Phasic Stimulation of Dopaminergic Neurons of the Lateral Substantia Nigra Increases Open Field Exploratory Behaviour and Reduces Habituation Over Time.

Transient nigrostriatal dopaminergic signalling is well known for its role in reinforcement learning and increasingly so for its role in the initiation of voluntary movement. However, how transient bursts of dopamine modulate voluntary movement remains unclear, likely due to the heterogeneity of the nigrostriatal system, the focus of optogenetic studies on locomotion at sub-sec time intervals, and the overlapping roles of phasic dopamine in behaviour and novelty signalling. In this study we investigated how phasic activity in the lateral substantia nigra pars compacta (lateral SNc) over time affects voluntary behaviours during exploration. Using a transgenic mouse model of both sexes expressing channelrhodopsin (ChR2) in dopamine transporter-expressing cells, we stimulated the lateral SNc while mice explored an open field over two consecutive days. We found that phasic activation of the lateral SNc induced an increase in exploratory behaviours including horizontal movement activity, locomotion initiation, and rearing specifically on the first open field exposure, but not on the second day. In addition, stimulated animals did not habituate to the same extent as their ChR2-negative counterparts, as indicated by a lack of decrease in baseline activity. These findings suggest that rather than prompting voluntary movement in general, phasic nigrostriatal dopamine prompts context-appropriate behaviours. In addition, dopamine signalling that modulates movement acts over longer timescales than the transient signal, affecting behaviour even after the signal has ended.

Differential Subcellular Distribution and Release Dynamics of Cotransmitted Cholinergic and GABAergic Synaptic Inputs Modify Dopaminergic Neuronal Excitability.

We identified three types of monosynaptic cholinergic inputs spatially arranged onto medial substantia nigra dopaminergic neurons in male and female mice: cotransmitted acetylcholine (ACh)/GABA, GABA-only, and ACh only. There was a predominant GABA-only conductance along lateral dendrites and soma-centered ACh/GABA cotransmission. In response to repeated stimulation, the GABA conductance found on lateral dendrites decremented less than the proximally located GABA conductance, and was more effective at inhibiting action potentials. While soma-localized ACh/GABA cotransmission showed depression of the GABA component with repeated stimulation, ACh-mediated nicotinic responses were largely maintained. We investigated whether this differential change in inhibitory/excitatory inputs leads to altered neuronal excitability. We found that a depolarizing current or glutamate preceded by cotransmitted ACh/GABA was more effective in eliciting an action potential compared with current, glutamate, or ACh/GABA alone. This enhanced excitability was abolished with nicotinic receptor inhibitors, and modulated by T- and L-type calcium channels, thus establishing that activity of multiple classes of ion channels integrates to shape neuronal excitability.SIGNIFICANCE STATEMENT Our laboratory has previously discovered a population of substantia nigra dopaminegic neurons (DA) that receive cotransmitted ACh and GABA. This study used subcellular optogenetic stimulation of cholinergic presynaptic terminals to map the functional ACh and GABA synaptic inputs across the somatodendritic extent of substantia nigra DA neurons. We determined spatially clustered GABA-only inputs on the lateral dendrites while cotransmitted ACh and GABA clustered close to the soma. We have shown that the action of GABA and ACh in cotransmission spatially clustered near the soma play a critical role in enhancing glutamate-mediated neuronal excitability through the activation of T- and L-type voltage-gated calcium channels.

An opinion on the debatable function of brain resident immune protein, T-cell receptor beta subunit in the central nervous system.

In recent years scientific research has established that the nervous and immune systems have shared molecular signaling components. Proteins native to immune cells, which are also found in the brain, have neuronal functions in the nervous system where they affect synaptic plasticity, axonal regeneration, neurogenesis, and neurotransmission. Certain native immune molecules like major histocompatibility complex I (MHC-I), paired immunoglobulin receptor B (PirB), toll-like receptor (TLR), cluster of differentiation-3 zeta (CD3ζ), CD4 co-receptor, and T-cell receptor beta (TCR-ß) expression in neurons have been extensively documented. In this review, we provide our opinion and discussed the possible roles of T-cell receptor beta subunits in modulating the function of neurons in the central nervous system. Based on the previous findings of Syken and Shatz., 2003; Nishiyori et al., 2004; Rodriguez et., 1993 and Komal et., 2014; we discuss whether restrictive expression of TCR-ß subunits in selected brain regions could be involved in the pathology of neurological disorders and whether their aberrant enhancement in expression may be considered as a suitable biomarker for aging or neurodegenerative diseases like Huntington's disease (HD).

