Search

Endocannabinoids have been shown to mediate depolarization-induced suppression of GABAergic inhibition (DSI), possibly via release and retrograde diffusion following moderate to severe depolarization of hippocampal pyramidal neurons. However, it is not clear how hippocampal neurons, which have relatively low firing rates in vivo, achieve the degree of depolarization required to release endocannabinoids. Here it is demonstrated that DSI is not dependent on the occurrence of action potentials in the postsynaptic neuron, but is mediated by depolarization-induced calcium entry via voltage-controlled calcium channels (VCCs). The optimal level of calcium entry, and subsequent DSI, are directly related to the frequency of depolarizing pulses, which differs between immature and adult hippocampus. However, it is shown via modeled spike train inputs that the frequency dependence of DSI is overcome if two or more convergent spike trains from different neurons with normal in vivo firing rates converge and overlap in time. In these modeled circumstances, endocannabinoid-mediated DSI occurs most often when converging synaptic inputs from multiple neurons fire in synchrony to allow temporal summation of local membrane events in postsynaptic cells to exceed threshold for calcium entry. It is therefore possible that such suppression of inhibition would only occur during the time that recipient hippocampal neurons receive multiple coincident excitatory synaptic inputs.

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 μM). WIN 55,212-2 was also shown to inhibit the NMDA-induced increase in intracellular calcium concentration ([Ca 2+ ] i ) indicated by FURA-2 fluorescence imaging in the same cultured neurons. Changes in [Ca 2+ ] 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 [Ca 2+ ] 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 [Ca 2+ ] i via a cAMP/PKA-dependent process during the neurotoxic event.

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 γ-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.

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.

Kittler, Josef T., Elena V. Grigorenko, Chris Clayton, Shou-Yuan Zhuang, Sarah C. Bundey, Michael M. Trower, Don Wallace, Robert Hampson, and Sam Deadwyler. Large-scale analysis of gene expression changes during acute and chronic exposure to Δ9-THC in rats. Physiol Genomics 3: 175–185, 2000.—Large-scale cDNA microarrays were employed to assess transient changes in gene expression levels following acute and chronic exposure to cannabinoids in rats. A total of 24,456 cDNA clones were randomly selected from a rat brain cDNA library, amplified by PCR, and arrayed at high density to investigate differential gene expression profiles following acute (24 h), intermediate (7 days), and chronic (21 days) exposure to Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive ingredient of marijuana. Hippocampal mRNA probes labeled with 33P obtained from both vehicle and Δ9-THC-treated animals were hybridized with identical cDNA microarrays. Results revealed a total of 49 different genes altered by Δ9-THC exposure; of these, 28 were identified, 10 had homologies to expressed sequence tags (ESTs), and 11 had no homology to known sequences in the GenBank database. Chronic or acute cannabinoid receptor activation altered expression of several genes (i.e., prostaglandin D synthase, calmodulin) involved in biochemical cascades of cannabinoid synthesis or cannabinoid effector systems. Other genes [i.e., neural cell adhesion molecule (NCAM), myelin basic protein], whose relation to cannabinoid system function was not immediately obvious, were also significantly altered. Verification of the changes obtained with the large-scale screen was determined by RNA dot blots in different groups of animals treated the same as those in the large-scale screen. Results are discussed in terms of the different types of genes affected at different times during chronic Δ9-THC exposure.

