Smoking-Relevant Nicotine Concentration Attenuates the Unfolded Protein Response in Dopaminergic Neurons.

Retrospective epidemiological studies show an inverse correlation between susceptibility to Parkinson's disease and a person's history of tobacco use. Animal model studies suggest nicotine as a neuroprotective agent and nicotinic acetylcholine (ACh) receptors (nAChRs) as targets for neuroprotection, but the underlying neuroprotective mechanism(s) are unknown. We cultured mouse ventral midbrain neurons for 3 weeks. Ten to 20% of neurons were dopaminergic (DA), revealed by tyrosine hydroxylase (TH) immunoreactivity. We evoked mild endoplasmic reticulum (ER) stress with tunicamycin (Tu), producing modest increases in the level of nuclear ATF6, phosphorylated eukaryotic initiation factor 2α, nuclear XBP1, and the downstream proapoptotic effector nuclear C/EBP homologous protein. We incubated cultures for 2 weeks with 200 nm nicotine, the approximate steady-state concentration between cigarette smoking or vaping, or during nicotine patch use. Nicotine incubation suppressed Tu-induced ER stress and the unfolded protein response (UPR). Study of mice with fluorescent nAChR subunits showed that the cultured TH+ neurons displayed α4, α6, and ß3 nAChR subunit expression and ACh-evoked currents. Gene expression profile in cultures from TH-eGFP mice showed that the TH+ neurons also express several other genes associated with DA release. Nicotine also upregulated ACh-induced currents in DA neurons by ∼2.5-fold. Thus, nicotine, at a concentration too low to activate an appreciable fraction of plasma membrane nAChRs, induces two sequelae of pharmacological chaperoning in the ER: UPR suppression and nAChR upregulation. Therefore, one mechanism of neuroprotection by nicotine is pharmacological chaperoning, leading to UPR suppression. Measuring this pathway may help in assessing neuroprotection. SIGNIFICANCE STATEMENT: Parkinson's disease (PD) cannot yet be cured or prevented. However, many retrospective epidemiological studies reveal that PD is diagnosed less frequently in tobacco users. Existing programs attempting to develop nicotinic drugs that might exert this apparent neuroprotective effect are asking whether agonists, antagonists, partial agonists, or channel blockers show the most promise. The underlying logic resembles the previous development of varenicline for smoking cessation. We studied whether, and how, nicotine produces neuroprotective effects in cultured dopaminergic neurons, an experimentally tractable, mechanistically revealing neuronal system. We show that nicotine, operating via nicotinic receptors, does protect these neurons against endoplasmic reticulum stress. However, the mechanism is probably "inside-out": pharmacological chaperoning in the endoplasmic reticulum. This cellular-level insight could help to guide neuroprotective strategies.

Nicotinic receptor subtype-selective circuit patterns in the subthalamic nucleus.

The glutamatergic subthalamic nucleus (STN) exerts control over motor output through nuclei of the basal ganglia. High-frequency electrical stimuli in the STN effectively alleviate motor symptoms in movement disorders, and cholinergic stimulation boosts this effect. To gain knowledge about the mechanisms of cholinergic modulation in the STN, we studied cellular and circuit aspects of nicotinic acetylcholine receptors (nAChRs) in mouse STN. We discovered two largely divergent microcircuits in the STN; these are regulated in part by either α4ß2 or α7 nAChRs. STN neurons containing α4ß2 nAChRs (α4ß2 neurons) received more glutamatergic inputs, and preferentially innervated GABAergic neurons in the substantia nigra pars reticulata. In contrast, STN neurons containing α7 nAChRs (α7 neurons) received more GABAergic inputs, and preferentially innervated dopaminergic neurons in the substantia nigra pars compacta. Interestingly, local electrical stimuli excited a majority (79%) of α4ß2 neurons but exerted strong inhibition in 58% of α7 neurons, indicating an additional diversity of STN neurons: responses to electrical stimulation. Chronic exposure to nicotine selectively affects α4ß2 nAChRs in STN: this treatment increased the number of α4ß2 neurons, upregulated α4-containing nAChR number and sensitivity, and enhanced the basal firing rate of α4ß2 neurons both ex vivo and in vivo. Thus, chronic nicotine enhances the function of the microcircuit involving α4ß2 nAChRs. This indicates chronic exposure to nicotinic agonist as a potential pharmacological intervention to alter selectively the balance between these two microcircuits, and may provide a means to inhibit substantia nigra dopaminergic neurons.

