Carl R. Lupica

Independent Presynaptic and Postsynaptic Mechanisms Regulate Endocannabinoid Signaling at Multiple Synapses in the Ventral Tegmental Area

Dopamine (DA) neurons in the ventral tegmental area have been implicated in psychiatric disorders and drug abuse. Understanding the mechanisms through which their activity is regulated via the modulation of afferent input is imperative to understanding their roles in these conditions. Here we demonstrate that endocannabinoids liberated from DA neurons activate cannabinoid CB1 receptors located on glutamatergic axons and on GABAergic terminals targeting GABAB receptors located on these cells. Endocannabinoid release was initiated by inhibiting either presynaptic type-III metabotropic glutamate receptors or postsynaptic calcium-activated potassium channels, two conditions that also promote enhanced DA neuron excitability and bursting. Thus, activity-dependent release of endocannabinoids may act as a regulatory feedback mechanism to inhibit synaptic inputs in response to DA neuron bursting, thereby regulating firing patterns that may fine-tune DA release from afferent terminals.

Antagonists of the Receptor-G Protein Interface Block Gi-coupled Signal Transduction

The carboxyl terminus of heterotrimeric G protein α subunits plays an important role in receptor interaction. We demonstrate that peptides corresponding to the last 11 residues of Gαi1/2 or Gαo1 impair agonist binding to A1 adenosine receptors, whereas Gαs or Gαt peptides have no effect. Previously, by using a combinatorial library we identified a series of Gαtpeptide analogs that bind rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996)J. Biol. Chem. 271, 361–366). Native Gαi1/2 peptide as well as several analogs were tested for their ability to modulate agonist binding or antagonist-agonist competition using cells overexpressing human A1 adenosine receptors. Three peptide analogs decreased the K i , suggesting that they disrupt the high affinity receptor-G protein interaction and stabilize an intermediate affinity state. To study the ability of the peptides to compete with endogenous Gαiproteins and block signal transduction in a native setting, we measured activation of G protein-coupled K+ channels through A1 adenosine or γ-aminobutyric acid, type B, receptors in hippocampal CA1 pyramidal neurons. Native Gαi1/2, peptide, and certain analog peptides inhibited receptor-mediated K+ channel gating, dependent on which receptor was activated. This differential perturbation of receptor-G protein interaction suggests that receptors that act on the same G protein can be selectively disrupted.

Control of cannabinoid CB1 receptor function on glutamate axon terminals by endogenous adenosine acting at A1 receptors

Marijuana is a widely used drug that impairs memory through interaction between its psychoactive constituent, Δ-9-tetrahydrocannabinol (Δ9-THC), and CB1 receptors (CB1Rs) in the hippocampus. CB1Rs are located on Schaffer collateral (Sc) axon terminals in the hippocampus, where they inhibit glutamate release onto CA1 pyramidal neurons. This action is shared by adenosine A1 receptors (A1Rs), which are also located on Sc terminals. Furthermore, A1Rs are tonically activated by endogenous adenosine (eADO), leading to suppressed glutamate release under basal conditions. Colocalization of A1Rs and CB1Rs, and their coupling to shared components of signal transduction, suggest that these receptors may interact. We examined the roles of A1Rs and eADO in regulating CB1R inhibition of glutamatergic synaptic transmission in the rodent hippocampus. We found that A1R activation by basal or experimentally increased levels of eADO reduced or eliminated CB1R inhibition of glutamate release, and that blockade of A1Rs with caffeine or other antagonists reversed this effect. The CB1R–A1R interaction was observed with the agonists WIN55,212-2 and Δ9-THC and during endocannabinoid-mediated depolarization-induced suppression of excitation. A1R control of CB1Rs was stronger in the C57BL/6J mouse hippocampus, in which eADO levels were higher than in Sprague Dawley rats, and the eADO modulation of CB1R effects was absent in A1R knock-out mice. Since eADO levels and A1R activation are regulated by homeostatic, metabolic, and pathological factors, these data identify a mechanism in which CB1R function can be controlled by the brain adenosine system. Additionally, our data imply that caffeine may potentiate the effects of marijuana on hippocampal function.

Nogo receptor 1 regulates formation of lasting memories

Formation of lasting memories is believed to rely on structural alterations at the synaptic level. We had found that increased neuronal activity down-regulates Nogo receptor-1 (NgR1) in brain regions linked to memory formation and storage, and postulated this to be required for formation of lasting memories. We now show that mice with inducible overexpression of NgR1 in forebrain neurons have normal long-term potentiation and normal 24-h memory, but severely impaired month-long memory in both passive avoidance and swim maze tests. Blocking transgene expression normalizes these memory impairments. Nogo, Lingo-1, Troy, endogenous NgR1, and BDNF mRNA expression levels were not altered by transgene expression, suggesting that the impaired ability to form lasting memories is directly coupled to inability to down-regulate NgR1. Regulation of NgR1 may therefore serve as a key regulator of memory consolidation. Understanding the molecular underpinnings of synaptic rearrangements that carry lasting memories may facilitate development of treatments for memory dysfunction.

