George Lees

Sodium channel inhibition by anandamide and synthetic cannabimimetics in brain

Anandamide is a prominent member of the endocannabinoids, a group of diffusible lipid molecules which influences neuronal excitability. In this context, endocannabinoids are known to modulate certain presynaptic Ca(2+) and K(+) channels, either through cannabinoid (CB1) receptor stimulation and second messenger pathway activation or by direct action. We investigated the susceptibility of voltage-sensitive sodium channels to anandamide and other cannibimimetics using both biochemical and electrophysiological approaches. Here we report that anandamide, AM 404 and WIN 55,212-2 inhibit veratridine-dependent depolarization of synaptoneurosomes (IC(50)s, respectively 21.8, 9.3 and 21.1 microM) and veratridine-dependent release of L-glutamic acid and GABA from purified synaptosomes [IC(50)s: 5.1 microM (L-glu) and 16.5 microM (GABA) for anandamide; 1.6 microM (L-glu) and 3.3 microM (GABA) for AM 404, and 12.2 (L-glu) and 14.4 microM (GABA) for WIN 55,212-2]. The binding of [3H]batrachotoxinin A 20-alpha-benzoate to voltage-sensitive sodium channels was also inhibited by low to mid micromolar concentrations of anandamide, AM 404 and WIN 55,212-2. In addition, anandamide (10 microM), AM 404 (10 microM) and WIN 55,212-2 (1 microM) were found to markedly block TTX-sensitive sustained repetitive firing in cortical neurones without altering primary spikes, consistent with a state-dependent mechanism. None of the inhibitory effects we demonstrate on voltage-sensitive sodium channels are attenuated by the potent CB1 antagonist AM 251 (1-2 microM). Anandamides action is reversible and its effects are enhanced by fatty acid amidohydrolase inhibition. We propose that voltage-sensitive sodium channels may participate in a novel signaling pathway involving anandamide. This mechanism has potential to depress synaptic transmission in brain by damping neuronal capacity to support action potentials and reducing evoked release of both excitatory and inhibitory transmitters.

Immunological identification of the mammalian H3 histamine receptor in the mouse brain

Affinity-purified antibodies raised against the peptide sequence H3 (349–358) receptor specifically recognized two protein species with Mr 62 000 and 93 000 in adult mouse forebrain membranes. Both immunoreactive species were suppressed greatly by preincubation of the antibody with the respective peptide. Immunohistochemical analysis using affinity-purified anti-H3 (349–358) antibodies yielded a high degree of coincidence with ligand-autoradiographical information, with high levels detected in the CA3 and dentate gyrus of the hippocampus, laminae V of the cerebral cortex, the olfactory tubercle, Purkinje cell layer of the cerebellum, substantia nigra, globus pallidus, thalamus and striatum. This study suggests further biochemical evidence for multiple H3 receptor subtypes and the widespread distribution of the H3 receptor in the mammalian brain.

Cholinergic receptors on cultured neurones from the central nervous system of embryonic cockroaches

Cultured neurones from the cockroach, Periplaneta americana, have been used to investigate putative acetylcholine receptors. Ligand-binding experiments revealed that these neurones possessed an alpha-bungarotoxin binding site that was saturable, had an apparent affinity constant of 3.51 nM and was predominantly nicotinic in nature. An individual culture of 50,000 neurones had a maximum of 4200 pmol. binding sites per gram of protein. [I125]alpha-BTX autoradiography showed the binding sites to be distributed over both the neuronal cell bodies and their associated axonal processes. Both acetylcholine and nicotine applied by pressure ejection to the neuronal soma induced depolarizing responses and in the majority of cells tested the response was blocked by alpha-BTX at a concentration of 25 nM in a time dependent manner. Some of the neurones, however, were depolarized by acetylcholine and nicotine after 3 h incubation in alpha-BTX. These experiments suggest that two populations of cells possessing extrajunctional nicotinic receptors were present in these cultures. In the majority of cells these receptors were sensitive to alpha-BTX but in a subpopulation the receptors were unaffected by this toxin.

Stereoselective modulatory actions of oleamide on GABAA receptors and voltage-gated Na+ channels in vitro: a putative endogenous ligand for depressant drug sites in CNS

