Carl R. Lupica - US grants

Opioids are known to have important behavioral and physiological actions that are mediated by interactions with neurons in the central nervous system. However, while the behavioral actions of the opioids, including analgesia and euphoria, are well-known, the functional roles these molecules play as neuromodulators in the mammalian brain remain poorly understood. The following experiments will evaluate the actions of opioids, acting at pharmacologically defined mu-, delta- and kappa-opioid receptor subtypes, as modulators of synaptic transmission in the CA1 and dentate gyrus regions of the hippocampus, and in another brain region known as the periaqueductal gray. The experiments described in this proposal will utilize whole-cell patch clamp electrophysiological techniques to examine the effects of opioids on individual neurons in rat brain slice preparations. The first set of experiments will focus on the mechanisms through which mu-, delta-, and kappa-opioid receptor agonists act to decrease evoked and spontaneous synaptic transmission in the hippocampal slice. In particular, these experiments will examine the contributions made by distinct voltage-dependent calcium channels, intracellular calcium stores, and nitric oxide in supporting synaptic transmission and the modulation of this process. The second set of experiments will determine the effects of opioid agonists directly on subpopulations of gamma-aminobutyric acid (GABA)-containing neurons (interneurons) in the CA1 region of the hippocampus, and the cellular mechanisms these receptors utilize to affect these cells. These experiments will be aided through the use of differential interference contrast microscopy to identify these neurons in living brain slices. The third set of experiments will determine what mechanisms account for the ability of certain non-opioid peptides to attenuate the behavioral and cellular actions of the opioids. Thus, we will determine the effects of the peptides cholecystokinin (CCK) and neuropeptide FF on neurons found in the hippocampus, and the periaqueductal gray. The investigations described in this grant proposal should improve our understanding of the specific receptor subtypes that the opioids act upon, the cellular mechanisms mediating their responses, the roles opioids may play as neuromodulators in the brain, and the mechanism of their interaction with other non-opioid peptides. in addition, since limbic structures like the hippocampus have been shown to participate in normal and abnormal behavioral states such as learning, epilepsy, and emotional behavior, and the periaqueductal gray has been shown to be important in mediating opioid analgesia, these studies may yield new insights as to the interaction between opioids and these phenomena.

This project was initiated to fill a void in our knowledge regarding the neurobiological substrates of the the adverse effects of chronic marijuana use on cognition in humans. It is well-known that both acute and chronic marijuana use in humans impairs short-term memory, reaction times, and general higher-order cognitive processing. These studies seek to utilize animal models to explore the effects of both acute and chronic exposure to the main psychoactive ingredient in marijuana, delta9-tetrahydrocannabinol (THC) on the neurophysiology of the hippocampus and now the ventral tegmanetal area (VTA). One series of experiments involve repeated i.p. injections with THC for varying periods of time, followed by varying periods of withdrawal from the drug, and then evaluation of electrophysiological parameters in brain slices containing the rodent hippocampus. Results from these studies, published earlier this year, indicate that following a 1d withdrawal from a 7d treatment with THC, the drug was undetectable in hippocampus using LC-MS. However, the 7d exposure to THC produced tolerance to the inhibition of GABA, but not glutamate release by the cannabinoid agonist WIN55,212-2. Additionally, unlike controls, the hippocampal slices from the chronic THC animals did not demonstrate long-term potentiation (LTP), a cellular correlate of learning and memory. A single injection of THC was insufficient to block LTP, the LTP blockade persisted for 3d after the last THC injection, and it was prevented by pretreatment of the animals before each THC injection with the antagonist AM251 (2 mg/kg). Additional experiments now under way will examine the hypothesis that the CB1 cannabinoid receptor is necessary to permit normal cognition and learning and memory over the life span of an organism. For these studies, we are comparing the level of LTP in hippocampal brain slices obtained from CB1+/+ and -/- animals at various ages. In addition, we are defining the actions of acute THC exposure on individual neurons in hippocampal brain slices using whole-cell recordings. The majority of studies to date have utilized synthetic CB agonists to assess the role of CB1 receptors in modulating hippocampal synaptic function. By comparing the effects of THC to those of these synthetic agonists, we hope to identify putative molecular targets of THC that may help explain memory impairments in humans following chronic marijuana use. These experiments will also define the consequences of both acute and repeated THC exposure in the rodent hippocampus and will define the importance of the CB1 receptor in cognition throughout the lifespan of the mouse. New experiments will examine the consequences of long-term exposure to THC on dopamine neurons in the ventral tegmental area (VTA). These studies will determine whether THC exposure alters the function of dopamine neurons, and results in impaired forms of synaptic plasticity.

