Two epilepsy-associated variants in KCNA2 (KV 1.2) at position H310 oppositely affect channel functional expression.

Two KCNA2 variants (p.H310Y and p.H310R) were discovered in paediatric patients with epilepsy and developmental delay. KCNA2 encodes KV 1.2-channel subunits, which regulate neuronal excitability. Both gain and loss of KV 1.2 function cause epilepsy, precluding the prediction of variant effects; and while H310 is conserved throughout the KV -channel superfamily, it is largely understudied. We investigated both variants in heterologously expressed, human KV 1.2 channels by immunocytochemistry, electrophysiology and voltage-clamp fluorometry. Despite affecting the same channel, at the same position, and being associated with severe neurological disease, the two variants had diametrically opposite effects on KV 1.2 functional expression. The p.H310Y variant produced 'dual gain of function', increasing both cell-surface trafficking and activity, delaying channel closure. We found that the latter is due to the formation of a hydrogen bond that stabilizes the active state of the voltage-sensor domain. Additionally, H310Y abolished 'ball and chain' inactivation of KV 1.2 by KV ß1 subunits, enhancing gain of function. In contrast, p.H310R caused 'dual loss of function', diminishing surface levels by multiple impediments to trafficking and inhibiting voltage-dependent channel opening. We discuss the implications for KV -channel biogenesis and function, an emergent hotspot for disease-associated variants, and mechanisms of epileptogenesis. KEY POINTS: KCNA2 encodes the subunits of KV 1.2 voltage-activated, K+ -selective ion channels, which regulate electrical signalling in neurons. We characterize two KCNA2 variants from patients with developmental delay and epilepsy. Both variants affect position H310, highly conserved in KV channels. The p.H310Y variant caused 'dual gain of function', increasing both KV 1.2-channel activity and the number of KV 1.2 subunits on the cell surface. H310Y abolished 'ball and chain' (N-type) inactivation of KV 1.2 by KV ß1 subunits, enhancing the gain-of-function phenotype. The p.H310R variant caused 'dual loss of function', diminishing the presence of KV 1.2 subunits on the cell surface and inhibiting voltage-dependent channel opening. As H310Y stabilizes the voltage-sensor active conformation and abolishes N-type inactivation, it can serve as an investigative tool for functional and pharmacological studies.

Inhibition of CD40-TRAF6-dependent inflammatory activity halts the onset of diabetic retinopathy in streptozotocin-diabetic mice.

Diabetes initiates inflammation that can impair the retinal vasculature, and lead to diabetic retinopathy; one of the leading causes of blindness. Inflammatory pathways have been examined as potential therapeutic targets for diabetic retinopathy, but there is still a need for early-stage treatments. We hypothesized that the CD40-TNF Receptor Associated Factor 6 (TRAF6) axis plays a pivotal role in the onset of diabetic retinopathy, and that the CD40-TRAF6 axis would be a prime therapeutic target for early-stage non-proliferative diabetic retinopathy. The CD40-TRAF6 complex can initiate NFκB activation, inflammation, and tissue damage. Further, CD40 and TRAF6 are constitutively expressed on Muller glia, and upregulated in the diabetic retina. Yet the role of the CD40-TRAF6 complex in the onset of diabetic retinopathy is still unclear. In the current study, we examined the CD40-TRAF6 axis in diabetic retinopathy using a small molecule inhibitor (SMI-6877002) on streptozotocin-induced diabetic mice. When CD40-TRAF6-dependent inflammation was inhibited, retinal vascular leakage and capillary degeneration was ameliorated in diabetic mice. Collectively, these data suggest that the CD40-TRAF6 axis plays a pivotal role in the onset of diabetic retinopathy, and could be a novel therapeutic target for early diabetic retinopathy.

An epilepsy-associated KV1.2 charge-transfer-center mutation impairs KV1.2 and KV1.4 trafficking.

