Improved expression of halorhodopsin for light-induced silencing of neuronal activity

The ability to control and manipulate neuronal activity within an intact mammalian brain is of key importance for mapping functional connectivity and for dissecting the neural circuitry underlying behaviors. We have previously generated transgenic mice that express channelrhodopsin-2 for light-induced activation of neurons and mapping of neural circuits. Here we describe transgenic mice that express halorhodopsin (NpHR), a light-driven chloride pump that can be used to silence neuronal activity via light. Using the Thy-1 promoter to target NpHR expression to neurons, we found that neurons in these mice expressed high levels of NpHR-YFP and that illumination of cortical pyramidal neurons expressing NpHR-YFP led to rapid, reversible photoinhibition of action potential firing in these cells. However, NpHR-YFP expression led to the formation of numerous intracellular blebs, which may disrupt neuronal function. Labeling of various subcellular markers indicated that the blebs arise from retention of NpHR-YFP in the endoplasmic reticulum. By improving the signal peptide sequence and adding an ER export signal to NpHR-YFP, we eliminated the formation of blebs and dramatically increased the membrane expression of NpHR-YFP. Thus, the improved version of NpHR should serve as an excellent tool for neuronal silencing in vitro and in vivo.     

3H]acetylcholine synthesis in cultured ciliary ganglion neurons: effects of myotube membranes

Avian ciliary ganglion neurons in cell culture were examined for the capacity to synthesize acetylcholine (ACh) from the exogenously supplied precursor, choline. Relevant kinetic parameters of the ACh synthetic system in cultured neurons were found to be virtually the same as those of the ganglionic terminals in the intact iris. Neurons were cultured in the presence of and allowed to innervate pectoral muscle; this results in an capacity for ACh synthesis. In particular, the ability to increase ACh synthesis upon demand after stimulation is affected by interaction with the target. This effect is shown to be an acceleration of the maturation of the cultured neurons. Lysed and washed membrane remnants of the muscle target were able to duplicate, in part, this effect of live target tissue on neuronal transmitter metabolism. Culture medium conditioned by muscle, and by the membrane remnants of muscle, was without significant effect. Thus, substances secreted into the medium do not play a major role in this interaction. Neurons cultured with either muscle or muscle membrane remnants formed large, elongate structures on the target membrane surface. These were not seen in the absence of the target at the times examined. This morphological difference in terminal-like structures may parallel the developmental increases in size and vesicular content of ciliary ganglion nerve terminals in the chick iris, and may relate to the increased ACh synthetic activity. The results suggest that direct contact with an appropriate target membrane has a profound, retrograde influence upon neuronal metabolic and morphological maturation.     

Transport-dependent maturation of organelles in neurons

Some organelles show a spatial gradient of maturation along the neuronal process where more mature organelles are found closer to the cell body. This gradient is set up by progressive maturation steps that are aided by differential organelle distribution as well as transport. Autophagosomes and endosomes mature as they acquire lysosomal membrane proteins and decrease their luminal pH as they are retrogradely transported towards the cell body. The acquisition of lysosomal proteins along the neuronal processes likely occurs through fusion or membrane exchange events with Golgi-derived donor transport carriers that are transported anterogradely from the cell body. The mechanisms by which endosomes and autophagosomes mature might be applicable to other organelles that are transported along neuronal processes. Defects in axonal transport may also contribute to the accumulation of immature organelles in neurons. Such accumulations have been seen in neurons of neurodegenerative models.     

Identification of a Group of GABAergic Neurons in the Dorsomedial Area of the Locus Coeruleus