Loss of tyrosine hydroxylase, motor deficits and elevated iron in a mouse model of phospholipase A2G6-associated neurodegeneration (PLAN).

Phospholipase A2G6-associated neurodegeneration (PLAN) is a rare early-onset monogenic neurodegenerative movement disorder which targets the basal ganglia and other regions in the central and peripheral nervous system; presenting as a series of heterogenous subtypes in patients. We describe here a B6.C3-Pla2g6m1J/CxRwb mouse model of PLAN which presents with early-onset neurodegeneration at 90 days which is analogous of the disease progression that is observed in PLAN patients. Homozygous mice had a progressively worsening motor deficit, which presented as tremors starting at 65 days and progressed to severe motor dysfunction and increased falls on the wire hang test at 90 days. This motor deficit positively correlated with a reduction in tyrosine hydroxylase (TH) protein expression in dopaminergic neurons of the substantia nigra (SN) without any neuronal loss. Fluorescence imaging of Thy1-YFP revealed spheroid formation in the SN. The spheroids in homozygous mice strongly mirrors those observed in patients and were demonstrated to correlate strongly with the motor deficits as measured by the wire hang test. The appearance of spheroids preceded TH loss and increased spheroid numbers negatively correlated with TH expression. Perls/DAB staining revealed the presence of iron accumulation within the SN of mice. This mouse model captures many of the major hallmarks of PLAN including severe-early onset neurodegeneration, a motor deficit that correlates directly to TH levels, spheroid formation and iron accumulation within the basal ganglia. Thus, this mouse line is a useful tool for further research efforts to improve understanding of how these disease mechanisms give rise to the disease presentations seen in PLAN patients as well as to test novel therapies.

Cell-Genotype Specific Effects of Mecp2 Mutation on Spontaneous and Nicotinic Acetylcholine Receptor-Evoked Currents in Medial Prefrontal Cortical Pyramidal Neurons in Female Rett Model Mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutation in the X-linked MECP2 gene. Random X-inactivation produces a mosaic of mutant (MT) and wild-type (WT) neurons in female Mecp2+/- (het) mice. Many RTT symptoms are alleviated by increasing activity in medial prefrontal cortex (mPFC) in RTT model mice (Howell et al., 2017). Using a GFP-MeCP2 fusion protein to distinguish WT from MT pyramidal neurons in mPFC we found cell autonomous (cell genotype specific) and non-autonomous effects of MeCP2 deficiency on spontaneous excitatory/inhibitory balance, nicotinic acetylcholine receptor (nAChR) currents and evoked activity. MT Layer 5 and 6 (L5, L6) neurons of male nulls, and MT L6 of het mice had reduced spontaneous excitatory synaptic input compared to WT in wild-type male (WTm), female (WTf) and het mice. Inhibitory synaptic charge in MT L6 equaled WT in 2-4-month hets. At 6-7 months inhibitory charge in WT in het slices was increased compared to both MT in het and WT in WTf; however, in hets the excitatory/inhibitory charge ratio was still greater in WT compared to MT. nAChR currents were reduced in L6 of nulls and MT L6 in het slices compared to WT neurons of het, WTm and WTf. At 2-4 months, ACh perfusion increased frequency of inhibitory currents to L6 neurons equally in all genotypes but increased excitatory inputs to MT and WT in hets less than WT in WTfs. Unexpectedly ACh perfusion evoked greater sustained IPSC and EPSC input to L5 neurons of nulls compared to WTm.

Differential Control of Dopaminergic Excitability and Locomotion by Cholinergic Inputs in Mouse Substantia Nigra.

