Mark G. Rae

Functional expression of cell surface cannabinoid CB1 receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons

At present, little is known about the mechanisms by which cannabinoids exert their effects on the central nervous system. In this study, fluorescence imaging and electrophysiological techniques were used to investigate the functional relationship between cell surface cannabinoid type 1 (CB(1)) receptors and GABAergic synaptic transmission in cultured hippocampal neurons. CB(1) receptors were labelled on living neurons using a polyclonal antibody directed against the N-terminal 77 amino acid residues of the rat cloned CB(1) receptor. Highly punctate CB(1) receptor labelling was observed on fine axons and at axonal growth cones, with little somatic labelling. The majority of these sites were associated with synaptic terminals, identified either with immunohistochemical markers or by using the styryl dye FM1-43 to label synaptic vesicles that had undergone active turnover. Dual labelling of neurons for CB(1) receptors with either the inhibitory neurotransmitter GABA or its synthesising enzyme glutamate decarboxylase, demonstrated a strong correspondence. The immunocytochemical data was supported by functional studies using whole-cell patch-clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs). The cannabinoid agonist WIN55,212-2 (100nM) markedly inhibited (by 77+/-6.3%) the frequency of pharmacologically-isolated GABAergic mIPSCs. The effects of WIN55,212-2 were blocked in the presence of the selective CB(1) receptor antagonist SR141716A (100nM).In conclusion, the present data show that cell surface CB(1) receptors are expressed at presynaptic GABAergic terminals, where their activation inhibits GABA release. Their presence on growth cones could indicate a role in the targeting of inhibitory connections during development.

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.

BOTH MGLUR1 AND MGLUR5 MEDIATE CA2+ RELEASE AND INWARD CURRENTS IN HIPPOCAMPAL CA1 PYRAMIDAL NEURONS

Using combined whole-cell voltage-clamp recording and Ca2+ imaging we have investigated further the characteristics and pharmacology of group I metabotropic l-glutamate receptor (mGluR)-mediated responses in CA1 pyramidal neurons of the rat hippocampus. The selective group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG), evoked a transient increase in intracellular Ca2+ levels ([Ca2+]i), within neuronal somas and apical dendrites, together with a relatively long lasting inward current (IDHPG). Both types of response were enhanced by depolarisation (-30 mV), and this condition was used for their characterisation. The DHPG-induced [Ca2+]i rise was much more sensitive to manipulations of Ca2+ homeostasis, such as using the Ca2+ store depleting agent, cyclopiazonic acid (50-100 μM), the fast Ca2+ buffer, BAPTA (intracellular; 20-40 mM) and Ca2+-free/EGTA (1 mM) bath solution, than IDHPG, suggesting that these responses are, in the main part, mediated by distinct processes. The selective mGluR1 and mGluR5 antagonists, (S)-(+)-α-amino-a-methylbenzeneacetic acid (LY367385; 100 μM) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP;10 μM), respectively, markedly inhibited both IDHPG and the DHPG-evoked increase in [Ca2+]i. Moreover, these antagonists inhibited the Ca2+ response by more than 50% suggesting a synergistic interaction between mGluR1 and mGluR5. This study demonstrates that in CA1 pyramidal neurons group I mGluR-mediated inward currents and Ca2+ release from intracellular stores are enhanced under depolarising conditions and that mGluR1 and mGluR5 both contribute to these phenomena.Using combined whole-cell voltage-clamp recording and Ca2+ imaging we have investigated further the characteristics and pharmacology of group I metabotropic l-glutamate receptor (mGluR)-mediated responses in CA1 pyramidal neurons of the rat hippocampus. The selective group I mGluR agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG), evoked a transient increase in intracellular Ca2+ levels ([Ca2+]i), within neuronal somas and apical dendrites, together with a relatively long lasting inward current (I(DHPG)). Both types of response were enhanced by depolarisation (-30 mV), and this condition was used for their characterisation. The DHPG-induced [Ca2+]i rise was much more sensitive to manipulations of Ca2+ homeostasis, such as using the Ca2+ store depleting agent, cyclopiazonic acid (50-100 microM), the fast Ca2+ buffer, BAPTA (intracellular; 20-40 mM) and Ca(2+)-free/EGTA (1 mM) bath solution, than I(DHPG), suggesting that these responses are, in the main part, mediated by distinct processes. The selective mGluR1 and mGluR5 antagonists, (S)-(+)-alpha-amino-a-methylbenzeneacetic acid (LY367385; 100 microM) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP;10 microM), respectively, markedly inhibited both I(DHPG) and the DHPG-evoked increase in [Ca2+]i. Moreover, these antagonists inhibited the Ca2+ response by more than 50% suggesting a synergistic interaction between mGluR1 and mGluR5. This study demonstrates that in CA1 pyramidal neurons group I mGluR-mediated inward currents and Ca2+ release from intracellular stores are enhanced under depolarising conditions and that mGluR1 and mGluR5 both contribute to these phenomena.

Modulation of enteric neurons by interleukin-6 and corticotropin-releasing factor contributes to visceral hypersensitivity and altered colonic motility in a rat model of irritable bowel syndrome.