The locus coeruleus (LC)-norepinephrine (NE) system in the brainstem plays a critical role in a variety of behaviors is an important target of pharmacological intervention to several neurological disorders. Although GABA is the major inhibitory neurotransmitter of LC neurons, the modulation of LC neuronal firing activity by local GABAergic interneurons remains poorly understood with respect to their precise location, intrinsic membrane properties and synaptic modulation. Here, we took an optogenetic approach to address these questions. Channelrhodopsin (ChR2) in a tandem with the yellow fluorescent protein (YFP) was expressed in GABAergic neurons under the control of glutamic acid decarboxylase 2 (GAD2) promoter. Immediately dorsomedial to the LC nucleus, a group of GABAergic neurons was observed. They had small soma and were densely packed in a small area, which we named the dorsomedial LC or dmLC nucleus. These GABAergic neurons showed fast firing activity, strong inward rectification and spike frequency adaptation. Lateral inhibition among these GABAergic neurons was observed. Optostimulation of the dmLC area drastically inhibited LC neuronal firing frequency, expanded the spike intervals, and reset their pacemaking activity. Analysis of the light evoked inhibitory postsynaptic currents (IPSCs) indicated that they were monosynaptic. Such light evoked IPSCs were not seen in slices where this group of GABAergic neurons was absent. Thus, an isolated group of GABAergic neurons is demonstrated in the LC area, whose location, somatic morphology and intrinsic membrane properties are clearly distinguishable from adjacent LC neurons. They interact with each and may inhibit LC neurons as well as a part of local neuronal circuitry in the LC.     

Lipid rafts act as specialized domains for tetanus toxin binding and internalization into neurons

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons.     

Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures

A prerequisite for many studies of neurons in culture is a means of determining their original identity. We needed such a technique to study the interactions in vitro between a class of spinal cord neurons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes, which differ in color but are otherwise similar, to identify neurons in culture. The long carbon chain carbocyanine dyes we use are lipid-soluble and so become incorporated into the plasma membrane. Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks in culture. These dyes do not affect the survival, development, or basic physiological properties of neurons and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane. If so, carbocyanine dyes may be most useful for retrograde labeling over relatively short distances. An additional feature of carbocyanine labeling is that neuronal processes are brightly fluorescent for the first few days in culture, presumably because dye rapidly diffuses into newly inserted membrane. We have used carbocyanine dyes to identify sympathetic preganglionic neurons in culture. Our results indicate that preganglionic neurons can survive in the absence of their target cells and that several aspects of their differentiation in the absence of target appear normal.     

Siva is an apoptosis-selective p53 target gene important for neuronal cell death

p53 plays a central role in neuronal cell death resulting from acute injury or disease. To define the pathway by which p53 triggers apoptosis, we used microarray analysis to identify p53 target genes specifically upregulated during apoptosis but not cell cycle arrest. This analysis identified a small subset of targets highly selective for the p53 apoptotic response, including Siva, a proapoptotic protein whose function is not well understood. Siva's expression pattern suggests that it plays an instructive role in apoptosis, and accordingly, we demonstrate that Siva is essential for p53-dependent apoptosis in cerebellar granule neurons. In addition, we determine that endogenous Siva is associated with the plasma membrane and that Caspase-8 and Bid are important for neuronal apoptosis. Our studies highlight the participation of membrane signaling events in p53's apoptotic program in primary neurons and have significant implications for understanding the mechanisms underlying pathogenesis after neuronal injury and in neurodegenerative diseases.     

Bridging behavior and physiology: ion-channel perspective on mushroom body-dependent olfactory learning and memory in Drosophila

An important body of evidence documents the differential expression of ion channels in brains, suggesting they are essential to endow particular brain structures with specific physiological properties. Because of their role in correlating inputs and outputs in neurons, modulation of voltage-dependent ion channels (VDICs) can profoundly change neuronal network dynamics and performance, and may represent a fundamental mechanism for behavioral plasticity, one that has received less attention in learning and memory studies. Revisiting three paradigmatic mutations altering olfactory learning and memory in Drosophila (dunce, leonardo, amnesiac) a link was established between each mutation and the operation of VDICs in Kenyon cells, the intrinsic neurons of the mushroom bodies (MBs). In Drosophila, MBs are essential to the emergence of olfactory associative learning and retention. Abnormal ion channel operation might underlie failures in neuronal physiology, and be crucial to understand the abnormal associative learning and retention phenotypes the mutants display. We also discuss the only case in which a mutation in an ion channel gene (shaker) has been directly linked to olfactory learning deficits. We analyze such evidence in light of recent discoveries indicating an unusual ion current profile in shaker mutant MB intrinsic neurons. We anticipate that further studies of acquisition and retention mutants will further confirm a link between such mutations and malfunction of specific ion channel mechanisms in brain structures implicated in learning and memory.     

