Biomedical subjects
Publications and source records attributed to Bradley E Alger.
Endocannabinoids can mediate neuroprotection, but it is not known how. In this issue of Neuron, Monory et al. use mutant mice and localized viral targeting to produce conditional knockouts of the cannabinoid CB1 receptor. They show that protection against kainic acid-induced seizures and cell death is conferred by CB1Rs on hippocampal glutamatergic nerve terminals.
Endogenous cannabinoids (eCBs) are produced and mobilized in a cytosolic calcium ([Ca2+]i)-dependent manner, and they regulate excitatory and inhibitory neurotransmitter release by acting as retrograde messengers. An indirect but real-time bioassay for this process on GABAergic transmission is DSI (depolarization-induced suppression of inhibition). The magnitude of DSI correlates linearly with depolarization-induced increase of [Ca2+]i that is thought to be initiated by Ca2+ influx through voltage-gated Ca2+ channels. However, the identity of Ca2+ sources involved in eCB mobilization in DSI remains undetermined. Here we show that, in CA1 pyramidal cells, DSI-inducing depolarizing voltage steps caused Ca2+-induced Ca2+ release (CICR) by activating the ryanodine receptor (RyR) Ca2+-release channel. CICR was reduced, and the remaining increase in [Ca2+]i was less effective in generating DSI, when the RyR antagonists, ryanodine or ruthenium red, were applied intracellularly, or the Ca2+ stores were depleted by the Ca2+-ATPase inhibitors, cyclopiazonic acid or thapsigargin. The CICR-dependent effects were most prominent in cultured or immature acute slices, but were also detectable in slices from adult tissue. Thus we suggest that voltage-gated Ca2+ entry raises local [Ca2+]i sufficiently to activate nearby RyRs and that the resulting CICR plays a critical role in initiating eCB mobilization. RyR may be a key molecule for the depolarization-induced production of eCBs that inhibit GABA release in the hippocampus.
Endocannabinoids are a class of fatty acid derivatives defined by their ability to interact with the specific cannabinoid receptors that were originally identified as the targets of Delta9-tetrahydocannabinol (Delta9-THC), the psychoactive component of cannabis. Endocannabinoids have been implicated in a growing number of important physiological and behavioral events. A full understanding of the functions of endocannabinoids will involve knowing which ones are active, and how they are produced, during any given physical event. However, studying these small lipids in the brain presents many technical challenges. New selective pharmacological tools promise to be very useful in unraveling the complexities of endocannabinoid signaling, but parallel developments from the investigation of the cellular neurophysiology of the endocannabinoid systems highlight the difficulties remaining.
Intercellular signaling dynamics critically influence the functional roles that the signals can play. Small lipids are synthesized and released from neurons, acting as intercellular signals in regulating neurotransmitter release, modulating ion channels on target cells, and modifying synaptic plasticity. The repertoire of biological effects of lipids such as endocannabinoids (eCBs) is rapidly expanding, yet lipid signaling dynamics have not been studied. The eCB system constitutes a powerful tool for bioassaying the dynamics of lipid signaling. The eCBs are synthesized in, and released from, postsynaptic somatodendritic domains that are readily accessible to whole-cell patch electrodes. The dramatic effects of these lipid signals are detected electrophysiologically as CB1-dependent alterations in conventional synaptic transmission, which therefore serve as a sensitive reporter of eCB actions. We used electrophysiological recording, photolytic release of caged glutamate and a newly developed caged AEA (anandamide), together with rapid [Ca2+]i measurements, to investigate the dynamics of retrograde eCB signaling between CA1 pyramidal cells and GABAergic synapses in rat hippocampus in vitro. We show that, at 22 degrees C, eCB synthesis and release must occur within 75-190 ms after the initiating stimulus, almost an order of magnitude faster than previously thought. At 37 degrees C, the time could be < 50 ms. Activation of CB1 and downstream processes constitute a significant fraction of the total delay and are identified as major rate-limiting steps in retrograde signaling. Our findings imply that lipid messenger dynamics are comparable with those of metabotropic neurotransmitters and can modulate neuronal interactions on a similarly fast time scale.
