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Cognitive deficits are long-lasting consequences of drug use, yet the convergent mechanism by which classes of drugs with different pharmacological properties cause similar deficits is unclear. We find that both phencyclidine and methamphetamine, despite differing in their targets in the brain, cause the same glutamatergic neurons in the medial prefrontal cortex of male mice to gain a GABAergic phenotype and decrease expression of their glutamatergic phenotype. Suppressing drug-induced gain of GABA with RNA-interference prevents appearance of memory deficits. Stimulation of dopaminergic neurons in the ventral tegmental area is necessary and sufficient to produce this gain of GABA. Drug-induced prefrontal hyperactivity drives this change in transmitter identity. Returning prefrontal activity to baseline, chemogenetically or with clozapine, reverses the change in transmitter phenotype and rescues the associated memory deficits. This work reveals a shared and reversible mechanism that regulates the appearance of cognitive deficits upon exposure to different drugs., (© 2024. The Author(s).)

Autism spectrum disorders (ASD) can be caused by environmental factors. These factors act early in the development of the nervous system and induce stereotyped repetitive behaviors and diminished social interactions, among other outcomes. Little is known about how these behaviors are produced. In pregnant women, delivery of valproic acid (VPA) (to control seizure activity or stabilize mood) or immune activation by a virus increases the incidence of ASD in offspring. We found that either VPA or Poly Inosine:Cytosine (which mimics a viral infection), administered at mouse embryonic day 12.5, induced a neurotransmitter switch from GABA to glutamate in PV- and CCK-expressing interneurons in the medial prefrontal cortex by postnatal day 10. The switch was present for only a brief period during early postnatal development, observed in male and female mice at postnatal day 21 and reversed in both males and females by postnatal day 30. At postnatal day 90, male mice exhibited stereotyped repetitive behaviors and diminished social interaction while female mice exhibited only stereotyped repetitive behavior. Transfecting GAD1 in PV- and CCK-expressing interneurons at postnatal day 10, to reintroduce GABA expression, overrode the switch and prevented expression of autistic-like behavior. These findings point to an important role of neurotransmitter switching in mediating the environmental causes of autism., Competing Interests: Competing interests statement:The authors declare no competing interest.

Breast cancer metastasis to the brain is a clinical challenge rising in prevalence. However, the underlying mechanisms, especially how cancer cells adapt a distant brain niche to facilitate colonization, remain poorly understood. A unique metabolic feature of the brain is the coupling between neurons and astrocytes through glutamate, glutamine, and lactate. Here we show that extracellular vesicles from breast cancer cells with a high potential to develop brain metastases carry high levels of miR-199b-5p, which shows higher levels in the blood of breast cancer patients with brain metastases comparing to those with metastatic cancer in other organs. miR-199b-5p targets solute carrier transporters (SLC1A2/EAAT2 in astrocytes and SLC38A2/SNAT2 and SLC16A7/MCT2 in neurons) to hijack the neuron-astrocyte metabolic coupling, leading to extracellular retention of these metabolites and promoting cancer cell growth. Our findings reveal a mechanism through which cancer cells of a non-brain origin reprogram neural metabolism to fuel brain metastases., (© 2024. The Author(s).)

Stable matching of neurotransmitters with their receptors is fundamental to synapse function and reliable communication in neural circuits. Presynaptic neurotransmitters regulate the stabilization of postsynaptic transmitter receptors. Whether postsynaptic receptors regulate stabilization of presynaptic transmitters has received less attention. Here, we show that blockade of endogenous postsynaptic acetylcholine receptors (AChR) at the neuromuscular junction destabilizes the cholinergic phenotype in motor neurons and stabilizes an earlier, developmentally transient glutamatergic phenotype. Further, expression of exogenous postsynaptic gamma-aminobutyric acid type A receptors (GABA A receptors) in muscle cells stabilizes an earlier, developmentally transient GABAergic motor neuron phenotype. Both AChR and GABA A receptors are linked to presynaptic neurons through transsynaptic bridges. Knockdown of specific components of these transsynaptic bridges prevents stabilization of the cholinergic or GABAergic phenotypes. Bidirectional communication can enforce a match between transmitter and receptor and ensure the fidelity of synaptic transmission. Our findings suggest a potential role of dysfunctional transmitter receptors in neurological disorders that involve the loss of the presynaptic transmitter., Competing Interests: Competing interests statement:The authors declare no competing interest.

Overgeneralization of fear to harmless situations is a core feature of anxiety disorders resulting from acute stress, yet the mechanisms by which fear becomes generalized are poorly understood. In this study, we show that generalized fear in mice results from a transmitter switch from glutamate to γ-aminobutyric acid (GABA) in serotonergic neurons of the lateral wings of the dorsal raphe. Similar change in transmitter identity was found in the postmortem brains of individuals with posttraumatic stress disorder (PTSD). Overriding the transmitter switch in mice prevented the acquisition of generalized fear. Corticosterone release and activation of glucocorticoid receptors mediated the switch, and prompt antidepressant treatment blocked the cotransmitter switch and generalized fear. Our results provide important insight into the mechanisms involved in fear generalization.