We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events along the biosynthesis and secretion pathway impair trafficking. These studies suggested that F233S causes a depolarizing shift of ∼48 mV on KV1.2 voltage dependence. Optical tracking of the KV1.2(F233S) voltage-sensing domain (rescued by wild-type KV1.2 or KV1.4) revealed that it operates with modestly perturbed voltage dependence and retains pore coupling, evidenced by off-charge immobilization. The equivalent mutation in the Shaker K+ channel (F290S) was reported to modestly affect trafficking and strongly affect function: an ∼80-mV depolarizing shift, disrupted voltage sensor activation and pore coupling. Our work exposes the multigenic, molecular etiology of a variant associated with epilepsy and reveals that charge-transfer-center disruption has different effects in KV1.2 and Shaker, the archetypes for potassium channel structure and function.

Retinal Inflammation, Oxidative Stress, and Vascular Impairment Is Ablated in Diabetic Mice Receiving XMD8-92 Treatment.

The global number of diabetics continues to rise annually. As diabetes progresses, almost all of Type I and more than half of Type II diabetics develop diabetic retinopathy. Diabetic retinopathy is a microvascular disease of the retina, and is the leading cause of blindness in the working-age population worldwide. With such a significant health impact, new drugs are required to halt the blinding threat posed by this visual disorder. The cause of diabetic retinopathy is multifactorial, and an optimal therapeutic would halt inflammation, cease photoreceptor cell dysfunction, and ablate vascular impairment. XMD8-92 is a small molecule inhibitor that blocks inflammatory activity downstream of ERK5 (extracellular signal-related kinase 5) and BRD4 (bromodomain 4). ERK5 elicits inflammation, is increased in Type II diabetics, and plays a pathologic role in diabetic nephropathy, while BRD4 induces retinal inflammation and plays a role in retinal degeneration. Further, we provide evidence that suggests both pERK5 and BRD4 expression are increased in the retinas of our STZ (streptozotocin)-induced diabetic mice. Taken together, we hypothesized that XMD8-92 would be a good therapeutic candidate for diabetic retinopathy, and tested XMD8-92 in a murine model of diabetic retinopathy. In the current study, we developed an in vivo treatment regimen by administering one 100 µL subcutaneous injection of saline containing 20 µM of XMD8-92 weekly, to STZ-induced diabetic mice. XMD8-92 treatments significantly decreased diabetes-mediated retinal inflammation, VEGF production, and oxidative stress. Further, XMD8-92 halted the degradation of ZO-1 (zonula occludens-1), which is a tight junction protein associated with vascular permeability in the retina. Finally, XMD8-92 treatment ablated diabetes-mediated vascular leakage and capillary degeneration, which are the clinical hallmarks of non-proliferative diabetic retinopathy. Taken together, this study provides strong evidence that XMD8-92 could be a potentially novel therapeutic for diabetic retinopathy.

Feasibility of simple exercise interventions for men with osteoporosis - A prospective randomized controlled pilot study.

