RESUMO
Homeostatic matching of pre- and postsynaptic function has been observed in many species and neural structures, but whether transcriptional changes contribute to this form of trans-synaptic coordination remains unknown. To identify genes whose expression is altered in presynaptic neurons as a result of perturbing postsynaptic excitability, we applied a transcriptomics-friendly, temperature-inducible Kir2.1-based activity clamp at the first synaptic relay of the Drosophila olfactory system, a central synapse known to exhibit trans-synaptic homeostatic matching. Twelve hours after adult-onset suppression of activity in postsynaptic antennal lobe projection neurons of males and females, we detected changes in the expression of many genes in the third antennal segment, which houses the somata of presynaptic olfactory receptor neurons. These changes affected genes with roles in synaptic vesicle release and synaptic remodeling, including several implicated in homeostatic plasticity at the neuromuscular junction. At 48 h and beyond, the transcriptional landscape tilted toward protein synthesis, folding, and degradation; energy metabolism; and cellular stress defenses, indicating that the system had been pushed to its homeostatic limits. Our analysis suggests that similar homeostatic machinery operates at peripheral and central synapses and identifies many of its components. The presynaptic transcriptional response to genetically targeted postsynaptic perturbations could be exploited for the construction of novel connectivity tracing tools.SIGNIFICANCE STATEMENT Homeostatic feedback mechanisms adjust intrinsic and synaptic properties of neurons to keep their average activity levels constant. We show that, at a central synapse in the fruit fly brain, these mechanisms include changes in presynaptic gene expression that are instructed by an abrupt loss of postsynaptic excitability. The trans-synaptically regulated genes have roles in synaptic vesicle release and synapse remodeling; protein synthesis, folding, and degradation; and energy metabolism. Our study establishes a role for transcriptional changes in homeostatic synaptic plasticity, points to mechanistic commonalities between peripheral and central synapses, and potentially opens new opportunities for the development of connectivity-based gene expression systems.
Assuntos
Homeostase/fisiologia , Plasticidade Neuronal/fisiologia , Terminações Pré-Sinápticas/metabolismo , Sinapses/genética , Sinapses/metabolismo , Animais , Animais Geneticamente Modificados , Antenas de Artrópodes/inervação , Antenas de Artrópodes/metabolismo , Drosophila , Feminino , Expressão GênicaRESUMO
Sleep-promoting neurons in the dorsal fan-shaped body (dFB) of Drosophila are integral to sleep homeostasis, but how these cells impose sleep on the organism is unknown. We report that dFB neurons communicate via inhibitory transmitters, including allatostatin-A (AstA), with interneurons connecting the superior arch with the ellipsoid body of the central complex. These "helicon cells" express the galanin receptor homolog AstA-R1, respond to visual input, gate locomotion, and are inhibited by AstA, suggesting that dFB neurons promote rest by suppressing visually guided movement. Sleep changes caused by enhanced or diminished allatostatinergic transmission from dFB neurons and by inhibition or optogenetic stimulation of helicon cells support this notion. Helicon cells provide excitation to R2 neurons of the ellipsoid body, whose activity-dependent plasticity signals rising sleep pressure to the dFB. By virtue of this autoregulatory loop, dFB-mediated inhibition interrupts processes that incur a sleep debt, allowing restorative sleep to rebalance the books. VIDEO ABSTRACT.
Assuntos
Drosophila melanogaster/fisiologia , Interneurônios/fisiologia , Sono/fisiologia , Animais , Encéfalo/fisiologia , Ritmo Circadiano , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Homeostase , Hormônios de Inseto/fisiologia , Luz , Locomoção/efeitos da radiação , Masculino , Potenciais da Membrana , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Optogenética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Visão OcularRESUMO
Sleep disconnects animals from the external world, at considerable risks and costs that must be offset by a vital benefit. Insight into this mysterious benefit will come from understanding sleep homeostasis: to monitor sleep need, an internal bookkeeper must track physiological changes that are linked to the core function of sleep. In Drosophila, a crucial component of the machinery for sleep homeostasis is a cluster of neurons innervating the dorsal fan-shaped body (dFB) of the central complex. Artificial activation of these cells induces sleep, whereas reductions in excitability cause insomnia. dFB neurons in sleep-deprived flies tend to be electrically active, with high input resistances and long membrane time constants, while neurons in rested flies tend to be electrically silent. Correlative evidence thus supports the simple view that homeostatic sleep control works by switching sleep-promoting neurons between active and quiescent states. Here we demonstrate state switching by dFB neurons, identify dopamine as a neuromodulator that operates the switch, and delineate the switching mechanism. Arousing dopamine caused transient hyperpolarization of dFB neurons within tens of milliseconds and lasting excitability suppression within minutes. Both effects were transduced by Dop1R2 receptors and mediated by potassium conductances. The switch to electrical silence involved the downregulation of voltage-gated A-type currents carried by Shaker and Shab, and the upregulation of voltage-independent leak currents through a two-pore-domain potassium channel that we term Sandman. Sandman is encoded by the CG8713 gene and translocates to the plasma membrane in response to dopamine. dFB-restricted interference with the expression of Shaker or Sandman decreased or increased sleep, respectively, by slowing the repetitive discharge of dFB neurons in the ON state or blocking their entry into the OFF state. Biophysical changes in a small population of neurons are thus linked to the control of sleep-wake state.