The Endocannabinoid Anandamide Inhibits the Function of α4β2 Nicotinic Acetylcholine Receptors

The effects of the endocannabinoid anandamide (arachidonylethanolamide, AEA) on the function of α4β2 nicotinic acetylcholine receptors (nAChR) stably expressed in SH-EP1 cells were investigated using the whole-cell patch-clamp technique. In the concentration range of 200 nM to 2 μM, AEA significantly reduced the maximal amplitudes and increased the desensitization of acetylcholine (ACh)-induced currents. The effects of AEA could be neither replicated by the exogenous cannabinoid Δ9-tetrahydrocannabinol (1 μM) nor reversed by the selective CB1 receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (SR-141716A) (1 μM). The actions of AEA were apparent when applied extracellularly but not during intracellular dialysis. Furthermore, the effects of AEA ACh currents were not altered by the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. The onset and washout of the AEA effects required several minutes (10–30 min), but the latter was significantly decreased in the presence of lipid-free bovine serum albumin (BSA). Moreover, BSA alone increased peak ACh current amplitudes and diminished desensitization rates in naive cells, suggesting a tonic modulation of α4β2 nAChR function by an endogenous AEA-like lipid. Further analysis of AEA effects on α4β2 nAChR-mediated currents, using a two-stage desensitization model, indicated that the first forward rate constant leading to desensitization, k1, increased nearly 30-fold as a linear function of the AEA concentration. In contrast, the observation that the other three rate constants were unaltered by AEA suggested that AEA raised the energy of the activated state. These results indicate that AEA directly inhibits the function of α4β2 nAChRs in a CB1 receptor-independent manner.

Afferent-Specific AMPA Receptor Subunit Composition and Regulation of Synaptic Plasticity in Midbrain Dopamine Neurons by Abused Drugs

Ventral tegmental area (VTA) dopamine (DA) neurons play a pivotal role in processing reward-related information and are involved in drug addiction and mental illness in humans. Information is conveyed to the VTA in large part by glutamatergic afferents that arise in various brain nuclei, including the pedunculopontine nucleus (PPN). Using a unique rat brain slice preparation, we found that PPN stimulation activates afferents targeting GluR2-containing AMPA receptors (AMPAR) on VTA DA neurons, and these afferents did not exhibit long-term depression (LTD). In contrast, activation of glutamate afferents onto the same DA neurons via stimulation within the VTA evoked EPSCs mediated by GluR2-lacking AMPARs that demonstrated LTD or EPSCs mediated by GluR2-containing AMPA receptors that did not express LTD. Twenty-four hours after single cocaine injections to rats, GluR2-lacking AMPARs were increased at both PPN and local VTA projections, and this permitted LTD expression in both pathways. Single injections with the main psychoactive ingredient of marijuana, Δ9-tetrahydrocannabinol (Δ9-THC), increased GluR2-lacking AMPA receptors and permitted LTD in only the PPN pathway, and these effects were prevented by in vivo pretreatment with the cannabinoid CB1 receptor antagonist AM251. These results demonstrate that cocaine more globally increases GluR2-lacking AMPA receptors at all glutamate synapses on VTA dopamine neurons, whereas Δ9-THC selectively increased GluR2-lacking AMPA receptors at subcortical PPN synapses. This suggests that different abused drugs may exert influence over distinct sets of glutamatergic afferents to VTA DA neurons which may be associated with different reinforcing or addictive properties of these drugs.

Dopamine D4 Receptor Excitation of Lateral Habenula Neurons via Multiple Cellular Mechanisms

Glutamatergic lateral habenula (LHb) output communicates negative motivational valence to ventral tegmental area (VTA) dopamine (DA) neurons via activation of the rostromedial tegmental nucleus (RMTg). However, the LHb also receives a poorly understood DA input from the VTA, which we hypothesized constitutes an important feedback loop regulating DA responses to stimuli. Using whole-cell electrophysiology in rat brain slices, we find that DA initiates a depolarizing inward current (IDAi) and increases spontaneous firing in 32% of LHb neurons. IDAi was also observed upon application of amphetamine or the DA uptake blockers cocaine or GBR12935, indicating involvement of endogenous DA. IDAi was blocked by D4 receptor (D4R) antagonists (L745,870 or L741,742), and mimicked by a selective D4R agonist (A412997). IDAi was associated with increased whole-cell conductance and was blocked by Cs+ or a selective blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel, ZD7288. IDAi was also associated with a depolarizing shift in half-activation voltage for the hyperpolarization-activated cation current (Ih) mediated by HCN channels. Recordings from LHb neurons containing fluorescent retrograde tracers revealed that IDAi was observed only in cells projecting to the RMTg and not the VTA. In parallel with direct depolarization, DA also strongly increased synaptic glutamate release and reduced synaptic GABA release onto LHb cells. These results demonstrate that DA can excite glutamatergic LHb output to RMTg via multiple cellular mechanisms. Since the RMTg strongly inhibits midbrain DA neurons, activation of LHb output to RMTg by DA represents a negative feedback loop that may dampen DA neuron output following activation.