Endocannabinoids (eCBs) act as retrograde messengers at inhibitory synapses of the hippocampal CA1 region. Current models place eCB synthesis in the postsynaptic pyramidal cell and the site of eCB action at cannabinoid receptors located on presynaptic interneuron terminals. Four responses at the CA1-interneuron synapse are attributed to eCBs: depolarization-induced suppression of inhibition (DSI), G-protein-coupled receptor-mediated enhancement of DSI (DeltaDSI), persistent suppression of evoked inhibitory postsynaptic currents (eIPSCs), and finally, mGluR-dependent long-term depression (iLTD). It has been proposed that all are mediated by the eCB, 2-arachidonoyl glycerol, yet there is evidence that DSI does not arise from the same underlying biochemical processes as the other responses. In view of the increasing importance of eCB effects in the brain, it will be essential to understand the mechanisms by which eCB effects are produced. Our results reveal new differences in the biochemical pathways by which the eCB-dependent responses are initiated. Both U73122, a phospholipase C antagonist, and RHC-80267, a diacylglycerol (DAG) lipase antagonist, prevented eCB-dependent iLTD induction by 3,5-dihydroxyphenylglycine (DHPG). However, mAChR activation does not cause eCB-dependent iLTD. Neither enzyme inhibitor affects DSI, and persistent eCB-dependent eIPSC suppression induced by either mGluRs or mAChRs is unaffected by U73122. On the other hand, inhibition of DAG lipase prevents persistent eCB-dependent eIPSC suppression triggered by mAChRs. The results show that the biochemical pathways for the various eCB-dependent responses differ and might therefore be independently manipulated.
Theta rhythms are behaviorally relevant electrical oscillations in the mammalian brain, particularly the hippocampus. In many cases, theta oscillations are shaped by inhibitory postsynaptic potentials (IPSPs) that are driven by glutamatergic and/or cholinergic inputs. Here we show that hippocampal theta rhythm IPSPs induced in the CA1 region by muscarinic acetylcholine receptors independent of all glutamate receptors can be briefly interrupted by action potential-induced, retrograde endocannabinoid release. Theta IPSPs can be recorded in CA1 pyramidal cell somata surgically isolated from CA3, subiculum, and even from their own apical dendrites. These results suggest that perisomatic-targeting interneurons whose output is subject to inhibition by endocannabinoids are the likely source of theta IPSPs. Interneurons having these properties include the cholecystokinin-containing cells. Simultaneous recordings from pyramidal cell pairs reveal synchronous theta-frequency IPSPs in neighboring pyramidal cells, suggesting that these IPSPs may help entrain or modulate small groups of pyramidal cells.
The dentate gyrus is a key input gateway for the hippocampus, and dentate function is potently regulated by GABAergic inhibition. GABAergic inhibition is plastic and modulated by many factors. Cytoplasmic calcium ([Ca(+)](i)) is one of these factors, and its elevation inhibits GABA-mediated transmission in the hippocampus including the dentate gyrus granule cells (DGCs). We examined whether the [Ca(+)](i)-dependent decrease of GABA(A) receptor-mediated inhibitory postsynaptic current (IPSC) is explained by the retrograde suppression of GABA release caused by the depolarization-induced elevation of [Ca(+)](i) in DGCs (DSI: depolarization-induced suppression of inhibition). Repeated brief depolarizations or a single long depolarization inhibited spontaneous IPSCs with amplitudes over 25 pA for up to a minute, and reduced the amplitude of IPSCs evoked by direct stimulation in the molecular layer, suggesting that DGCs are susceptible to DSI. The magnitude of DSI correlated linearly with the duration of depolarization, and so did the increase of [Ca(+)](i). DSI was blocked by intrapipette application of BAPTA. In addition, bath application of thapsigargin and ryanodine, and intrapipette application of ryanodine and ruthenium red reduced the [Ca(+)](i) increase caused by the DSI-inducing depolarization, and substantially reduced the magnitude of DSI. Finally, the cannabinoid receptor agonists, CP55,942 and WIN55,212-2, mimicked DSI and prevented further IPSC reduction by DSI. DSI was blocked by the antagonist, SR141716A. We conclude that GABAergic inhibition in DGCs is subject to endogenous cannabinoid (eCB)-mediated retrograde regulation, and this process involves a depolarization-initiated release of Ca(+) from ryanodine-sensitive stores. Our findings suggest eCBs probably have physiological functions in the regulation of GABAergic plasticity in the dentate gyrus.
In hippocampal pyramidal cells, a rise in Ca(2+) releases endocannabinoids that activate the presynaptic cannabinoid receptor (CB1R) and transiently reduce GABAergic transmission-a process called depolarization-induced suppression of inhibition (DSI). The mechanism that limits the duration of endocannabinoid action in intact cells is unknown. Here we show that inhibition of cyclooxygenase-2 (COX-2), not fatty acid amide hydrolase (FAAH), prolongs DSI, suggesting that COX-2 limits endocannabinoid action.