Bioelectronic Medicine stands as an emerging field that rapidly evolves and offers distinctive clinical benefits, alongside unique challenges. It consists of the modulation of the nervous system by precise delivery of electrical current for the treatment of clinical conditions, such as post-stroke movement recovery or drug-resistant disorders. The unquestionable clinical impact of Bioelectronic Medicine is underscored by the successful translation to humans in the last decades, and the long list of preclinical studies. Given the emergency of accelerating the progress in new neuromodulation treatments (i.e., drug-resistant hypertension, autoimmune and degenerative diseases), collaboration between multiple fields is imperative. This work intends to foster multidisciplinary work and bring together different fields to provide the fundamental basis underlying Bioelectronic Medicine. In this review we will go from the biophysics of the cell membrane, which we consider the inner core of neuromodulation, to patient care. We will discuss the recently discovered mechanism of neurotransmission switching and how it will impact neuromodulation design, and we will provide an update on neuronal and glial basis in health and disease. The advances in biomedical technology have facilitated the collection of large amounts of data, thereby introducing new challenges in data analysis. We will discuss the current approaches and challenges in high throughput data analysis, encompassing big data, networks, artificial intelligence, and internet of things. Emphasis will be placed on understanding the electrochemical properties of neural interfaces, along with the integration of biocompatible and reliable materials and compliance with biomedical regulations for translational applications. Preclinical validation is foundational to the translational process, and we will discuss the critical aspects of such animal studies. Finally, we will focus on the patient point-of-care and challenges in neuromodulation as the ultimate goal of bioelectronic medicine. This review is a call to scientists from different fields to work together with a common endeavor: accelerate the decoding and modulation of the nervous system in a new era of therapeutic possibilities., Competing Interests: JC is employee of RBI and founder of the Luxi Group. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. MAG-G was coordinator of the research topic collection “Women in neuroscience of Bioelectronic Medicine”, and SCT, MET and SVC were editors of the same research topic collection. This had no impact on the peer review process and the final decision., (Copyright © 2024 González-González, Conde, Latorre, Thébault, Pratelli, Spitzer, Verkhratsky, Tremblay, Akcora, Hernández-Reynoso, Ecker, Coates, Vincent and Ma.)

A comprehensive understanding of neuronal diversity and connectivity is essential for understanding the anatomical and cellular mechanisms that underlie functional contributions. With the advent of single-cell analysis, growing information regarding molecular profiles leads to the identification of more heterogeneous cell types. Therefore, the need for additional orthogonal recombinase systems is increasingly apparent, as heterogeneous tissues can be further partitioned into increasing numbers of specific cell types defined by multiple features. Critically, new recombinase systems should work together with pre-existing systems without cross-reactivity in vivo. Here, we introduce novel site-specific recombinase systems based on ΦC31 bacteriophage recombinase for labeling multiple cell types simultaneously and a novel viral strategy for versatile and robust intersectional expression of any transgene. Together, our system will help researchers specifically target different cell types with multiple features in the same animal., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Neurotransmitter switching is a form of brain plasticity in which an environmental stimulus causes neurons to replace one neurotransmitter with another, often resulting in changes in behavior. This raises the possibility of applying a specific environmental stimulus to induce a switch that can enhance a desirable behavior or ameliorate symptoms of a specific pathology. For example, a stimulus inducing an increase in the number of neurons expressing dopamine could treat Parkinson's disease, or one affecting the number expressing serotonin could alleviate depression. This may already be producing successful treatment outcomes without our knowing that transmitter switching is involved, with improvement of motor function through physical activity and cure of seasonal depression with phototherapy. This review presents prospects for future investigation of neurotransmitter switching, considering opportunities and challenges for future research and describing how the investigation of transmitter switching is likely to evolve with new tools, thus reshaping our understanding of both normal brain function and mental illness., (Copyright © 2020 the authors.)

Physical exercise promotes motor skill learning in normal individuals and those with neurological disorders but its mechanism of action is unclear. We find that one week of voluntary wheel running enhances the acquisition of motor skills in normal adult mice. One week of running also induces switching from ACh to GABA expression in neurons in the caudal pedunculopontine nucleus (cPPN). Consistent with regulation of motor skills, we show that the switching neurons make projections to the substantia nigra (SN), ventral tegmental area (VTA) and ventrolateral-ventromedial nuclei of the thalamus (VL-VM). Use of viral vectors to override transmitter switching blocks the beneficial effect of running on motor skill learning. We suggest that neurotransmitter switching provides the basis by which sustained running benefits motor skill learning, presenting a target for clinical treatment of movement disorders.