BACKGROUND: Aging is associated with progressive loss of musculoskeletal performance. Exercise interventions can improve physical function in the elderly but there is a paucity of comparative assessments in order to understand what specific goals can be achieved particularly with less demanding exercise interventions readily accessible for untrained men. METHODS: Prospective randomized, controlled, single center exploratory trial to compare four distinct exercise interventions, i.e. Resistance Training (RT), Whole Body Vibration Exercise (WBV), Qi Gong (QG) and wearing a Spinal orthosis (SO) for 6 months in men at risk for osteoporosis aged 65-90 years. Primary endpoint was change in isometric one repetition maximum force trunk strength for extension (TSE) and flexion (TSF) compared to baseline, secondary endpoints covered key parameters of geriatric functional assessment, including Handgrip Strength (HS), Chair-Rise-Test (CRT), Usual Gait Speed (UGS) and Timed-Up-and-Go (TUG). RESULTS: Altogether 47 men (mean age 77 ±6.1 years) were randomized to RT, (n = 11) WBV (n = 13), QG (n = 10) and SO(n = 13). RT, defined as reference exercise intervention, lead to significant improvements for TSE (p = 0.009) and TSF (p = 0.013) and was significantly superior in the between-group analysis for TSE (p = 0.038). Vibration exercise caused sign. Improvements in TSE (p = 0.014) and CRT (p = 0.005), the Spinal orthosis improved CRT (p = 0.003) and Gait Speed (p = 0.027), while the QG intervention did not attain any sig. Developments.Subgroup analyses revealed most pronounced musculoskeletal progress in vulnerable patients (age ≥ 80 years, pre-sarcopenia, multimorbidity ≥3chronic diseases). Irrespective of the type of exercise, participants ≥80 years experienced significant gains in TSE (p = 0.029) and CRT (p = 0.017). Presarcopenic subjects (Skeletal muscle Index (SMI) ≤10.75 kg/m2) improved in TSE (p = 0.003), CRT (p = 0.001) and UGS (p = 0.016). Multimorbid participants achieved sig. Gains in TSE (p < 0.001), TSF (p = 0.002), UGS (p = 0.036) and HS (p = 0.046). CONCLUSIONS: In this exploratory trial we found that simple exercise interventions are feasible in elderly men eliciting specific benefits, i.e. improvements are attained in those tasks addressed with the respective exercise modality. While targeted resistance training is superior in increasing TSE, alternative simple exercise interventions also appear to elicit beneficial effects, even in vulnerable patients, i.e. those with low muscle mass, above 80 years of age or multimorbidity.

Aryl Hydrocarbon Receptor Agonist VAF347 Impedes Retinal Pathogenesis in Diabetic Mice.

Diabetic retinopathy is the leading cause of blindness in the working-age population worldwide. Although the cause of diabetic retinopathy is multifactorial, IL-17A is a prevalent inflammatory cytokine involved in the promotion of diabetes-mediated retinal inflammation and the progression of diabetic retinopathy. The primary source of IL-17A is Th17 cells, which are T helper cells that have been differentiated by dendritic cells in a proinflammatory cytokine environment. Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can manipulate dendritic cell maturation, halt the production of IL-6 (a proinflammatory cytokine), and suppress Th17 cell differentiation. In the current study, we examined the efficacy of an AhR agonist, VAF347, as a potential therapeutic for the onset of non-proliferative diabetic retinopathy in streptozotocin (STZ)-induced diabetic C57BL/6 mice. We determined that diabetes-mediated leukostasis, oxidative stress, and inflammation in the retina of STZ-diabetic mice were all significantly lower when treated with the AhR agonist VAF347. Furthermore, when VAF347 was subcutaneously injected into STZ-diabetic mice, retinal capillary degeneration was ameliorated, which is the hallmark of non-proliferative diabetic retinopathy in this diabetes murine model. Collectively, these findings provide evidence that the AhR agonist VAF347 could be a potentially novel therapeutic for non-proliferative diabetic retinopathy.

Tracking the motion of the KV1.2 voltage sensor reveals the molecular perturbations caused by a de novo mutation in a case of epilepsy.

