Degassing of early-formed planetesimals restricted water delivery to Earth.

The timing of delivery and the types of body that contributed volatiles to the terrestrial planets remain highly debated1,2. For example, it is unknown if differentiated bodies, such as that responsible for the Moon-forming giant impact, could have delivered substantial volatiles3,4 or if smaller, undifferentiated objects were more probable vehicles of water delivery5-7. Here we show that the water contents of minerals in achondrite meteorites (mantles or crusts of differentiated planetesimals) from both the inner and outer portions of the early Solar System are ≤2 µg g-1 H2O. These are among the lowest values ever reported for extraterrestrial minerals. Our results demonstrate that differentiated planetesimals efficiently degassed before or during melting. This finding implies that substantial amounts of water could only have been delivered to Earth by means of unmelted material.

From cannabis to the endocannabinoid system: refocussing attention on potential clinical benefits.

Cannabis sativa is one of the oldest herbal remedies known to man. Over the past four thousand years, it has been used for the treatment of numerous diseases but due to its psychoactive properties, its current medicinal usage is highly restricted. In this review, we seek to highlight advances made over the last forty years in the understanding of the mechanisms responsible for the effects of cannabis on the human body and how these can potentially be utilized in clinical practice. During this time, the primary active ingredients in cannabis have been isolated, specific cannabinoid receptors have been discovered and at least five endogenous cannabinoid neurotransmitters (endocannabinoids) have been identified. Together, these form the framework of a complex endocannabinoid signalling system that has widespread distribution in the body and plays a role in regulating numerous physiological processes within the body. Cannabinoid ligands are therefore thought to display considerable therapeutic potential and the drive to develop compounds that can be targeted to specific neuronal systems at low enough doses so as to eliminate cognitive side effects remains the 'holy grail' of endocannabinoid research.

Leptin enhances NR2B-mediated N-methyl-D-aspartate responses via a mitogen-activated protein kinase-dependent process in cerebellar granule cells.

It is well documented that the hormone leptin regulates energy balance via its actions in the hypothalamus. However, evidence is accumulating that leptin plays a key role in numerous CNS functions. Indeed, leptin receptors are expressed in many extrahypothalamic brain regions, with high levels found in the hippocampus and cerebellum. In the hippocampus leptin has been shown to facilitate N-methyl-D-aspartate receptor function and modulate synaptic plasticity. A role for leptin in cerebellar function is also indicated as leptin-deficient rodents display reduced mobility that is unrelated to obesity. Here we show that leptin receptor immunolabeling can be detected in cultured cerebellar granule cells, being expressed at the somatic plasma membrane and also concentrated at synapses. Furthermore, leptin facilitated NR2B N-methyl-D-aspartate receptor-mediated Ca2+ influx in cerebellar granule cells via a mitogen-activated protein kinase-dependent pathway. These findings provide the first direct evidence for a cellular action of leptin in cerebellar neurons. In addition, given that N-methyl-D-aspartate receptor activity in the cerebellum is crucial for normal locomotor function, these data also have important implications for the potential role of leptin in the control of movement.

Leptin enhances NMDA receptor function and modulates hippocampal synaptic plasticity.

The obese gene product leptin is an important signaling protein that regulates food intake and body weight via activation of the hypothalamic leptin receptor (Ob-Rb; Jacob et al., 1997). However, there is growing evidence that Ob-Rb is also expressed in CNS regions, not directly associated with energy homeostasis (Mercer et al., 1996; Hakansson et al., 1998). In the hippocampus, an area of the brain involved in learning and memory, we have found that leptin facilitates the induction of synaptic plasticity. Leptin converts short-term potentiation of synaptic transmission induced by primed burst stimulation of the Schaffer collateral commissural pathway into long-term potentiation. The mechanism underlying this effect involves facilitation of NMDA receptor function because leptin rapidly enhances NMDA-induced increases in intracellular Ca(2+) levels ([Ca(2+)](i)) and facilitates NMDA, but not AMPA, receptor-mediated synaptic transmission. The signaling mechanism underlying these effects involves activation of phosphoinositide 3-kinase, mitogen-activated protein kinase, and Src tyrosine kinases. These data indicate that a novel action of leptin in the CNS is to facilitate hippocampal synaptic plasticity via enhanced NMDA receptor-mediated Ca(2+) influx. Impairment of this process may contribute to the cognitive deficits associated with diabetes mellitus.

Role of Ca2+ stores in metabotropic L-glutamate receptor-mediated supralinear Ca2+ signaling in rat hippocampal neurons.

