GABAA receptor RDL inhibits Drosophila olfactory associative learning

In both mammals and insects, neurons involved in learning are strongly modulated by the inhibitory neurotransmitter GABA. The GABAA receptor, resistance to dieldrin (Rdl), is highly expressed in the Drosophila mushroom bodies (MBs), a group of neurons playing essential roles in insect olfactory learning. Flies with increased or decreased expression of Rdl in the MBs were generated. Olfactory associative learning tests showed that Rdl overexpression impaired memory acquisition but not memory stability. This learning defect was due to disrupting the physiological state of the adult MB neurons rather than causing developmental abnormalities. Remarkably, Rdl knockdown enhanced memory acquisition but not memory stability. Functional cellular imaging experiments showed that Rdl overexpression abolished the normal calcium responses of the MBs to odors while Rdl knockdown increased these responses. Together, these data suggest that RDL negatively modulates olfactory associative learning, possibly by gating the input of olfactory information into the MBs.     

Molecular analysis of vesicular amine transporter function and targeting to secretory organelles.

Vesicular transporters are responsible for the loading of neurotransmitters into specialized secretory organelles in neurons and neuroendocrine cells to make them available for regulated neurosecretion. The exocytotic release of neurotransmitter therefore depends on the functional activity of the vesicular transporters and their efficient sorting to these secretory organelles. Molecular analysis of vesicular transport proteins has revealed important information regarding structural domains responsible for their functional properties, including substrate specificity and trafficking to various classes of secretory vesicles. These studies have established the existence of an important functional relationship between transporter activity and presynaptic quantal neurosecretion.     

Testing odor response stereotypy in the Drosophila mushroom body

The mushroom body is an insect brain structure required for olfactory learning. Its principal neurons, the Kenyon cells (KCs), form a large cell population. The neuronal populations from which their olfactory input derives (olfactory sensory and projection neurons) can be identified individually by genetic, anatomical, and physiological criteria. We ask whether KCs are similarly identifiable individually, using genetic markers and whole-cell patch-clamp in vivo. We find that across-animal responses are as diverse within the genetically labeled subset as across all KCs in a larger sample. These results combined with those from a simple model, using projection neuron odor responses as inputs, suggest that the precise circuit specification seen at earlier stages of odor processing is likely absent among the mushroom body KCs.     

Glutamate, GABA and acetylcholine signaling components in the lamina of the Drosophila visual system

Synaptic connections of neurons in the Drosophila lamina, the most peripheral synaptic region of the visual system, have been comprehensively described. Although the lamina has been used extensively as a model for the development and plasticity of synaptic connections, the neurotransmitters in these circuits are still poorly known. Thus, to unravel possible neurotransmitter circuits in the lamina of Drosophila we combined Gal4 driven green fluorescent protein in specific lamina neurons with antisera to gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, a GABA(B) type of receptor, L-glutamate, a vesicular glutamate transporter (vGluT), ionotropic and metabotropic glutamate receptors, choline acetyltransferase and a vesicular acetylcholine transporter. We suggest that acetylcholine may be used as a neurotransmitter in both L4 monopolar neurons and a previously unreported type of wide-field tangential neuron (Cha-Tan). GABA is the likely transmitter of centrifugal neurons C2 and C3 and GABA(B) receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABA(A) receptor subunit RDL may be expressed by L4 neurons and a type of tangential neuron (rdl-Tan). Strong vGluT immunoreactivity was detected in alpha-processes of amacrine neurons and possibly in the large monopolar neurons L1 and L2. These neurons also express glutamate-like immunoreactivity. However, antisera to ionotropic and metabotropic glutamate receptors did not produce distinct immunosignals in the lamina. In summary, this paper describes novel features of two distinct types of tangential neurons in the Drosophila lamina and assigns putative neurotransmitters and some receptors to a few identified neuron types.     