Understanding how dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc) govern movements requires a detailed knowledge of how different neurotransmitter systems modulate DA neuronal excitability. We report a heterogeneity of electrophysiological properties between medial and lateral SNc neurons modulated by cholinergic neurotransmission. Lateral DA neurons received mainly excitatory (nicotinic or glutamatergic) mediated cholinergic neurotransmission. Medial DA neurons received predominantly GABAergic currents mediated by presynaptic nicotinic receptors or biphasic GABAergic and nicotinic neurotransmission conveyed by GABA and ACh corelease, which inhibited DA neurons. To examine whether cholinergic signaling in the SNc controls mouse behavior, we used optogenetics in awake behaving mice and found that activation of cholinergic terminals in the medial SNc decreased locomotion, whereas activation in the lateral SNc increased locomotion. Our findings provide novel insights on how cholinergic inputs to subregions of the SNc regulate the excitability of DA neurons differentially, resulting in different patterns of motor behavior.

Vulnerability to nicotine self-administration in adolescent mice correlates with age-specific expression of α4* nicotinic receptors.

The majority of smokers begin during adolescence, a developmental period with a high susceptibility to substance abuse. Adolescents are affected differently by nicotine compared to adults, with adolescents being more vulnerable to nicotine's rewarding properties. It is unknown if the age-dependent molecular composition of a younger brain contributes to a heightened susceptibility to nicotine addiction. Nicotine, the principle pharmacological component of tobacco, binds and activates nicotinic acetylcholine receptors (nAChRs) in the brain. The most prevalent is the widely expressed α4-containing (α4*) subtype which mediates reward and is strongly implicated in nicotine dependence. Exposing different age groups of mice, postnatal day (P) 44-86 days old, to a two bottle-choice oral nicotine self-administration paradigm for five days yielded age-specific consumption levels. Nicotine self-administration was elevated in the P44 group, peaked at P54-60 and was drastically lower in the P66 through P86 groups. We also quantified α4* nAChR expression via spectral confocal imaging of brain slices from α4YFP knock-in mice, in which the α4 nAChR subunit is tagged with a yellow fluorescent protein. Quantitative fluorescence revealed age-specific α4* nAChR expression in dopaminergic and GABAergic neurons of the ventral tegmental area. Receptor expression showed a strong positive correlation with daily nicotine dose, suggesting that α4* nAChR expression levels are age-specific and may contribute to the propensity to self-administer nicotine.

T-cell receptors modify neuronal function in the central nervous system.

Recent published findings have shown that many proteins discovered in the immune system and residing on immune cells with well established immune-related functions are also found in neurons of the central nervous system. These studies have uncovered a rich variety of neuronal functions attributed to these immune proteins. This review will focus on two key interacting protein complexes that previously were known for adaptive immune reactions, the major histocompatability complex and the T-cell receptor complex. We will review where these immune proteins are expressed in the CNS and their neuronal function.

cAMP-dependent protein kinase inhibits α7 nicotinic receptor activity in layer 1 cortical interneurons through activation of D1/D5 dopamine receptors.

KEY POINTS: Protein kinases can modify the function of many proteins including ion channels. However, the role of protein kinase A in modifying nicotinic receptors in the CNS has never been investigated. We showed through whole-cell recordings of layer 1 prefrontal cortical interneurons that α7 nicotinic responses are negatively modulated by protein kinase A. Furthermore, we show that stimulation of dopamine receptors can similarly attenuate α7 nicotinic responses through the activation of protein kinase A. These results suggest how the interaction of the cholinergic and dopaminergic systems may influence neuronal excitability in the brain. ABSTRACT: Phosphorylation of ion channels, including nicotinic acetylcholine receptors (nAChRs), by protein kinases plays a key role in the modification of synaptic transmission and neuronal excitability. α7 nAChRs are the second most prevalent nAChR subtype in the CNS following α4ß2. Serine 365 in the M3-M4 cytoplasmic loop of the α7 nAChR is a phosphorylation site for protein kinase A (PKA). D1/D5 dopamine receptors signal through the adenylate cyclase-PKA pathway and play a key role in working memory and attention in the prefrontal cortex. Thus, we examined whether the dopaminergic system, mediated through PKA, functionally interacts with the α7-dependent cholinergic neurotransmission. In layer 1 interneurons of mouse prefrontal cortex, α7 nicotinic currents were decreased upon stimulation with 8-Br-cAMP, a PKA activator. In HEK 293T cells, dominant negative PKA abolished 8-Br-cAMP's effect of diminishing α7 nicotinic currents, while a constitutively active PKA catalytic subunit decreased α7 currents. In brain slices, the PKA inhibitor KT-5720 nullified 8-Br-cAMP's effect of attenuating α7 nicotinic responses, while applying a PKA catalytic subunit in the pipette solution decreased α7 currents. 8-Br-cAMP stimulation reduced surface expression of α7 nAChRs, but there was no change in single-channel conductance. The D1/D5 dopamine receptor agonist SKF 83822 similarly attenuated α7 nicotinic currents from layer 1 interneurons and this attenuation of nicotinic current was prevented by KT-5720. These results demonstrate that dopamine receptor-mediated activation of PKA negatively modulates nicotinic neurotransmission in prefrontal cortical interneurons, which may be a contributing mechanism of dopamine modulation of cognitive behaviours such as attention or working memory.