The role of metabotropic l-glutamate (mGlu) receptors in supralinear Ca(2+) signaling was investigated in cultured hippocampal cells using Ca(2+) imaging techniques and whole-cell voltage-clamp recording. In neurons, but not glia, global supralinear Ca(2+) release from intracellular stores was observed when the mGlu receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) was combined with elevated extracellular K(+) levels (10.8 mm), moderate depolarization (15-30 mV), or NMDA (3 micrometer). There was a delay (2-8 min) before the stores were fully charged, and the enhancement persisted for a short period (up to 10 min) after removal of the store-loading stimulus. Studies with the mGlu receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine demonstrated that these effects were mediated by activation of the mGlu(5) receptor subtype. The L-type voltage-gated Ca(2+) channel antagonist nifedipine (10 micrometer) substantially reduced responses to DHPG obtained in the presence of elevated extracellular K(+) but not NMDA. This suggests that the Ca(2+) that is required to load the stores can enter either through L-type voltage-gated Ca(2+) channels or directly through NMDA receptors. The findings that both depolarization and NMDA receptor activation can facilitate mGlu receptor Ca(2+) signaling adds considerable flexibility to the processes that underlie activity-dependent changes in synaptic strength. In particular, a temporal separation between the store-loading stimulus and the activation of mGlu receptors could be used as a recency detector in neurons.

Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons.

Agonist-induced internalization of G-protein-coupled receptors is an important mechanism for regulating receptor abundance and availability at the plasma membrane. In this study we have used immunolabeling techniques and confocal microscopy to investigate agonist-induced internalization and trafficking of CB(1) receptors in rat cultured hippocampal neurons. The levels of cell surface CB(1) receptor immunoreactivity associated with presynaptic GABAergic terminals decreased markedly (by up to 84%) after exposure to the cannabinoid agonist (+)-WIN55212, in a concentration-dependent (0.1-1 microm) and stereoselective manner. Inhibition was maximal at 16 hr and abolished in the presence of SR141716A, a selective CB(1) receptor antagonist. Methanandamide (an analog of an endogenous cannabinoid, anandamide) also reduced cell surface labeling (by 43% at 1 microm). Differential labeling of cell surface and intracellular pools of receptor demonstrated that the reduction in cell surface immunoreactivity reflects agonist-induced internalization and suggests that the internalized CB(1) receptors are translocated toward the soma. The internalization process did not require activated G-protein alpha(i) or alpha(o) subunits. A different pattern of cell surface CB(1) receptor expression was observed using an undifferentiated F-11 cell line, which had pronounced somatic labeling. In these cells substantial CB(1) receptor internalization was also observed after exposure to (+)-WIN55212 (1 microm) for relatively short periods (30 min) of agonist exposure. In summary, this dynamic modulation of CB(1) receptor expression may play an important role in the development of cannabinoid tolerance in the CNS. Agonist-induced internalization at presynaptic terminals has important implications for the modulatory effects of G-protein-coupled receptors on neurotransmitter release.

L-glutamate and acetylcholine mobilise Ca2+ from the same intracellular pool in cerebellar granule cells using transduction mechanisms with different Ca2+ sensitivities.

Ca2+ mobilisation induced by L-glutamate (Glu) and acetylcholine (ACh) has been studied in cultured cerebellar granule cells using digital fluorescence microscopy. The ability of Glu-receptor activation to mobilise Ca2+ was decreased when [Ca2+]o was lowered to 10 microM (from 1.8 mM). It was enhanced when [Ca2+]i was raised using 25 mM external K+ or by N-methyl-D-aspartate (NMDA), which selectively activates a distinct Glu-receptor subtype. The enhancement was dependent on entry of external Ca2+. In contrast, the ability of ACh receptor activation to mobilise Ca2+ was not affected by these conditions. Furthermore, pretreatment with pertussis toxin inhibited Ca2+ mobilisation in response to Glu-receptor activation without affecting mobilisation in response to ACh. However, activation of both receptors mobilised Ca2+ from a common, thapsigargin-sensitive pool. We conclude that there are differences in the Ca2+ mobilization pathways for the two receptor systems in cerebellar granule cells. The Ca(2+)-sensitivity of this Ca2+ mobilizing Glu receptor may have implications for its function in neuronal synaptogenesis and plasticity.

Activation of the NO-cGMP signalling pathway depresses hippocampal synaptic transmission through an adenosine receptor-dependent mechanism.