The nucleus accumbens (NAc) represents a critical site for the rewarding and addictive properties of several classes of abused drugs. Therefore, it is necessary to understand the actions of abused drugs such as marijuana, cocaine, and opioids on physiology of this system. In addition, this brain nucleus is known to mediate motivational aspects of behavior. For this reason it has been implicated in a variety of psychiatric disorders that involve alterations in mood and motivation, as well as in the process of drug addiction. The NAc medium spiny GABAergic output neurons (MSNs) receive innervation from other intrinsic MSNs, and glutamatergic innervation from extrinsic sources. Both GABAergic and glutamatergic synapses onto MSNs are inhibited by drugs of abuse, suggesting that this action may contribute to the rewarding properties of these drugs. In addition, drugs of abuse are known to increase NAc dopamine (DA). One role of DA in regulating NAc activity may be to contribute to the long-term changes in excitatory transmission observed following repetitive activation of glutamatergic afferents. However, the precise mechanisms through which such synaptic plasticity develops, and how drugs of abuse, including cannabinoids (CBs), alter such synaptic plasticity, remain poorly understood. To investigate the actions of CBs in the NAc, we are utilizing both electrophysiological and voltammetric recording techniques in brain slices. By combining these approaches, we hope to be able to simultaneously monitor changes in DA levels and the development of synaptic plasticity. We are also collaborating with Dr. Bruce Hope investigating the effects of repeated cocaine treatment on synaptic inputs to MSNs. To do this, we are using rats that express a fluorescent marker in a discrete population of MSNs as a result of cocaine sensitization. By visualizing these specific neurons in our slice preparation, we can perform electrophysiological recordings from these cells and assess the mechanisms supporting cocaine sensitization.

During this year, we have examined the interactions between the mu opioid and CCK- B receptors in acutely isolated living neurons obtained from brains of rat pups. The method begins with living slices of rat brain, which are dissociated by gently approaching the slice with a small vibrating glass probe. The beauty of this method is that very discrete regions can be dissociated. Thus for example, it is easy to confine the dissociation to say the pyramidal layer of the CA1 region in the rostral hippocampus. Another great advantage to the method is that, as no enzymes need be used, presynaptic fragments remain attached, and their presence is evident by spontaneous synaptic activity that can be recorded in the isolated neuron. We found that both CCK and normorphine slow the rate of this activity in hippocampal pyramidal neurons, and that CCK also diminishes the amplitudes. Therefore, both CCK and opioid receptors are active in this preparation. To detect a cell-level interaction of these receptors, however, they should both be expressed on the same cell. The hippocampus is of interest because of it's well known cellular architecture, the vast accumulation of knowledge about it, and its role in declarative memory and spacial mapping. The cell type of interest for our purpose of expressing both CCK and mu opioid receptors would then be an interneuron, and a CCK-type basket cell interneuron in particular. As only about 10% of hippocampal neurons are interneurons, the yield of candidate cells is low to begin with. Presynaptic fragments are unnecessary in this model, so the slices were treated with low concentrations of enzymes, which gave better yields of healthy cells. However, interneurons were very scarce, and usable ones even more scarce. Some data showed that CCK increased high voltage activated calcium currents, but the cells disintegrated before a test of normorphine could be made on the same one. Given the bad odds, I switched to periaqueductal gray (PAG) cells. The PAG is known to be involved in, among others, fear and defense reactions and in endogenous analgesia. In contrast to the hippocampus, PAG cells are mostly (80%) interneurons, and they are known to express both CCK and mu opioid receptors. However, using variables in enzyme (protease XIV or papain) dissociation, trituration vs. vibrating probe, employing variations in "perforated patch" (beta escin or nystatin) technique to preserve the viability of recorded cells, using pyruvate or ascorbate to enhance the lifetime of dissociated cells, the yield of data was still very poor. In late July I began using a new, more stable micromanipulator, retiring the 20 year old hydraulic one. It is hoped that the enhanced mechanical stability will permit longer and more reliable recordings.