The potent cannabinoid receptor agonist WIN 55,212-2 produces positive shifts in steady-state inactivation of the potassium A current (IA) in rat hippocampal neurons via an adenosine 3',5'-cyclic monophosphate (cAMP)-, protein kinase A (PKA)-dependent process. This effect is probably mediated by phosphorylation or dephosphorylation of the IA channel protein. The role of protein phosphorylation in this cascade was tested by testing cannabinoid actions in cultured hippocampal neurons (pyramidal cells) that were exposed also to either the catalytic subunit of PKA (PKAc), a PKA-specific phosphorylation inhibitor (IP-20, Walsh peptide), or a potent protein phosphatase inhibitor (okadaic acid). Cannabinoids such as WIN 55,212-2 produce a positive (rightwards) shift in the steady-state inactivation of IA, thus providing increased current at a given membrane voltage. Cells dialyzed with PKAc showed a negative shift in IA inactivation, opposite to that produced by cannabinoids, and similar to that produced by increased levels of cAMP. In addition, PKAc completely blocked the positive shift produced by WIN 55,212-2. In contrast, dialysis of cells with IP-20 produced a positive shift in steady state inactivation of IA, similar to that produced by WIN, but the effects were not additive with cannabinoid receptor activation. The phosphatase inhibitor, okadaic acid produced a small negative shift in IA steady-state inactivation when administered alone, and blocked the positive shift produced by WIN 55,212-2. Okadaic acid also enhanced the negative shift in IA inactivation when co-administered with forskolin. The effects of okadaic acid and WIN 55,212-2 were not additive, suggesting a common pathway. These results demonstrate that IA is altered by direct manipulations of the phosphorylation status of the channel protein, and that cannabinoid effects on IA are probably mediated by dephosphorylation of the IA channel.

The time course of changes across 21 days of continuous exposure to Delta9-tetrahydrocannabinol (Delta9-THC) was assessed for the level of cannabinoid receptor (CB1) mRNA expression in three different rat brain regions: cerebellum, hippocampus and corpus striatum. Expression levels of CB1 mRNA were determined using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) following a protocol which included a gene standard, 28S ribonucleic acid protein (rRNA), for normalization of levels of RNA in the three different brain regions. The levels of CB1 mRNA were assessed in four different rats at each of seven time points (6 h, and 1, 2, 3, 7, 14 and 21 days) during a 21-day Delta9-THC one dose day-1 (10 mg kg-1) treatment regimen. In the cerebellum and hippocampus, CB1 mRNA levels were increased above vehicle control animals at 7 and 14 days of treatment. In the striatum the levels of CB1 transcripts were severely reduced from days 2-14. CB1 message expression in all three brain areas returned to vehicle control levels by day 21 of Delta9-THC treatment, a time at which behavioral tolerance has been previously reported. An additional measure, receptor stimulated GTPgammaS binding, performed over the same time period revealed differential desensitization within the 3 brain areas as a function of chronic exposure to Delta9-THC. Hippocampus was the earliest to desensitize decreasing to 35% of control by treatment day 7, followed by a decrease in the cerebellum to that same level on day 14 of treatment. The striatum showed only half that degree of desensitization (65%) over the entire 21-day treatment period. Comparisons suggests that CB1 message may be regulated by different effector systems in each of the three areas during chronic Delta9-THC exposure.

Cannabinoid receptor agonists have been previously shown to enhance a potassium A-current (IA) in cultured rat hippocampal neurons. This effect has been further demonstrated to be dependent on G-protein linkage to adenylyl cyclase and levels of intracellular cyclic AMP (cAMP). The present study extends this analysis to the involvement of cAMP-dependent protein kinase (PKA) in this cascade. Specific activators and inhibitors of PKA were shown to have differential effects on the voltage dependence of IA. Specific activators of PKA produced a negative shift in voltage dependence of IA, whereas PKA inhibitors produced a positive shift in IA voltage dependence, the latter similar to that effected by the cannabinoid agonist WIN 55,212-2. Although the negative shift in IA induced by PKA stimulation could be reversed by PKA inhibitors, the positive shift produced by the PKA inhibitors alone was only 50–60% of the cannabinoid-produced shift in IA voltage dependence. This partial effect of PKA inhibition was confirmed by biochemical assays in the same cultured neurons that showed a similar 50–60% decrement in in vitro protein phosphorylation produced by PKA inhibitors. Results are discussed in terms of a diffusible second messenger linkage of the cannabinoid receptor to the A-current channel via the role of protein phosphorylation in modulation of IA.