Synaptic communication requires the expression of functional postsynaptic receptors that match the presynaptically released neurotransmitter. The ability of neurons to switch the transmitter they release is increasingly well documented, and these switches require changes in the postsynaptic receptor population. Although the activity-dependent molecular mechanism of neurotransmitter switching is increasingly well understood, the basis of specification of postsynaptic neurotransmitter receptors matching the newly expressed transmitter is unknown. Using a functional assay, we show that sustained application of glutamate to embryonic vertebrate skeletal muscle cells cultured before innervation is necessary and sufficient to up-regulate ionotropic glutamate receptors from a pool of different receptors expressed at low levels. Up-regulation of these ionotropic receptors is independent of signaling by metabotropic glutamate receptors. Both imaging of glutamate-induced calcium elevations and Western blots reveal ionotropic glutamate receptor expression prior to immunocytochemical detection. Sustained application of glutamate to skeletal myotomes in vivo is necessary and sufficient for up-regulation of membrane expression of the GluN1 NMDA receptor subunit. Pharmacological antagonists and morpholinos implicate p38 and Jun kinases and MEF2C in the signal cascade leading to ionotropic glutamate receptor expression. The results suggest a mechanism by which neuronal release of transmitter up-regulates postsynaptic expression of appropriate transmitter receptors following neurotransmitter switching and may contribute to the proper expression of receptors at the time of initial innervation., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Neuroplasticity has classically been understood to arise through changes in synaptic strength or synaptic connectivity. A newly discovered form of neuroplasticity, neurotransmitter switching, involves changes in neurotransmitter identity. Chronic exposure to different photoperiods alters the number of dopamine (tyrosine hydroxylase, TH+) and somatostatin (SST+) neurons in the paraventricular nucleus (PaVN) of the hypothalamus of adult rats and results in discrete behavioral changes. Here, we investigate whether photoperiod-induced neurotransmitter switching persists during aging and whether epigenetic mechanisms of histone acetylation and DNA methylation may contribute to this neurotransmitter plasticity. We show that this plasticity in rats is robust at 1 and at 3 months but reduced in TH+ neurons at 12 months and completely abolished in both TH+ and SST+ neurons by 18 months. De novo expression of DNMT3a catalyzing DNA methylation and anti-AcetylH3 assessing histone 3 acetylation were observed following short-day photoperiod exposure in both TH+ and SST+ neurons at 1 and 3 months while an overall increase in DNMT3a in SST+ neurons paralleled neuroplasticity reduction at 12 and 18 months. Histone acetylation increased in TH+ neurons and decreased in SST+ neurons following short-day exposure at 3 months while the total number of anti-AcetylH3+ PaVN neurons remained constant. Reciprocal histone acetylation in TH+ and SST+ neurons indicates the importance of studying epigenetic regulation at the circuit level for identified cell phenotypes. The findings may be useful for developing approaches for noninvasive treatment of disorders characterized by neurotransmitter dysfunction., (© 2019 Wiley Periodicals, Inc.)

Neurotransmitter switching in the adult mammalian brain occurs following photoperiod-induced stress, but the mechanism of regulation is unknown. Here, we demonstrate that elevated activity of dopaminergic neurons in the paraventricular nucleus of the hypothalamus (PaVN) in the adult rat is required for the loss of dopamine expression after long-day photoperiod exposure. The transmitter switch occurs exclusively in PaVN dopaminergic neurons that coexpress vesicular glutamate transporter 2 (VGLUT2), is accompanied by a loss of dopamine type 2 receptors (D2Rs) on corticotrophin-releasing factor (CRF) neurons, and can lead to increased release of CRF. Suppressing activity of all PaVN glutamatergic neurons decreases the number of inhibitory PaVN dopaminergic neurons, indicating homeostatic regulation of transmitter expression in the PaVN., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Changes in social preference of amphibian larvae result from sustained exposure to kinship odorants. To understand the molecular and cellular mechanisms of this neuroplasticity, we investigated the effects of olfactory system activation on neurotransmitter (NT) expression in accessory olfactory bulb (AOB) interneurons during development. We show that protracted exposure to kin or non-kin odorants changes the number of dopamine (DA)- or gamma aminobutyric acid (GABA)-expressing neurons, with corresponding changes in attraction/aversion behavior. Changing the relative number of dopaminergic and GABAergic AOB interneurons or locally introducing DA or GABA receptor antagonists alters kinship preference. We then isolate AOB microRNAs (miRs) differentially regulated across these conditions. Inhibition of miR-375 and miR-200b reveals that they target Pax6 and Bcl11b to regulate the dopaminergic and GABAergic phenotypes. The results illuminate the role of NT switching governing experience-dependent social preference. VIDEO ABSTRACT., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Neurotransmitter switching is the gain of one neurotransmitter and the loss of another in the same neuron in response to chronic stimulation. Neurotransmitter receptors on postsynaptic cells change to match the identity of the newly expressed neurotransmitter. Neurotransmitter switching often appears to change the sign of the synapse from excitatory to inhibitory or from inhibitory to excitatory. In these cases, neurotransmitter switching and receptor matching thus change the polarity of the circuit in which they take place. Neurotransmitter switching produces up or down reversals of behavior. It is also observed in response to disease. These findings raise the possibility that neurotransmitter switching contributes to depression, schizophrenia, and other illnesses. Many early discoveries of the single gain or loss of a neurotransmitter may have been harbingers of neurotransmitter switching.