Chronic nicotine pretreatment is sufficient to upregulate α4* nicotinic receptors and increase oral nicotine self-administration in mice.

BACKGROUND: Understanding the underlying causes of nicotine addiction will require a multidisciplinary approach examining the key molecular, cellular and neuronal circuit functional changes that drive escalating levels of nicotine self-administration. In this study, we examined whether mice pretreated with chronic nicotine, at a dosing regimen that results in maximal nicotinic acetylcholine receptor (nAChR) upregulation, would display evidence of nicotine-dependent behaviour during nicotine self-administration. RESULTS: We investigated oral self-administration of nicotine using a two-bottle choice paradigm in which one bottle contained the vehicle (saccharine-sweetened water), while the other contained nicotine (200 µg/ml) in vehicle. Knock-in mice with YFP-tagged α4 nAChR subunits (α4YFP) were implanted with osmotic pumps delivering either nicotine (2 mg/kg/hr) or saline for 10 days. After 10 days of pretreatment, mice were exposed to the nicotine self-administration paradigm, consisting of four days of choice followed by three days of nicotine abstinence repeated for five weeks. Mice pre-exposed to nicotine had upregulated α4YFP nAChR subunits in the hippocampal medial perforant path and on ventral tegmental area GABAergic neurons as compared to chronic saline mice. Compared to control saline-pretreated mice, in a two bottle-choice experiment, nicotine-primed mice ingested a significantly larger daily dose of nicotine and also exhibited post-abstinence binge drinking of nicotine. CONCLUSIONS: Chronic forced pre-exposure of nicotine is sufficient to induce elevated oral nicotine intake and supports the postulate that nAChR upregulation may be a key factor influencing nicotine self-administration.

GABAergic and glutamatergic efferents of the mouse ventral tegmental area.

The role of dopaminergic (DA) projections from the ventral tegmental area (VTA) in appetitive and rewarding behavior has been widely studied, but the VTA also has documented DA-independent functions. Several drugs of abuse, act on VTA GABAergic neurons, and most studies have focused on local inhibitory connections. Relatively little is known about VTA GABA projection neurons and their connections to brain sites outside the VTA. This study employed viral-vector-mediated cell-type-specific anterograde tracing, classical retrograde tracing, and immunohistochemistry to characterize VTA GABA efferents throughout the brain. We found that VTA GABA neurons project widely to forebrain and brainstem targets, including the ventral pallidum, lateral and magnocellular preoptic nuclei, lateral hypothalamus, and lateral habenula. Minor projections also go to central amygdala, mediodorsal thalamus, dorsal raphe, and deep mesencephalic nuclei, and sparse projections go to prefrontal cortical regions and to nucleus accumbens shell and core. These projections differ from the major VTA DA target regions. Retrograde tracing studies confirmed results from the anterograde experiments and differences in projections from VTA subnuclei. Retrogradely labeled GABA neurons were not numerous, and most non-tyrosine hydroxylase/retrogradely labeled cells lacked GABAergic markers. Many non-TH/retrogradely labeled cells projecting to several areas expressed VGluT2. VTA GABA and glutamate neurons project throughout the brain, most prominently to regions with reciprocal connections to the VTA. These data indicate that VTA GABA and glutamate neurons may have more DA-independent functions than previously recognized.