Hyperactivity of the stress system and low‐grade immune activation characterize the functional bowel disorder irritable bowel syndrome (IBS). These studies show that interleukin (IL)‐6 and IL‐8 and the stress hormone corticotropin‐releasing factor (CRF), present in IBS plasma, have functional effects on gastrointestinal activity by stimulating myenteric neurons and colonic contractions. Moreover, in the Wistar Kyoto rat model of IBS, which exhibits altered gastrointestinal motility and visceral pain sensitivity, blocking IL‐6 and/or CRF1 receptors alleviates these IBS‐like symptoms. Underlying these effects are altered colonic protein expression of tight junction proteins which regulate gut barrier function and the T‐type calcium channel CaV3.2, which has been linked to visceral pain. These findings demonstrate the importance of the enteric nervous system and intestinal physiology in bowel dysfunction.

Cannabinoids on the Brain

Cannabis has a long history of consumption both for recreational and medicinal uses. Recently there have been significant advances in our understanding of how cannabis and related compounds (cannabinoids) affect the brain and this review addresses the current state of knowledge of these effects. Cannabinoids act primarily via two types of receptor, CB1 and CB2, with CB1 receptors mediating most of the central actions of cannabinoids. The presence of a new type of brain cannabinoid receptor is also indicated. Important advances have been made in our understanding of cannabinoid receptor signaling pathways, their modulation of synaptic transmission and plasticity, the cellular targets of cannabinoids in different central nervous system (CNS) regions and, in particular, the role of the endogenous brain cannabinoid (endocannabinoid) system. Cannabinoids have widespread actions in the brain: in the hippocampus they influence learning and memory; in the basal ganglia they modulate locomotor activity and reward pathways; in the hypothalamus they have a role in the control of appetite. Cannabinoids may also be protective against neurodegeneration and brain damage and exhibit anticonvulsant activity. Some of the analgesic effects of cannabinoids also appear to involve sites within the brain. These advances in our understanding of the actions of cannabinoids and the brain endocannabinoid system have led to important new insights into neuronal function which are likely to result in the development of new therapeutic strategies for the treatment of a number of key CNS disorders.

Effect of reactive oxygen species and nitric oxide in the neural control of intrarenal haemodynamics in anaesthetized normotensive rats

This study examined the interaction between reactive oxygen species and nitric oxide (NO) in mediating the decrease in renal blood flow (RBF) evoked by sympathetic renal nerve stimulation (RNS).

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process

The interaction between interleukin IL-1 alpha and PGE2 on P388D1 cells has been investigated. Preincubation of murine macrophage-like cells, P388D1, with IL-1 alpha (0-73 pM) reduced the binding of PGE2 to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1 alpha decreased the PGE2 binding by lowering both the high and low affinity receptor binding capacities (from 0.31 +/- 0.02 to 0.12 +/- 0.01 fmol/10(6) cells for the high affinity receptor binding sites and from 2.41 +/- 0.12 to 1.51 +/- 0.21 fmol/10(6) cells for the low affinity receptor binding sites). However, the dissociation constants of the receptors of the IL-1 alpha-treated cells remained unchanged. Inhibition of PGE2 binding by IL-1 alpha did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1 alpha inhibits the binding of PGE2 to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE2.

Regulation of prostaglandin E2 binding to a murine macrophage cell line, P388D1, by insulin

Preincubation of murine macrophage-like P388D1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 +/- 0.02 to 0.63 +/- 0.03 fmol/10(6) cells for the high affinity receptor binding sites, and from 2.4 +/- 0.31 to 5.0 +/- 0.41 fmol/10(6) cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE2.

Cognitive dysfunction in Duchenne muscular dystrophy: a possible role for neuromodulatory immune molecules

Duchenne muscular dystrophy (DMD) is an X chromosome-linked disease characterized by progressive physical disability, immobility, and premature death in affected boys. Underlying the devastating symptoms of DMD is the loss of dystrophin, a structural protein that connects the extracellular matrix to the cell cytoskeleton and provides protection against contraction-induced damage in muscle cells, leading to chronic peripheral inflammation. However, dystrophin is also expressed in neurons within specific brain regions, including the hippocampus, a structure associated with learning and memory formation. Linked to this, a subset of boys with DMD exhibit nonprogressing cognitive dysfunction, with deficits in verbal, short-term, and working memory. Furthermore, in the genetically comparable dystrophin-deficient mdx mouse model of DMD, some, but not all, types of learning and memory are deficient, and specific deficits in synaptogenesis and channel clustering at synapses has been noted. Little consideration has been devoted to the cognitive deficits associated with DMD compared with the research conducted into the peripheral effects of dystrophin deficiency. Therefore, this review focuses on what is known about the role of full-length dystrophin (Dp427) in hippocampal neurons. The importance of dystrophin in learning and memory is assessed, and the potential importance that inflammatory mediators, which are chronically elevated in dystrophinopathies, may have on hippocampal function is also evaluated.

Effect of clazosentan, a selective endothelin A receptor antagonist, and tezosentan, a dual endothelin A/B antagonist, on pulsatile shear stress induced constriction of the iliac in the anaesthetized pig

1. The effects of changes in mean and pulsatile shear stress on the diameter of the iliac of the anaesthetized pig were investigated in the presence of clazosentan and tezosentan.