Maturation of rhythmic neural network: role of central modulatory inputs.

Modulatory systems are well known for their roles in tuning the cellular and synaptic properties in the adult neuronal networks, and play a major role in the control of the flexibility of functional outputs. However far less is known concerning their role in the maturation of neural networks during the development. In this review, using the stomatogastric nervous system of lobster, we will show that the neuromodulatory system exerts a powerful influence on developing neural networks. In the adult the number of both motor target neurons and their modulatory neurons is restricted to tens of identifiable cells. They are therefore well characterized in terms of cellular, synaptic and morphological properties. In the embryo, these target cells and their neuromodulatory population are already present from mid-embryonic life. However, the motor output generated by the system is quite different: while in the embryo all the target neurons are organized into a single network generating unique motor pattern, in the adult this population splits into two distinct networks generating separate patterns. This ontogenetic partitioning does not rely on progressive acquisition of adult properties but rather on a switch between two possible network operations. Indeed, adult networks are present early in the embryonic life but their expression is repressed by central modulatory neurons. Moreover, embryonic networks can be revealed in the adult system again by altering modulatory influences. Therefore, independently of the developmental age, two potential network phenotypes co-exist within the same neuronal architecture: when one is expressed, the other one is hidden and vice versa. These transitions do not necessarily need dramatic changes such as growth/retraction of processes, acquisition of new intra-membrane proteins etc. but rather, as shown by modelling studies, it may simply rely on a subtle tuning of pre-existing intercellular electrical coupling. This in turn suggests that progressive ontogenetic alteration may not take place at the level of the target network but rather at the level of modulatory input neurons.     

A membrane glycoprotein associated with the limbic system mediates the formation of the septo-hippocampal pathway in vitro

The ability of a neuronal surface glycoprotein to mediate the formation of neuronal connections was tested in an explant culture system. A monoclonal antibody against the limbic system-associated membrane protein (LAMP) was used in co-cultures containing cholinergic neurons of the septum and their hippocampal target neurons. Antibody treatment had no effect on general axon outgrowth, but significantly diminished the ability of septal cholinergic axons to invade and collateralize in the hippocampus. The results suggest that factors regulating general axon outgrowth may be distinct from those regulating the patterns of outgrowth that define the formation of neural circuits.     

Changes of the neuronal membrane excitability as cellular mechanisms of learning and memory

In the presented review given literature and results of own studies of dynamics of electrical characteristics of neurons, which change are included in processes both an elaboration of learning, and retention of the long-term memory. Literary datas and our results allow to conclusion, that long-term retention of behavioural reactions during learning is accompanied not only by changing efficiency of synaptic transmission, as well as increasing of excitability of command neurons of the defensive reflex. This means, that in the process of learning are involved long-term changes of the characteristics a membrane of certain elements of neuronal network, dependent from the metabolism of the cells. see text). Thou phenomena possible mark as cellular (electrophysiological) correlates of long-term plastic modifications of the behaviour. The analyses of having results demonstrates an important role of membrane characteristics of neurons (their excitability) and parameters an synaptic transmission not only in initial stage of learning, as well as in long-term modifications of the behaviour (long-term memory).     

Regulatory mechanisms that govern nicotinic synapse formation in neurons

Individual cholinoceptive neurons express high levels of different neuronal nicotinic acetylcholine receptor (nAChR) subtypes, and target them to the appropriate synaptic regions for proper function. This review focuses on the intercellular and intracellular processes that regulate nAChR expression in vertebrate peripheral nervous system (PNS) and central nervous system (CNS) neurons. Specifically, we discuss the cellular and molecular mechanisms that govern the induction and maintenance of nAChR expression-innervation, target tissue interactions, soluble factors, and activity. We define the regulatory principles of interneuronal nicotinic synapse differentiation that have emerged from these studies. We also discuss the molecular players that target nAChRs to the surface membrane and the interneuronal synapse.     

