Andrew J. Irving

Hardwiring the Brain: Endocannabinoids Shape Neuronal Connectivity

The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB1 cannabinoid receptors (CB1Rs) are enriched in the axonal growth cones of γ-aminobutyric acid–containing (GABAergic) interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB1R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB1Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.

The GPR55 ligand l-α-lysophosphatidylinositol promotes RhoA-dependent Ca2+ signaling and NFAT activation

The endogenous phospholipid l‐α‐lyso‐phosphatidylinositol (LPI) was recently identified as a novel ligand for the orphan G protein‐coupled receptor 55 (GPR55). In this study we define the downstream signaling pathways activated by LPI in a human embryonic kidney (HEK) 293 cell line engineered to stably express recombinant human GPR55. We find that treatment with LPI induces marked GPR55 internalization and stimulates a sustained, oscillatory Ca2+ release pathway, which is dependent on Gα13 and requires RhoA activation. We then establish that this signaling cascade leads to the efficient activation of NFAT (nu‐clear factor of activated T cells) family transcription factors and their nuclear translocation. Analysis of cannabinoid ligand activity at GPR55 revealed no clear effect of the endocannabinoids anandamide and 2‐arachidonoylglycerol;however, the classical CB1 antagonist AM251 evoked GPR55‐mediated Ca2+ signaling. Thus, LPI is a potent and efficacious ligand at GPR55, which is likely to be a key plasma membrane mediator of LPI‐mediated signaling events and changes in gene expression.—Henstridge, C. M., Balenga, N. A. B., Ford, L. A., Ross, R. A., Waldhoer, M., Irving, A. J. The GPR55 ligand l‐α‐lysophosphatidylinositol promotes RhoA‐dependent Ca2+ signaling and NFAT activation. FASEB J. 23, 183‐193 (2009)

Leptin inhibits epileptiform-like activity in rat hippocampal neurones via PI 3-kinase-driven activation of BK channels

The obese gene product, leptin is an important circulating satiety factor that regulates energy balance via its actions in the hypothalamus. However, leptin receptors are also expressed in brain regions not directly associated with energy homeostasis, such as the hippocampus. Here, leptin inhibits hippocampal neurones via activation of large conductance Ca2+‐activated K+ (BK) channels, a process that may be important in regulating neuronal excitability. We now show that leptin receptor labelling is expressed on somata, dendrites and axons, and is also concentrated at synapses in hippocampal cultures. In functional studies, leptin potently and reversibly reduces epileptiform‐like activity evoked in lean, but not leptin‐resistant Zucker fa/fa rats. Furthermore, leptin also depresses enhanced Ca2+ levels evoked following Mg2+ removal in hippocampal cultures. The ability of leptin to modulate this activity requires activation of BK, but not KATP, channels as the effects of leptin were mimicked by the BK channel activator NS‐1619, and inhibited by the BK channel inhibitors, iberiotoxin and charybdotoxin. The signalling mechanisms underlying this process involve stimulation of phosphoinositide 3‐kinase (PI 3‐kinase), but not mitogen‐activated protein kinase (MAPK), as two structurally unrelated inhibitors of PI 3‐kinase, LY294002 and wortmannin, blocked the actions of leptin. These data indicate that leptin, via PI 3‐kinase‐driven activation of BK channels, elicits a novel mechanism for controlling neuronal excitability. As uncontrolled excitability in the hippocampus is one underlying cause of temporal lobe epilepsy, this novel action of leptin could provide an alternative therapeutic target in the management of epilepsy.

Localisation of cannabinoid CB1 receptor immunoreactivity in the guinea pig and rat myenteric plexus

Activation of cannabinoid CB1 receptors inhibits gastrointestinal motility, propulsion, and transit, whereas selective antagonism of these receptors has the opposite effects, suggesting the presence of endocannabinoid tone. Supporting evidence for presynaptic CB1 receptors on myenteric neurons has been found in vitro. In this study, selective CB1 receptor antibodies and neuronal markers were used to identify and characterise myenteric neurons expressing cannabinoid receptors. Whole mounts of rat and guinea pig myenteric preparations were dually labelled with antibodies against the CB1 receptor and choline acetyltransferase, neurofilament proteins, calbindin, calretinin, synapsin I, microtubule‐associated protein‐2, calcitonin gene‐related peptide, or substance P. The pattern of CB1 receptor labelling and the neurochemical classification of CB1 receptor‐positive cells were markedly influenced by the species and fixation procedure. Virtually all choline acetyltransferase‐immunoreactive myenteric neurons expressed CB1 receptors in ganglia from both species. Subpopulations of neurons identified with calbindin, calretinin, and microtubule‐associated protein‐2 did not express CB1 receptors. A few calcitonin gene‐related peptide‐ and substance P‐positive somata coexpressed CB1 receptor immunoreactivity but showed little colocalisation on individual fibres. There was a close association between CB1 receptor immunoreactivity and fibres labelled for synaptic protein, suggesting a role in the modulation of transmitter release. Functional responses to cannabinoids in the presence of hexamethonium suggest further that CB1 receptors occur on excitatory motoneurons. In conclusion, CB1 receptors are expressed on a variety of cholinergic sensory, interneuronal, and motor neurons in myenteric ganglia. J. Comp. Neurol. 448:410–422, 2002.