Synaptic glutamate release by ventromedial hypothalamic neurons is part of the neurocircuitry that prevents hypoglycemia

The importance of neuropeptides in the hypothalamus has been experimentally established. Due to difficulties in assessing function in vivo, the roles of the fast-acting neurotransmitters glutamate and GABA are largely unknown. Synaptic vesicular transporters (VGLUTs for glutamate and VGAT for GABA) are required for vesicular uptake and, consequently, synaptic release of neurotransmitters. Ventromedial hypothalamic (VMH) neurons are predominantly glutamatergic and express VGLUT2. To evaluate the role of glutamate release from VMH neurons, we generated mice lacking VGLUT2 selectively in SF1 neurons (a major subset of VMH neurons). These mice have hypoglycemia during fasting secondary to impaired fasting-induced increases in the glucose-raising pancreatic hormone glucagon and impaired induction in liver of mRNAs encoding PGC-1alpha and the gluconeogenic enzymes PEPCK and G6Pase. Similarly, these mice have defective counterregulatory responses to insulin-induced hypoglycemia and 2-deoxyglucose (an antimetabolite). Thus, glutamate release from VMH neurons is an important component of the neurocircuitry that functions to prevent hypoglycemia.     

A glial variant of the vesicular monoamine transporter is required to store histamine in the Drosophila visual system

Unlike other monoamine neurotransmitters, the mechanism by which the brain's histamine content is regulated remains unclear. In mammals, vesicular monoamine transporters (VMATs) are expressed exclusively in neurons and mediate the storage of histamine and other monoamines. We have studied the visual system of Drosophila melanogaster in which histamine is the primary neurotransmitter released from photoreceptor cells. We report here that a novel mRNA splice variant of Drosophila VMAT (DVMAT-B) is expressed not in neurons but rather in a small subset of glia in the lamina of the fly's optic lobe. Histamine contents are reduced by mutation of dVMAT, but can be partially restored by specifically expressing DVMAT-B in glia. Our results suggest a novel role for a monoamine transporter in glia that may be relevant to histamine homeostasis in other systems.     

The transport of neurotransmitters into synaptic vesicles.

As investigations identify additional plasma membrane neurotransmitter transporters, attention has focused on the molecular basis of neurotransmitter transport into synaptic vesicles. The transport of biogenic amines into chromaffin granules has served as the paradigm for understanding vesicular transport. Recent work now describes the vesicular transport of other classical neurotransmitters, which occur by distinct but related mechanisms. To determine their biochemical basis, several of the transporters have been functionally reconstituted in liposomes. The ability of vesicular amine transport to protect against the neurotoxin MPP+ has permitted the isolation of the first cDNA clone for a member of this family, and the sequence establishes a relationship with drug-resistance transporters in bacteria.     

Export of Erwinia chrysanthemi (EC16) protease by Escherichia coli

Abstract During exponential growth, Erwinia chrysanthemi (EC16) exports 99% of the protease (PRT) into the growth medium. By screening an EC16 genomic library in Escherichia coli HB101, several Prt⁺ clones were identified. A 16-kb Eco RI fragment, carrying the prt gene, was subcloned into pBR322 (pAKC326). E. coli HB101[pAKC326] cells exported PRT into the growth medium during exponential growth. PRT export was not accompanied by periplasmic leakage. E. coli HB101 carrying EC16 prt and pel genes (encoding pectate lyase) exported PRT but retained PEL in the periplasm. These findings indicate the occurrence of a PRT-specific export system in EC16, which is also functional in an E. coli strain carrying the prt ⁺ DNA segment.     