The nAChR was stably expressed in the cell line SH-EP1, obtained from R. Lucas (Phoenix, AZ). The cells were studied under whole cell voltage clamp conditions using 100% series resistance compensation. Rapid superfusion of ACh generated currents that rose rapidly to a peak (in about 80 ms) and desensitized as a double exponential function of time in the continued presence of ACh (5 s). This decay was modeled as two sequential desensitized states with forward rate constants k1 and k2 and backward rate constants k-1 and k-2. The rate constant k1 increased with the ACh response, in conformity with the sequential mechanism, while the others remained unchanged. [unreadable] When AEA was superfused onto the SH-EP1 cells, the peak response to ACh was diminished and continued to decrease with time up to about 50 min. This slow time course is expected for a lipophilic compound such as AEA. The recovery also required tens of minutes, but was accelerated to under 10 min by using the lipid scavenger bovine serum albumin. More strikingly, AEA increased k1 in direct proportion to the AEA concentration without evidence of saturation. The increase in k1, up to 25-fold at 2 micromolar AEA concentration, generated the spike-like responses to ACh. Simulations showed that the very rapid entry into desensitization caused by AEA could account for about 80% of the decrease in the peak amplitude because responses closer to the spreading ACh front desensitized before summating with the responses at a further diffusional distance. The rest of the decrease was postulated to occur through desensitization of the inactive state of the AChR.[unreadable] Confocal microscopy of the fluorescent Ca++ indicator fluro-3 showed that ACh caused transient increases in Ca++ concentration. To test if lipophilic AEA accelerated desensitization through an intracellular Ca++ dependent mechanism, we first showed that AEA dialyzed into the cell through the patch pipette had no effect on ACh currents. Next we used the fast acting Ca++ chelator BAPTA in the patch pipette and showed that it failed to oppose the AEA effects.[unreadable] To explore pharmacological specificity of the AEA effects, we showed that the cannabinoid agonist delta-9-tetrahydrocannabinol (THC) had no effects whatever at the nAChR. The CB1 antagonist SR-141716A, co-administered with AEA, failed to antagonize the AEA depression of peak amplitude, though it did antagonize the increase in k1 slightly. Finally, in a preliminary test of the structural requirements of the AEA effect, we tested the octahydro analogue, arachidoyl ethanolamide (H-8-AEA). Despite the structural similarity to AEA, H-8-AEA at 1 micromolar concentration was devoid of activity at the nAChR.[unreadable] We conclude that AEA, at physiologically relevant concentrations, directly blocks ACh responses at the nAChR primarily by greatly increasing its rate of desensitization. As this rate increased linearly with the AEA concentration, we suspect that it acts through graded alterations of the membrane. Experiments to test this hypothesis are planned.

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. One of the objectives of these studies is 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. The primary focus of this laboratory is to examine the mechanisms through which abused drugs alter the electrical activity of neurons and the ways in which these neurons communicate with each other via synaptic connections. Therefore, one of our goals is to identify specific ion channels whose activity is modified by abused drugs such as marijuana, nicotine, heroin, and cocaine. To achieve these goals we will utilize rat brain slices acutely obtained from discrete brain areas involved in processing information regarding pleasurable and unpleasant environmental stimuli. We utilize whole-cell electrophysiological recordings, and cellular anatomical techniques to reconstruct the neurons from which we record. In these ongoing studies we are examining the mechanisms through which these drugs affect neurons and their connections in the ventral tegmental area (VTA). This brain area and its connections are strongly implicated in the reinforcing and rewarding actions of all abused drugs, as well as in mediating the rewarding effects of natural environmental stimuli, such as food, water, etc. The VTA is also involved in processing information regarding the physiological stress responses, mood and affect, and mental alertness. Because of its central role in these processes, the VTA is a brain area that contributes to disorders such as addiction, psychiatric stress disorders, clinical depression, and psychiatric anxiety disorders. Recent studies in the laboratory have focused on delineating the relative contribution of synaptic inputs to the