Tanya L. Daigle

Acute Brain Slice Methods for Adult and Aging Animals: Application of Targeted Patch Clamp Analysis and Optogenetics

The development of the living acute brain slice preparation for analyzing synaptic function roughly a half century ago was a pivotal achievement that greatly influenced the landscape of modern neuroscience. Indeed, many neuroscientists regard brain slices as the gold-standard model system for detailed cellular, molecular, and circuitry level analysis and perturbation of neuronal function. A critical limitation of this model system is the difficulty in preparing slices from adult and aging animals, and over the past several decades few substantial methodological improvements have emerged to facilitate patch clamp analysis in the mature adult stage. In this chapter we describe a robust and practical protocol for preparing brain slices from mature adult mice that are suitable for patch clamp analysis. This method reduces swelling and damage in superficial layers of the slices and improves the success rate for targeted patch clamp recordings, including recordings from fluorescently labeled populations in slices derived from transgenic mice. This adult brain slice method is suitable for diverse experimental applications, including both monitoring and manipulating neuronal activity with genetically encoded calcium indicators and optogenetic actuators, respectively. We describe the application of this adult brain slice platform and associated methods for screening kinetic properties of Channelrhodopsin (ChR) variants expressed in genetically defined neuronal subtypes.

GRK2: multiple roles beyond G protein-coupled receptor desensitization

G protein-coupled receptor kinases (GRKs) regulate numerous G protein-coupled receptors (GPCRs) by phosphorylating the intracellular domain of the active receptor, resulting in receptor desensitization and internalization. GRKs also regulate GPCR trafficking in a phosphorylation-independent manner via direct protein-protein interactions. Emerging evidence suggests that GRK2, the most widely studied member of this family of kinases, modulates multiple cellular responses in various physiological contexts by either phosphorylating non-receptor substrates or interacting directly with signaling molecules. In this review, we discuss traditional and newly discovered roles of GRK2 in receptor internalization and signaling as well as its impact on non-receptor substrates. We also discuss novel exciting roles of GRK2 in the regulation of dopamine receptor signaling and in the activation and trafficking of the atypical GPCR, Smoothened (Smo).

A Gαs DREADD Mouse for Selective Modulation of cAMP Production in Striatopallidal Neurons

Here, we describe a newly generated transgenic mouse in which the Gs DREADD (rM3Ds), an engineered G protein-coupled receptor, is selectively expressed in striatopallidal medium spiny neurons (MSNs). We first show that in vitro, rM3Ds can couple to Gαolf and induce cAMP accumulation in cultured neurons and HEK-T cells. The rM3Ds was then selectively and stably expressed in striatopallidal neurons by creating a transgenic mouse in which an adenosine2A (adora2a) receptor-containing bacterial artificial chromosome was employed to drive rM3Ds expression. In the adora2A-rM3Ds mouse, activation of rM3Ds by clozapine-N-oxide (CNO) induces DARPP-32 phosphorylation, consistent with the known consequence of activation of endogenous striatal Gαs-coupled GPCRs. We then tested whether CNO administration would produce behavioral responses associated with striatopallidal Gs signaling and in this regard CNO dose-dependently decreases spontaneous locomotor activity and inhibits novelty induced locomotor activity. Last, we show that CNO prevented behavioral sensitization to amphetamine and increased AMPAR/NMDAR ratios in transgene-expressing neurons of the nucleus accumbens shell. These studies demonstrate the utility of adora2a-rM3Ds transgenic mice for the selective and noninvasive modulation of Gαs signaling in specific neuronal populations in vivo.This unique tool provides a new resource for elucidating the roles of striatopallidal MSN Gαs signaling in other neurobehavioral contexts.

A dopamine D1 receptor-dependent β-arrestin signaling complex potentially regulates morphine-induced psychomotor activation but not reward in mice.

