Jimok Kim

Inhibition of cyclooxygenase-2 potentiates retrograde endocannabinoid effects in hippocampus

In hippocampal pyramidal cells, a rise in Ca2+ 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.

Supply and demand for endocannabinoids

The endocannabinoid system consists of G-protein-coupled cannabinoid receptors that can be activated by cannabis-derived drugs and small lipids termed endocannabinoids (eCBs) plus associated biochemical machinery (precursors, synthetic and degradative enzymes, transporters). The eCB system in the brain primarily influences neuronal synaptic communication, and affects biological functions - including eating, anxiety, learning and memory, growth and development - via an array of actions throughout the nervous system. Although many aspects of synaptic regulation by eCBs are becoming clear, details of the subcellular organization and regulation of the eCB system are less well understood. This review focuses on recent investigations that illuminate fundamental issues of eCB storage, release, and functional roles.

Reduction in endocannabinoid tone is a homeostatic mechanism for specific inhibitory synapses

When chronic alterations in neuronal activity occur, network gain is maintained by global homeostatic scaling of synaptic strength, but the stability of microcircuits can be controlled by unique adaptations that differ from the global changes. It is not understood how specificity of synaptic tuning is achieved. We found that, although a large population of inhibitory synapses was homeostatically scaled down after chronic inactivity, decreased endocannabinoid tone specifically strengthened a subset of GABAergic synapses that express cannabinoid receptors. In rat hippocampal slice cultures, a 3–5-d blockade of neuronal firing facilitated uptake and degradation of anandamide. The consequent reduction in basal stimulation of cannabinoid receptors augmented GABA release probability, fostering rapid depression of synaptic inhibition and on-demand disinhibition. This regulatory mechanism, mediated by activity-dependent changes in tonic endocannabinoid level, permits selective local tuning of inhibitory synapses in hippocampal networks.

Synapse-Specific Adaptations to Inactivity in Hippocampal Circuits Achieve Homeostatic Gain Control while Dampening Network Reverberation

Synaptic homeostasis, induced by chronic changes in neuronal activity, is well studied in cultured neurons, but not in more physiological networks where distinct synaptic circuits are preserved. We characterized inactivity-induced adaptations at three sets of excitatory synapses in tetrodotoxin-treated organotypic hippocampal cultures. The adaptation to inactivity was strikingly synapse specific. Hippocampal throughput synapses (dentate-to-CA3 and CA3-to-CA1) were upregulated, conforming to homeostatic gain control in order to avoid extreme limits of neuronal firing. However, chronic inactivity decreased mEPSC frequency at CA3-to-CA3 synapses, which were isolated pharmacologically or surgically. This downregulation of recurrent synapses was opposite to that expected for conventional homeostasis, in apparent conflict with typical gain control. However, such changes contributed to an inactivity-induced shortening of reverberatory bursts generated by feedback excitation among CA3 pyramids, safeguarding the network from possible runaway excitation. Thus, synapse-specific adaptations of synaptic weight not only contributed to homeostatic gain control, but also dampened epileptogenic network activity.

Neuronal expression of CB2 cannabinoid receptor mRNAs in the mouse hippocampus.

In the brain, CB1 cannabinoid receptors primarily mediate the effects of cannabinoids, but CB2 cannabinoid receptors (CB2Rs) have recently been discovered in the nervous system and also implicated in neuromodulatory roles. To understand the mechanisms of CB2R functions in the brain, it is essential to localize CB2Rs, but the types of cells expressing CB2Rs have been controversial. Unequivocal localization of CB2Rs in the brain has been impeded in part by the low expression levels of CB2Rs and poor specificity of detection methods. Here, we used an ultrasensitive and specific in situ hybridization method called the RNAscope to determine the spatial pattern of CB2R mRNA expression in the mouse hippocampus. CB2R mRNAs were mostly expressed in a subset of excitatory and inhibitory neurons in the CA1, CA3 and dentate gyrus areas, but rarely in microglia. CB2R knock-out mice were used as a negative control. Using the quantitative real-time polymerase chain reaction, we also found that the temporal pattern of CB2R mRNA expression was stable during postnatal development. Consistent with previous reports, the immunological detection of CB2Rs was not reliable, implying extremely low levels of the protein expression and/or insufficient specificity of the current anti-CB2R antibodies. Our findings of the expression patterns of CB2R mRNAs may help determine the cell types involved in, and hence the mechanisms of, the CB2R-mediated neuromodulation.

Chronic activation of CB2 cannabinoid receptors in the hippocampus increases excitatory synaptic transmission.

The effects of cannabinoids are primarily mediated by two types of cannabinoid receptors, CB1 receptors in the nervous system and CB2 receptors in the immune system. Recent evidence indicates that CB2 receptors are also widely expressed in the brain and involved in neuropsychiatric functions, such as schizophrenia‐like behaviours, anxiety, memory, vomiting and pain. The cellular mechanisms by which CB2 receptors regulate neuronal functions are unknown. We show that chronic activation of CB2 receptors in the hippocampus for 7–10 days increases excitatory synaptic transmission, whereas short‐term activation of CB2 receptors has little effect on synaptic activity. This study reveals a novel role of CB2 receptors in the brain, which is clearly distinct from that of CB1 receptors, and thus, will help us to understand better the diverse effects of cannabinoids in the nervous system.