GABAA Receptors Modulate Early Spontaneous Excitatory Activity in Differentiating P19 Neurons

Abstract: P19 embryonic carcinoma (EC) stem cells are pluripotent and are efficiently induced to differentiate into neurons and glia with retinoic acid (RA) treatment. Within 5 days, a substantial number of differentiating P19 cells express gene products that are characteristic of a neuronal phenotype. P19 neurons were used as a model to explore the relationship between neuronal “differentiation” in vitro and the acquisition of γ-aminobutyric acid (GABA_A) receptors and functional GABA responses. Pulse-labeling experiments using bromodeoxyuridine indicated that all neurons had become postmitotic within 3–4 days after treatment with RA. This was confirmed by a reduction in the immunocytochemical detection of the undifferentiated stem cell antigen SSEA-1. Subsequently, a transient expression of nestin was observed during the first 5 days in vitro (DIV) after exposure to RA. By 5–10 DIV after RA, a significant number of neurons (～80–90%) expressed immunocytochemically detectable glutamate decarboxylase and GABA coincident with the acquisition of membrane binding sites for tetanus toxin. These phenotypic markers were maintained for >30 DIV after RA. Under current-clamp conditions, random, low-amplitude, spontaneous electrical activity appeared in neurons within the first few days after RA treatment and this was blocked by the specific GABA_A receptor antagonist bicuculline. Thereafter, the appearance and progressive increases in the frequency of spontaneous action potentials in P19 neurons were observed that were similarly attenuated by bicuculline. In neurons > 5 DIV after RA, exogenous application of GABA elicited similar action potentials. The onset of excitatory responses to GABA or muscimol in voltage-clamped neurons appeared immediately after the cessation of neuronal mitosis and before the previously reported acquisition of responses to glutamate. In fura-2 imaging studies, the exogenous application of GABA resulted in neuron-specific increases in intracellular Ca²⁺. Thus, P19 neurons provide an in vitro model for the study of the early acquisition and properties of electrical excitability to GABA and the expression of functional GABA_A receptors.     

Cell and molecular analysis of long-term sensitization in Aplysia

We have found that one cellular locus for the storage of the memory underlying short-term sensitization of the gill and siphon withdrawal reflex in Aplysia is the set of monosynaptic connections between the siphon sensory cells and the gill and siphon motor neurons. These connections also participate in the storage of memory underlying long-term sensitization. In animals that have undergone long-term sensitization, the amplitudes of the monosynaptic connections are significantly larger (2.2x) than the ones in control animals. To study the mechanisms of onset and retention of long-term synaptic facilitation that underly long-term sensitization and the role of protein synthesis in long-term memory, we have developed two types of reduced preparations: the intact reflex isolated from the remainder of the animal, and a dissociated cell culture system in which the monosynaptic component (sensory neurons and motor neurons) of the neuronal circuit mediating the withdrawal reflex is reconstituted. We found that protein synthesis inhibitors, such as anisomycin or emetine, and RNA synthesis inhibitors, such as actinomycin D or alpha-amanitin, blocked long-term facilitation without interfering with short-term facilitation. These results suggest that the acquisition of long-term memory may require the expression of genes and the synthesis of proteins not needed for short-term memory.     

The endocannabinoid system controls key epileptogenic circuits in the hippocampus

Balanced control of neuronal activity is central in maintaining function and viability of neuronal circuits. The endocannabinoid system tightly controls neuronal excitability. Here, we show that endocannabinoids directly target hippocampal glutamatergic neurons to provide protection against acute epileptiform seizures in mice. Functional CB1 cannabinoid receptors are present on glutamatergic terminals of the hippocampal formation, colocalizing with vesicular glutamate transporter 1 (VGluT1). Conditional deletion of the CB1 gene either in cortical glutamatergic neurons or in forebrain GABAergic neurons, as well as virally induced deletion of the CB1 gene in the hippocampus, demonstrate that the presence of CB1 receptors in glutamatergic hippocampal neurons is both necessary and sufficient to provide substantial endogenous protection against kainic acid (KA)-induced seizures. The direct endocannabinoid-mediated control of hippocampal glutamatergic neurotransmission may constitute a promising therapeutic target for the treatment of disorders associated with excessive excitatory neuronal activity.     