Morphine is a widely used analgesic in humans that is associated with multiple untoward effects, such as addiction and physical dependence. In rodent models, morphine also induces locomotor activity. These effects likely involve functionally selective mechanisms. Indeed, G protein-coupled receptor desensitization and adaptor protein β-arrestin 2 (βarr2) through its interaction with the μ-opioid receptor regulates the analgesic but not the rewarding properties of morphine. However, βarr2 is also required for morphine-induced locomotor activity in mice, but the exact cellular and molecular mechanisms that mediate this arrestin-dependent behavior are not understood. In this study, we show that βarr2 is required for morphine-induced locomotor activity in a dopamine D1 receptor (D1R)-dependent manner and that a βarr2/phospho-ERK (βarr2/pERK) signaling complex may mediate this behavior. Systemic administration of SL327, an MEK inhibitor, inhibits morphine-induced locomotion in wild-type mice in a dose-dependent manner. Acute morphine administration to mice promotes the formation of a βarr2/pERK signaling complex. Morphine-induced locomotor activity and formation of the βarr2/pERK signaling complex is blunted in D1R knockout (D1-KO) mice and is presumably independent of D2 dopamine receptors. However, D1Rs are not required for morphine-induced reward as D1-KO mice show the same conditioned place preference for morphine as do control mice. Taken together, these results suggest a potential role for a D1R-dependent βarr2/pERK signaling complex in selectively mediating the locomotor-stimulating but not the rewarding properties of morphine.

Targeting β-arrestin2 in the treatment of l-DOPA–induced dyskinesia in Parkinson’s disease

Significance β-Arrestins are unique proteins that have multiple cellular functions such as G protein-coupled receptor signal desensitization, protein trafficking and signaling molecule scaffolding. Treatment of Parkinson’s disease (PD) motor symptoms by l-3,4-dihydroxyphenylalanine (l-DOPA) has been hampered by abnormal involuntary movements or dyskinetic side effects. The cause of these dyskinesias has been attributed to receptor supersensitivity and uncontrolled neuronal excitability. Here we demonstrate in multiple preclinical models of l-DOPA–induced dyskinesias and PD that expression levels of β-arrestin2 can alter manifestation of these dyskinesias by reducing receptor supersensitivity while maintaining the therapeutic effect of l-DOPA. Thus novel drugs that increase β-arrestin–dependent function at dopamine receptors may be useful in ameliorating PD motor symptoms without inducing dyskinesias. Parkinson’s disease (PD) is characterized by severe locomotor deficits and is commonly treated with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (l-DOPA), but its prolonged use causes dyskinesias referred to as l-DOPA–induced dyskinesias (LIDs). Recent studies in animal models of PD have suggested that dyskinesias are associated with the overactivation of G protein-mediated signaling through DA receptors. β-Arrestins desensitize G protein signaling at DA receptors (D1R and D2R) in addition to activating their own G protein-independent signaling events, which have been shown to mediate locomotion. Therefore, targeting β-arrestins in PD l-DOPA therapy might prove to be a desirable approach. Here we show in a bilateral DA-depletion mouse model of Parkinson’s symptoms that genetic deletion of β-arrestin2 significantly limits the beneficial locomotor effects while markedly enhancing the dyskinesia-like effects of acute or chronic l-DOPA treatment. Viral rescue or overexpression of β-arrestin2 in knockout or control mice either reverses or protects against LIDs and its key biochemical markers. In other more conventional animal models of DA neuron loss and PD, such as 6-hydroxydopamine–treated mice or rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–treated nonhuman primates, β-arrestin2 overexpression significantly reduced dyskinesias while maintaining the therapeutic effect of l-DOPA. Considerable efforts are being spent in the pharmaceutical industry to identify therapeutic approaches to block LIDs in patients with PD. Our results point to a potential therapeutic approach, whereby development of either a genetic or pharmacological intervention to enhance β-arrestin2- or limit G protein-dependent D1/D2R signaling could represent a more mechanistically informed strategy.