Recent evidence indicates the number of dopaminergic neurons in the adult rodent hypothalamus and midbrain is regulated by environmental cues, including photoperiod, and that this occurs via up- or down-regulation of expression of genes and proteins that are important for dopamine (DA) synthesis in extant neurons ('DA neurotransmitter switching'). If the same occurs in humans, it may have implications for neurological symptoms associated with DA imbalances. Here we tested whether there are differences in the number of tyrosine hydroxylase (TH, the rate-limiting enzyme in DA synthesis) and DA transporter (DAT) immunoreactive neurons in the midbrain of people who died in summer (long-day photoperiod, n = 5) versus winter (short-day photoperiod, n = 5). TH and DAT immunoreactivity in neurons and their processes was qualitatively higher in summer compared with winter. The density of TH immunopositive (TH+) neurons was significantly (~6-fold) higher whereas the density of TH immunonegative (TH-) neurons was significantly (~2.5-fold) lower in summer compared with winter. The density of total neurons (TH+ and TH- combined) was not different. The density of DAT+ neurons was ~2-fold higher whereas the density of DAT- neurons was ~2-fold lower in summer compared with winter, although these differences were not statistically significant. In contrast, midbrain nuclear volume, the density of supposed glia (small TH- cells), and the amount of TUNEL staining were the same in summer compared with winter. This study provides the first evidence of an association between environmental stimuli (photoperiod) and the number of midbrain DA neurons in humans, and suggests DA neurotransmitter switching underlies this association.

Among the many forms of brain plasticity, changes in synaptic strength and changes in synapse number are particularly prominent. However, evidence for neurotransmitter respecification or switching has been accumulating steadily, both in the developing nervous system and in the adult brain, with observations of transmitter addition, loss, or replacement of one transmitter with another. Natural stimuli can drive these changes in transmitter identity, with matching changes in postsynaptic transmitter receptors. Strikingly, they often convert the synapse from excitatory to inhibitory or vice versa, providing a basis for changes in behavior in those cases in which it has been examined. Progress has been made in identifying the factors that induce transmitter switching and in understanding the molecular mechanisms by which it is achieved. There are many intriguing questions to be addressed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Activity-dependent neurotransmitter switching engages genetic programs regulating transmitter synthesis, but the mechanism by which activity is transduced is unknown. We suppressed activity in single neurons in the embryonic spinal cord to determine whether glutamate-gamma-aminobutyric acid (GABA) switching is cell autonomous. Transmitter respecification did not occur, suggesting that it is homeostatically regulated by the level of activity in surrounding neurons. Graded increase in the number of silenced neurons in cultures led to graded decrease in the number of neurons expressing GABA, supporting non-cell-autonomous transmitter switching. We found that brain-derived neurotrophic factor (BDNF) is expressed in the spinal cord during the period of transmitter respecification and that spike activity causes release of BDNF. Activation of TrkB receptors triggers a signaling cascade involving JNK-mediated activation of cJun that regulates tlx3, a glutamate/GABA selector gene, accounting for calcium-spike BDNF-dependent transmitter switching. Our findings identify a molecular mechanism for activity-dependent respecification of neurotransmitter phenotype in developing spinal neurons., (Copyright © 2014 Elsevier Inc. All rights reserved.)

erbb4 is a susceptibility gene for schizophrenia and ErbB4 signals have been hypothesized to function in a number of cortical developmental processes (Silberberg et al., 2006; Mei and Xiong, 2008). Several recent studies show that the expression of ErbB4 is mainly restricted to GABAergic interneurons (Yau et al., 2003; Woo et al., 2007), specifically, to parvalbumin-positive (PV) fast-spiking (FS) interneurons (Vullhorst et al., 2009; Fazzari et al., 2010), a large majority of which are PV FS basket cells (Kawaguchi, 1995; Taniguchi et al., 2013). However, in the medial prefrontal cortex (mPFC), a brain region that is closely associated with neuropsychiatric disorders including schizophrenia, little is known about the roles of ErbB4 signals during the development of GABAergic circuitry particularly that associated with PV FS basket cells. Here, using molecular genetics, biochemistry, and electrophysiology, we deleted ErbB4 receptors in GABAergic forebrain neurons during the embryonic period and demonstrated that in the mouse mPFC, ErbB4 signals were dispensable for the development of GABAergic synapses by PV FS basket cells. Interestingly, they were required for the final maturation rather than the initial formation of glutamatergic synapses on PV FS basket cells. Furthermore, activity-dependent GABAergic PV FS pyramidal neuron transmission was decreased, whereas activity of pyramidal neurons was increased in KO mice. Together, these data indicate that ErbB4 signals contribute to the development of GABAergic circuitry associated with FS basket cells in component- and stage-dependent manners in the mPFC in vivo, and may suggest a mechanism for neuropsychiatric disorders including schizophrenia.