Differential expression and function of nicotinic acetylcholine receptors in subdivisions of medial habenula.

Neuronal nAChRs in the medial habenula (MHb) to the interpeduncular nucleus (IPN) pathway are key mediators of nicotine's aversive properties. In this paper, we report new details regarding nAChR anatomical localization and function in MHb and IPN. A new group of knock-in mice were created that each expresses a single nAChR subunit fused to GFP, allowing high-resolution mapping. We find that α3 and ß4 nAChR subunit levels are strong throughout the ventral MHb (MHbV). In contrast, α6, ß2, ß3, and α4 subunits are selectively found in some, but not all, areas of MHbV. All subunits were found in both ChAT-positive and ChAT-negative cells in MHbV. Next, we examined functional properties of neurons in the lateral and central part of MHbV (MHbVL and MHbVC) using brain slice patch-clamp recordings. MHbVL neurons were more excitable than MHbVC neurons, and they also responded more strongly to puffs of nicotine. In addition, we studied firing responses of MHbVL and MHbVC neurons in response to bath-applied nicotine. Cells in MHbVL, but not those in MHbVC, increased their firing substantially in response to 1 µm nicotine. Additionally, MHbVL neurons from mice that underwent withdrawal from chronic nicotine were less responsive to nicotine application compared with mice withdrawn from chronic saline. Last, we characterized rostral and dorsomedial IPN neurons that receive input from MHbVL axons. Together, our data provide new details regarding neurophysiology and nAChR localization and function in cells within the MHbV.

Bidirectional dopamine modulation of excitatory and inhibitory synaptic inputs to subthalamic neuron subsets containing α4ß2 or α7 nAChRs.

The subthalamic nucleus (STN) possesses microcircuits distinguished by subtypes of nicotinic acetylcholine receptors (nAChRs). Although dysfunction of the STN is well-known in Parkinson's disease, there is still little information about whether dopamine differentially modulates excitatory and inhibitory synaptic inputs to STN neurons expressing different nAChR subtypes. To address this issue, we performed brain slice patch-clamp recordings on STN neurons, while we pharmacologically manipulated dopaminergic inputs. In STN neuron subsets containing either α4ß2 or α7 nAChRs, D1 and D2 receptors respectively enhanced and inhibited spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs) and firing rates. The elevation of dopamine levels resulted in diverse regulations of synaptic transmission in these two neuron subsets, and interestingly, the dopamine regulation of sIPSCs significantly correlated with that of sEPSCs. Surprisingly, depletion of dopamine either by reserpine treatment or by unilateral 6-OHDA lesion of nigrostriatal dopaminergic neurons did not alter synaptic inputs to STN neurons, but STN neurons in the 6-OHDA-lesioned side exhibited hyperactivity. In summary, dopamine regulated both GABAergic and glutamatergic synaptic inputs to STN neuron subsets containing either α4ß2 or α7 nAChRs, forming a balancing machinery to control neuronal activity. In parkinsonian mice, postsynaptic mechanisms may exist and contribute to the hyperactivity of STN neurons.

Chronic nicotine cell specifically upregulates functional alpha 4* nicotinic receptors: basis for both tolerance in midbrain and enhanced long-term potentiation in perforant path.

Understanding effects of chronic nicotine requires identifying the neurons and synapses whose responses to nicotine itself, and to endogenous acetylcholine, are altered by continued exposure to the drug. To address this problem, we developed mice whose alpha4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased alpha4 fluorescence in several regions; among these, midbrain and hippocampus were assessed functionally. Although the midbrain dopaminergic system dominates reward pathways, chronic nicotine does not change alpha4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional alpha4* receptors localize to the GABAergic neurons of the VTA and substantia nigra pars reticulata. In consequence, GABAergic neurons from chronically nicotine-treated mice have a higher basal firing rate and respond more strongly to nicotine; because of the resulting increased inhibition, dopaminergic neurons have lower basal firing and decreased response to nicotine. In hippocampus, chronic exposure to nicotine also increases alpha4* fluorescence on glutamatergic axons of the medial perforant path. In hippocampal slices from chronically treated animals, acute exposure to nicotine during tetanic stimuli enhances induction of long-term potentiation in the medial perforant path, showing that the upregulated alpha4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional alpha4* receptors therefore provides a possible explanation for two effects of chronic nicotine: sensitization of synaptic transmission in forebrain and tolerance of dopaminergic neuron firing in midbrain.