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We tested the hypothesis that transient, partial inhibition of the Na,K-pumps could produce lasting effects on synaptic efficacy in brain tissue by applying a low concentration of the ouabain analogue, dihydroouabain (DHO), to hippocampal slices for 15 min and studying the effects on field excitatory postsynaptic potentials (fEPSPs). DHO caused a suppression of fEPSPs during the application period, but this recovered only partially, to approximately 80% of control levels, after washout lasting as long as 2 h. The lasting suppression had several properties in common with low-frequency stimulation induced long-term depression (LFS-LTD), including an ability to depotentiate long-term potentiated responses. However, DHO-LTD was insensitive to blockade of N-methyl-d-aspartate or mGlu receptors or to inhibitors of protein kinase C or p38 MAP kinase. DHO-LTD did not co-occlude with LFS-LTD and therefore appears to represent a novel form of LTD. Interestingly, DHO-LTD could be prevented by pretreating slices with iberiotoxin, the selective blocker of large, Ca(2+)-dependent K+ channels ("big K," BK channels), although this toxin did not affect basal fEPSPs. Certain pathological conditions, including hypoxia and ischemia, are associated with a decrease in Na,K-pump activity and hence DHO-LTD may serve as a model for the effects on neuronal function in these conditions.
Regulation of GABA release is crucial for normal brain functioning, and GABAA-mediated IPSCs are strongly influenced by repetitive stimulation and neuromodulation. However, GABA exocytosis has not been examined directly in organized tissue. Important issues remain outside the realm of electrophysiological techniques or are complicated by postsynaptic factors. For example, it is not known whether all presynaptic modulators affect release from all boutons in the same way, or whether modulator effects depend on the presence of certain types of voltage-gated calcium channels (VGCCs). To address such issues, we used confocal imaging and styryl dyes to monitor exocytosis from identified GABAergic boutons in organotypic hippocampal slice cultures. Repetitively evoked IPSCs declined more rapidly and completely than exocytosis, suggesting that depletion of filled vesicles cannot fully account for IPSC depression and underscoring the usefulness of directly imaging exocytosis. Stimulation at 10 Hz produced a transient facilitation of exocytosis that was dependent on L-type VGCCs. Using specific toxins, we found that release mediated via N-type and P-type VGCCs had similar properties. Neither baclofen nor a cannabinoid receptor agonist, CP55940, affected all boutons uniformly; they slowed release from some but completely prevented detectable release from others. Increasing stimulus frequency overcame this blockade of release. However, baclofen and CP55940 did not act identically, because only baclofen reduced facilitation and affected bouton releasing via P/Q-type VGCCs. Direct observation thus revealed novel features of GABAergic exocytosis and its regulation that would have been difficult or impossible to detect electrophysiologically. These features advance the understanding of the regulation of synapses and networks by presynaptic inhibition.
Endogenous cannabinoids (endocannabinoids) are endogenous compounds that resemble the active ingredient of marijuana and activate the cannabinoid receptor in the brain. They mediate retrograde signaling from principal cells to both inhibitory ["depolarization-induced suppression of inhibition" (DSI)] and excitatory ("depolarization-induced suppression of excitation") afferent fibers. Transient endocannabinoid release is triggered by voltage-dependent Ca(2+) influx and is upregulated by group I metabotropic glutamate receptor activation. Here we show that muscarinic acetylcholine receptor (mAChR) activation also enhances transient endocannabinoid release (DSI) and induces persistent release. Inhibitory synapses in the rat hippocampal CA1 region of acute slices were studied using whole-cell patch-clamp techniques. We found that low concentrations (0.2-0.5 microm) of carbachol (CCh) enhanced DSI without affecting basal evoked IPSCs (eIPSCs) by activating mAChRs on postsynaptic cells. Higher concentrations of CCh (> or =1 microm) enhanced DSI and also persistently depressed basal eIPSCs, mainly by releasing endocannabinoids. Persistent CCh-induced endocannabinoid release did not require an increase in [Ca2+]i but was dependent on G-proteins. Although they were independent at the receptor level, muscarinic and glutamatergic mechanisms of endocannabinoid release shared intracellular machinery. Replication of the effects of CCh by blocking acetylcholinesterase with eserine suggests that mAChR-mediated endocannabinoid release is physiologically relevant. This study reveals a new role of the muscarinic cholinergic system in mammalian brain.