Trafficking of vesicular neurotransmitter transporters

Vesicular neurotransmitter transporters are required for the storage of all classical and amino acid neurotransmitters in secretory vesicles. Transporter expression can influence neurotransmitter storage and release, and trafficking targets the transporters to different types of secretory vesicles. Vesicular transporters traffic to synaptic vesicles (SVs) as well as large dense core vesicles and are recycled to SVs at the nerve terminal. Some of the intrinsic signals for these trafficking events have been defined and include a dileucine motif present in multiple transporter subtypes, an acidic cluster in the neural isoform of the vesicular monoamine transporter (VMAT) 2 and a polyproline motif in the vesicular glutamate transporter (VGLUT) 1. The sorting of VMAT2 and the vesicular acetylcholine transporter to secretory vesicles is regulated by phosphorylation. In addition, VGLUT1 uses alternative endocytic pathways for recycling back to SVs following exocytosis. Regulation of these sorting events has the potential to influence synaptic transmission and behavior.     

Hypoxia. 3. Hypoxia and neurotransmitter synthesis

Central and peripheral neurons as well as neuroendocrine cells express a variety of neurotransmitters/modulators that play critical roles in regulation of physiological systems. The synthesis of several neurotransmitters/modulators is regulated by O(2)-requiring rate-limiting enzymes. Consequently, hypoxia resulting from perturbations in O(2) homeostasis can affect neuronal functions by altering neurotransmitter synthesis. Two broad categories of hypoxia are frequently encountered: continuous hypoxia (CH) and intermittent hypoxia (IH). CH is often seen during high altitude sojourns, whereas IH is experienced in sleep-disordered breathing with recurrent apneas (i.e., brief, repetitive cessations of breathing). This article presents what is currently known on the effects of both forms of hypoxia on neurotransmitter levels and neurotransmitter synthesizing enzymes in the central and peripheral nervous systems.     

Regulated arrest of cell proliferation mediated by yeast prt1 mutations

Several temperature-sensitive cell-division-cycle (cdc) mutations differentially affect the regulatory step for cell proliferation in the yeast. Saccharomyces cerevisiae, including one mutation termed cdc63-1, which resides in a previously known gene called PRT1. Other mutations in the PRT1 gene have been shown by others to affect an initiation step in protein synthesis. Here we show that at the appropriate nonpermissive temperature each prt1 mutation can produce a uniform and concerted arrest of cell division; the prt1-1 mutation, like cdc63-1, is shown to arrest cells specifically at the regulatory step for cell proliferation. This response of cessation of cell division is different from the response of cells to an equivalent limitation of protein synthesis using cycloheximide or verrucarin A, which implies that the PRT1 gene product could separately influence both cellular growth via protein synthesis and events in the regulation of cell proliferation.     

Vesicular neurotransmitter transporter trafficking in vivo: Moving from cells to flies

During exocytosis, classical and amino acid neurotransmitters are released from the lumen of synaptic vesicles to allow signaling at the synapse. The storage of neurotransmitters in synaptic vesicles and other types of secretory vesicles requires the activity of specific vesicular transporters. Glutamate and monoamines such as dopamine are packaged by VGLUTs and VMATs respectively. Changes in the localization of either protein have the potential to up- or down regulate neurotransmitter release, and some of the mechanisms for sorting these proteins to secretory vesicles have been investigated in cultured cells in vitro. We have used Drosophila molecular genetic techniques to study vesicular transporter trafficking in an intact organism and have identified a motif required for localizing Drosophila VMAT (DVMAT) to synaptic vesicles in vivo. In contrast to DVMAT, large deletions of Drosophila VGLUT (DVGLUT) show relatively modest deficits in localizing to synaptic vesicles, suggesting that DVMAT and DVGLUT may undergo different modes of trafficking at the synapse. Further in vivo studies of DVMAT trafficking mutants will allow us to determine how changes in the localization of vesicular transporters affect the nervous system as a whole and complex behaviors mediated by aminergic circuits.     

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.     

The blu Blur: mutation of a vesicular glutamate transporter reduces the resolution of zebrafish vision

Vesicular transporters mediate the packaging of neurotransmitters into synaptic vesicles and can therefore control the amount of neurotransmitter released into the synaptic cleft. In this issue of Neuron, Smear et al. demonstrate that mutation of a vesicular glutamate transporter (Vglut) found in the retinal ganglion cells (RGCs) of zebrafish alters both the synaptic transmission and connectivity between RGCs and their targets, limiting the transfer of visually evoked activity from RGCs and degrading behaviors that depend on high-acuity vision.     

