutations in MC4R are the single most common cause of monogenic obesity. Investigating the way in which these receptors signal and traffic to the cell membrane is vital in understanding disease processes related to MCR dysfunction. MRAP is an MC2R accessory protein, responsible for adrenal MC2R trafficking and function. Here we identify MRAP2 as a unique homologue of MRAP, expressed in brain and the adrenal gland. We report that MRAP and MRAP2 can interact with all 5 MCRs. This interaction results in MC2R surface expression and signaling. In contrast, MRAP and MRAP2 can reduce MC1R, MC3R, MC4R, and MC5R responsiveness to [Nle4,D-Phe7]alpha-melanocyte-stimulating hormone (NDP-MSH). Collectively, our data identify MRAP and MRAP2 as unique bidirectional regulators of the MCR family.

Localization of N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) expression in mouse brain: A new perspective on N-acylethanolamines as Neural Signaling Molecules

N‐acylethanolamines (NAEs) are membrane‐derived lipids that are utilized as signaling molecules in the nervous system (e.g., the endocannabinoid anandamide). An N‐acyl phosphatidylethanolamine phospholipase D (NAPE‐PLD) that catalyzes formation of NAEs was recently identified as a member of the zinc metallohydrolase family of enzymes. NAPE‐PLD−/− mice have greatly reduced brain levels of long‐chain saturated NAEs but wild‐type levels of polyunsaturated NAEs (e.g., anandamide), suggesting an important role for NAPE‐PLD in the biosynthesis of at least a subset of endogenous NAEs in the mammalian nervous system. To provide a neuroanatomical basis for investigation of NAPE‐PLD function, here we have analyzed expression of NAPE‐PLD in the mouse brain using mRNA in situ hybridization and immunocytochemistry. NAPE‐PLD−/− mice were utilized to establish the specificity of probes/antibodies used. The most striking feature of NAPE‐PLD expression in the brain was in the dentate gyrus, where a strong mRNA signal was detected in granule cells. Accordingly, immunocytochemical analysis revealed intense NAPE‐PLD immunoreactivity in the axons of granule cells (mossy fibers). Intense NAPE‐PLD immunoreactivity was also detected in axons of the vomeronasal nerve that project to the accessory olfactory bulb. NAPE‐PLD expression was detected in other brain regions (e.g., hippocampus, cortex, thalamus, hypothalamus), but the intensity of immunostaining was weaker than in mossy fibers. Collectively, the data obtained indicate that NAPE‐PLD is expressed by specific populations of neurons in the brain and targeted to axonal processes. We suggest that NAEs generated by NAPE‐PLD in axons may act as anterograde synaptic signaling molecules that regulate the activity of postsynaptic neurons. J. Comp. Neurol. 506:604–615, 2008.

CB1 cannabinoid receptor activity is modulated by the cannabinoid receptor interacting protein CRIP 1a.

The CB1 cannabinoid receptor is a G-protein coupled receptor that has important physiological roles in synaptic plasticity, analgesia, appetite, and neuroprotection. We report the discovery of two structurally related CB1 cannabinoid receptor interacting proteins (CRIP1a and CRIP1b) that bind to the distal C-terminal tail of CB1. CRIP1a and CRIP1b are generated by alternative splicing of a gene located on chromosome 2 in humans, and orthologs of CRIP1a occur throughout the vertebrates, whereas CRIP1b seems to be unique to primates. CRIP1a coimmunoprecipitates with CB1 receptors derived from rat brain homogenates, indicating that CRIP1a and CB1 interact in vivo. Furthermore, in superior cervical ganglion neurons coinjected with CB1 and CRIP1a or CRIP1b cDNA, CRIP1a, but not CRIP1b, suppresses CB1-mediated tonic inhibition of voltage-gated Ca2+ channels. Discovery of CRIP1a provides the basis for a new avenue of research on mechanisms of CB1 regulation in the nervous system and may lead to development of novel drugs to treat disorders where modulation of CB1 activity has therapeutic potential (e.g., chronic pain, obesity, and epilepsy).

Fatty acid amide hydrolase expression in rat choroid plexus: possible role in regulation of the sleep-inducing action of oleamide

The enzyme fatty acid amide hydrolase (FAAH) catalyses hydrolysis of oleamide, a sleep-inducing lipid whose concentration in the cerebrospinal fluid (CSF) is elevated in sleep-deprived mammals. Previous studies have reported expression of FAAH by distinct populations of neurons in the rat brain. Here we demonstrate using immunocytochemical methods that FAAH is also expressed by non-neuronal epithelial cells of the rat choroid plexus. The choroid plexus is formed by invaginations of the pia mater into the ventricle cavities of the brain and an important function of the choroidal epithelium is to regulate production and composition of CSF. Therefore, the role of FAAH in epithelial cells of the choroid plexus may be to control the concentration of oleamide in the CSF and as such FAAH may exert an important regulatory role in shaping the duration and magnitude of the sleep-inducing effect of endogenously or exogenously derived oleamide.

Cannabinoid Receptor–Interacting Protein 1a Modulates CB1 Receptor Signaling and Regulation

Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor–interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca2+ channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide–binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [35S]GTPγS (guanylyl-5′-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA–mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [35S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist-stimulated CB1R signaling and downregulation of CB1Rs. Thus, CRIP1a appears to act as a broad negative regulator of CB1R function.