Norepinephrine release in the cerebellum contributes to aversive learning.

The modulation of dopamine release from midbrain projections to the striatum has long been demonstrated in reward-based learning, but the synaptic basis of aversive learning is far less characterized. The cerebellum receives axonal projections from the locus coeruleus, and norepinephrine release is implicated in states of arousal and stress, but whether aversive learning relies on plastic changes in norepinephrine release in the cerebellum is unknown. Here we report that in mice, norepinephrine is released in the cerebellum following an unpredicted noxious event (a foot-shock) and that this norepinephrine release is potentiated powerfully with fear acquisition as animals learn that a previously neutral stimulus (tone) predicts the aversive event. Importantly, both chemogenetic and optogenetic inhibition of the locus coeruleus-cerebellum pathway block fear memory without impairing motor function. Thus, norepinephrine release in the cerebellum is modulated by experience and underlies aversive learning.

Development of a Dual Fluorescent and Magnetic Resonance False Neurotransmitter That Reports Accumulation and Release from Dopaminergic Synaptic Vesicles.

Myriad neuropsychiatric disorders are due to dopamine dysfunction. However, understanding these disorders is limited by our ability to measure dopamine storage and release. Fluorescent false neurotransmitters (FFNs), small-molecule dyes that co-transit through the synaptic vesicle cycle, have allowed us to image dopamine in cell culture and acute brain slice, but in vivo microscopy is constrained by the biopenetrance of light. Here, we adapt FFNs into magnetic resonance false neurotransmitters (MFNs). The design principles guiding MFNs are (1) the molecule is a valid false neurotransmitter and (2) it has a 19F-substituent near a pH-sensing functional group, which (3) has pKa close to 6 so that the probe within vesicles is protonated. We demonstrate that MFN103 meets these criteria. While a magnetic resonance spectroscopy (MRS) signal was too low for measurement in vivo with the current technology, in principle, MFNs can quantify neurotransmitters within and without synaptic vesicles, which may underlie noninvasive in vivo analysis of dopamine neurotransmission.

The chemical tools for imaging dopamine release.

Dopamine is a modulatory neurotransmitter involved in learning, motor functions, and reward. Many neuropsychiatric disorders, including Parkinson's disease, autism, and schizophrenia, are associated with imbalances or dysfunction in the dopaminergic system. Yet, our understanding of these pervasive public health issues is limited by our ability to effectively image dopamine in humans, which has long been a goal for chemists and neuroscientists. The last two decades have witnessed the development of many molecules used to trace dopamine. We review the small molecules, nanoparticles, and protein sensors used with fluorescent microscopy/photometry, MRI, and PET that shape dopamine research today. None of these tools observe dopamine itself, but instead harness the biology of the dopamine system-its synthetic and metabolic pathways, synaptic vesicle cycle, and receptors-in elegant ways. Their advantages and weaknesses are covered here, along with recent examples and the chemistry and biology that allow them to function.

Coordinated Postnatal Maturation of Striatal Cholinergic Interneurons and Dopamine Release Dynamics in Mice.

Dynamic changes in motor abilities and motivated behaviors occur during the juvenile and adolescent periods. The striatum is a subcortical nucleus critical to action selection, motor learning, and reward processing. Its tonically active cholinergic interneuron (ChI) is an integral regulator of the synaptic activity of other striatal neurons, as well as afferent axonal projections of midbrain dopamine (DA) neurons; however, little is known about its development. Here, we report that ChI spontaneous activity increases during postnatal development of male and female mice, concomitant with a decreased afterhyperpolarization (AHP). We characterized the postnatal development of four currents that contribute to the spontaneous firing rate of ChIs, including ISK, IA, Ih, and INaP We demonstrated that the developmental increase in INaP drives increased ChI firing rates during the postnatal period and can be reversed by the INaP inhibitor, ranolazine. We next addressed whether immature cholinergic signaling may lead to functional differences in DA release during the juvenile period. In the adult striatum, nicotinic acetylcholine receptors (nAChRs) prevent linear summation of DA release in response to trains of high-frequency stimuli. We show that, in contrast, during the second postnatal week, DA release linearly sums with trains of high-frequency stimuli. Consistently, nAChR antagonists exert little effect on dopamine release at postnatal day (P)10, but enhance the summation of evoked DA release in mice older than postnatal day P28. Together, these results reveal that postnatal maturation of ChI activity is due primarily to enhanced INaP and identify an interaction between developing cholinergic signaling and DA neurotransmission in the juvenile striatum.SIGNIFICANCE STATEMENT Motor skills and motivated behavior develop rapidly in juvenile rodents. Recent work has highlighted processes that contribute to the postnatal maturation of striatal principal neurons during development. The functional development of the striatal cholinergic interneuron (ChI), however, has been unexplored. In this study, we tracked the ontogeny of ChI activity and cellular morphology, as well as the developmental trajectory of specific conductances that contribute to the activity of these cells. We further report a link between cholinergic signaling and dopamine (DA) release, revealing a change in the frequency-dependence of DA release during the early postnatal period that is mediated by cholinergic signaling. This study provides evidence that striatal microcircuits are dynamic during the postnatal period and that they undergo coordinated maturation.

mTOR Suppresses Macroautophagy During Striatal Postnatal Development and Is Hyperactive in Mouse Models of Autism Spectrum Disorders.