This review covers recent developments in the cellular neurophysiology of retrograde signaling in the mammalian central nervous system. Normally at a chemical synapse a neurotransmitter is released from the presynaptic element and diffuses to the postsynaptic element, where it binds to and activates receptors. In retrograde signaling a diffusible messenger is liberated from the postsynaptic element, and travels "backwards" across the synaptic cleft, where it activates receptors on the presynaptic cell. Receptors for retrograde messengers are usually located on or near the presynaptic nerve terminals, and their activation causes an alteration in synaptic transmitter release. Although often considered in the context of long-term synaptic plasticity, retrograde messengers have numerous roles on the short-term regulation of synaptic transmission. The focus of this review will be on a group of molecules from different chemical classes that appear to act as retrograde messengers. The evidence supporting their candidacy as retrograde messengers is considered and evaluated. Endocannabinoids have recently emerged as one of the most thoroughly investigated, and widely accepted, classes of retrograde messenger in the brain. The study of the endocannabinoids can therefore serve as a model for the investigation of other putative messengers, and most attention is devoted to a discussion of systems that use these new messenger molecules.
Exogenous cannabinoids disrupt behavioral learning and impede induction of long-term potentiation (LTP) in the hippocampus, yet endogenous cannabinoids (endocannabinoids) transiently suppress inhibitory post-synaptic currents (IPSCs) by activating cannabinoid CB1 receptors on GABAergic interneurons. We found that release of endocannabinoids by a rat CA1 pyramidal cell during this depolarization-induced suppression of inhibition (DSI) enabled a normally ineffective train of excitatory post-synaptic currents (EPSCs) to induce LTP in that cell, but not in neighboring cells. By showing that endocannabinoids facilitate LTP induction and help target LTP to single cells, these data shed new light on the physiological roles of endocannabinoids and may lead to a greater understanding of their effects on behavior and potential clinical use.
Extra- and intracellular records were made from rat acute hippocampal slices to examine the effects of partial inhibition of Na(+)-K(+)-ATPases (Na(+)-K(+) pumps) on neuronal hyperexcitability. Bath application of the low-affinity cardiac glycoside, dihydroouabain (DHO), reversibly induced interictal-like epileptiform bursting activity in the CA1 region. Burst-firing was correlated with inhibition of the pumps, which was assayed by changes in [K(+)](o) uptake rates measured with K(+)-ion-sensitive microelectrodes. Large increases in resting [K(+)](o) did not occur. DHO induced a transient depolarization (5-6 mV) followed by a long-lasting hyperpolarization (approximately 6 mV) in CA1 pyramidal neurons, which was accompanied by a 30% decrease in resting input resistance. Block of an electrogenic pump current could explain the depolarization but not the hyperpolarization of the membrane. Increasing [K(+)](o) from 3 to 5.5 mM minimized these transient shifts in passive membrane properties without preventing DHO-induced hyperexcitability. DHO decreased synaptic transmission, but increased the coupling between excitatory postsynaptic potentials and spike firing (E-S coupling). Monosynaptic inhibitory postsynaptic potential (IPSP) amplitudes declined to approximately 25% of control at the peak of bursting activity; however, miniature TTX-resistant inhibitory postsynaptic current amplitudes were unaffected. DHO also reduced the initial slope of the intracellular excitatory postsynaptic potential (EPSP) to approximately 40% of control. The conductances of pharmacologically isolated IPSPs and EPSPs in high-Ca/high-Mg-containing saline were also reduced by DHO by approximately 50%. The extracellular fiber volley amplitude was reduced by 15-20%, suggesting that the decrease in neurotransmission was partly due to a reduction in presynaptic fiber excitability. DHO enhanced a late depolarizing potential that was superimposed on the EPSP and could obscure it. This potential was not blocked by antagonists of NMDA receptors, and blockade of NMDA, mGlu, or GABA(A) receptors did not affect burst firing. The late depolarizing component enabled the pyramidal cells to reach spike threshold without changing the actual voltage threshold for firing. We conclude that reduced GABAergic potentials and enhanced E-S coupling are the primary mechanisms underlying the hyperexcitability associated with impaired Na(+)-K(+) pump activity.
This review covers the main features of a newly discovered intercellular signaling system in which endogenous ligands of the brain's cannabinoid receptors, or endocannabinoids, serve as retrograde messengers that enable a cell to control the strength of its own synaptic inputs. Endocannabinoids are released by bursts of action potentials, including events resembling interictal spikes, and probably by seizures as well. Activation of cannabinoid receptors has been implicated in neuroprotection against excitotoxicity and can help explain the anticonvulsant properties of cannabinoids that have been known since antiquity.