The main psychoactive component of marijuana is known as delta9-tetrahydrocannabinol (THC). In addition, it has recently been discovered that endogenous substances are synthesized in the brain that can activate cannabinoid receptors, and these substances are referred to as endocannabinoids. All drugs, both natural and synthetic, that act at receptors for this substance are known collectively as cannabinoids (CBs). Cannabinoid drugs obtained by the smoking or ingestion of marijuana are used illicitly presumably because they are reinforcing or rewarding to humans. The objectives of this study are to gain knowledge about the underlying mechanisms through which cannabinoids alter brain cell function, and ultimately the mechanisms that produce the pleasurable effects of these drugs that sustain their illicit use. Cannabinoids act in discrete brain areas to produce their effects. The focus of this laboratory is to examine the effects of drugs of abuse on the electrical activity of individual nerve cells (neurons) and the ways in which these neurons communicate with each other via synaptic connections. Therefore, one of our goals is to identify specific electrically conducting ion channels whose activity is modified by CBs. To achieve these goals we will utilize rat brain slices acutely obtained from discrete brain areas, perform whole-cell electrophysiological recordings, and, using cellular anatomical techniques, morphologically reconstruct the neurons from which we record. The brain slice technique permits continuous and visually clear access to these areas of relatively intact nerve cell circuits while allowing perfusion with control saline or various solutions of drugs. In addition, because of the excellent access to the neurons in this preparation we can, through the use of both biophysical techniques and ion channel blocking agents, identify the ion channels acted upon by abused drugs. In the present study we will examine cannabinoid actions on nerve cells and their connections in an area of the midbrain known as the ventral tegmental area (VTA). This brain area and its connections have been strongly implicated in the reinforcing and rewarding actions of virtually all abused drugs. We will also examine the effects of these drugs in the nucleus accumbens, a brain area that is also known to be involved in the rewarding effects of CBs. Cannabinoids are known to be abused and are self-administered by laboratory animals. However, their actions in the VTA have only been recently investigated. It is known that injection of CBs into anesthetized rats causes an increase in firing rate of nerve cells in the VTA, and that activity of these nerve cells is important in producing pleasurable sensations. However, the mechanism by which this increase in firing is incompletely understood. Therefore, the present study will be one of the first to examine the mechanisms of action of cannabinoids on neurons in this important region of the brain. Therefore, these studies will allow greater understanding of the neural substrates that support the abuse of this class of drugs, as well as describe the basic mechanisms of drug abuse in general. It is hoped that these studies will produce knowledge that can be used to develop treatments for the abuse of CBs. Brain slice preparations will be used in conjuction with confocal microscopy to answer basic questions regarding the synthesis and release of endocannabinoids in VTA and NAc neurons. Previously, we demonstrated that by blocking a potassium ion channel (sK) that normally keeps DANs relatively quiescent, we can increase their level of spontaneous activity, and this results in endocannabinoid release that inhibits GABA synaptic inputs to DANs, that arise from neurons in the nucleus accumbens. We have recently followed these studies up by demonstrating that other manipulations that can increase DAN activity, such as glutamatergic excitation of these neurrons, can also promote endocannabinoid release. Therefore, these studies are the first to demonstrate this novel circuit that both THC, and endocannabinoids can act upon. Based on our data, we hypothesize that activation of cannabinoid receptors will result in a decrease in inhibitory GABA release onto DANs, and that this will increase the output of the DANs, and increase dopamine release in the nucleus accumbens. A manuscript detailing these results has been submitted for review at the Journal of Neuroscience. As the next step in these ongoing studies, we have set up a confocal microscope equipped to perform calcium imaging in brain slices, and to further examine the role of endocannabinoids in regulating neurotransmitter release in dopamine-containing neurons (DANs) in the VTA. The basic strategy that will be used is to evoke the release of calcium from intracellular stores using the technique of flash photolysis, which causes the uncaging of a molecule such as phosphatidylinositol. We will then assess whether this rise in intracellular calcium can subsequently result in an increase in the release of endocannabinoid that will then inhibit the release of the inhibitory neurotransmitter GABA. The rise in intracellular calcium levels will be monitored using ratiometric calcium detection techniques and the level of calcium will be related to the degree of synaptic inhibition that can be reversed by cannabinoid receptor antagonist application. These studies will be the first to directly relate rises in intracellular calcium with the release of endocannabinoids in the mammalian CNS.