Leptin promotes rapid dynamic changes in hippocampal dendritic morphology

Recent studies have implicated the hormone leptin in synaptic plasticity associated with neuronal development and learning and memory. Indeed, leptin facilitates hippocampal long-term potentiation and leptin-insensitive rodents display impaired hippocampal synaptic plasticity suggesting a role for endogenous leptin. Structural changes are also thought to underlie activity-dependent synaptic plasticity and this may be regulated by specific growth factors. As leptin is reported to have neurotrophic actions, we have examined the effects of leptin on the morphology and filopodial outgrowth in hippocampal neurons. Here, we demonstrate that leptin rapidly enhances the motility and density of dendritic filopodia and subsequently increases the density of hippocampal synapses. This process is dependent on the synaptic activation of NR2A-containing NMDA receptors and is mediated by the MAPK (ERK) signaling pathway. As dendritic morphogenesis is associated with activity-dependent changes in synaptic strength, the rapid structural remodeling of dendrites by leptin has important implications for its role in regulating hippocampal synaptic plasticity and neuronal development.

GPR55 ligands promote receptor coupling to multiple signalling pathways

Background and purpose:  Although GPR55 is potently activated by the endogenous lysophospholipid, L‐α‐lysophosphatidylinositol (LPI), it is also thought to be sensitive to a number of cannabinoid ligands, including the prototypic CB1 receptor antagonists AM251 and SR141716A (Rimonabant®). In this study we have used a range of functional assays to compare the pharmacological activity of selected cannabinoid ligands, AM251, AM281 and SR141716A with LPI in a HEK293 cell line engineered to stably express recombinant, human GPR55.

A role for L-α-lysophosphatidylinositol and GPR55 in the modulation of migration, orientation and polarization of human breast cancer cells

Background and purpose:  Increased circulating levels of L‐α‐lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G‐protein‐coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55.

Minireview: Recent Developments in the Physiology and Pathology of the Lysophosphatidylinositol-Sensitive Receptor GPR55

Emerging data suggest that off-target cannabinoid effects may be mediated via novel seven-transmembrane spanning/G protein-coupled receptors. Due to its cannabinoid sensitivity, the G protein-coupled receptor 55 (GPR55) was recently proposed as a candidate; however, GPR55 is phylogenetically distinct from the traditional cannabinoid receptors, and the conflicting pharmacology, signaling, and functional data have prevented its classification as a novel cannabinoid receptor. Indeed, the most consistent and potent agonist to date is the noncannabinoid lysophospholipid, lysophosphatidylinositol. Here we present new human GPR55 mRNA expression data, providing supportive evidence of GPR55 expression in a vast array of tissues and cell types. Moreover, we summarize major recent developments in GPR55 research and aim to update the reader in the rapidly expanding fields of GPR55 pharmacology, physiology, and pathology.

Leptin inhibits rat hippocampal neurons via activation of large conductance calcium-activated K+ channels.

Leptin is an important circulating factor that regulates energy balance via the leptin receptor Ob-Rb in the hypothalamus. Ob-Rb activation may inhibit hypothalamic neurons by activating ATP-sensitive K+ channels (KATP channels). Here we show that leptin inhibits hippocampal neurons via phosphoinositide 3-kinase (PI3-kinase)–driven activation of large conductance, calcium-activated K+ channels (BK channels), but not KATP channels. This may be an important mechanism for regulating hippocampal excitability.

Leptin induces a novel form of NMDA receptor-dependent long-term depression.

It is becoming apparent that the hormone leptin plays an important role in modulating hippocampal function. Indeed, leptin enhances NMDA receptor activation and promotes hippocampal long‐term potentiation (LTP). Furthermore, obese rodents with dysfunctional leptin receptors display impairments in hippocampal synaptic plasticity. Here we demonstrate that under conditions of enhanced excitability (evoked in Mg2+‐free medium or following blockade of GABAA receptors), leptin induces a novel form of long‐term depression (LTD) in area CA1 of the hippocampus. Leptin‐induced LTD was markedly attenuated in the presence of D‐(‐)‐2‐Amino‐5‐Phosphonopentanoic acid (D‐AP5), suggesting that it is dependent on the synaptic activation of NMDA receptors. In addition, low‐frequency stimulus‐evoked LTD occluded the effects of leptin. In contrast, metabotropic glutamate receptors (mGluRs) did not contribute to leptin‐induced LTD as mGluR antagonists failed to either prevent or reverse this process. The signalling mechanisms underlying leptin‐induced LTD were independent of the Ras‐Raf‐mitogen‐activated protein kinase signalling pathway, but were markedly enhanced following inhibition of either phosphoinositide 3‐kinase or protein phosphatases 1 and 2A. These data indicate that under conditions of enhanced excitability, leptin induces a novel form of homosynaptic LTD, which further underscores the proposed key role for this hormone in modulating NMDA receptor‐dependent hippocampal synaptic plasticity.