Endocannabinoid-mediated signaling in the cerebral cortex represents a unique communication channel between neurons: in contrast to the conventional direction of information flow, neurons can release a retrograde messenger that acts backward to suppress transmitter release from afferent synaptic terminals. The signaling molecule is believed to be an endocannabinoid, which selectively activates CB1 cannabinoid receptors that are located on axons of cholecystokinin (CCK)-containing inhibitory neurons. These cells are involved in the generation of high-frequency oscillations, thereby ensuring sufficient synchrony among active neurons to assist associative synaptic plasticity (learning). Concurrently, these neurons collect and convey emotional and motivational information to boost the process of synaptic plasticity. Consequently, malfunctioning of the CCK cell network, or its disturbance by exogenous cannabinoids, results in learning and memory deficits as well as mood disorders, most notably anxiety. We aim to uncover the molecular machinery of retrograde signaling, which likely involves a cascade of nitric oxide (NO) and endocannabinoids, and the location and operational principles of the implicated receptors, enzymes, and signal molecules, together with the functional role of inhibitory neurons under endocannabinoid control. Results from this research may lead to a novel approach to the pharmacotherapy of anxiety and drug dependence.
Our experiments primarily explore the mechanism and function of endocannabinoid signaling and, in addition, are designed to test the hypothesis that there is a division of labor between major inhibitory (GABA-containing) cell types in cortical information processing. The cellular components of the circuits are called basket cells; they control the ensemble activity and synchronization of principal neurons and are divided into two major types with different connectivities and functions: one characterized by the expression of the calcium-binding protein parvalbumin (PV) while the other contains the neuropeptide CCK. The electrically and synaptically coupled ensembles of PV-containing basket cells are indispensable components of the oscillating cortical hardware; they represent the rigid (nonplastic) precision clockwork without which no cortical operations are possible. The activity of a similar syncytium of CCK-containing basket cells is superimposed on the PV basket cell-entrained network, conveying the emotional and motivational effects carried by subcortical pathways (containing serotonin, for example). Actions of the CCK cell ensemble are highly modifiable also by local neuromodulators and retrograde signal molecules, which may allow further fine-tuning of principal cell cooperation. Impairment of this tuning system likely results in mood disorders such as anxiety. Endocannabinoid signaling plays a crucial role in regulating postsynaptic effects of the CCK cell ensemble (i.e., the fine-tuning device) and therefore is an ideal target for the pharmacotherapy of anxiety-like behaviors.
We aim to analyze the mechanisms of endocannabinoid control of specific inhibitory circuits by using a multidisciplinary approach that combines the generation of transgenic animal models, in vitro and in vivo electrophysiology, state-of-the-art molecular anatomical techniques, and behavioral studies. We will generate mice overexpressing CB1 cannabinoid receptors in the entire GABAergic system of the central nervous system by using the GAD65 promoter, mice in which the CB1 cannabinoid receptors are expressed in PV-containing neurons using an in vivo modified PV-BAC clone, and mice in which the CB1 cannabinoid receptors are expressed in PV-containing neurons but not in the CCK-containing cells. Electrophysiological and behavioral studies of the modulation of inhibitory (GABAergic) transmission in these animal lines not only will uncover the functional roles of endocannabinoid signaling but also will assist in revealing the functional differences—in other words, the division of labor—between major inhibitory cell types in cortical information processing.
In vitro electrophysiology will be used to investigate the cellular mechanisms of cannabinoid actions and the pharmacological separation of cannabinoid actions mediated by CB1 receptors present on CCK-containing GABAergic axon terminals from the novel cannabinoid-sensitive receptors that regulate glutamatergic synaptic transmission and which we described in our laboratory. We will also clarify the possible selectivity of the two endocannabinoids (anandamide and 2-arachidonoyl glycerol or 2-AG) for CB1 and the novel receptor, investigating their physiological roles by using inhibitors of their specific metabolizing enzymes. We have preliminary evidence that, in addition to endocannabinoids, the gaseous messenger molecule NO is also involved in retrograde synaptic signaling. Such a mechanism is particularly attractive, given that endocannabinoids are lipid soluble and thus are unlikely to cross the extracellular space. We aim to uncover the link between NO-mediated cGMP generation and endocannabinoid synthesis in presynaptic GABAergic terminals. The relative contribution of the CCK versus the PV cell network to hippocampal fast oscillations will be examined by distinct CB1-mediated modulation of the two basket cell types in normal and transgenic animals (expressing CB1 only in CCK or only in PV cells). In vivo electrophysiological studies will identify the roles of endocannabinoid-mediated synaptic signaling in hippocampal network activity patterns (theta and gamma oscillations, sharp waves), the possible differential involvement of the two receptors and/or the two endocannabinoids, using agonists and antagonists selective either for CB1 alone or for both CB1 and the new CB receptor, and selective inhibitors of anandamide and 2-AG metabolism. The relative contributions of the two basket cell types to hippocampal theta and gamma oscillations revealed in the in vitro studies will be reexamined in vivo in the same transgenic animal lines.
Molecular anatomical studies will focus on describing the regional, cellular, and subcellular distribution of the synthesizing (DAG-lipase, PLD) and metabolizing enzymes of endocannabinoids (FAAH and MGL) in the hippocampus, amygdala, neocortex, and basal ganglia in pre- and postsynaptic elements and their relationship to CB1 receptor distribution and on confirming their redistribution in the transgenic animals as well as in epileptic human hippocampus (lobectomy) samples. Such anatomical data are indispensable for the correct planning and interpretation of any functional studies.
At the behavioral level, we will differentiate the role in anxiety of CB1 and the novel cannabinoid receptors as well as the two endocannabinoids by the combined use of selective ligands, metabolizing enzyme inhibitors, and the CB1-KO mice in the elevated plus-maze, social interaction, and light/dark box tasks. We will also determine the role of the NO-endocannabinoid signal cascade in the generation of anxiety-like behavior using NO donors and NO synthase inhibitors in combination with agonists and antagonists of CB1 and the new CB receptor.
Understanding the mechanisms of the anxiety-related activity of the CCK cell network as well as its modulation by endocannabinoids and subcortical transmitter systems, together with the precise location and operation of the implicated receptors, enzymes, and signal molecules, are of major importance for the design of new strategies in the pharmacotherapy of anxiety as well as in the treatment of drug dependence. Earlier studies in the field of anxiety and addiction rarely considered the precise molecular and synaptic organization of cortical circuits in the interpretation of psychopharmacological and behavioral data. A novel aspect of this work is that our working hypotheses are based on the synthesis of knowledge from anatomical neuropharmacology as well as cellular and network electrophysiology of the cerebral cortex. This has allowed us to ask more focused questions and to design transgenic animal models that, in addition to the psychopathological implications, will shed light on the physiological roles of the CCK- and PV-containing interneuron ensembles in the generation of cortical electrical activity patterns involved in cognitive functions.
Last updated August 2008