T-cell receptor activation decreases excitability of cortical interneurons by inhibiting α7 nicotinic receptors.

Many proteins in the immune system are also expressed in the brain. One such class of immune proteins are T-cell receptors (TCRs), whose functions in T lymphocytes in adaptive immunity are well characterized. In the brain, TCRs are confined to neocortical neurons, but their functional role has not been determined. In mouse layer 1 neocortical neurons, TCR activation inhibited α7 nicotinic currents. TCRs modulated α7 currents via tyrosine phosphorylation of α7 nicotinic receptors (nAChRs) through src tyrosine kinases because eliminating lck kinase expression, coexpressing fyn kinase dead, or mutating tyrosine to alanine in α7 blocked the effect of TCR activation. We found that TCR stimulation decreased surface α7 nAChRs and reduced single-channel conductance. These results reveal that TCRs play a major role in the modulation of cholinergic neurotransmission in the brain mediated by α7 nAChRs and that this has a profound effect on regulating neuronal excitability.

RIC-3 differentially modulates α4ß2 and α7 nicotinic receptor assembly, expression, and nicotine-induced receptor upregulation.

BACKGROUND: Recent work has shown that the chaperone resistant to inhibitors of acetylcholinesterase (RIC-3) is critical for the folding, maturation and functional expression of a variety of neuronal nicotinic acetylcholine receptors. α7 nicotinic receptors can only assemble and functionally express in select lines of cells, provided that RIC-3 is present. In contrast, α4ß2 nicotinic receptors can functionally express in many cell lines even without the presence of RIC-3. Depending on the cell line, RIC-3 has differential effects on α4ß2 receptor function - enhancement in mammalian cells but inhibition in Xenopus oocytes. Other differences between the two receptor types include nicotine-induced upregulation. When expressed in cell lines, α4ß2 receptors readily and robustly upregulate with chronic nicotine exposure. However, α7 nicotinic receptors appear more resistant and require higher concentrations of nicotine to induce upregulation. Could the coexpression of RIC-3 modulate the extent of nicotine-induced upregulation not only for α7 receptors but also α4ß2 receptors? We compared and contrasted the effects of RIC-3 on assembly, trafficking, protein expression and nicotine-induced upregulation on both α7 and α4ß2 receptors using fluorescent protein tagged nicotinic receptors and Förster resonance energy transfer (FRET) microscopy imaging. RESULTS: RIC-3 increases assembly and cell surface trafficking of α7 receptors but does not alter α7 protein expression in transfected HEK293T cells. In contrast, RIC-3 does not affect assembly of α4ß2 receptors but increases α4 and ß2 subunit protein expression. Acute nicotine (30 min exposure) was sufficient to upregulate FRET between α4 and ß2 subunits. Surprisingly, when RIC-3 was coexpressed with α4ß2 receptors nicotine-induced upregulation was prevented. α7 receptors did not upregulate with acute nicotine in the presence or absence of RIC-3. CONCLUSIONS: These results provide interesting novel data that RIC-3 differentially regulates assembly and expression of different nicotinic receptor subunits. These results also show that nicotine-mediated upregulation of α4ß2 receptors can be dynamically regulated by the presence of the chaperone, RIC-3. This could explain a novel mechanism why high affinity α4ß2 receptors are upregulated in specific neuronal subtypes in the brain and not others.

Spectral confocal imaging of fluorescently tagged nicotinic receptors in knock-in mice with chronic nicotine administration.

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates. Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology. By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using Förster Resonance Energy Transfer (FRET) in transfected cultured cells. More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged α4 nAChR subunits (α4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS. In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from α4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue. We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain α4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of α4YFP receptors in the medial perforant path of the hippocampus.

α4* Nicotinic acetylcholine receptors modulate experience-based cortical depression in the adult mouse somatosensory cortex.