Orbitofrontal activation restores insight lost after cocaine use

Addiction is characterized by a lack of insight into the likely outcomes of ones behavior. Insight, or the ability to imagine outcomes, is evident when outcomes have not been directly experienced. Using this concept, work in both rats and humans has recently identified neural correlates of insight in the medial and orbital prefrontal cortices. We found that these correlates were selectively abolished in rats by cocaine self-administration. Their abolition was associated with behavioral deficits and reduced synaptic efficacy in orbitofrontal cortex, the reversal of which by optogenetic activation restored normal behavior. These results provide a link between cocaine use and problems with insight. Deficits in these functions are likely to be particularly important for problems such as drug relapse, in which behavior fails to account for likely adverse outcomes. As such, our data provide a neural target for therapeutic approaches to address these defining long-term effects of drug use.

CYP3A5 Mediates Effects of Cocaine on Human Neocorticogenesis: Studies using an In Vitro 3D Self-Organized hPSC Model with a Single Cortex-Like Unit.

Because of unavoidable confounding variables in the direct study of human subjects, it has been difficult to unravel the effects of prenatal cocaine exposure on the human fetal brain, as well as the cellular and biochemical mechanisms involved. Here, we propose a novel approach using a human pluripotent stem cell (hPSC)-based 3D neocortical organoid model. This model retains essential features of human neocortical development by encompassing a single self-organized neocortical structure, without including an animal-derived gelatinous matrix. We reported previously that prenatal cocaine exposure to rats during the most active period of neural progenitor proliferation induces cytoarchitectural changes in the embryonic neocortex. We also identified a role of CYP450 and consequent oxidative ER stress signaling in these effects. However, because of differences between humans and rodents in neocorticogenesis and brain CYP metabolism, translation of the research findings from the rodent model to human brain development is uncertain. Using hPSC 3D neocortical organoids, we demonstrate that the effects of cocaine are mediated through CYP3A5-induced generation of reactive oxygen species, inhibition of neocortical progenitor cell proliferation, induction of premature neuronal differentiation, and interruption of neural tissue development. Furthermore, knockdown of CYP3A5 reversed these cocaine-induced pathological phenotypes, suggesting CYP3A5 as a therapeutic target to mitigate the deleterious neurodevelopmental effects of prenatal cocaine exposure in humans. Moreover, 3D organoid methodology provides an innovative platform for identifying adverse effects of abused psychostimulants and pharmaceutical agents, and can be adapted for use in neurodevelopmental disorders with genetic etiologies.

Attenuated response to methamphetamine sensitization and deficits in motor learning and memory after selective deletion of β-catenin in dopamine neurons

In the present study, we analyzed mice with a targeted deletion of β-catenin in DA neurons (DA-βcat KO mice) to address the functional significance of this molecule in the shaping of synaptic responses associated with motor learning and following exposure to drugs of abuse. Relative to controls, DA-βcat KO mice showed significant deficits in their ability to form long-term memories and displayed reduced expression of methamphetamine-induced behavioral sensitization after subsequent challenge doses with this drug, suggesting that motor learning and drug-induced learning plasticity are altered in these mice. Morphological analyses showed no changes in the number or distribution of tyrosine hydroxylase-labeled neurons in the ventral midbrain. While electrochemical measurements in the striatum determined no changes in acute DA release and uptake, a small but significant decrease in DA release was detected in mutant animals after prolonged repetitive stimulation, suggesting a possible deficit in the DA neurotransmitter vesicle reserve pool. However, electron microscopy analyses did not reveal significant differences in the content of synaptic vesicles per terminal, and striatal DA levels were unchanged in DA-βcat KO animals. In contrast, striatal mRNA levels for several markers known to regulate synaptic plasticity and DA neurotransmission were altered in DA-βcat KO mice. This study demonstrates that ablation of β-catenin in DA neurons leads to alterations of motor and reward-associated memories and to adaptations of the DA neurotransmitter system and suggests that β-catenin signaling in DA neurons is required to facilitate the synaptic remodeling underlying the consolidation of long-term memories.