VTA dopamine neurons arising from distinct brain regions. One of the sub-cortical inputs to the VTA that we are currently studying is that from the pedunculopontine nucleus (PPN). This brain nucleus provides strong acetylcholinergic (Ach) input to the VTA, and therefore is likely involved in regulating the reinforcing and addictive properties of the drug nicotine. Therefore, these studies will provide information that will be useful in the treatment of nicotine addiction, as well as in the prevention of respiratory disorders, such as emphysema, and lung cancer, resulting from nicotine addiction. Moreover, since the PPN is known to be critical to setting states of alertness, and physiological arousal, it is strongly implicated as a subcortical brain structure involved in anxiety, and chronic stress disorders. Our most recent studies examine the properties of the PPN input to the VTA, with regard to nicotine sensitivity, as well as the ability of this pathway to undergo a long-term change know as long-term depression (LTD) following exposure to either environmental stress or cocaine. We have found that the PPN inputs to the VTA can be weakened if animals are exposed to stress, or cocaine, and that nicotine exposure can alter the ability of the PPN to activate the reward-relevant dopamine neurons in the VTA. In order to further these studies we have also developed a genetically altered mouse in which the green fluorescent protein (GFP) is expressed only in dopamine neurons. The use of confocal microscopy in brain slices containing the VTA permits us to identify these neurons prior to performing electrophysiological studies. This has greatly increased the speed of data collection in the laboratory.

We have begun to examine the neurodegeneration that occurs in a genetically modified mouse that was developed by our collaborators at the Karolinska Institute in Sweden. We have now established a successful breeding colony of these mice at our institute, and we have made them available to our local collaborators. These mice possess a mutation in the mitochondrial gene known as mitochondrial transcription factor A (tFam). This gene regulates mitochondrial DNA transcription in all cells, and is necessary for continued oxidative phosphorylation. However, by targeting this mutation to dopamine neurons using the promoter that drives dopamine transporter expression, only these neurons are affected by the mutation. Our present work shows that the DA neurons degenerate slowly over a 30 week period, and that these MitoPark mice display many hallmarks of Parkinsons disease in humans. This includes sensitivity to pharmacological treatments, such as L-Dopa therapy, and the loss of this therapeutic benefit as the neurodegeneration progresses. Our studies have also shown that expression of glial cell line-derived neurotrophic factor (GDNF) through adeno-associated virus (AAV) can spare these dopamine neurons, and protect against either neurotoxin or genetically induced parkinsonism in mice. In addition, in pilot studies conducted with the dopamine neurotoxin MPTP, we have found that the loss of dopamine causes profound changes in the physiological properties of the striatum that were also prevented by AAV-mediated gene expression of GDNF. We will now begin to test this form of gene therapy in the tFam genetic model of Parkinsons disease, and attempt to reverse the neurodegeneration at various time points during the disease progression. Our most recent work with the MitoPark mice reveals an interesting change in DA neuron physiology at a time during development at which the animals are behaviorally asymptomatic. We find a dramatic reduction in the presence of a membrane current known as Ih in the DA neurons located in the substantia nigra, This current is normally involved in re-setting the neuronal membrane potential near action potential threshold following a large inhibition. Therefore, Ih may be involved in maintaining normal pacemaking activity in DA neurons. We are currently assessing the relevance of this change in Ih density in MitoPark mice, and theorize that a loss of Ih may represent one of the early consequences of mitochondrial impairment in DA neurons degenerating in Parkinson's disease. Our current studies show that the mRNAs encoding Ih are not altered in MitoPark mice suggesting that the down-regulation of these ion channels is post-translational. In the most recent reporting period we have found that DA neurons in MitoPark mice are impaired compared to control mice, and that this impairment leads to a reduction in the available pool of DA that is releasable. The most surprising aspect of these studies is that the observed impairment in DA neuron function occurs at an age where parkinsonian symptoms are absent in these mice. Thus, we believe that this model may permit for the first time the ability to track changes in the nigrostriatal DA system over time, which should lead to promising targets for therapeutic intervention. A further developmental characterization of the dopamine neurons in the substantia nigra of these mice is currently underway to determine whether there is a progression in ion channel dysfunction as the animals begin to display more severe parkinsonian behavioral symptoms. In addition, a detailed anatomical analysis is underway to determine whether the ion channel changes we have observed are related to changes in neuronal morphology, and the loss of synaptic function