Deletion of CB2 cannabinoid receptors reduces synaptic transmission and long-term potentiation in the mouse hippocampus.

The effects of cannabinoids are mostly mediated by two types of cannabinoid receptors—CB1 receptors in the nervous system and CB2 receptors in the immune system. However, CB2 cannabinoid receptors have recently been discovered in the brain and also implicated in neurophysiological functions. The deletion of CB2 receptors in mice induces long‐term memory deficits and schizophrenia‐like behaviors, implying that endogenous activity of CB2 receptors might be involved in neuropsychiatric effects. Little is known about the cellular mechanisms by which physiological activation of CB2 receptors modulates neuronal functions. We aimed to determine how deletion of CB2 receptors in mice affects synaptic transmission and plasticity. Electrophysiological and morphological studies indicated that CB2 receptor knockout resulted in decreases in excitatory synaptic transmission, long‐term potentiation, and dendritic spine density in the hippocampus. Our data imply that endogenous activity of CB2 receptors might contribute to the maintenance of synaptic functions and the expression of normal long‐term potentiation. This study provides insights into the role of CB2 cannabinoid receptors in regulating cognitive functions such as long‐term memory.

Neuregulin-1 Impairs the Long-term Depression of Hippocampal Inhibitory Synapses by Facilitating the Degradation of Endocannabinoid 2-AG

Endocannabinoids play essential roles in synaptic plasticity; thus, their dysfunction often causes impairments in memory or cognition. However, it is not well understood whether deficits in the endocannabinoid system account for the cognitive symptoms of schizophrenia. Here, we show that endocannabinoid-mediated synaptic regulation is impaired by the prolonged elevation of neuregulin-1, the abnormality of which is a hallmark in many patients with schizophrenia. When rat hippocampal slices were chronically treated with neuregulin-1, the degradation of 2-arachidonoylglycerol (2-AG), one of the major endocannabinoids, was enhanced due to the increased expression of its degradative enzyme, monoacylglycerol lipase. As a result, the time course of depolarization-induced 2-AG signaling was shortened, and the magnitude of 2-AG-dependent long-term depression of inhibitory synapses was reduced. Our study reveals that an alteration in the signaling of 2-AG contributes to hippocampal synaptic dysfunction in a hyper-neuregulin-1 condition and thus provides novel insights into potential schizophrenic therapeutics that target the endocannabinoid system.

An Improved Test for Detecting Multiplicative Homeostatic Synaptic Scaling

Homeostatic scaling of synaptic strengths is essential for maintenance of network “gain”, but also poses a risk of losing the distinctions among relative synaptic weights, which are possibly cellular correlates of memory storage. Multiplicative scaling of all synapses has been proposed as a mechanism that would preserve the relative weights among them, because they would all be proportionately adjusted. It is crucial for this hypothesis that all synapses be affected identically, but whether or not this actually occurs is difficult to determine directly. Mathematical tests for multiplicative synaptic scaling are presently carried out on distributions of miniature synaptic current amplitudes, but the accuracy of the test procedure has not been fully validated. We now show that the existence of an amplitude threshold for empirical detection of miniature synaptic currents limits the use of the most common method for detecting multiplicative changes. Our new method circumvents the problem by discarding the potentially distorting subthreshold values after computational scaling. This new method should be useful in assessing the underlying neurophysiological nature of a homeostatic synaptic scaling transformation, and therefore in evaluating its functional significance.

Distinct roles of neuronal and microglial CB2 cannabinoid receptors in the mouse hippocampus

The effects of cannabinoids are primarily mediated by type-1 cannabinoid receptors in the brain and type-2 cannabinoid receptors (CB2Rs) in the peripheral immune system. However, recent evidence demonstrates that CB2Rs are also expressed in the brain and implicated in neuropsychiatric effects. Diverse types of cells in various regions in the brain express CB2Rs but the cellular loci of CB2Rs that induce specific behavioral effects have not been determined. To manipulate CB2R expression in specific types of cells in the dorsal hippocampus of adult mice, we used Cre-dependent overexpression and CRISPR-Cas9 genome-editing techniques in combination with adeno-associated viruses and transgenic mice. Elevation and disruption of CB2R expression in microglia in the CA1 area increased and decreased, respectively, contextual fear memory. In CA1 pyramidal neurons, disruption of CB2R expression enhanced spatial working memory, whereas their overexpression reduced anxiety levels assessed asan increase in the exploration time in the central area of open field. Interneuronal CB2Rs were not involved in the modulation of cognitive or emotional behaviors tested in this study. The targeted manipulation of CB2R expression in pyramidal neurons and microglia suggests that CB2Rs in different types of cells in the mature hippocampus play distinct roles in the regulation of memory and anxiety.