Cannabinoids produce neuroprotection by reducing intracellular calcium release from ryanodine-sensitive stores.

Exogenously administered cannabinoids are neuroprotective in several different cellular and animal models. In the current study, two cannabinoid CB1 receptor ligands (WIN 55,212-2, CP 55,940) markedly reduced hippocampal cell death, in a time-dependent manner, in cultured neurons subjected to high levels of NMDA (15 microM). WIN 55,212-2 was also shown to inhibit the NMDA-induced increase in intracellular calcium concentration ([Ca2+](i)) indicated by FURA-2 fluorescence imaging in the same cultured neurons. Changes in [Ca2+](i) occurred with similar concentrations (25-100 nM) and in the same time-dependent manner (pre-exposure 1-15 min) as CB1 receptor mediated neuroprotective actions. Both effects were blocked by the CB1 receptor antagonist SR141716A. An underlying mechanism was indicated by the fact that (1) the NMDA-induced increase in [Ca2+](i) was inhibited by ryanodine, implicating a ryanodine receptor (RyR) coupled intracellular calcium channel, and (2) the cannabinoid influence involved a reduction in cAMP cAMP-dependent protein kinase (PKA) dependent phosphorylation of the same RyR levels that regulate channel. Moreover the time course of CB1 receptor mediated inhibition of PKA phosphorylation was directly related to effective pre-exposure intervals for cannabinoid neuroprotection. Control studies ruled out the involvement of inositol-trisphosphate (IP3) pathways, enhanced calcium reuptake and voltage sensitive calcium channels in the neuroprotective process. The results suggest that cannabinoids prevent cell death by initiating a time and dose dependent inhibition of adenylyl cyclase, that outlasts direct action at the CB1 receptor and is capable of reducing [Ca2+](i) via a cAMP/PKA-dependent process during the neurotoxic event.

Functional significance of cannabinoid-mediated, depolarization-induced suppression of inhibition (DSI) in the hippocampus.

A number of recent studies have demonstrated that a well-known form of short-term plasticity at hippocampal GABAergic synapses, called depolarization-induced suppression of inhibition (DSI), is in fact mediated by the retrograde actions of endocannabinoids released in response to depolarization of the postsynaptic cells. These studies suggest that endogenous cannabinoids may play an important role in regulating inhibitory tone in the mammalian CNS. Despite the widespread interest and potential physiological importance of DSI, many questions regarding the physiological relevance of DSI remain. To that end, this study set out to define the specific limiting conditions that could elicit DSI at GABAergic synapses in CA1 hippocampal pyramidal neurons and to determine if DSI could be elicited with pulse trains that mimic hippocampal cell-firing patterns that occur in vivo. Whole cell recordings from hippocampal neurons under voltage-clamp configuration were made in rat hippocampal slices. Spontaneous and evoked gamma-aminobutyric acid-A (GABAA) receptor-mediated inhibitory postsynaptic currents (sIPSCs and eIPSCs, respectively) were recorded prior to and following depolarization of CA1 hippocampal pyramidal cells. Depolarizing voltage pulses were shaped to evoke currents in QX-314-treated cells similar to those accompanying single spontaneous voltage-clamped action potentials recorded from the soma. Attempts were made to elicit DSI with trains of these pulses that mimicked hippocampal cell firing patterns in vivo, for instance, when animals traverse place fields or are performing a short-term memory task. DSI could not be elicited by such pulse trains or by a number of other combinations of behaviorally specific firing parameters. The minimum duration of depolarization necessary to elicit DSI in hippocampal neurons determined by paired-pulse manipulation was 50 -75 ms at a critical interval of 20 -30 ms between pulse pairs. Under the conditions tested, the normal firing patterns of hippocampal neurons that occur in vivo do not appear to elicit DSI.

Assessment of cannabinoid induced gene changes: tolerance and neuroprotection.

The analysis of gene changes associated with exposure to cannabinoids is critical due to the multiple possible signaling pathways potentially affected by cannabinoid receptor activation. A comparison of altered gene profiles under two different conditions, one in vivo (chronic exposure to delta-9-THC) and the other in vitro (neuroprotection mediated by WIN55212-2), was made to determine whether it was possible to identify common genes that were affected. Up and down-regulated sets of genes are described. Genes affected in one or the other circumstance include alterations in a 14-3-3 regulator protein of PKC, CREB, BDNF and GABA receptor subunit proteins, as well as several genes associated with known cannabinoid receptor-coupled signaling pathways. Unexpectedly, several genes that were altered in both circumstances were associated with synaptic and membrane structure, motility and neuron growth. These included, neuronal cell adhesion molecule (NCAM), hyloronidan motility receptor, and myelin proteolipid protein. While the basis for involvement of these particular genes in cannabinoid receptor activated functional processes within the cell is still not well understood, awareness that significant numbers of genes and presumably proteins are changed following either acute or long-term exposure may provide new insight into their effects.