KEY POINTS: KV1.2 channels, encoded by the KCNA2 gene, regulate neuronal excitability by conducting K+ upon depolarization. A new KCNA2 missense variant was discovered in a patient with epilepsy, causing amino acid substitution F302L at helix S4, in the KV1.2 voltage-sensing domain. Immunocytochemistry and flow cytometry showed that F302L does not impair KCNA2 subunit surface trafficking. Molecular dynamics simulations indicated that F302L alters the exposure of S4 residues to membrane lipids. Voltage clamp fluorometry revealed that the voltage-sensing domain of KV1.2-F302L channels is more sensitive to depolarization. Accordingly, KV1.2-F302L channels opened faster and at more negative potentials; however, they also exhibited enhanced inactivation: that is, F302L causes both gain- and loss-of-function effects. Coexpression of KCNA2-WT and -F302L did not fully rescue these effects. The proband's symptoms are more characteristic of patients with loss of KCNA2 function. Enhanced KV1.2 inactivation could lead to increased synaptic release in excitatory neurons, steering neuronal circuits towards epilepsy. ABSTRACT: An exome-based diagnostic panel in an infant with epilepsy revealed a previously unreported de novo missense variant in KCNA2, which encodes voltage-gated K+ channel KV1.2. This variant causes substitution F302L, in helix S4 of the KV1.2 voltage-sensing domain (VSD). F302L does not affect KCNA2 subunit membrane trafficking. However, it does alter channel functional properties, accelerating channel opening at more hyperpolarized membrane potentials, indicating gain of function. F302L also caused loss of KV1.2 function via accelerated inactivation onset, decelerated recovery and shifted inactivation voltage dependence to more negative potentials. These effects, which are not fully rescued by coexpression of wild-type and mutant KCNA2 subunits, probably result from the enhancement of VSD function, as demonstrated by optically tracking VSD depolarization-evoked conformational rearrangements. In turn, molecular dynamics simulations suggest altered VSD exposure to membrane lipids. Compared to other encephalopathy patients with KCNA2 mutations, the proband exhibits mild neurological impairment, more characteristic of patients with KCNA2 loss of function. Based on this information, we propose a mechanism of epileptogenesis based on enhanced KV1.2 inactivation leading to increased synaptic release preferentially in excitatory neurons, and hence the perturbation of the excitatory/inhibitory balance of neuronal circuits.

RORγt Inhibitor-SR1001 Halts Retinal Inflammation, Capillary Degeneration, and the Progression of Diabetic Retinopathy.

Diabetic retinopathy is a diabetes-mediated retinal microvascular disease that is the leading cause of blindness in the working-age population worldwide. Interleukin (IL)-17A is an inflammatory cytokine that has been previously shown to play a pivotal role in the promotion and progression of diabetic retinopathy. Retinoic acid-related orphan receptor gammaT (RORγt) is a ligand-dependent transcription factor that mediates IL-17A production. However, the role of RORγt in diabetes-mediated retinal inflammation and capillary degeneration, as well as its potential therapeutic attributes for diabetic retinopathy has not yet been determined. In the current study, we examined retinal inflammation and vascular pathology in streptozotocin-induced diabetic mice. We found RORγt expressing cells in the retinal vasculature of diabetic mice. Further, diabetes-mediated retinal inflammation, oxidative stress, and retinal endothelial cell death were all significantly lower in RORγt-/- mice. Finally, when a RORγt small molecule inhibitor (SR1001) was subcutaneously injected into diabetic mice, retinal inflammation and capillary degeneration were ameliorated. These findings establish a pathologic role for RORγt in the onset of diabetic retinopathy and identify a potentially novel therapeutic for this blinding disease.

VGluT1 Deficiency Impairs Visual Attention and Reduces the Dynamic Range of Short-Term Plasticity at Corticothalamic Synapses.

The most common excitatory neurotransmitter in the central nervous system, glutamate, is loaded into synaptic vesicles by vesicular glutamate transporters (VGluTs). The primary isoforms, VGluT1 and 2, are expressed in complementary patterns throughout the brain and correlate with short-term synaptic plasticity. VGluT1 deficiency is observed in certain neurological disorders, and hemizygous (VGluT1+/-) mice display increased anxiety and depression, altered sensorimotor gating, and impairments in learning and memory. The synaptic mechanisms underlying these behavioral deficits are unknown. Here, we show that VGluT1+/- mice had decreased visual processing speeds during a sustained visual-spatial attention task. Furthermore, in vitro recordings of corticothalamic (CT) synapses revealed dramatic reductions in short-term facilitation, increased initial release probability, and earlier synaptic depression in VGluT1+/- mice. Our electron microscopy results show that VGluT1 concentration is reduced at CT synapses of hemizygous mice, but other features (such as vesicle number and active zone size) are unchanged. We conclude that VGluT1-haploinsuficiency decreases the dynamic range of gain modulation provided by CT feedback to the thalamus, and this deficiency contributes to the observed attentional processing deficit. We further hypothesize that VGluT1 concentration regulates release probability by applying a "brake" to an unidentified presynaptic protein that typically acts as a positive regulator of release.