Assuntos
Drosophila melanogaster/fisiologia , Homeostase , Sono/fisiologia , Animais , Membrana Celular/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Condutividade Elétrica , Feminino , Masculino , Neurotransmissores/metabolismo , Optogenética , Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Transporte Proteico , Receptores Dopaminérgicos/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Privação do Sono , Distúrbios do Início e da Manutenção do Sono/fisiopatologia , Fatores de Tempo , Vigília/fisiologiaRESUMO
Sleep is under homeostatic control, but the mechanisms that sense sleep need and correct sleep deficits remain unknown. Here, we report that sleep-promoting neurons with projections to the dorsal fan-shaped body (FB) form the output arm of Drosophila's sleep homeostat. Homeostatic sleep control requires the Rho-GTPase-activating protein encoded by the crossveinless-c (cv-c) gene in order to transduce sleep pressure into increased electrical excitability of dorsal FB neurons. cv-c mutants exhibit decreased sleep time, diminished sleep rebound, and memory deficits comparable to those after sleep loss. Targeted ablation and rescue of Cv-c in sleep-control neurons of the dorsal FB impair and restore, respectively, normal sleep patterns. Sleep deprivation increases the excitability of dorsal FB neurons, but this homeostatic adjustment is disrupted in short-sleeping cv-c mutants. Sleep pressure thus shifts the input-output function of sleep-promoting neurons toward heightened activity by modulating ion channel function in a mechanism dependent on Cv-c.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Proteínas Ativadoras de GTPase/genética , Homeostase/fisiologia , Mutação/genética , Neurônios/metabolismo , Sono/genética , Animais , Proteínas Ativadoras de GTPase/metabolismo , Privação do Sono/genética , Privação do Sono/metabolismoRESUMO
One defining characteristic of the mammalian brain is its neuronal diversity. For a given region, substructure, layer or even cell type, variability in neuronal morphology and connectivity persists. Although it is well known that such cellular properties vary considerably according to neuronal type, the substantial biophysical diversity of neurons of the same morphological class is typically averaged out and ignored. Here we show that the amplitude of hyperpolarization-evoked sag of membrane potential recorded in olfactory bulb mitral cells is an emergent, homotypic property of local networks and sensory information processing. Simultaneous whole-cell recordings from pairs of cells show that the amount of hyperpolarization-evoked sag potential and current (Ih) is stereotypic for mitral cells belonging to the same glomerular circuit. This is corroborated by a mosaic, glomerulus-based pattern of expression of the HCN2 (hyperpolarization-activated cyclic nucleotide-gated channel 2) subunit of the Ih channel. Furthermore, inter-glomerular differences in both membrane potential sag and HCN2 protein are diminished when sensory input to glomeruli is genetically and globally altered so that only one type of odorant receptor is universally expressed. Population diversity in this intrinsic property therefore reflects differential expression between local mitral cell networks processing distinct odour-related information.
Assuntos
Rede Nervosa/fisiologia , Bulbo Olfatório/citologia , Bulbo Olfatório/fisiologia , Olfato/fisiologia , Animais , Feminino , Perfilação da Expressão Gênica , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Neurológicos , Canais de Potássio , Receptores Odorantes/metabolismoRESUMO
Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms--GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs) was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during excitotoxicity and the first molecular target of the UPS affected in this cell death process.
Assuntos
Glutamato Descarboxilase/metabolismo , Hipocampo/citologia , Neurônios/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Células Cultivadas , Ácido Glutâmico/farmacologia , Hidrólise/efeitos dos fármacos , Ratos , UbiquitinaçãoRESUMO
In the olfactory bulb the sets of mitral cells that project their apical dendrite to the same glomerulus represent unique functional networks. While it is known that mitral cells release vesicular glutamate from their apical tuft it is believed that the resultant self-excitation (SE), transmitted via dendritic gap junctions, is the main form of lateral transmission within the mitral cell assembly. In this study we used simultaneous whole-cell recordings from mitral cell pairs to show that a direct form of chemical lateral excitation (LE) provides a means of mitral cell-mitral cell communication. In contrast to the ubiquitous expression and robust nature of SE, the efficacy of glutamatergic LE between mitral cells is highly variable and mediated by calcium-impermeable AMPA receptors. We also find that the strength of LE is bi-directionally modulated, in a homeostatic manner, by sniffing-like patterns of presynaptic activity. Since these changes last many minutes we suggest that such mitral cell-mitral cell interactions provide the glomerular network with a locus for olfactory plasticity and a potential mechanism for receptive field modulation.