The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative has focused scientific attention on the necessary tools to understand the human brain and mind. Here, we outline our collective vision for what we can achieve within a decade with properly targeted efforts and discuss likely technological deliverables and neuroscience progress., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Many forms of electrical excitability expressed in the embryonic nervous system depend on Ca(2+) influx. This discovery has stimulated investigation of the functions of spontaneous elevations of intracellular Ca(2+) and their roles in neuronal development. We present a protocol for imaging different classes of intracellular Ca(2+) transients in embryonic Xenopus (amphibian) spinal neurons grown in dissociated cell culture and in the intact neural tube (the developing spinal cord), focusing on early stages of neuronal differentiation around the time of neural tube closure. The protocol describes methods for gain-of-function and loss-of-function experiments to reveal the functions of these Ca(2+) transients. The methods can also be applied to explant and organotypic cultures. The procedures are sufficiently simple that they can be further adapted for dissociated neuronal cell cultures from other developing embryos, embryonic spinal cords of vertebrates such as zebrafish, and ganglia in the developing nervous systems of invertebrates.

Neurotransmitters have been thought to be fixed throughout life, but whether sensory stimuli alter behaviorally relevant transmitter expression in the mature brain is unknown. We found that populations of interneurons in the adult rat hypothalamus switched between dopamine and somatostatin expression in response to exposure to short- and long-day photoperiods. Changes in postsynaptic dopamine receptor expression matched changes in presynaptic dopamine, whereas somatostatin receptor expression remained constant. Pharmacological blockade or ablation of these dopaminergic neurons led to anxious and depressed behavior, phenocopying performance after exposure to the long-day photoperiod. Induction of newly dopaminergic neurons through exposure to the short-day photoperiod rescued the behavioral consequences of lesions. Natural stimulation of other sensory modalities may cause changes in transmitter expression that regulate different behaviors.

The role of electrical activity in axon guidance has been extensively studied in vitro. To better understand its role in the intact nervous system, we imaged intracellular Ca(2+) in zebrafish primary motor neurons (PMN) during axon pathfinding in vivo. We found that PMN generate specific patterns of Ca(2+) spikes at different developmental stages. Spikes arose in the distal axon of PMN and were propagated to the cell body. Suppression of Ca(2+) spiking activity in single PMN led to stereotyped errors, but silencing all electrical activity had no effect on axon guidance, indicating that an activity-based competition rule regulates this process. This competition was not mediated by synaptic transmission. Combination of PlexinA3 knockdown with suppression of Ca(2+) activity in single PMN produced a synergistic increase in the incidence of pathfinding errors. However, expression of PlexinA3 transcripts was not regulated by activity. Our results provide an in vivo demonstration of the intersection of spontaneous electrical activity with the PlexinA3 guidance molecule receptor in regulation of axon pathfinding.

The identity of the neurotransmitters expressed by neurons has been thought to be fixed and immutable, but recent studies demonstrate that changes in electrical activity can rapidly and reversibly reconfigure the transmitters and corresponding transmitter receptors that neurons express. Induction of transmitter expression can be achieved by selective activation of afferents recruited by a physiological range of sensory input. Strikingly, neurons acquiring an additional transmitter project to appropriate targets prior to transmitter respecification in some cases, indicating the presence of reserve pools of neurons that can boost circuit function. We discuss the evidence for such reserve pools, their likely locations and ways to test for their existence, and the potential clinical value of such circuit-specific neurotransmitter respecification for treatments of neurological disorders., (Copyright © 2011 Wiley Periodicals, Inc.)

For many years it has been assumed that the identity of the transmitters expressed by neurons is stable and unchanging. Recent work, however, shows that electrical activity can respecify neurotransmitter expression during development and in the mature nervous system, and an understanding is emerging of the molecular mechanisms underlying activity-dependent transmitter respecification. Changes in postsynaptic neurotransmitter receptor expression accompany and match changes in transmitter specification, thus enabling synaptic transmission. The functional roles of neurotransmitter respecification are beginning to be understood and appear to involve homeostatic synaptic regulation, which in turn influences behaviour. Activation of this novel form of plasticity by sensorimotor stimuli may provide clinical benefits.