Neutrophil elastase contributes to the pathological vascular permeability characteristic of diabetic retinopathy.

AIMS/HYPOTHESIS: Levels of neutrophil elastase, a serine protease secreted by neutrophils, are elevated in diabetes. The purpose of this study was to determine whether neutrophil elastase (NE) contributes to the diabetes-induced increase in retinal vascular permeability in mice with streptozotocin-induced diabetes, and, if so, to investigate the potential role of IL-17 in this process. METHODS: In vivo, diabetes was induced in neutrophil elastase-deficient (Elane-/-), Il-17a-/- and wild-type mice. After 8 months of diabetes, Elane-/- mice and wild-type age-matched control mice were injected with FITC-BSA. Fluorescence microscopy was used to assess leakage of FITC-BSA from the retinal vasculature into the neural retina. The level of NE in Il-17a-/- diabetic retina and sera were determined by ELISA. In vitro, the effect of NE on the permeability and viability of human retinal endothelial cells and the expression of junction proteins and adhesion molecules were studied. RESULTS: Eight months of diabetes resulted in increased retinal vascular permeability and levels of NE in retina and plasma of wild-type animals. All of these abnormalities were significantly inhibited in mice lacking the elastase. The diabetes-induced increase in NE was inhibited in mice lacking IL-17. In vitro, NE increased retinal endothelial cell permeability, which was partially inhibited by a myeloid differentiation primary response 88 (MyD88) inhibitor, NF-κB inhibitor, and protease-activated receptor (PAR)2 inhibitor. NE degraded vascular endothelial-cadherin (VE-cadherin) in a concentration-dependent manner. CONCLUSIONS/INTERPRETATION: IL-17 regulates NE expression in diabetes. NE contributes to vascular leakage in diabetic retinopathy, partially through activation of MyD88, NF-κB and PAR2 and degradation of VE-cadherin.

Diabetes induces IL-17A-Act1-FADD-dependent retinal endothelial cell death and capillary degeneration.

PURPOSE: Diabetes leads to progressive complications such as diabetic retinopathy, which is the leading cause of blindness within the working-age population worldwide. Interleukin (IL)-17A is a cytokine that promotes and progresses diabetes. The objective of this study was to determine the role of IL-17A in retinal capillary degeneration, and to identify the mechanism that induces retinal endothelial cell death. These are clinically meaningful abnormalities that characterize early-stage non-proliferative diabetic retinopathy. METHODS: Retinal capillary degeneration was examined in vivo using the streptozotocin (STZ) diabetes murine model. Diabetic-hyperglycemia was sustained for an 8-month period in wild type (C57BL/6) and IL-17A-/- mice to elucidate the role of IL-17A in retinal capillary degeneration. Further, ex vivo studies were performed in retinal endothelial cells to identify the IL-17A-dependent mechanism that induces cell death. RESULTS: It was determined that diabetes-induced retinal capillary degeneration was significantly lower in IL-17A-/- mice. Further, retinal endothelial cell death occurred through an IL-17A/IL-17R ➔ Act1/FADD signaling cascade, which caused caspase-mediated apoptosis. CONCLUSION: These are the first findings that establish a pathologic role for IL-17A in retinal capillary degeneration. Further, a novel IL-17A-dependent apoptotic mechanism was discovered, which identifies potential therapeutic targets for the early onset of diabetic retinopathy.

Diabetes-mediated IL-17A enhances retinal inflammation, oxidative stress, and vascular permeability.