Macroautophagy (hereafter referred to as autophagy) plays a critical role in neuronal function related to development and degeneration. Here, we investigated whether autophagy is developmentally regulated in the striatum, a brain region implicated in neurodevelopmental disease. We demonstrate that autophagic flux is suppressed during striatal postnatal development, reaching adult levels around postnatal day 28 (P28). We also find that mTOR signaling, a key regulator of autophagy, increases during the same developmental period. We further show that mTOR signaling is responsible for suppressing autophagy, via regulation of Beclin-1 and VPS34 activity. Finally, we discover that autophagy is downregulated during late striatal postnatal development (P28) in mice with in utero exposure to valproic acid (VPA), an established mouse model of autism spectrum disorder (ASD). VPA-exposed mice also display deficits in striatal neurotransmission and social behavior. Correction of hyperactive mTOR signaling in VPA-exposed mice restores social behavior. These results demonstrate that neurons coopt metabolic signaling cascades to developmentally regulate autophagy and provide additional evidence that mTOR-dependent signaling pathways represent pathogenic signaling cascades in ASD mouse models that are active during specific postnatal windows.

Can Interactions Between α-Synuclein, Dopamine and Calcium Explain Selective Neurodegeneration in Parkinson's Disease?

Several lines of evidence place alpha-synuclein (aSyn) at the center of Parkinson's disease (PD) etiology, but it is still unclear why overexpression or mutated forms of this protein affect some neuronal populations more than others. Susceptible neuronal populations in PD, dopaminergic neurons of the substantia nigra pars compacta (SNpc) and the locus coeruleus (LC), are distinguished by relatively high cytoplasmic concentrations of dopamine and calcium ions. Here we review the evidence for the multi-hit hypothesis of neurodegeneration, including recent papers that demonstrate synergistic interactions between aSyn, calcium ions and dopamine that may lead to imbalanced protein turnover and selective susceptibility of these neurons. We conclude that decreasing the levels of any one of these toxicity mediators can be beneficial for the survival of SNpc and LC neurons, providing multiple opportunities for targeted drug interventions aimed at modifying the course of PD.

Secondary Ammonium Agonists Make Dual Cation-π Interactions in α4ß2 Nicotinic Receptors.

A cation-π interaction between the ammonium group of an agonist and a conserved tryptophan termed TrpB is a near universal feature of agonist binding to nicotinic acetylcholine receptors (nAChRs). TrpB is one of five residues that form the aromatic box of the agonist binding site, and for the prototype agonists ACh and nicotine, only TrpB makes a functional cation-π interaction. We report that, in addition to TrpB, a significant cation-π interaction is made to a second aromatic, TyrC2, by the agonists metanicotine, TC299423, varenicline, and nornicotine. A common structural feature of these agonists, and a distinction from ACh and nicotine, is a protonated secondary amine that provides the cation for the cation-π interaction. These results indicate a distinction in binding modes between agonists with subtly different structures that may provide guidance for the development of subtype-selective agonists of nAChRs.

Probing for and Quantifying Agonist Hydrogen Bonds in α6ß2 Nicotinic Acetylcholine Receptors.

Designing subtype-selective agonists for neuronal nicotinic acetylcholine receptors is a challenging and significant goal aided by intricate knowledge of each subtype's binding patterns. We previously reported that in α6ß2 receptors, acetylcholine makes a functional cation-π interaction with Trp149, but nicotine and TC299423 do not, suggesting a distinctive binding site. This work explores hydrogen binding at the backbone carbonyl associated with α6ß2 Trp149. Substituting residue i + 1, Thr150, with its α-hydroxy analogue (Tah) attenuates the carbonyl's hydrogen bond accepting ability. At α6(T150Tah)ß2, nicotine shows a 24-fold loss of function, TC299423 shows a modest loss, and acetylcholine shows no effect. Nicotine was further analyzed via a double-mutant cycle analysis utilizing N'-methylnicotinium, which indicated a hydrogen bond in α6ß2 with a ΔΔG of 2.6 kcal/mol. Thus, even though nicotine does not make the conserved cation-π interaction with Trp149, it still makes a functional hydrogen bond to its associated backbone carbonyl.

The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors.

Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish the relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical and temporomandibular pain.

Heterologous expression and nonsense suppression provide insights into agonist behavior at α6ß2 nicotinic acetylcholine receptors.

The α6-containing subtypes of the nicotinic acetylcholine receptor (nAChR) are localized to presynaptic terminals of the dopaminergic pathways of the central nervous system. Selective ligands for these nAChRs are potentially useful in both Parkinson's disease and addiction. For these and other goals, it is important to distinguish the binding behavior of agonists at the α6-ß2 binding site versus other subtypes. To study this problem, we apply nonsense suppression-based non-canonical amino acid mutagenesis. We report a combination of four mutations in α6ß2 that yield high-level heterologous expression in Xenopus oocytes. By varying mRNA injection ratios, two populations were observed with unique characteristics, likely due to differing stoichiometries. Responses to nine known nAChR agonists were analyzed at the receptor, and their corresponding EC50 values and efficacies are reported. The system is compatible with nonsense suppression, allowing structure-function studies between Trp149 - a conserved residue on loop B found to make a cation-π interaction at several nAChR subtypes - and several agonists. These studies reveal that acetylcholine forms a strong cation-π interaction with the conserved tryptophan, while nicotine and TC299423 do not, suggesting a unique pharmacology for the α6ß2 nAChR.