N-Aryl Piperazine Metabotropic Glutamate Receptor 5 Positive Allosteric Modulators Possess Efficacy in Preclinical Models of NMDA Hypofunction and Cognitive Enhancement

Impaired transmission through glutamatergic circuits has been postulated to play a role in the underlying pathophysiology of schizophrenia. Furthermore, inhibition of the N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors (NMDAR) induces a syndrome that recapitulates many of the symptoms observed in patients with schizophrenia. Selective activation of metabotropic glutamate receptor subtype 5 (mGlu5) may provide a novel therapeutic approach for treatment of symptoms associated with schizophrenia through facilitation of transmission through central glutamatergic circuits. Here, we describe the characterization of two novel N-aryl piperazine mGlu5 positive allosteric modulators (PAMs): 2-(4-(2-(benzyloxy)acetyl)piperazin-1-yl)benzonitrile (VU0364289) and 1-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-((4-fluorobenzyl)oxy)ethanone (DPFE). VU0364289 and DPFE induced robust leftward shifts in the glutamate concentration-response curves for Ca2+ mobilization and extracellular signal-regulated kinases 1 and 2 phosphorylation. Both PAMs displayed micromolar affinity for the common mGlu5 allosteric binding site and high selectivity for mGlu5. VU0364289 and DPFE possessed suitable pharmacokinetic properties for dosing in vivo and produced robust dose-related effects in reversing amphetamine-induced hyperlocomotion, a preclinical model predictive of antipsychotic-like activity. In addition, DPFE enhanced acquisition of contextual fear conditioning in rats and reversed behavioral deficits in a mouse model of NMDAR hypofunction. In contrast, DPFE had no effect on reversing apomorphine-induced disruptions of prepulse inhibition of the acoustic startle reflex. These mGlu5 PAMs also increased monoamine levels in the prefrontal cortex, enhanced performance in a hippocampal-mediated memory task, and elicited changes in electroencephalogram dynamics commensurate with procognitive effects. Collectively, these data support and extend the role for the development of novel mGlu5 PAMs for the treatment of psychosis and cognitive deficits observed in individuals with schizophrenia.

Selective Deletion of GRK2 Alters Psychostimulant-Induced Behaviors and Dopamine Neurotransmission

GRK2 is a G protein-coupled receptor kinase (GRK) that is broadly expressed and is known to regulate diverse types of receptors. GRK2 null animals exhibit embryonic lethality due to a severe developmental heart defect, which has precluded the study of this kinase in the adult brain. To elucidate the specific role of GRK2 in the brain dopamine (DA) system, we used a conditional gene knockout approach to selectively delete GRK2 in DA D1 receptor (D1R)-, DA D2 receptor (D2R)-, adenosine 2A receptor (A2AR)-, or DA transporter (DAT)-expressing neurons. Here we show that select GRK2-deficient mice display hyperactivity, hyposensitivity, or hypersensitivity to the psychomotor effects of cocaine, altered striatal signaling, and DA release and uptake. Mice with GRK2 deficiency in D2R-expressing neurons also exhibited increased D2 autoreceptor activity. These findings reveal a cell-type-specific role for GRK2 in the regulation of normal motor behavior, sensitivity to psychostimulants, dopamine neurotransmission, and D2 autoreceptor function.

Elimination of GRK2 from Cholinergic Neurons Reduces Behavioral Sensitivity to Muscarinic Receptor Activation

Although G-protein-coupled receptor kinase 2 (GRK2) is the most widely studied member of a family of kinases that has been shown to exert powerful influences on a variety of G-protein-coupled receptors, its role in the brain remains largely unknown. Here we report the localization of GRK2 in the mouse brain and generate novel conditional knock-out (KO) mice to assess the physiological importance of this kinase in cholinergic neurons. Mice with the selective deletion of GRK2 in this cell population (ChATIRES-creGrk2f/f KO mice) exhibit reduced behavioral responsiveness to challenge with oxotremorine-M (Oxo-M), a nonselective muscarinic acetylcholine receptor agonist. Specifically, Oxo-M-induced hypothermia, hypolocomotion, and salivation were markedly reduced in these animals, while analgesic responses were unaltered. In contrast, we found that GRK2 deficiency in cholinergic neurons does not alter cocaine-induced psychomotor activation, behavioral sensitization, or conditioned place preference. These results demonstrate that the elimination of GRK2 in cholinergic neurons reduces sensitivity to select muscarinic-mediated behaviors, while dopaminergic effects remain intact and further suggests that GRK2 may selectively impair muscarinic acetylcholine receptor-mediated function in vivo.