Enhanced expression of hypersensitive alpha4* nAChR in adult mice increases the loss of midbrain dopaminergic neurons.

We describe an inducible genetic model for degeneration of midbrain dopaminergic neurons in adults. In previous studies, knock-in mice expressing hypersensitive M2 domain Leu9'Ser (L9'S) alpha4 nicotinic receptors (nAChR) at near-normal levels displayed dominant neonatal lethality and dopaminergic deficits in embryonic midbrain, because the hypersensitive nAChR is excitotoxic. However, heterozygous L9'S mice that retain the neomycin resistance cassette (neo) in a neighboring intron express low levels of the mutant allele (approximately 25% of normal levels), and these neo-intact mice are therefore viable and fertile. The neo cassette is flanked by loxP sites. In adult animals, we locally injected helper-dependent adenovirus (HDA) expressing cre recombinase. Local excision of the neo cassette, via cre-mediated recombination, was verified by genomic analysis. In L9'S HDA-cre injected animals, locomotion was reduced both under baseline conditions and after amphetamine application. There was no effect in L9'S HDA-control treated animals or in wild-type (WT) littermates injected with either virus. Immunocytochemical analyses revealed marked losses (> 70%) of dopaminergic neurons in L9'S HDA-cre injected mice compared to controls. At 20-33 days postinjection in control animals, the coexpressed marker gene, yellow fluorescent protein (YFP), was expressed in many neurons and few glial cells near the injection, emphasizing the neurotropic utility of the HDA. Thus, HDA-mediated gene transfer into adult midbrain induced sufficient functional expression of cre in dopaminergic neurons to allow for postnatal deletion of neo. This produced increased L9'S mutant nAChR expression, which in turn led to nicotinic cholinergic excitotoxicity in dopaminergic neurons.

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression.

In Parkinson's Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated "tagmentation" to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

TC299423, a Novel Agonist for Nicotinic Acetylcholine Receptors.

(E)-5-(Pyrimidin-5-yl)-1,2,3,4,7,8-hexahydroazocine (TC299423) is a novel agonist for nicotinic acetylcholine receptors (nAChRs). We examined its efficacy, affinity, and potency for α6ß2∗ (α6ß2-containing), α4ß2∗, and α3ß4∗ nAChRs, using [125I]-epibatidine binding, whole-cell patch-clamp recordings, synaptosomal 86Rb+ efflux, [3H]-dopamine release, and [3H]-acetylcholine release. TC299423 displayed an EC50 of 30-60 nM for α6ß2∗ nAChRs in patch-clamp recordings and [3H]-dopamine release assays. Its potency for α6ß2∗ in these assays was 2.5-fold greater than that for α4ß2∗, and much greater than that for α3ß4∗-mediated [3H]-acetylcholine release. We observed no major off-target binding on 70 diverse molecular targets. TC299423 was bioavailable after intraperitoneal or oral administration. Locomotor assays, measured with gain-of-function, mutant α6 (α6L9'S) nAChR mice, show that TC299423 elicits α6ß2∗ nAChR-mediated responses at low doses. Conditioned place preference assays show that low-dose TC299423 also produces significant reward in α6L9'S mice, and modest reward in WT mice, through a mechanism that probably involves α6(non-α4)ß2∗ nAChRs. However, TC299423 did not suppress nicotine self-administration in rats, indicating that it did not block nicotine reinforcement in the dosage range that was tested. In a hot-plate test, TC299423 evoked antinociceptive responses in mice similar to those of nicotine. TC299423 and nicotine similarly inhibited mouse marble burying as a measure of anxiolytic effects. Taken together, our data suggest that TC299423 will be a useful small-molecule agonist for future in vitro and in vivo studies of nAChR function and physiology.

Novel seizure phenotype and sleep disruptions in knock-in mice with hypersensitive alpha 4* nicotinic receptors.

A leucine to alanine substitution (L9'A) was introduced in the M2 region of the mouse alpha4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, alpha4(L9'A)beta2 nAChRs were > or =30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9'A mutation and studied their cellular responses, seizure phenotype, and sleep-wake cycle. Seizure studies on alpha4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of alpha4or beta2 subunits. Thalamic cultures and synaptosomes from L9'A mice were hypersensitive to nicotine-induced ion flux. L9'A mice were approximately 15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9'A mice differed qualitatively from those in WT: L9'A seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9'A mice were partial, whereas WT seizures were generalized. When L9'A homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure approximately 20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9'A mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive alpha4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotine-induced seizures resembling ADNFLE.