Diabetic retinopathy is a prevailing diabetes complication, and one of the leading causes of blindness worldwide. IL-17A is a cytokine involved in the onset of diabetic complications. In the current study, we examined the role of IL-17A in the development of retinal inflammation and long-term vascular pathology in diabetic mice. We found IL-17A expressing T cells and neutrophils in the retinal vasculature. Further, the IL-17A receptor was expressed on Muller glia, retinal endothelial cells, and photoreceptors. Finally, diabetes-mediated retinal inflammation, oxidative stress, and vascular leakage were all significantly lower in IL-17A-/- mice. These are all clinically meaningful abnormalities that characterize the onset of diabetic retinopathy.

Muscarinic M4 Receptors on Cholinergic and Dopamine D1 Receptor-Expressing Neurons Have Opposing Functionality for Positive Reinforcement and Influence Impulsivity.

The neurotransmitter acetylcholine has been implicated in reward learning and drug addiction. However, the roles of the various cholinergic receptor subtypes on different neuron populations remain elusive. Here we study the function of muscarinic M4 receptors (M4Rs) in dopamine D1 receptor (D1R) expressing neurons and cholinergic neurons (expressing choline acetyltransferase; ChAT), during various reward-enforced behaviors and in a "waiting"-impulsivity test. We applied cell-type-specific gene deletions targeting M4Rs in D1RCre or ChATCre mice. Mice lacking M4Rs in D1R-neurons displayed greater cocaine seeking and drug-primed reinstatement than their littermate controls in a Pavlovian conditioned place preference (CPP) paradigm. Furthermore, the M4R-D1RCre mice initiated significantly more premature responses (PRs) in the 5-choice-serial-reaction-time-task (5CSRTT) than their littermate controls, indicating impaired waiting impulse control. In contrast, mice lacking M4Rs in cholinergic neurons did not acquire cocaine Pavlovian conditioning. The M4R-ChATCre mice were also unable to learn positive reinforcement to either natural reward or cocaine in an operant runway paradigm. Immediate early gene (IEG) expression (cFos and FosB) induced by repeated cocaine injections was significantly increased in the forebrain of M4R-D1RCre mice, whereas it remained normal in the M4R-ChATCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality. Furthermore, we found that neurons expressing both M4Rs and D1Rs are important for signaling impulse control.

Indispensable Amino Acid-Deficient Diets Induce Seizures in Ketogenic Diet-Fed Rodents, Demonstrating a Role for Amino Acid Balance in Dietary Treatments for Epilepsy.

Background: Low protein amounts are used in ketogenic diets (KDs), where an essential (indispensable) amino acid (IAA) can become limiting. Because the chemically sensitive, seizurogenic, anterior piriform cortex (APC) is excited by IAA limitation, an imbalanced KD could exacerbate seizure activity. Objective: We questioned whether dietary IAA depletion worsens seizure activity in rodents fed KDs. Methods: In a series of 6 trials, male rats or gerbils of both sexes (6-8/group) were given either control diets (CDs) appropriate for each trial, a KD, or a threonine-devoid (ThrDev) diet for ≥7 d, and tested for seizures using various stimuli. Microchip analysis of rat APCs was also used to determine if changes in transcripts for structures relevant to seizurogenesis are affected by a ThrDev diet. Glutamate release was measured in microdialysis samples from APCs during the first meal after 7 d on a CD or a ThrDev diet. Results: Adult rats showed increased susceptibility to seizures in both chemical (58%) and electroshock (doubled) testing after 7 d on a ThrDev diet compared with CD (each trial, P ≤ 0.05). Seizure-prone Mongolian gerbils had fewer seizures after receiving a KD, but exacerbated seizures (68%) after 1 meal of KD minus Thr (KD-T compared with CD, P < 0.05). In kindled rats fed KD-T, both counts (19%) and severities (77%) of seizures were significantly elevated (KD-T compared with CD, P < 0.05). Gene transcript changes were consistent with enhanced seizure susceptibility (7-21 net-fold increases, P = 0.045-0.001) and glutamate release into the APC was increased acutely (4-fold at 20 min, 2.6-fold at 60 min, P < 0.05) after 7 d on a ThrDev diet. Conclusion: Seizure severity in rats and gerbils was reduced after KDs and exacerbated by ThrDev, both in KD- and CD-fed animals, consistent with the mechanistic studies. We suggest that a complete protein profile in KDs may improve IAA balance in the APC, thereby lowering the risk of seizures.