This project was initiated to fill a void in our knowledge regarding the neurobiological substrates of the the adverse effects of chronic marijuana use on cognition in humans. It is well-known that both acute and chronic marijuana use in humans impairs short-term memory, reaction times, and general higher-order cognitive processing. These studies seek to utilize animal models to explore the effects of both acute and chronic exposure to the main psychoactive ingredient in marijuana, delta9-tetrahydrocannabinol (THC) on the neurophysiology of the hippocampus and now the ventral tegmental area (VTA). Our prior published study showed that repeated injections of THC blocked long-term potentiation (LTP), a cellular correlate of learning and memory. Furthermore, a single injection of THC was insufficient to block LTP, the LTP blockade persisted for 3d after the last THC injection, and it was prevented by pretreatment of the animals before each THC injection with the antagonist AM251 (2 mg/kg). We are also defining the actions of acute THC exposure on individual neurons in hippocampal brain slices using whole-cell recordings. The majority of studies to date have utilized synthetic CB agonists to assess the role of CB1 receptors in modulating hippocampal synaptic function. By comparing the effects of THC to those of these synthetic agonists, we hope to identify putative molecular targets of THC that may help explain memory impairments in humans following chronic marijuana use. Our most recent work has found that whereas THC acts as a partial agonist in the inhibition of glutamate release in the hippocampus, it is a full agonist when its effects are measured on the inhibition of GABA release in the hippocampus. We believe that this difference is due to a much higher CB1 receptor density on GABAergic axon terminals versus glutamate terminals in this brain structure, and we have proposed that this provides strong evidence that the primary site of THC's interaction to disrupt hippocampal-dependent memory is on GABAergic systems. In a separate study, we have reported that the effects of cannabinoids on hippocampal glutamate release can be greatly potentiated when adenosine A1 receptors are blocked. This suggests that endogenous adenosine is involved in regulating the strength of signaling through CB1 receptors in the hippocampus, and that endocannabinoid function is under control of the adenosine system. This is important because it is well known that the neuromodulator, adenosine, is released during periods of cellular and metabolic stress, and that it plays an important role in terminating seizures. Therefore, we predict that the conditions that regulate adenosine release will also modify the effects of endogenous cannabinoids, and the effects of marijuana in the hippocampus. Current experiments are involved in defining the source and site of action of endogenous adenosine in the hippocampus, with the ultimate goal of identifying how adenosine A1 receptors and endogenous adenosine interfere with cannabinoid receptor signaling. Preliminary data indicate that endogenous adenosine are released from mossy fibers in in area CA3 and this is inhibiting impulse activity in CA3 neuron axons that express cannabinoid CB1 receptors in area CA1. Through this mechanism we hypothesize that endogenous adenosine released during states of high metabolic demand can regulate the functions of endogenous cannabinoids and THC in area CA1 of the hippocampus. Ultimately, this will affect cognition, mood and learning by disrupting hippocampal function. An additional pertinent observation from these studies is that the commonly consumed substance, caffeine, which is available in many forms, can increase the ability of marijuana to disrupt hippocampal function. This could have important implications for the developing brain, either in utero, before birth, or in the adolescent brain. Relevant public health concerns may therefore involve the use of marijuana and beverages containing high levels of caffeine in adolescents, and the adverse cognitive and neurodevelopmental consequences.

