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 α4β2 or α7 nAChRs. STN neurons containing α4β2 nAChRs (α4β2 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 α4β2 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 α4β2 nAChRs in STN: this treatment increased the number of α4β2 neurons, upregulated α4-containing nAChR number and sensitivity, and enhanced the basal firing rate of α4β2 neurons both ex vivo and in vivo. Thus, chronic nicotine enhances the function of the microcircuit involving α4β2 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 β4 nAChR subunit levels are strong throughout the ventral MHb (MHbV). In contrast, α6, β2, β3, 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

Increased Sensitivity to Agonist-Induced Seizures, Straub Tail, and Hippocampal Theta Rhythm in Knock-In Mice Carrying Hypersensitive α4 Nicotinic Receptors

We studied a strain of exon replacement mice (“L9′S knock-in”) whose α4 nicotinic receptor subunits have a leucine to serine mutation in the M2 region, 9′ position (Labarca et al., 2001); this mutation renders α4-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_Areceptor 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. [125I]Epibatidine binding to brain membranes showed that the mutant allele was expressed at ∼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 α4 receptors in autosomal dominant nocturnal frontal-lobe epilepsy

Nicotine Activation of α4* 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 α4 nicotinic subunits containing a single point mutation, Leu^(9′) → Ala^(9′) in the pore-forming M2 domain, rendering α4* receptors hypersensitive to nicotine. Selective activation of α4* 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 α4* receptors is sufficient for nicotine-induced reward, tolerance, and sensitization

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 ∼0.5 μm. 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

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 ∼0.5 μm. 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

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 β3 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

Chronic Nicotine Cell Specifically Upregulates Functional α4* 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 α4 nicotinic receptor subunits are replaced by normally functioning fluorescently tagged subunits, providing quantitative studies of receptor regulation at micrometer resolution. Chronic nicotine increased α4 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 α4* receptor levels in dopaminergic neurons of ventral tegmental area (VTA) or substantia nigra pars compacta. Instead, upregulated, functional α4* 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 α4* 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 α4* receptors in this pathway are also functional. The pattern of cell-specific upregulation of functional α4* 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

Novel Seizure Phenotype and Sleep Disruptions in Knock-In Mice with Hypersensitive α4* Nicotinic Receptors

A leucine to alanine substitution (L9′A) was introduced in the M2 region of the mouse α4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, α4(L9′A)β2 nAChRs were ≥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 α4-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 α4or β2 subunits. Thalamic cultures and synaptosomes from L9′A mice were hypersensitive to nicotine-induced ion flux. L9′A mice were ∼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 ∼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 α4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotine-induced seizures resembling ADNFLE