Functional α6ß4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties.

The α6ß4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6ß4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6ß4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6ß4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6ß4 receptor assembly. Effects on α6ß4 involve BARP's N-terminal region and IRE1α's splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6ß4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6ß4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6ß4 receptor.

Hair cell α9α10 nicotinic acetylcholine receptor functional expression regulated by ligand binding and deafness gene products.

Auditory hair cells receive olivocochlear efferent innervation, which refines tonotopic mapping, improves sound discrimination, and mitigates acoustic trauma. The olivocochlear synapse involves α9α10 nicotinic acetylcholine receptors (nAChRs), which assemble in hair cells only coincident with cholinergic innervation and do not express in recombinant mammalian cell lines. Here, genome-wide screening determined that assembly and surface expression of α9α10 require ligand binding. Ion channel function additionally demands an auxiliary subunit, which can be transmembrane inner ear (TMIE) or TMEM132e. Both of these single-pass transmembrane proteins are enriched in hair cells and underlie nonsyndromic human deafness. Inner hair cells from TMIE mutant mice show altered postsynaptic α9α10 function and retain α9α10-mediated transmission beyond the second postnatal week associated with abnormally persistent cholinergic innervation. Collectively, this study provides a mechanism to link cholinergic input with α9α10 assembly, identifies unexpected functions for human deafness genes TMIE/TMEM132e, and enables drug discovery for this elusive nAChR implicated in prevalent auditory disorders.

Polyamine regulation of ion channel assembly and implications for nicotinic acetylcholine receptor pharmacology.

Small molecule polyamines are abundant in all life forms and participate in diverse aspects of cell growth and differentiation. Spermidine/spermine acetyltransferase (SAT1) is the rate-limiting enzyme in polyamine catabolism and a primary genetic risk factor for suicidality. Here, using genome-wide screening, we find that SAT1 selectively controls nicotinic acetylcholine receptor (nAChR) biogenesis. SAT1 specifically augments assembly of nAChRs containing α7 or α4ß2, but not α6 subunits. Polyamines are classically studied as regulators of ion channel gating that engage the nAChR channel pore. In contrast, we find polyamine effects on assembly involve the nAChR cytosolic loop. Neurological studies link brain polyamines with neurodegenerative conditions. Our pharmacological and transgenic animal studies find that reducing polyamines enhances cortical neuron nAChR expression and augments nicotine-mediated neuroprotection. Taken together, we describe a most unexpected role for polyamines in regulating ion channel assembly, which provides a new avenue for nAChR neuropharmacology.

Cocaine-Induced Structural Plasticity in Input Regions to Distinct Cell Types in Nucleus Accumbens.

BACKGROUND: The nucleus accumbens (NAc) is a brain region implicated in pathological motivated behaviors such as drug addiction and is composed predominantly of two discrete populations of neurons, dopamine receptor-1- and dopamine receptor-2-expressing medium spiny neurons (D1-MSNs and D2-MSNs, respectively). It is unclear whether these populations receive inputs from different brain areas and whether input regions to these cell types undergo distinct structural adaptations in response to the administration of addictive drugs such as cocaine. METHODS: Using a modified rabies virus-mediated tracing method, we created a comprehensive brain-wide monosynaptic input map to NAc D1- and D2-MSNs. Next, we analyzed nearly 2000 dendrites and 125,000 spines of neurons across four input regions (the prelimbic cortex, medial orbitofrontal cortex, basolateral amygdala, and ventral hippocampus) at four separate time points during cocaine administration and withdrawal to examine changes in spine density in response to repeated intraperitoneal cocaine injection in mice. RESULTS: D1- and D2-MSNs display overall similar input profiles, with the exception that D1-MSNs receive significantly more input from the medial orbitofrontal cortex. We found that neurons in distinct brain areas projecting to D1- and D2-MSNs display different adaptations in dendritic spine density at different stages of cocaine administration and withdrawal. CONCLUSIONS: While NAc D1- and D2-MSNs receive input from similar brain structures, cocaine-induced spine density changes in input regions are quite distinct and dynamic. While previous studies have focused on input-specific postsynaptic changes within NAc MSNs in response to cocaine, these findings emphasize the dramatic changes that occur in the afferent input regions as well.

Circuit-based frameworks of depressive behaviors: The role of reward circuitry and beyond.