A shared vesicular carrier allows synaptic corelease of GABA and glycine

The type of vesicular transporter expressed by a neuron is thought to determine its neurotransmitter phenotype. We show that inactivation of the vesicular inhibitory amino acid transporter (Viaat, VGAT) leads to embryonic lethality, an abdominal defect known as omphalocele, and a cleft palate. Loss of Viaat causes a drastic reduction of neurotransmitter release in both GABAergic and glycinergic neurons, indicating that glycinergic neurons do not express a separate vesicular glycine transporter. This loss of GABAergic and glycinergic synaptic transmission does not impair the development of inhibitory synapses or the expression of KCC2, the K+ -Cl- cotransporter known to be essential for the establishment of inhibitory neurotransmission. In the absence of Viaat, GABA-synthesizing enzymes are partially lost from presynaptic terminals. Since GABA and glycine compete for vesicular uptake, these data point to a close association of Viaat with GABA-synthesizing enzymes as a key factor in specifying GABAergic neuronal phenotypes.     

Why glycine transporters have different stoichiometries

In the brain, neurons and glial cells compete for the uptake of the fast neurotransmitters, glutamate, GABA and glycine, through specific transporters. The relative contributions of glia and neurons to the neurotransmitter uptake depend on the kinetic properties, thermodynamic coupling and density of transporters but also on the intracellular metabolization or sequestration of the neurotransmitter. In the case of glycine, which is both an inhibitory transmitter and a neuromodulator of the excitatory glutamatergic transmission as a co-agonist of N-methyl D-aspartate receptors, the glial (GlyT1b) and neuronal (GlyT2a) transporters differ at least in three aspects: (i) stoichiometries, (ii) reverse uptake capabilities and (iii) pre-steady-state kinetics. A 3 Na(+)/1 Cl(-)/gly stoichiometry was established for GlyT2a on the basis of a 2 charges/glycine flux ratio and changes in the reversal potential of the transporter current as a function of the extracellular glycine, Na(+) and Cl(-) concentrations. Therefore, the driving force available for glycine uphill transport in neurons is about two orders of magnitude larger than for glial cells. In addition, GlyT2a shows a severe limitation for reverse uptake, which suggests an essential role of GlyT2a in maintaining a high intracellular glycine pool, thus facilitating the refilling of synaptic vesicles by the low affinity, low specificity vesicular transporter VGAT/VIAAT. In contrast, the 2 Na(+)/1 Cl(-)/gly stoichiometry and bi-directional transport properties of GlyT1b are appropriate for the control of the extracellular glycine concentration in a submicromolar range that can modulate N-methyl D-aspartate receptors effectively. Finally, analysis of the pre-steady-state kinetics of GlyT1b and GlyT2a revealed that at the resting potential neuronal transporters are preferentially oriented outward, ready to bind glycine, which suggests a kinetic advantage in the uptake contest.     

Elimination of the vesicular acetylcholine transporter in the striatum reveals regulation of behaviour by cholinergic-glutamatergic co-transmission