Ultrastructural characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death in embryonic dopaminergic neurons

Developing neuronal populations undergo significant attrition by natural cell death. Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis during synaptogenesis. Following this time window, destruction of the anatomic target of dopaminergic neurons results in dopaminergic cell death but the morphology is no longer apoptotic. We describe ultrastructural changes that appear unique to dying embryonic dopaminergic neurons. In primary cultures of mesencephalon, death of dopaminergic neurons is triggered by activation of glutamate receptors sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and differs ultrastructurally from both neuronal apoptosis or typical excitotoxicity. AMPA causes morphological changes selectively in dopaminergic neurons, without affecting other neurons in the same culture dishes. Two hours after the onset of treatment swelling of Golgi complexes is apparent. At 3 h, dopaminergic neurons display loss of membrane asymmetry (coinciding with commitment to die), as well as nuclear membrane invagination, irregular aggregation of chromatin, and mitochondrial swelling. Nuclear changes continue to worsen until loss of cytoplasmic structures and cell death begins to occur after 12 h. These changes are different from those described in neurons undergoing either apoptosis or excitotoxic death, but are similar to ultrastructural changes observed in spontaneous death of dopaminergic neurons in the natural mutant weaver mouse.     

A longitudinal study of the effects of prenatal ethanol exposure on neuronal acquisition and death in the principal sensory nucleus of the trigeminal nerve: interaction with changes induced by transection of the infraorbital nerve

The present study determines (1) whether ethanol-induced microencephaly results from reductions in neuronal acquisition (i.e., cell proliferation and neuronal migration) and/or increases in neuronal death and (2) whether ethanol exacerbates death by the same mode as that for naturally occurring or lesion-induced neuronal death. Pregnant rats were exposed to a diet containing 6.7% (v/v) ethanol or an isocaloric control diet during the last two weeks of gestation. At birth, the right infraorbital nerves of the pups were transected. The numbers of neurons in the principal sensory nucleus of the trigeminal nerve (PSN) on both sides of the pons were examined at various prenatal and early postnatal timepoints. The numbers of pyknotic and argyrophilic PSN cells were also counted. Ethanol delayed and reduced (19.9%) the prenatal acquisition of PSN neurons. The postnatal decline in neuronal number (indicative of neuronal death) was significantly increased (10.6%) by ethanol. Likewise, the numbers of pyknotic and silver-stained cells were significantly higher in ethanol-treated rats. Lesion of the infraorbital nerve induced significant transsynaptic neuronal death in the control rats. Ethanol increased the amount of death caused by the lesion; however, it altered neither the timing of the neuronal loss nor the incidence of pyknosis or silver-staining. Therefore, ethanol affects both neuronal acquisition and survival; the greater effect being on neuronal acquisition. The timing and morphology of dying cells indicate that regardless of the cause (natural processes, ethanol-induced, or lesion-induced), neurons die in the developing PSN by the same mode.     

Optical calcium imaging in the nervous system of Drosophila melanogaster

BACKGROUND: Drosophila melanogaster is one of the best-studied model organisms in biology, mainly because of the versatility of methods by which heredity and specific expression of genes can be traced and manipulated. Sophisticated genetic tools have been developed to express transgenes in selected cell types, and these techniques can be utilized to target DNA-encoded fluorescence probes to genetically defined subsets of neurons. Neuroscientists make use of this approach to monitor the activity of restricted types or subsets of neurons in the brain and the peripheral nervous system. Since membrane depolarization is typically accompanied by an increase in intracellular calcium ions, calcium-sensitive fluorescence proteins provide favorable tools to monitor the spatio-temporal activity across groups of neurons. SCOPE OF REVIEW: Here we describe approaches to perform optical calcium imaging in Drosophila in consideration of various calcium sensors and expression systems. In addition, we outline by way of examples for which particular neuronal systems in Drosophila optical calcium imaging have been used. Finally, we exemplify briefly how optical calcium imaging in the brain of Drosophila can be carried out in practice. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: Drosophila provides an excellent model organism to combine genetic expression systems with optical calcium imaging in order to investigate principles of sensory coding, neuronal plasticity, and processing of neuronal information underlying behavior. This article is part of a Special Issue entitled Biochemical, Biophysical and Genetic Approaches to Intracellular Calcium Signaling.