Activation of the NO-cGMP pathway or adenosine receptors depresses reversibly synaptic transmission in the hippocampus. Here we demonstrate, using the selective A1 receptor antagonist DPCPX, a convergence in the mechanisms of action of the NO donor SNAP, the cGMP phosphodiesterase inhibitor zaprinast and adenosine.

The group I mGlu receptor agonist DHPG induces a novel form of LTD in the CA1 region of the hippocampus.

The group I specific metabotropic glutamate (mGlu) receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) (100 microM, 10 min) induced long-term depression (LTD) of synaptic transmission in the CA1 region of adult rat hippocampal slices, measured using a grease-gap recording technique. In "normal" (1 mM Mg2+-containing) medium, LTD (measured 30 min after washout of DHPG) was small (13+/-3%), but LTD was enhanced if DHPG was applied when the tissue was made hyperexcitable, either by omitting Mg2+ from the perfusate (35+/-3%) or by adding the GABA(A) receptor antagonist picrotoxin (29+/-2%). The N-methyl-D-aspartate (NMDA) receptor antagonist AP5 (100 microM) substantially reduced the generation of DHPG-induced LTD in Mg2+-free medium, but had little effect on LTD induced in the presence of picrotoxin. In Mg2+-free medium, the threshold concentration of DHPG required to induce LTD was between 1 and 3 microM. Neither agonists specific for group II (100 nM DCG-IV or 1 microM LY354740) or group III (10 microM L-AP4) mGlu receptors or a combined group I and II agonist (30-100 microM (1S,3R)-ACPD) induced LTD. However, an agonist (1 mM CHPG) which activates mGlu5 but not mGlu1 receptors did induce LTD. Surprisingly, DHPG-induced LTD was reversed by mGlu receptor antagonists, applied hours after washout of DHPG. DHPG-induced LTD did not occlude with LTD induced by synaptic activation (1200 stimuli delivered at 2 Hz), in Mg2+-free medium. These data show that activation of group I mGlu receptors (probably mGlu5) can induce LTD and that this mGlu receptor-mediated LTD may, or may not, require activation of NMDA receptors, depending on the experimental conditions.

Actions of cannabinoid receptor ligands on rat cultured sensory neurones: implications for antinociception.

Cannabinoids modulate nociceptive processing in models of acute, inflammatory and neuropathic pain. We have investigated the location and function of cannabinoid receptors on cultured neonatal dorsal root ganglion (DRG) neurones and F-11 cells, a dorsal root ganglionxneuroblastoma hybridoma which displays several of the features of authentic DRG neurones. CB(1) receptor immunolabelling was observed on the cell bodies and as fine puncta on processes of both cultured DRG neurones and F-11 cells. Additionally, fluorescence-activated cell sorting (FACS) analysis provided evidence that both CB(1) and CB(2) receptors are expressed on populations of cells within the cultured DRG and F-11 cells. The cannabinoid receptor agonist (+)-WIN55212 (10 and 100 nM) inhibited the mean voltage-activated Ca(2+) current in DRG neurones by 21% and 30%, respectively. The isomer, (-)-WIN55212 (10 and 100 nM) produced significantly less inhibition of 6% and 10% respectively. The CB(1) selective receptor antagonist SR141716A (100 nM) enhanced the peak high voltage-activated Ca(2+) current by 24% and simultaneous application of SR141716A (100 nM) and (+)-WIN55212 (100 nM) resulted in a significant attenuation of the inhibition obtained with (+)-WIN55212 alone. These data give functional evidence for the hypothesis that the analgesic actions of cannabinoids may be mediated by presynaptic inhibition of transmitter release in sensory neurones.

Localization of the glutamate receptor subunit GluR1 on the surface of living and within cultured hippocampal neurons.

The distribution of the glutamate receptor subunit GluR1 was investigated in cultured hippocampal neurons by confocal microscopy, using polyclonal antibodies directed against either the N- or C-terminal region. On living neurons, GluR1 immunofluorescence was detected with the N-terminal antibody only. GluR1 was localized in a highly punctate manner on the surface of neuronal soma and throughout the dendritic tree. Many GluR1 puncta co-localized with the synaptic marker synaptophysin, although extrasynaptic GluR1 puncta were also observed. A comparison of GluR1 subunit distribution of living neurons labelled with N-terminal antibody with that obtained after the cells had been fixed, permeabilized and subsequently reacted with C-terminal or additional N-terminal antibody showed a number of differences. In permeabilized cells additional, diffuse labelling was observed which was very pronounced in the soma and extended into the proximal dendrites. Furthermore, some spines showed little or no labelling of their membrane surface, but labelled strongly after the cells had been fixed and permeabilized. Such spines may be the postsynaptic components of silent or suboptimal synapses.