Cre-expressing neurons in visual cortex of Ntsr1-Cre GN220 mice are corticothalamic and are depolarized by acetylcholine.

The Ntsr1-Cre GN220 mouse expresses Cre-recombinase in corticothalamic (CT) neurons in neocortical layer 6. It is not known if the other major types of pyramidal neurons in this layer also express this enzyme. By electrophysiological recordings in slices and histological analysis of the uptake of retrogradely transported beads we show that Cre-positive neurons are CT and not corticocortical or corticoclaustral types. Furthermore, we show that Ntsr1-Cre-positive cells are immuno-positive for the nuclear transcription factor Forkhead box protein P2 (FoxP2). We conclude that Cre-expression is limited to a specific type of pyramidal neuron: CT. However, it appears as not all CT neurons are Cre-expressing; there are indications that the penetrance of the gene is about 90%. We demonstrate the utility of assigning a specific identity to individual neurons by determining that the CT neurons are potently modulated by acetylcholine acting on both nicotinic and muscarinic acetylcholine receptors. These results corroborate the suggested function of these neurons in regulating the gain of thalamocortical transfer of sensory information depending on attentional demand and state of arousal.

The Sigma-2 Receptor Selective Agonist Siramesine (Lu 28-179) Decreases Cocaine-Reinforced Pavlovian Learning and Alters Glutamatergic and Dopaminergic Input to the Striatum.

Drug addiction is a chronic, debilitating disease that affects millions of people around the world causing a substantial societal burden. Despite decades of research efforts, treatment possibilities remain limited and relapse represents the most treatment-resistant element. Neurosteroid sigma-1 receptors have been meticulously studied in psychostimulant reinforced Pavlovian learning, while the sigma-2 receptor subtype has remained unexplored. Recent development of selective sigma-2 receptor ligands have now made it possible to investigate if the sigma-2 receptor system is a potential target to treat drug addiction. We examined the effect of the sigma-2 receptor agonist Siramesine (Lu 28-179) on cocaine-associated locomotion, Pavlovian learning, and reward neurocircuitry using electrophysiology recordings and in vivo microdialysis. We found that Siramesine significantly attenuated conditioned place preference acquisition and expression, as well as it completely blocked cocaine-primed reinstatement. Siramesine, in a similar manner as the selective sigma-1 receptor antagonist BD 1063, decreased acute locomotor responses to cocaine. Immunohistochemistry suggests co-expression of progesterone receptor membrane component 1/sigma-2 receptors and vesicular glutamate transporter 1 in presynaptic boutons of the nucleus accumbens (NAc). Whole-cell voltage clamp recordings of neurons in the NAc indicated that Siramesine decreases the presynaptic release probability of glutamate. Further, we demonstrated, via in vivo microdialysis, that Siramesine significantly decreased cocaine-evoked dopamine release in the striatum of freely moving mice. Collectively, these findings demonstrate that sigma-2 receptors regulate neurocircuitry responsible for positive reinforcement and thereby play a role in cocaine-reinforced Pavlovian behaviors.

Kainate receptor subunit diversity underlying response diversity in retinal off bipolar cells.