Using whole-cell electrophysiology in brain slices, we find that physiological effects of 5-HT1A and 5-HT2A receptor activation were dramatically reduced following withdrawal of either cocaine self-administration (CSA) or yoked cocaine administration (CYA) in pyramidal neurons. Moreover, these reduced effects of 5-HT at these receptors persisted for many weeks after cocaine withdrawal, suggesting they may be involved in long-term aspects of cocaine addiction, such as drug craving. As 5-HT in the OFC is implicated in behavioral flexibility, learning and other cognitive abilities, our experiments provide novel information as to the role that 5-HT plays in cocaine addiction, and in the impaired decision-making exhibited during cocaine addiction and withdrawal. In addition, since 5-HT is linked to several neuropsychiatric disorders, such as depression, anxiety, dementia, impulsive-aggression disorder (IAD), obsessive-compulsive disorder (OCD), and post-traumatic stres disorder (PTSD), these experiments provide valuable information as to the clinical utility of targeting the 5-HT system in the OFC as potential therapies. Our recent efforts in this project have focused on developing a transgenic rat model that will permit us to examine the role of 5-HT in the regulation of a class of important neurons in the OFC, known as fast-spiking (FS), parvalbumin (PV), interneurons. These neurons constitute a minority of the total cell population in the OFC, but are important because they use the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), to coordinate the activity of the much larger population of pyramidal neurons, via extensive synaptic connections. Therefore, these neurons are integral to the computational processes that are necessary for normal OFC function, and are likely to be involved in psychiatric and addiction disorders in which this brain are contributes. To study these neurons our transgenic core has developed a rat model in which the PV gene promoter drives the expression of cre recombinase. This permits selective expression fluorescent proteins and opsins, such as channelrhodopsin-2 (ChR2), that allows us to study these neurons, and manipulate their activity with light. Our current experiments have focused on the characterization of these PV cells in brain slices, and in defining the effects of 5-HT receptors in the regulation of their activity. We have also begun to determine whether CSA alters the basal function of these PV neurons, and whether CSA alters the effects of 5-HT on this class of cell. It is hoped that the identification of the roles of these neurons in psychiatric illness and addiction will aid in developing novel targeted therapeutic approaches for treatment in humans.

In our ongoing studies to identify the mechanisms through which cannabinoids alter brain function, we have begun collaborating with the Designer Drug Research Unit (DDRU) at the NIDA Intramural Research Program, to compare the pharmacological effects of designer cannabinoids with those of conventional natural cannabinoids such at delta-9-tetrahydrocannabinol (delta-9-THC), found in the marijuana plant. Designer cannabinoids are psychoactive molecules that are often marketed as incense, spice or other plant-related formulations. The psychoactive components of these drugs are typically synthesized in clandestine laboratories by amateur chemists, and in most cases structurally resemble cannabinoid molecules. The synthetic cannabinoids are made in bulk and sprayed onto plant material. The fact that these molecules are made in illicit laboratories without regulatory control often leads to exposure to adulterants and contaminants that can result in unintended toxicity. In addition, the structure of these synthetic cannabinoids is such that they have stronger effects, and longer durations of action at the same cannabinoid receptors that are activated by delta-9-THC. Although many of these drugs are widely consumed, their safety is generally untested, and their complete pharmacological sites of action remain unknown. The illicit nature of these compounds and their incompletely understood pharmacological actions has resulted in a large increase in world-wide emergency room visits by individuals using these drugs. Our initial studies have examined 3 compounds that were isolated from material seized by the U.S. Drug Enforcement Agency (DEA), and subsequently synthesized by professional chemists. These studies show that the compounds AM-2201, and XLR-11 are full agonists at CB1 receptors that inhibit glutamate release in the hippocampus. This is in contrast to delta-9-THC, which purportedly acts as a partial agonist, demonstrating approximately one-half of the ability to inhibit glutamate responses as the synthetic molecules. In addition, these synthetic cannabinoids were much more potent than delta-9-THC on this measure. Another compound that we tested, known as JWH-018, was also more efficacious than delta-9-THC, but less potent than the other 2 synthetic compounds. Our general conclusion thus far is that the synthetic cannabinoids can bind to the cannabinoid CB1 receptor with much greater potency and efficacy than delta-9-THC. We predict that this will lead to a much greater inhibition of neurotransmitter release, and a greater disruption of hippocampus-dependent cognition, and perhaps result in much higher levels of anxiety in humans. Additionally, these pharmacological properties of the synthetic cannabinoids would result in much longer durations of action, compared to delta-9-THC, because of the higher potency. Ongoing studies are designed to look for off site effects of these compounds at non-cannabinoid receptor targets in the brain. Currently, we are evaluating potential interactions of these ligands with serotonin type 1 receptors in the cerebral cortex, as well as at calcium-activated potassium channels in the nucleus accumbens in rat brain slices. Preliminary data suggest that there may be actions of these synthetic cannabinoids at other molecular sites in the CNS.