Functional expression of cell surface cannabinoid CB(1) 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.

NMDA receptor dependence of mGlu-mediated depression of synaptic transmission in the CA1 region of the rat hippocampus.

1. The depression of synaptic transmission by the specific metabotropic glutamate receptor (mGlu) agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD) was investigated in area CA1 of the hippocampus of 4-10 week old rats, by use of grease-gap and intracellular recording techniques. 2. In the presence of 1 mM Mg2+, (1S,3R)-ACPD was a weak synaptic depressant. In contrast, in the absence of added Mg2+, (1S,3R)-ACPD was much more effective in depressing both the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated components of synaptic transmission. At 100 microM, (1S,3R)-ACPD depressed the slope of the field excitatory postsynaptic potential (e.p.s.p.) by 96 +/- 1% (mean +/- s.e.mean; n = 7) compared with 23 +/- 4% in 1 mM Mg(2+)-containing medium (n = 17). 3. The depressant action of 100 microM (1S,3R)-ACPD in Mg(2+)-free medium was reduced from 96 +/- 1 to 46 +/- 6% (n = 7) by the specific NMDA receptor antagonist (R)-2-amino-5-phosphonopentanoate (AP5; 100 microM). 4. Blocking both components of GABA receptor-mediated synaptic transmission with picrotoxin (50 microM) and CGP 55845A (1 microM) in the presence of 1 mM Mg2+ also enhanced the depressant action of (1S,3R)-ACPD (100 microM) from 29 +/- 5 to 67 +/- 6% (n = 6). 5. The actions of (1S,3R)-ACPD, recorded in Mg(2+)-free medium, were antagonized by the mGlu antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG). Thus, depressions induced by 30 microM (1S,3R)-ACPD were reversed from 48 +/- 4 to 8 +/- 6% (n = 4) by 1 mM (+)-MCPG. 6. In Mg(2+)-free medium, a group I mGlu agonist, (RS)-3, 5-dihydroxyphenylglycine (DHPG; 100 microM) depressed synaptic responses by 74 +/- 2% (n = 18). In contrast, neither the group II agonists ((2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine; L-CCG-1; 10 microM; n = 4) and ((2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine; DCG-IV; 100 nM; n = 3) nor the group III agonist ((S)-2-amino-4-phosphonobutanoic acid; L-AP4; 10 microM; n = 4) had any effect. 7. The depolarizing action of (1S,3R)-ACPD, recorded intracellularly, was similar in the presence and absence of Mg(2+)-AP5 did not affect the (1S,3R)-ACPD-induced depolarization in Mg(2+)-free medium. Thus, 50 microM (1S,3R)-ACPD induced depolarizations of 9 +/- 3 mV (n = 5), 10 +/- 2 mV (n = 4) and 8 +/- 2 mV (n = 5) in the three respective conditions. 8. On resetting the membrane potential in the presence of 50 microM (1S,3R)-ACPD to its initial level, the e.p.s.p. amplitude was enhanced by 8 +/- 3% in 1 mM Mg2+ (n = 5) compared with a depression of 37 +/- 11% in the absence of Mg2+ (n = 4). Addition of AP5 prevented the (1S,3R)-ACPD-induced depression of the e.p.s.p. (depression of 4 +/- 5% (n = 5)). 9. It is concluded that activation by group 1 mGlu agonists results in a depression of excitatory synaptic transmission in an NMDA receptor-dependent manner.

Low-frequency activation of the NMDA receptor system can prevent the induction of LTP.

In rat hippocampal slices bathed in Mg2(+)-free medium tetanic stimulation generally failed to elicit long-term potentiation (LTP) in the Schaffer collateral-commissural pathway. However, LTP could be induced in the same slices following the re-introduction of 1 mM Mg2+. In Mg2(+)-free medium, LTP could be induced in the presence of 20 microM D-2-amino-5-phosphonovalerate (APV) but was blocked by increasing the APV concentration to 200 microM. The lower concentration of APV is sufficient to prevent N-methyl-D-aspartate (NMDA) receptor activation during low-frequency transmission but not during the tetanus. We suggest therefore, that certain types of activation of the NMDA receptor system can disable the LTP induction mechanism.

Activation of group I mGluRs potentiates NMDA responses in rat hippocampal slices.