The transmitter phenotype of a neuron has long been thought to be stable for the lifespan. Much as eyes have one color and do not change it over time, neurons have been thought to have one neurotransmitter and retain it for their lifetime. Both principles, exclusivity and stability, are challenged by recent data. More and more neurons in different regions of the brain appear to coexpress two or more neurotransmitters. Moreover, the profile of neurotransmitter expression of a given neuron has been shown to change over time, both during development and in response to changes in activity. The present review summarizes recent studies of this neurotransmitter phenotype plasticity (NPP). Homeostatic mechanisms of plasticity are aimed at maintaining the system within a functional range. They appear to be critical for optimal network operations and have been thought to operate largely by regulating intrinsic excitability, synapse number and synaptic strength. NPP provides a new and unexpected level of regulation of network homeostasis. We propose that it provides the basis for NT coexpression and discuss emerging issues and new questions for further studies in coming years., (Copyright © 2011 Wiley Periodicals, Inc.)

The development of the nervous system involves the generation of a stunningly diverse array of neuronal subtypes that enable complex information processing and behavioral outputs. Deciphering how the nervous system acquires and interprets information and orchestrates behaviors will be greatly enhanced by the identification of distinct neuronal circuits and by an understanding of how these circuits are formed, changed, and/or maintained over time. Addressing these challenging questions depends in part on the ability to accurately identify and characterize the unique neuronal subtypes that comprise individual circuits. Distinguishing characteristics of neuronal subgroups include but are not limited to neurotransmitter phenotype, dendritic morphology, the identity of synaptic partners, and the expression of constellations of subgroup-specific proteins, including ion channel subtypes.

Cyclic AMP (cAMP) and calcium are ubiquitous, interdependent second messengers that regulate a wide range of cellular processes. During development of neuronal networks they are critical for the first step of circuit formation, transducing signals required for axon pathfinding. Surprisingly, the spatial and temporal cAMP and calcium codes used by axon guidance molecules are unknown. Here, we identify characteristics of cAMP and calcium transients generated in growth cones during Netrin-1-dependent axon guidance. In filopodia, Netrin-1-dependent Deleted in Colorectal Cancer (DCC) receptor activation induces a transient increase in cAMP that causes a brief increase in calcium transient frequency. In contrast, activation of DCC in growth cone centers leads to a transient calcium-dependent cAMP increase and a sustained increase in frequency of calcium transients. We show that filopodial cAMP transients regulate spinal axon guidance in vitro and commissural axon pathfinding in vivo. These growth cone codes provide a basis for selective activation of specific downstream effectors.

Background: cAMP is a ubiquitous second messenger involved in a wide spectrum of cellular processes including gene transcription, cell proliferation, and axonal pathfinding. Precise spatiotemporal manipulation and monitoring in live cells are crucial for investigation of cAMP-dependent pathways, but existing tools have several limitations., Findings: We have improved the suitability of cAMP manipulating and monitoring tools for live cell imaging. We attached a red fluorescent tag to photoactivated adenylyl cyclase (PACα) that enables reliable visualization of this optogenetic tool for cAMP manipulation in target cells independently of its photoactivation. We show that replacement of CFP/YFP FRET pair with GFP/mCherry in the Epac2-camps FRET probe reduces photobleaching and stabilizes the noise level during imaging experiments., Conclusions: The modifications of PACα and Epac2-camps enhance these tools for in vitro cAMP studies in cultured living cells and in vivo studies in live animals in a wide range of experiments, and particularly for long term time-lapse imaging.

Little is known about genetic influences on the volume of subcortical brain structures in adult humans, particularly whether there is regional specificity of genetic effects. Understanding patterns of genetic covariation among volumes of subcortical structures may provide insight into the development of individual differences that have consequences for cognitive and emotional behavior and neuropsychiatric disease liability. We measured the volume of 19 subcortical structures (including brain and ventricular regions) in 404 twins (110 monozygotic and 92 dizygotic pairs) from the Vietnam Era Twin Study of Aging and calculated the degree of genetic correlation among these volumes. We then examined the patterns of genetic correlation through hierarchical cluster analysis and by principal components analysis. We found that a model with four genetic factors best fit the data: a Basal Ganglia/Thalamus factor; a Ventricular factor; a Limbic factor; and a Nucleus Accumbens factor. Homologous regions from each hemisphere loaded on the same factors. The observed patterns of genetic correlation suggest the influence of multiple genetic influences. There is a genetic organization among structures which distinguishes between brain and cerebrospinal fluid spaces and between different subcortical regions. Further study is needed to understand this genetic patterning and whether it reflects influences on early development, functionally dependent patterns of growth or pruning, or regionally specific losses due to genes involved in aging, stress response, or disease., (Copyright © 2010 Wiley-Liss, Inc.)