The molecular mechanisms that mediate experience-based changes in the function of the cerebral cortex, particularly in the adult animal, are poorly understood. Here we show using in vivo voltage-sensitive dye imaging, that whisker trimming leads to depression of whisker-evoked sensory responses in primary, secondary and associative somatosensory cortical regions. Given the importance of cholinergic neurotransmission in cognitive and sensory functions, we examined whether α4-containing (α4*) nicotinic acetylcholine receptors (nAChRs) mediate cortical depression. Using knock-in mice that express YFP-tagged α4 nAChRs subunits, we show that whisker trimming selectively increased the number α4*-YFP nAChRs in layer 4 of deprived barrel columns within 24 h, which persisted until whiskers regrew. Confocal and electron microscopy revealed that these receptors were preferentially increased on the cell bodies of GABAergic neurons. To directly link these receptors with functional cortical depression, we show that depression could be induced in normal mice by topical application or micro-injection of α4* nAChR agonist in the somatosensory cortex. Furthermore, cortical depression could be blocked after whisker trimming with chronic infusions of an α4* nAChR antagonist. Collectively, these results uncover a new role for α4* nAChRs in regulating rapid changes in the functional responsiveness of the adult somatosensory cortex.

Nicotinic α5 subunits drive developmental changes in the activation and morphology of prefrontal cortex layer VI neurons.

BACKGROUND: Nicotinic signaling in prefrontal layer VI pyramidal neurons is important to the function of mature attention systems. The normal incorporation of α5 subunits into α4ß2* nicotinic acetylcholine receptors augments nicotinic signaling in these neurons and is required for normal attention performance in adult mice. However, the role of α5 subunits in the development of the prefrontal cortex is not known. METHODS: We sought to answer this question by examining nicotinic currents and neuronal morphology in layer VI neurons of medial prefrontal cortex of wild-type and α5 subunit knockout (α5(-/-)) mice during postnatal development and in adulthood. RESULTS: In wild-type but not in α5(-/-) mice, there is a developmental peak in nicotinic acetylcholine currents in the third postnatal week. At this juvenile time period, the majority of neurons in all mice have long apical dendrites extending into cortical layer I. Yet, by early adulthood, wild-type but not α5(-/-) mice show a pronounced shift toward shorter apical dendrites. This cellular difference occurs in the absence of genotype differences in overall cortical morphology. CONCLUSIONS: Normal developmental changes in nicotinic signaling and dendritic morphology in prefrontal cortex depend on α5-comprising nicotinic acetylcholine receptors. It appears that these receptors mediate a specific developmental retraction of apical dendrites in layer VI neurons. This finding provides novel insight into the cellular mechanisms underlying the known attention deficits in α5(-/-) mice and potentially also into the pathophysiology of developmental neuropsychiatric disorders such as attention-deficit disorder and autism.

A rapid agonist application system for fast activation of ligand-gated ion channels.

The synaptic delay between neurotransmitter release across the synaptic cleft and activation of neurotransmitter gated ion channels is less than a ms. Nicotinic acetylcholine receptors (nAChRs), like many other classes of ligand-gated ion channels, are comprised of different protein subunits forming a variety of receptors with different activation and desensitization kinetics and pharmacological sensitivities. To measure and fully characterize ligand-gated ion channel currents accurately, one must apply agonists in a fraction of a ms and repeatedly at various concentrations without any prior desensitization of the receptors. In this paper, we describe an economical, easy to assemble and operate rapid drug application system. The drug applicator system consists of a parallel array of three pinch valves, which allow either agonist or wash solution into a theta tube. Solution exchanges of 0.16 ms can be achieved. In transfected cells, ACh elicited α4ß2 nicotinic currents with mean rise times of 55±13 ms. We recorded α7 nAChRs, which desensitize very rapidly, and obtained very fast rise times of 19±2 ms. With this novel drug applicator, agonists can be applied repeatedly without any loss of current. Hence, complete dose-response relations can be obtained for even α7 nAChRs, which are very sensitive to desensitization caused by agonist exposure on a ms time scale. The drug application system can also be extended to the study of ligand-gated ion channels in brain slices. The theta tube valve-driven drug applicator system can be applied to study other ligand-gated ion channels including glutamate and GABA receptors.

Developmental sex differences in nicotinic currents of prefrontal layer VI neurons in mice and rats.