The pharmacology of the metabotropic glutamate receptor (mGluR)-mediated potentiation of N-methyl-D-aspartate (NMDA)-evoked depolarisations in the CA1 region of rat hippocampal slices was investigated using an extracellular grease-gap method. The group I and II mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD; 10 mu M) potentiated responses to NMDA (15-25 mu M), giving a dose ratio of 0.84 +/- 0.02. The mGluR group I specific agonist (RS3,5-dihydroxyphenylglycine (DHPG) (3-10 mu M) also induced a dose-dependent and reversible enhancement of responses to NMDA (dose ratio for 10 mu M DHPG was 0.77 +/- 0.02). In contrast, the group II selective agonist (2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV; 0.5-1 mu M) and the group III specific agonist (S)-2-amino-4-phosphonobutanoate (L-AP4; 50 mu M) caused little or no potentiation of responses to NMDA. The potentiation induced by 3-5 mu M DHPG was reversibly antagonised by the group I and II antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG; 1 mM). The present findings demonstrate that activation of group I mGluRs enhance NMDA responses in the hippocampus.

Differential trafficking of adenosine receptors in hippocampal neurons monitored using GFP- and super-ecliptic pHluorin-tagged receptors.

Adenosine receptors (ARs) modulate many cellular and systems-level processes in the mammalian CNS. However, little is known about the trafficking of ARs in neurons, despite their importance in controlling seizure activity and in neuroprotection in cerebral ischaemia. To address this we examined the agonist-dependent internalisation of C-terminal GFP-tagged A(1)Rs, A(2A)Rs and A(3)Rs in primary hippocampal neurons. Furthermore, we developed a novel super-ecliptic pHluorin (SEP)-tagged A(1)R which, via the N-terminal SEP tag, reports the cell-surface expression and trafficking of A(1)Rs in real-time. We demonstrate the differential trafficking of ARs in neurons: A(3)Rs internalise more rapidly than A1Rs, with little evidence of appreciable A(2A)R trafficking over the time-course of the experiments. Furthermore, the novel SEP-A(1)R construct revealed the time-course of internalisation and recovery of cell-surface expression to occur within minutes of agonist exposure and removal, respectively. These observations highlight the labile nature of A(1)R and A(3)Rs when expressed at the neuronal plasma membrane. Given the high levels of adenosine in the brain during ischaemia and seizures, internalisation of the inhibitory A(1)R may result in hyperexcitability, increased brain damage and the development of chronic epileptic states.

Fluorescent ligand binding reveals heterogeneous distribution of adrenoceptors and 'cannabinoid-like' receptors in small arteries.

BACKGROUND AND PURPOSE: Pharmacological analysis of synergism or functional antagonism between different receptors commonly assumes that interacting receptors are located in the same cells. We have now investigated the distribution of alpha-adrenoceptors, beta-adrenoceptors and cannabinoid-like (GPR55) receptors in the mouse arteries. EXPERIMENTAL APPROACH: Fluorescence intensity from vascular tissue incubated with fluorescent ligands (alpha(1)-adrenoceptor ligand, BODIPY-FL-prazosin, QAPB; beta-adrenoceptor ligand, TMR-CGP12177; fluorescent angiotensin II; a novel diarylpyrazole cannabinoid ligand (Tocrifluor 1117, T1117) was measured with confocal microscopy. Small mesenteric and tail arteries of wild-type and alpha(1B/D)-adrenoceptor-KO mice were used. KEY RESULTS: T1117, a fluorescent form of the cannabinoid CB(1) receptor antagonist AM251, was a ligand for GPR55, with low affinity for CB(1) receptors. In mesenteric arterial smooth muscle cells, alpha(1A)-adrenoceptors were predominantly located in different cells from those with beta-adrenoceptors, angiotensin receptors or cannabinoid-like (GPR55) receptors. Cells with beta-adrenoceptors predominated at arterial branches. Endothelial cells expressed beta-adrenoceptors, alpha-adrenoceptors and cannabinoid-like receptors. Only endothelial alpha-adrenoceptors appeared in clusters. Adventitia was a rich source of G protein-coupled receptors (GPCRs), particularly fibroblasts and nerve tracts, where Schwann cells bound alpha-adrenoceptor, beta-adrenoceptor and CB-receptor ligands, with a mix of separate receptor locations and co-localization. CONCLUSIONS AND IMPLICATIONS: Within each cell type, each GPCR had a distinctive heterogeneous distribution with limited co-localization, providing a guide to the possibilities for functional synergism, and suggesting a new paradigm for synergism in which interactions may be either between cells or involve converging intracellular signalling processes.