cis‐9,10‐octadecenoamide (‘oleamide’) accumulates in CSF on sleep deprivation. It induces sleep in animals (the trans form is inactive) but its cellular actions are poorly characterized. We have used electrophysiology in cultures from embryonic rat cortex and biochemical studies in mouse nerve preparations to address these issues. Twenty μM cis‐oleamide (but not trans) reversibly enhanced GABAA currents and depressed the frequency of spontaneous excitatory and inhibitory synaptic activity in cultured networks. cis‐oleamide stereoselectively blocked veratridine‐induced (but not K+‐induced) depolarisation of mouse synaptoneurosomes (IC50, 13.9 μM). The cis isomer stereoselectively blocked veratridine‐induced (but not K+‐induced) [3H]‐GABA release from mouse synaptosomes (IC50, 4.6 μM). At 20 μM cis‐oleamide, but not trans, produced a marked inhibition of Na+ channel‐dependent rises in intrasynaptosomal Ca2+. The physiological significance of these observations was examined by isolating Na+ spikes in cultured pyramidal neurones. Sixty‐four μM cis‐oleamide did not significantly alter the amplitude, rate of rise or duration of unitary action potentials (1 Hz). cis‐Oleamide stereoselectively suppressed sustained repetitive firing (SRF) in these cells with an EC50 of 4.1 μM suggesting a frequency‐ or state‐dependent block of voltage‐gated Na+ channels. Oleamide is a stereoselective modulator of both postsynaptic GABAA receptors and presynaptic or somatic voltage‐gated Na+ channels which are crucial for synaptic inhibition and conduction. The modulatory actions are strikingly similar to those displayed by sedative or anticonvulsant barbiturates and a variety of general anaesthetics. Oleamide may represent an endogenous modulator for drug receptors and an important regulator of arousal.

Excitable properties of insect neurones in culture: A developmental study

Abstract The passive and excitable electrical properties of cockroach neurones growing in vitro have been investigated using intracellular recording techniques. The resting membrane potentials of the neurones are similar to those of their in vivo counterparts but the input resistances and membrane capacitive properties are more typical of embryonic insect neurones. During the first 12 days of growth in vitro the neurones exhibit delayed rectification in response to the injection of depolarising current steps. After this period “all or none” action potentials can be evoked by depolarising pulses in approximately half of the neurones tested. These spikes are abolised by 1 μM tetrodotoxin but are unaffected by 5 mM Co2+. Spontaneous excitatory activity develops in approx 25% of the neurones after 3 weeks in culture.

The sleep hormone oleamide modulates inhibitory ionotropic receptors in mammalian CNS in vitro

We examine the sensitivity of GABAA and glycine receptors (same ionotropic superfamily) to oleamide. We address subunit‐dependence/modulatory mechanisms and analogies with depressant drugs. Oleamide modulated human GABAA currents (α1β2γ2L) in oocytes (EC50, 28.94±s.e.mean of 1.4 μM; Maximum 216%±35 of control, n=4). Modulation of human α1 glycine homo‐oligomers (significant), was less marked, with a lower EC50 (P<0.05) than GABA receptors (EC50, 22.12±1.4 μM; Maximum 171%±30, n=11). Only the hypnogenic cis geometric isomer enhanced glycine currents (without altering slope or maximal current, it reduced the glycine EC50 from 322 to 239 μM: P<0.001). Modulation was not voltage‐dependent or associated with a shift in Er. β1 containing GABAA receptors (insensitive to many depressant drugs) were positively modulated by oleamide. Oleamide efficacy was circa 2× greater at α1β1γ2L than α1β2γ2L (P=0.007). Splice variation in γ subunits did not alter oleamide sensitivity. cis‐9,10‐Octadecenoamide had no effect on the equilibrium binding of [3H]‐muscimol or [3H]‐EBOB to mouse brain membranes. It does not directly mimic GABA, or operate as a neurosteroid‐, benzodiazepine‐ or barbiturate‐like modulator of GABAA‐receptors. The transport of [3H]‐GABA into mouse brain synaptoneurosomes was unaffected by high micromolar concentrations of cis‐9,10‐octadecenoamide. Oleamide does not enhance GABA‐ergic currents or prolong IPSCs by inhibiting GABA transport. Oleamide is a non‐selective modulator of inhibitory ionotropic receptors. The sleep lipid exerts its effects indirectly, or at a novel recognition site on the GABAA complex.

GABAA α6-Containing Receptors Are Selectively Compromised in Cerebellar Granule Cells of the Ataxic Mouse, Stargazer

Stargazer mice fail to express the γ2 isoform of transmembrane α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor regulatory proteins that has been shown to be absolutely required for the trafficking and synaptic targeting of excitatory AMPA receptors in adult murine cerebellar granule cells. Here we show that 30 ± 6% fewer inhibitory γ-aminobutyric acid, type A (GABAA), receptors were expressed in adult stargazer cerebellum compared with controls because of a specific loss of GABAA receptor expression in the cerebellar granule cell layer. Radioligand binding assays allied to in situ immunogold-EM analysis and furosemide-sensitive tonic current estimates revealed that expression of the extrasynaptic (α6βxδ) α6-containing GABAA receptor were markedly and selectively reduced in stargazer. These observations were compatible with a marked reduction in expression of GABAA receptor α6, δ (mature cerebellar granule cell-specific proteins), and β3 subunit expression in stargazer. The subunit composition of the residual α6-containing GABAA receptors was unaffected by the stargazer mutation. However, we did find evidence of an ∼4-fold up-regulation of α1βδ receptors that may compensate for the loss of α6-containing GABAA receptors. PCR analysis identified a dramatic reduction in the steady-state level of α6 mRNA, compatible with α6 being the primary target of the stargazer mutation-mediated GABAA receptor abnormalities. We propose that some aspects of assembly, trafficking, targeting, and/or expression of extrasynaptic α6-containing GABAA receptors in cerebellar granule cells are selectively regulated by AMPA receptor-mediated signaling.