Increased sensitivity to agonist-induced seizures, straub tail, and hippocampal theta rhythm in knock-in mice carrying hypersensitive alpha 4 nicotinic receptors.

We studied a strain of exon replacement mice ("L9'S knock-in") whose alpha4 nicotinic receptor subunits have a leucine to serine mutation in the M2 region, 9' position (Labarca et al., 2001); this mutation renders alpha4-containing receptors hypersensitive to agonists. Nicotine induced seizures at concentrations (1 mg/kg) approximately eight times lower in L9'S than in wild-type (WT) littermates. At these concentrations, L9'S but not WT showed increases in EEG amplitude and theta rhythm. L9'S mice also showed higher seizure sensitivity to the nicotinic agonist epibatidine, but not to the GABA(A) receptor blocker and proconvulsant bicuculline. Dorsiflexion of the tail (Straub tail) was the most sensitive nicotine effect found in L9'S mice (0.1 mg/kg). The L9'S mice were hypersensitive to galanthamine- and tacrine-induced seizures and Straub tails. There were no apparent neuroanatomical differences between L9'S and WT mice in several brain regions. [(125)I]Epibatidine binding to brain membranes showed that the mutant allele was expressed at approximately 25% of WT levels, presumably because of the presence of a neomycin selection cassette in a nearby intron. (86)Rb efflux experiments on brain synaptosomes showed an increased fraction of function at low agonist concentrations in L9'S mice. These data support the possible involvement of gain-of-function alpha4 receptors in autosomal dominant nocturnal frontal-lobe epilepsy.

Number, density, and surface/cytoplasmic distribution of GABA transporters at presynaptic structures of knock-in mice carrying GABA transporter subtype 1-green fluorescent protein fusions.

GABA transporter subtype 1 (GAT1) molecules were counted near GABAergic synapses, to a resolution of approximately 0.5 microm. Fusions between GAT1 and green fluorescent protein (GFP) were tested in heterologous expression systems, and a construct was selected that shows function, expression level, and trafficking similar to that of wild-type (WT) GAT1. A strain of knock-in mice was constructed that expresses this mGAT1-GFP fusion in place of the WT GAT1 gene. The pattern of fluorescence in brain slices agreed with previous immunocytochemical observations. [3H]GABA uptake, synaptic electrophysiology, and subcellular localization of the mGAT1-GFP construct were also compared with WT mice. Quantitative fluorescence microscopy was used to measure the density of mGAT1-GFP at presynaptic structures in CNS preparations from the knock-in mice. Fluorescence measurements were calibrated with transparent beads and gels that have known GFP densities. Surface biotinylation defined the fraction of transporters on the surface versus those in the nearby cytoplasm. The data show that the presynaptic boutons of GABAergic interneurons in cerebellum and hippocampus have a membrane density of 800-1300 GAT1 molecules per square micrometer, and the axons that connect boutons have a linear density of 640 GAT1 molecules per micrometer. A cerebellar basket cell bouton, a pinceau surrounding a Purkinje cell axon, and a cortical chandelier cell cartridge carry 9000, 7.8 million, and 430,000 GAT1 molecules, respectively; 61-63% of these molecules are on the surface membrane. In cultures from hippocampus, the set of fluorescent cells equals the set of GABAergic interneurons. Knock-in mice carrying GFP fusions of membrane proteins provide quantitative data required for understanding the details of synaptic transmission in living neurons.

Nicotine activation of alpha4* receptors: sufficient for reward, tolerance, and sensitization.

The identity of nicotinic receptor subtypes sufficient to elicit both the acute and chronic effects of nicotine dependence is unknown. We engineered mutant mice with a4 nicotinic subunits containing a single point mutation, Leu9' --> Ala9' in the pore-forming M2 domain, rendering a4* receptors hypersensitive to nicotine. Selective activation of a4* nicotinic acetylcholine receptors with low doses of agonist recapitulates nicotine effects thought to be important in dependence, including reinforcement in response to acute nicotine administration, as well as tolerance and sensitization elicited by chronic nicotine administration. These data indicate that activation of a4* receptors is sufficient for nicotine-induced reward, tolerance, and sensitization.