BACKGROUND: There is a large sex difference in the prevalence of attention deficit disorder; yet, relatively little is known about sex differences in the development of prefrontal attention circuitry. In male rats, nicotinic acetylcholine receptors excite corticothalamic neurons in layer VI, which are thought to play an important role in attention by gating the sensitivity of thalamic neurons to incoming stimuli. These nicotinic currents in male rats are significantly larger during the first postnatal month when prefrontal circuitry is maturing. The present study was undertaken to investigate whether there are sex differences in the nicotinic currents in prefrontal layer VI neurons during development. METHODOLOGY/PRINCIPAL FINDINGS: Using whole cell recording in prefrontal brain slice, we examined the inward currents elicited by nicotinic stimulation in male and female rats and two strains of mice. We found a prominent sex difference in the currents during the first postnatal month when males had significantly greater nicotinic currents in layer VI neurons compared to females. These differences were apparent with three agonists: acetylcholine, carbachol, and nicotine. Furthermore, the developmental sex difference in nicotinic currents occurred despite male and female rodents displaying a similar pattern and proportion of layer VI neurons possessing a key nicotinic receptor subunit. CONCLUSIONS/SIGNIFICANCE: This is the first illustration at a cellular level that prefrontal attention circuitry is differently affected by nicotinic receptor stimulation in males and females during development. This transient sex difference may help to define the cellular and circuit mechanisms that underlie vulnerability to attention deficit disorder.

Chronic nicotine selectively enhances alpha4beta2* nicotinic acetylcholine receptors in the nigrostriatal dopamine pathway.

These electrophysiological experiments, in slices and intact animals, study the effects of in vivo chronic exposure to nicotine on functional alpha4beta2* nAChRs in the nigrostriatal dopaminergic (DA) pathway. Recordings were made in wild-type and alpha4 nicotinic acetylcholine receptor (nAChR) subunit knock-out mice. Chronic nicotine enhanced methyllycaconitine citrate hydrate-resistant, dihydro-beta-erythroidine hydrobromide-sensitive nicotinic currents elicited by 3-1000 mum ACh in GABAergic neurons of the substantia nigra pars reticulata (SNr), but not in DA neurons of the substantia nigra pars compacta (SNc). This enhancement leads to higher firing rates of SNr GABAergic neurons and consequently to increased GABAergic inhibition of the SNc DA neurons. In the dorsal striatum, functional alpha4* nAChRs were not found on the neuronal somata; however, nicotine acts via alpha4beta2* nAChRs in the DA terminals to modulate glutamate release onto the medium spiny neurons. Chronic nicotine also increased the number and/or function of these alpha4beta2* nAChRs. These data suggest that in nigrostriatal DA pathway, chronic nicotine enhancement of alpha4beta2* nAChRs displays selectivity in cell type and in nAChR subtype as well as in cellular compartment. These selective events augment inhibition of SNc DA neurons by SNr GABAergic neurons and also temper the release of glutamate in the dorsal striatum. The effects may reduce the risk of excitotoxicity in SNc DA neurons and may also counteract the increased effectiveness of corticostriatal glutamatergic inputs during degeneration of the DA system. These processes may contribute to the inverse correlation between tobacco use and Parkinson's disease.

Demonstration of functional alpha4-containing nicotinic receptors in the medial habenula.

The medial habenula (MHb) exhibits exceptionally high levels of nicotinic acetylcholine receptors (nAChRs), but it remains unclear whether all expressed nAChR subunit mRNAs are translated to form functional receptors. In particular alpha4 subunits have not been reported to have any functional role, despite strong alpha4 mRNA expression in the ventrolateral MHb. We studied a strain of knock-in mice expressing fluorescent alpha4* nAChRs (alpha4YFP), as well as a knock-in strain expressing hypersensitive alpha4* nAChRs (alpha4L9'A). In alpha4YFP mice, there was strong fluorescence in the ventrolateral MHb. In hypersensitive alpha4L9'A mice, injections of a low dose of nicotine (0.1 mg/kg) led to strong c-fos expression in only the ventrolateral region of the MHb, but not in the MHb of wild-type (WT) mice. In MHb slice recordings, ventrolateral neurons from alpha4L9'A mice, but not from WT mice, responded robustly to nicotine (1 microM). Neurons in the medial aspect of the MHb had >10-fold smaller responses. Thus alpha4* nAChRs contribute to the selective activation of a subset of MHb neurons. Subunit composition analysis based on gain-of-function knock-in mice provides a useful experimental paradigm.