Cholinergic neurons in the striatum are thought to play major regulatory functions in motor behaviour and reward. These neurons express two vesicular transporters that can load either acetylcholine or glutamate into synaptic vesicles. Consequently cholinergic neurons can release both neurotransmitters, making it difficult to discern their individual contributions for the regulation of striatal functions. Here we have dissected the specific roles of acetylcholine release for striatal-dependent behaviour in mice by selective elimination of the vesicular acetylcholine transporter (VAChT) from striatal cholinergic neurons. Analysis of several behavioural parameters indicates that elimination of VAChT had only marginal consequences in striatum-related tasks and did not affect spontaneous locomotion, cocaine-induced hyperactivity, or its reward properties. However, dopaminergic sensitivity of medium spiny neurons (MSN) and the behavioural outputs in response to direct dopaminergic agonists were enhanced, likely due to increased expression/function of dopamine receptors in the striatum. These observations indicate that previous functions attributed to striatal cholinergic neurons in spontaneous locomotor activity and in the rewarding responses to cocaine are mediated by glutamate and not by acetylcholine release. Our experiments demonstrate how one population of neurons can use two distinct neurotransmitters to differentially regulate a given circuitry. The data also raise the possibility of using VAChT as a target to boost dopaminergic function and decrease high striatal cholinergic activity, common neurochemical alterations in individuals affected with Parkinson's disease.     

Emerging neuroskeletal signalling pathways: a review

Recent work has demonstrated that neurotransmitters, signalling molecules primarily associated with the nervous system, can have profound effects on the skeleton. Bone cells express a broad range of neurotransmitter receptors and transporters, and respond to receptor activation by initiating diverse intracellular signalling pathways, which modulate cellular function. Evidence of neuronal innervation in skeletal tissues, neurotransmitter release directly from bone cells and functional effects of pharmacological manipulation support the existence of a complex and functionally significant neurotransmitter-mediated signalling network in bone. This review aims to concisely summarise our current understanding of how neurotransmitters affect the skeletal system, focusing on their origin, cellular targets and functional effects in bone.     

Vesicular neurotransmitter transporters

Neurotransmission depends on the regulated release of chemical transmitter molecules. This requires the packaging of these substances into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific vesicular transporters. The family of genes encoding the vesicular transporters for biogenic amines and acetylcholine have recently been cloned. Direct comparison of their transport characteristics and pharmacology provides information about vesicular transport bioenergetics, substrate feature recognition by each transporter, and the role of vesicular amine storage in the mechanism of action of psychopharmacologic and neurotoxic agents. Regulation of vesicular transport activity may affect levels of neurotransmitter available for neurosecretion and be an important site for the regulation of synaptic function. Gene knockout studies have determined vesicular transport function is critical for survival and have enabled further evaluation of the role of vesicular neurotransmitter transporters in behavior and neurotoxicity. Molecular analysis is beginning to reveal the sites involved in vesicular transporter function and the sites that determine substrate specificity. In addition, the molecular basis for the selective targeting of these transporters to specific vesicle populations and the biogenesis of monoaminergic and cholinergic synaptic vesicles are areas of research that are currently being explored. This information provides new insights into the pharmacology and physiology of biogenic amine and acetylcholine vesicular storage in cardiovascular, endocrine, and central nervous system function and has important implications for neurodegenerative disease.     

Gamma neurons mediate dopaminergic input during aversive olfactory memory formation in Drosophila

Mushroom body (MB)-dependent olfactory learning in Drosophila provides a powerful model to investigate memory mechanisms. MBs integrate olfactory conditioned stimulus (CS) inputs with neuromodulatory reinforcement (unconditioned stimuli, US), which for aversive learning is thought to rely on dopaminergic (DA) signaling to DopR, a D1-like dopamine receptor expressed in MBs. A wealth of evidence suggests the conclusion that parallel and independent signaling occurs downstream of DopR within two MB neuron cell types, with each supporting half of memory performance. For instance, expression of the Rutabaga (Rut) adenylyl cyclase in γ neurons is sufficient to restore normal learning to rut mutants, whereas expression of Neurofibromatosis 1 (NF1) in α/β neurons is sufficient to rescue NF1 mutants. DopR mutations are the only case where memory performance is fully eliminated, consistent with the hypothesis that DopR receives the US inputs for both γ and α/β lobe traces. We demonstrate, however, that DopR expression in γ neurons is sufficient to fully support short- and long-term memory. We argue that DA-mediated CS-US association is formed in γ neurons followed by communication between γ and α/β neurons to drive consolidation.