Calcium-dependent electrical activity plays a significant role in neurotransmitter specification at early stages of development. To test the hypothesis that activity-dependent differentiation depends on molecular context, we investigated the development of dopaminergic neurons in the CNS of larval Xenopus laevis. We find that different dopaminergic nuclei respond to manipulation of this early electrical activity by ion channel misexpression with different increases and decreases in numbers of dopaminergic neurons. Focusing on the ventral suprachiasmatic nucleus and the spinal cord to gain insight into these differences, we identify distinct subpopulations of neurons that express characteristic combinations of GABA and neuropeptide Y as cotransmitters and Lim1,2 and Nurr1 transcription factors. We demonstrate that the developmental state of neurons identified by their spatial location and expression of these molecular markers is correlated with characteristic spontaneous calcium spike activity. Different subpopulations of dopaminergic neurons respond differently to manipulation of this early electrical activity. Moreover, retinohypothalamic circuit activation of the ventral suprachiasmatic nucleus recruits expression of dopamine selectively in reserve pool neurons that already express GABA and neuropeptide Y. The results are consistent with the hypothesis that spontaneously active neurons expressing GABA are most susceptible to activity-dependent expression of dopamine in both the spinal cord and brain. Because loss of dopaminergic neurons plays a role in neurological disorders such as Parkinson's disease, understanding how subpopulations of neurons become dopaminergic may lead to protocols for differentiation of neurons in vitro to replace those that have been lost in vivo.

Nervous system development proceeds by sequential gene expression mediated by cascades of transcription factors in parallel with sequences of patterned network activity driven by receptors and ion channels. These sequences are cell type- and developmental stage-dependent and modulated by paracrine actions of substances released by neurons and glia. How and to what extent these sequences interact to enable neuronal network development is not understood. Recent evidence demonstrates that CNS development requires intermediate stages of differentiation providing functional feedback that influences gene expression. We suggest that embryonic neuronal functions constitute a series of phenotypic checkpoint signatures; neurons failing to express these functions are delayed or developmentally arrested. Such checkpoints are likely to be a general feature of neuronal development and constitute presymptomatic signatures of neurological disorders when they go awry., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Neuronal differentiation is accomplished through cascades of intrinsic genetic factors initiated in neuronal progenitors by external gradients of morphogens. Activity has been thought to be important only late in development, but recent evidence suggests that activity also regulates early neuronal differentiation. Activity in post-mitotic neurons before synapse formation can regulate phenotypic specification, including neurotransmitter choice, but the mechanisms are not clear. We identified a mechanism that links endogenous calcium spike activity with an intrinsic genetic pathway to specify neurotransmitter choice in neurons in the dorsal embryonic spinal cord of Xenopus tropicalis. Early activity modulated transcription of the GABAergic/glutamatergic selection gene tlx3 through a variant cAMP response element (CRE) in its promoter. The cJun transcription factor bound to this CRE site, modulated transcription and regulated neurotransmitter phenotype via its transactivation domain. Calcium signaled through cJun N-terminal phosphorylation, which integrated activity-dependent and intrinsic neurotransmitter specification. This mechanism provides a basis for early activity to regulate genetic pathways at critical decision points, switching the phenotype of developing neurons.

Genetic programs, environmental factors, and electrical activity interact to drive the maturation of the brain. Although the cascade of transcription factors that leads to specification of the serotonergic phenotype has been well characterized, its interactions with electrical activity are not known. Here we show that spontaneous calcium spike activity in the hindbrain of developing Xenopus laevis larvae modulates the specification of serotonergic neurons via regulation of expression of the Lmx1b transcription factor. Activity acts downstream of Nkx2.2 but upstream of Lmx1b, leading to regulation of the serotonergic phenotype. Using global manipulation of activity and targeted alteration of Lmx1b expression, we also demonstrate that changes in the number of serotonergic neurons change larval swimming behavior. The results link activity-dependent regulation of a transcription factor to transmitter specification and altered behavior.

Neurotransmitter specification has been shown to depend on genetic programs and electrical activity; however, target-dependent regulation also plays important roles in neuronal development. We have investigated the impact of muscle targets on transmitter specification in Xenopus spinal neurons using a neuron-muscle coculture system. We find that neuron-muscle contact reduces the number of neurons expressing the noncholinergic transmitters GABA, glycine, and glutamate, while having no effect on the incidence of ChAT expression. We show that muscle activity is necessary for target-dependent reduction of noncholinergic transmitter expression. In addition, we demonstrate that coculture with muscle cells suppresses early spontaneous calcium spike activity in neurons and the presence of muscle cells abolishes activity-dependent transmitter specification. The results indicate that target-dependent regulation can be crucial in establishing neurotransmitter phenotypes and altering early neuronal excitability.