Opposite function of dopamine D1 and N-methyl-D-aspartate receptors in striatal cannabinoid-mediated signaling

It is well established that the cannabinoid and dopamine systems interact at various levels to regulate basal ganglia function. Although it is well known that acute administration of cannabinoids to mice can modify dopamine‐dependent behaviors, the intraneuronal signaling pathways employed by these agents in the striatum are not well understood. Here we used knockout mouse models to examine the regulation of striatal extracellular‐signal‐regulated kinases 1 and 2 (ERK1/2) signaling by behaviorally relevant doses of cannabinoids. This cellular pathway has been implicated as a central mediator of drug reward and synaptic plasticity. In C57BL/6J mice, acute administration of the cannabinoid agonists, (−)‐11‐hydroxydimethylheptyl‐Δ8‐tetrahydrocannabinol (HU‐210) and delta‐9‐tetrahydrocannabinol (Δ9‐THC), promoted a dose‐ and time‐dependent decrease in the phosphorylation of ERK1/2 in dorsal striatum. Co‐administration of the CB1 cannabinoid receptor antagonist N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide(AM251) with HU‐210 prevented ERK1/2 inactivation, indicating a requirement for activation of this receptor. In dopamine D1 receptor knockout animals treated with HU‐210, the magnitude of the HU‐210‐dependent decrease in striatal ERK1/2 signaling was greater than in wild‐type controls. In contrast, HU‐210 administration to N‐methyl‐D‐aspartate receptor knockdown mice was ineffective at promoting striatal ERK1/2 inactivation. Genetic deletion of other potential ERK1/2 mediators, the dopamine D2 receptors or β‐arrestin‐1 or ‐2, did not affect the HU‐210‐induced modulation of ERK1/2 signaling in the striatum. These results support the hypothesis that dopamine D1 receptors and N‐methyl‐D‐aspartate receptors act in an opposite manner to regulate striatal CB1 cannabinoid receptor signal transduction.

Opposite function of dopamine D1 and NMDA receptors in striatal cannabinoid-mediated signaling

It is well established that the cannabinoid and dopamine systems interact at various levels to regulate basal ganglia function. Although it is well known that acute administration of cannabinoids to mice can modify dopamine‐dependent behaviors, the intraneuronal signaling pathways employed by these agents in the striatum are not well understood. Here we used knockout mouse models to examine the regulation of striatal extracellular‐signal‐regulated kinases 1 and 2 (ERK1/2) signaling by behaviorally relevant doses of cannabinoids. This cellular pathway has been implicated as a central mediator of drug reward and synaptic plasticity. In C57BL/6J mice, acute administration of the cannabinoid agonists, (−)‐11‐hydroxydimethylheptyl‐Δ8‐tetrahydrocannabinol (HU‐210) and delta‐9‐tetrahydrocannabinol (Δ9‐THC), promoted a dose‐ and time‐dependent decrease in the phosphorylation of ERK1/2 in dorsal striatum. Co‐administration of the CB1 cannabinoid receptor antagonist N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide(AM251) with HU‐210 prevented ERK1/2 inactivation, indicating a requirement for activation of this receptor. In dopamine D1 receptor knockout animals treated with HU‐210, the magnitude of the HU‐210‐dependent decrease in striatal ERK1/2 signaling was greater than in wild‐type controls. In contrast, HU‐210 administration to N‐methyl‐D‐aspartate receptor knockdown mice was ineffective at promoting striatal ERK1/2 inactivation. Genetic deletion of other potential ERK1/2 mediators, the dopamine D2 receptors or β‐arrestin‐1 or ‐2, did not affect the HU‐210‐induced modulation of ERK1/2 signaling in the striatum. These results support the hypothesis that dopamine D1 receptors and N‐methyl‐D‐aspartate receptors act in an opposite manner to regulate striatal CB1 cannabinoid receptor signal transduction.