Major depressive disorder (MDD) is a common but serious neuropsychiatric affliction that comprises a diverse set of symptoms such as the inability to feel pleasure, lack of motivation, changes in appetite, and cognitive difficulties. Given the patient to patient symptomatic variability in MDD and differing severities of individual symptoms, it is likely that maladaptive changes in distinct brain areas may mediate discrete symptoms in MDD. The advent and recent surge of studies using viral-genetic approaches have allowed for circuit-specific dissection of networks underlying motivational behavior. In particular, areas such as the ventral tegmental area (VTA), nucleus accumbens (NAc), and ventral pallidum (VP) are thought to generally promote reward, with the medial prefrontal cortex (mPFC) providing top-down control of reward seeking. On the contrary, the lateral habenula (LHb) is considered to be the aversive center of the brain as it has been shown to encode negative valence. The behavioral symptoms of MDD may arise from a disruption in the reward circuitry, hyperactivity of aversive centers, or a combination of the two. Thus, gaining access to specific circuits within the brain and how separate motivational-relevant regions transmit and encode information between each other in the context of separate depression-related symptoms can provide critical knowledge towards symptom-specific treatment of MDD. Here, we review published literature emphasizing circuit- and cell type-specific dissection of depressive-like behaviors in animal models of depression with a particular focus on the chronic social defeat stress model of MDD.

Drd3 Signaling in the Lateral Septum Mediates Early Life Stress-Induced Social Dysfunction.

Early life stress (ELS) in the form of child abuse/neglect is associated with an increased risk of developing social dysfunction in adulthood. Little is known, however, about the neural substrates or the neuromodulatory signaling that govern ELS-induced social dysfunction. Here, we show that ELS-induced downregulation of dopamine receptor 3 (Drd3) signaling and its corresponding effects on neural activity in the lateral septum (LS) are both necessary and sufficient to cause social abnormalities in adulthood. Using in vivo Ca2+ imaging, we found that Drd3-expressing-LS (Drd3LS) neurons in animals exposed to ELS show blunted activity in response to social stimuli. In addition, optogenetic activation of Drd3LS neurons rescues ELS-induced social impairments. Furthermore, pharmacological treatment with a Drd3 agonist, which increases Drd3LS neuronal activity, normalizes the social dysfunctions of ELS mice. Thus, we identify Drd3 in the LS as a critical mediator and potential therapeutic target for the social abnormalities caused by ELS.

Distinct Ventral Pallidal Neural Populations Mediate Separate Symptoms of Depression.

Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression.

Group A Streptococcus intranasal infection promotes CNS infiltration by streptococcal-specific Th17 cells.

Group A streptococcal (GAS) infection induces the production of Abs that cross-react with host neuronal proteins, and these anti-GAS mimetic Abs are associated with autoimmune diseases of the CNS. However, the mechanisms that allow these Abs to cross the blood-brain barrier (BBB) and induce neuropathology remain unresolved. We have previously shown that GAS infection in mouse models induces a robust Th17 response in nasal-associated lymphoid tissue (NALT). Here, we identified GAS-specific Th17 cells in tonsils of humans naturally exposed to GAS, prompting us to explore whether GAS-specific CD4+ T cells home to mouse brains following i.n. infection. Intranasal challenge of repeatedly GAS-inoculated mice promoted migration of GAS-specific Th17 cells from NALT into the brain, BBB breakdown, serum IgG deposition, microglial activation, and loss of excitatory synaptic proteins under conditions in which no viable bacteria were detected in CNS tissue. CD4+ T cells were predominantly located in the olfactory bulb (OB) and in other brain regions that receive direct input from the OB. Together, these findings provide insight into the immunopathology of neuropsychiatric complications that are associated with GAS infections and suggest that crosstalk between the CNS and cellular immunity may be a general mechanism by which infectious agents exacerbate symptoms associated with other CNS autoimmune disorders.

Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke.

Brain endothelial cells form a paracellular and transcellular barrier to many blood-borne solutes via tight junctions (TJs) and scarce endocytotic vesicles. The blood-brain barrier (BBB) plays a pivotal role in the healthy and diseased CNS. BBB damage after ischemic stroke contributes to increased mortality, yet the contributions of paracellular and transcellular mechanisms to this process in vivo are unknown. We have created a transgenic mouse strain whose endothelial TJs are labeled with eGFP and have imaged dynamic TJ changes and fluorescent tracer leakage across the BBB in vivo, using two-photon microscopy in the t-MCAO stroke model. Although barrier function is impaired as early as 6 hr after stroke, TJs display profound structural defects only after 2 days. Conversely, the number of endothelial caveolae and transcytosis rate increase as early as 6 hr after stroke. Therefore, stepwise impairment of transcellular followed by paracellular barrier mechanisms accounts for the BBB deficits in stroke.