Anesthetic-like Interaction of the Sleep-inducing Lipid Oleamide with Voltage-gated Sodium Channels in Mammalian Brain

Backgroundcis- 9,10-Octadecenoamide (cOA) accumulates in cerebrospinal fluid during sleep deprivation and induces sleep in animals, but its cellular actions are poorly characterized. In earlier studies, like a variety of anesthetics, cOA modulated &ggr;-aminobutyric acidA receptors and inhibited transmitter release/burst firing in cultured neurones or synaptoneurosomes. MethodsHere, radioligand binding ([3H]batrachotoxinin A 20-&agr;-benzoate and mouse central nervous system synaptoneurosomes) and voltage clamp (whole cell recording from cultured NIE115 murine neuroblastoma) confirmed an interaction with neuronal voltage-gated sodium channels (VGSC). ResultscOA stereoselectively inhibited specific binding of toxin to VGSC (inhibitor concentration that displaces 50% of specifically bound radioligand, 39.5 &mgr;m). cOA increased (4×) the Kd of toxin binding without affecting its binding maximum. Rate of dissociation of radioligand was increased without altering association kinetics, suggesting an allosteric effect (indirect competition at site 2 on VGSC). cOA blocked tetrodotoxin-sensitive sodium currents (maximal effect and affinity were significantly greater at depolarized potentials;P < 0.01). Between 3.2 and 64 &mgr;m, the block was concentration-dependent and saturable, but cOA did not alter the V50 for activation curves or the measured reversal potential (P > 0.05). Inactivation curves were significantly shifted in the hyperpolarizing direction by cOA (maximum, −15.4 ± 0.9 mV at 32 &mgr;m). cOA (10 &mgr;m) slowed recovery from inactivation, with &tgr; increasing from 3.7 ± 0.4 ms to 6.4 ± 0.5 ms (P < 0.001). cOA did not produce frequency-dependent facilitation of block (up to 10 Hz). ConclusionsThese effects (and the capacity of oleamide to modulate &ggr;-aminobutyric acidA receptors in earlier studies) are strikingly similar to those of a variety of anesthetics. Oleamide may represent an endogenous ligand for depressant drug sites in mammalian brain.

Choline uptake by cultured neurones from the central nervous system of embryonic cockroaches

Abstract Cultured neurones from the cockroach, Periplaneta americana , have been used to investigate the uptake of [ 3 H]choline. The neurones take up choline from the extracellular medium by both high and low affinity transport systems. Analysis of the high affinity system showed it to be sodium-dependent, temperature-sensitive and largely inhibited by μ M hemicholinium-3, but relatively insensitive to metabolic inhibitors. Elevated potassium concentrations markedly decreased it, suggesting that high affinity uptake is dependent on membrane potential. This transport component is associated with a considerable degree of acetylcholine synthesis: the possibility that these results indicate the presence of presynaptic cholinergic neurones in this culture system is discussed.

The sleep lipid oleamide may represent an endogenous anticonvulsant: an in vitro comparative study in the 4-aminopyridine rat brain-slice model.

cis-Oleamide (cOA) is a putative endocannabinoid, which modulates GABA(A) receptors, Na+ channels and gap-junctions (important targets for clinical and experimental anticonvulsants). Here we address the hypothesis that cOA possesses seizure limiting properties and might represent an endogenous anticonvulsant. Field potentials were recorded from the rat hippocampus and visual cortex. The effects of cOA, were compared to carbamazepine (CBZ), pentobarbital (PB) and carbenoxolone (CRX) on 4-Aminopyridine(4AP)-induced epileptiform discharges. CBZ (100 microM), PB (50 microM) and CRX (100 microM), but not cOA (64 microM), significantly attenuated the duration of the evoked epileptiform discharges in CA1. Interictal activity in CA3 was significantly depressed by CRX and cOA (irreversible by AM251), increased by CBZ and remained unaffected by PB. CBZ, PB and CRX abolished spontaneous ictal events and attenuated evoked ictal discharges in the visual cortex. cOA did not abolish spontaneous ictal events, but significantly (albeit weakly) reduced the duration of evoked ictal events. cOA and CRX, in contrast to CBZ or PB, caused a significant delay in the development of the evoked (tonic phase) epileptiform discharges. The weak effects of cOA seem independent of cannabinoid (CB1) receptors. Enzymatic cleavage and lack of specific antagonists for cOA confound simple interpretations of its actions in slices. Its high lipophilicity, imposing a permeability barrier, may also explain the lack of anticonvulsant activity. The effects of cOA may well be masked by release of the endogenous ligand upon ictal depolarisation as we demonstrate here for established endocannabinoids. cOA does not possess profound antiepileptic actions in our hands compared to CBZ, PB or CRX.