The field of neuroscience is generating increased public appetite for information about exciting brain research and discoveries. As stewards of the discipline, together with FUN and others, the Society for Neuroscience (SfN) embraces public outreach and education as essential to its mission of promoting understanding of the brain and nervous system. The Society looks to its members, particularly the younger generation of neuroscientists, to inspire, inform and engage citizens of all ages, and most importantly our youth, in this important endeavor. Here we review SfN programs and resources that support public outreach efforts to inform, educate and tell the story of neuroscience. We describe the important role the Brain Awareness campaign has played in achieving this goal and highlight opportunities for FUN members and students to contribute to this growing effort. We discuss specific programs that provide additional opportunities for neuroscientists to get involved with K-12 teachers and students in ways that inspire youth to pursue further studies and possible careers in science. We draw attention to SfN resources that support outreach to broader audiences. Through ongoing partnerships such as that between SfN and FUN, the neuroscience community is well positioned to pursue novel approaches and resources, including harnessing the power of the Internet. These efforts will increase science literacy among our citizens and garner more robust support for scientific research.

Different types and patterns of spontaneous electrical activity drive many aspects of neuronal differentiation. Neurons in the developing Xenopus spinal cord exhibit calcium spikes, which regulate gene transcription and neurotransmitter specification. The ionic currents necessary for spike production have been described. However, the mechanisms that generate the onset of this activity and the basis of its regulation remain unclear. Although signaling molecules appear to act on plasma membrane receptors to trigger calcium spike activity, other mechanisms for spontaneous calcium spike regulation may exist as well. Here, we analyze the developmental expression of the Na(+), K(+)-ATPase beta3 subunit in Xenopus tropicalis embryos and show that its levels are downregulated at a time during embryonic development that coincides with the onset of prominent calcium spike activity in spinal neurons. Inhibition of an earlier increase in beta3 expression leads to more depolarized resting membrane potentials and results in later reduction of spike activity. This suppression of beta3 levels also reduces expression of the store-operated calcium channel subunit, Orai1. These findings suggest that the Na(+), K(+)-ATPase plays a role in initiating calcium spike activity and regulating calcium homeostasis.

Specification of the appropriate neurotransmitter is a crucial step in neuronal differentiation because it enables signalling among populations of neurons. Experimental manipulations demonstrate that both autonomous and activity-dependent genetic programs contribute to this process during development, but whether natural environmental stimuli specify transmitter expression in a neuronal population is unknown. We investigated neurons of the ventral suprachiasmatic nucleus that regulate neuroendocrine pituitary function in response to light in teleosts, amphibia and primates. Here we show that altering light exposure, which changes the sensory input to the circuit controlling adaptation of skin pigmentation to background, changes the number of neurons expressing dopamine in larvae of the amphibian Xenopus laevis in a circuit-specific and activity-dependent manner. Neurons newly expressing dopamine then regulate changes in camouflage colouration in response to illumination. Thus, physiological activity alters the numbers of behaviourally relevant amine-transmitter-expressing neurons in the brain at postembryonic stages of development. The results may be pertinent to changes in cognitive states that are regulated by biogenic amines.

Neurotransmitter signaling in the mature nervous system is well understood, but the functions of transmitters in the immature nervous system are less clear. Although transmitters released during embryogenesis regulate neuronal proliferation and migration, little is known about their role in regulating early neuronal differentiation. Here, we show that GABA and glutamate drive calcium-dependent embryonic electrical activity that regulates transmitter specification. The number of neurons expressing different transmitters changes when GABA or glutamate signaling is blocked chronically, either using morpholinos to knock down transmitter-synthetic enzymes or applying pharmacological receptor antagonists during a sensitive period of development. We find that calcium spikes are triggered by metabotropic GABA and glutamate receptors, which engage protein kinases A and C. The results reveal a novel role for embryonically expressed neurotransmitters.

Electrical activity has numerous roles in early neuronal development. Calcium transients generated at low frequencies regulate neural induction and neuronal proliferation, migration and differentiation. Recent work demonstrates that these signals participate in specification of the transmitters expressed in different classes of neurons. Matching of postsynaptic receptor expression with the novel expression of transmitters ensues. These findings have intriguing implications for development, mature function and evolution of the nervous system.

Axons and dendrites of developing neurons establish distributed innervation patterns enabling precise discrimination in sensory systems. We describe the role of the extracellular matrix molecule, laminin beta2, interacting with the Ca(V)2.2 calcium channel in establishing appropriate sensory innervation. In vivo, Ca(V)2.2 is expressed on the growth cones of Xenopus laevis sensory neurites and laminin beta2 is expressed in the skin. Culturing neurons on a laminin beta2 substrate inhibits neurite outgrowth in a specific and calcium-dependent manner. Blocking signaling between laminin beta2 and Ca(V)2.2 leads to increased numbers of sensory terminals in vivo. These findings suggest that interactions between extracellular matrix molecules and calcium channels regulate connectivity in the developing nervous system.