Postsynaptic kainate receptors mediate excitatory synaptic transmission over a broad range of temporal frequencies. In heterologous systems, the temporal responses of kainate receptors vary when different channel-forming and auxiliary subunits are co-expressed but how this variability relates to the temporal differences at central synapses is incompletely understood. The mammalian cone photoreceptor synapse provides advantages for comparing the different temporal signalling roles of kainate receptors, as cones release glutamate over a range of temporal frequencies, and three functionally distinct Off bipolar cell types receive cone signals at synapses that contain either AMPA or kainate receptors, all with different temporal properties. A disadvantage is that the different receptor subunits are not identified. We used in situ hybridization, immunocytochemistry, and pharmacology to identify the kainate receptor and auxiliary subunits in ground squirrel (Ictidomys tridecimlineatus) cb1a/b, cb2, and cb3a/b Off bipolar cell types. As expected, the types showed distinct subunit expression patterns. Kainate receptors mediated ∼80% of the synaptic response in cb3a/b cells and were heteromers of GluK1 and GluK5. Cb3a/b cells contained message for GluK1 and GluK5, and also GluK3 and the auxiliary subunit Neto1. The synaptic responses in cb1a/b cells were mediated by GluK1-containing kainate receptors that behaved differently from the receptors expressed by cb3a/b cells. AMPA receptors mediated the entire synaptic response in cb2 cells and the remaining synaptic response in cb3a/b cells. We conclude that GluK1 is the predominant kainate receptor subunit in cb1 and cb3 Off bipolar cells. Different temporal response properties may result from selective association with GluK3, GluK5, or Neto1.

Organizational motifs for ground squirrel cone bipolar cells.

In daylight vision, parallel processing starts at the cone synapse. Cone signals flow to On and Off bipolar cells, which are further divided into types according to morphology, immunocytochemistry, and function. The axons of the bipolar cell types stratify at different levels in the inner plexiform layer (IPL) and can interact with costratifying amacrine and ganglion cells. These interactions endow the ganglion cell types with unique functional properties. The wiring that underlies the interactions among bipolar, amacrine, and ganglion cells is poorly understood. It may be easier to elucidate this wiring if organizational rules can be established. We identify 13 types of cone bipolar cells in the ground squirrel, 11 of which contact contiguous cones, with the possible exception of short-wavelength-sensitive cones. Cells were identified by antibody labeling, tracer filling, and Golgi-like filling following transduction with an adeno-associated virus encoding for green fluorescent protein. The 11 bipolar cell types displayed two organizational patterns. In the first pattern, eight to 10 of the 11 types came in pairs with partially overlapping axonal stratification. Pairs shared morphological, immunocytochemical, and functional properties. The existence of similar pairs is a new motif that might have implications for how signals first diverge from a cone to bipolar cells and then reconverge onto a costratifying ganglion cell. The second pattern is a mirror symmetric organization about the middle of the IPL involving at least seven bipolar cell types. This anatomical symmetry may be associated with a functional symmetry in On and Off ganglion cell responses.

What the bird's brain tells the bird's eye: the function of descending input to the avian retina.

As Cajal discovered in the late 19th century, the bird retina receives a substantial input from the brain. Approximately 10,000 fibers originating in a small midbrain nucleus, the isthmo-optic nucleus (ION), terminate in each retina. The input to the ION is chiefly from the optic tectum which, in the bird, is the primary recipient of retinal input. These neural elements constitute a closed loop, the centrifugal visual system (CVS), beginning and ending in the retina, that delivers positive feedback to active ganglion cells. Several features of the system are puzzling. All fibers from the ION terminate in the ventral retina and an unusual axon-bearing amacrine cell, the target cell, is the postsynaptic partner of these fibers. While the rest of the CVS is orderly and retinotopic, target cell axons project seemingly at random, mostly to distant parts of the retina. We review here the most significant features of the anatomy and physiology of the CVS with a view to understanding its function. We suggest that many of the facts about this system, including some that are otherwise difficult to explain, can be accommodated within the hypothesis that the images of shadows cast on the ground or on objects in the environment, initiate a rapid and parallel search of the sky for a possible aerial predator. If a predator is located, shadow and predator would be temporarily linked together and tracked by the CVS.