RESUMO
Highly interconnected neural networks perform olfactory signal processing in the central nervous system. In insects, the first synaptic processing of the olfactory input from the antennae occurs in the antennal lobe, the functional equivalent of the olfactory bulb in vertebrates. Key components of the olfactory network in the antennal lobe are two main types of neurons: the local interneurons and the projection (output) neurons. Both neuron types have different physiological tasks during olfactory processing, which accordingly require specialized functional phenotypes. This review gives an overview of important cell type-specific functional properties of the different types of projection neurons and local interneurons in the antennal lobe of the cockroach Periplaneta americana, which is an experimental system that has elucidated many important biophysical and cellular bases of intrinsic physiological properties of these neurons.
Assuntos
Encéfalo/fisiologia , Odorantes , Condutos Olfatórios/fisiologia , Animais , BaratasRESUMO
Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.
Assuntos
Neurônios , Nociceptina , Peptídeos Opioides , Receptores Opioides , Animais , Peptídeos Opioides/metabolismo , Receptores Opioides/metabolismo , Receptores Opioides/genética , Neurônios/metabolismo , Humanos , Camundongos , Masculino , Área Tegmentar Ventral/metabolismo , Receptor de Nociceptina , Células HEK293 , Encéfalo/metabolismo , Camundongos Endogâmicos C57BL , Ligantes , Técnicas Biossensoriais/métodosRESUMO
Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fiber photometry enabled a direct recording of binding by N/OFQ receptor ligands, as well as the detection of natural or chemogenetically-evoked endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA). In summary, we show that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely-behaving animals.
RESUMO
Local interneurons (LNs) mediate complex interactions within the antennal lobe, the primary olfactory system of insects, and the functional analog of the vertebrate olfactory bulb. In the cockroach Periplaneta americana, as in other insects, several types of LNs with distinctive physiological and morphological properties can be defined. Here, we combined whole-cell patch-clamp recordings and Ca2+ imaging of individual LNs to analyze the role of spiking and nonspiking LNs in inter- and intraglomerular signaling during olfactory information processing. Spiking GABAergic LNs reacted to odorant stimulation with a uniform rise in [Ca2+]i in the ramifications of all innervated glomeruli. In contrast, in nonspiking LNs, glomerular Ca2+ signals were odorant specific and varied between glomeruli, resulting in distinct, glomerulus-specific tuning curves. The cell type-specific differences in Ca2+ dynamics support the idea that spiking LNs play a primary role in interglomerular signaling, while they assign nonspiking LNs an essential role in intraglomerular signaling.
Assuntos
Antenas de Artrópodes/inervação , Sinalização do Cálcio , Neurônios GABAérgicos/fisiologia , Interneurônios/fisiologia , Bulbo Olfatório/fisiologia , Condutos Olfatórios/fisiologia , Periplaneta/fisiologia , Olfato , Potenciais de Ação , Animais , Odorantes , Fatores de TempoRESUMO
Behavioral and physiological studies show that neuronal interactions among the glomeruli in the insect antennal lobe (AL) take place during the processing of odor information. These interactions are mediated by a complex network of inhibitory and excitatory local interneurons (LNs) that restructure the olfactory representation in the AL, thereby regulating the tuning profile of projection neurons. In Periplaneta americana, we characterized two LN types with distinctive physiological properties: (1) type I LNs that generated Na(+)-driven action potentials on odor stimulation and exhibited GABA-like immunoreactivity (GLIR) and (2) type II LNs, in which odor stimulation evoked depolarizations, but no Na(+)-driven action potentials (APs). Type II LNs did not express voltage-dependent transient Na(+) currents and accordingly would not trigger transmitter release by Na(+)-driven APs. Ninety percent of type II LNs did not exhibit GLIR. The distinct intrinsic firing properties were reflected in functional parameters of their voltage-activated Ca(2+) currents (I(Ca)). Consistent with graded synaptic release, we found a shift in the voltage for half-maximal activation of I(Ca) to more hyperpolarized membrane potentials in the type II LNs. These marked physiological differences between the two LN types imply consequences for their computational capacity, synaptic output kinetics, and thus their function in the olfactory circuit.
Assuntos
Potenciais de Ação/fisiologia , Cálcio/fisiologia , Interneurônios/classificação , Interneurônios/fisiologia , Bulbo Olfatório/citologia , Potenciais de Ação/efeitos dos fármacos , Análise de Variância , Animais , Fenômenos Biofísicos , Cádmio/farmacologia , Estimulação Elétrica , Técnicas In Vitro , Lisina/análogos & derivados , Lisina/metabolismo , Odorantes , Técnicas de Patch-Clamp , Periplaneta , Bloqueadores dos Canais de Potássio/farmacologia , Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Tetraetilamônio/farmacologia , Tetrodotoxina/farmacologia , Ácido gama-Aminobutírico/metabolismoRESUMO
A diverse population of local interneurons (LNs) helps to process, structure, and spatially represent olfactory information in the insect antennal lobe. In Periplaneta americana, we identified two subtypes of nonspiking local interneurons (type II LNs) by their distinct morphological and intrinsic electrophysiological properties. As an important step toward a better understanding of the cellular mechanisms that mediate odor information processing, we present a detailed analysis of their distinct voltage-activated Ca(2+) currents, which clearly correlated with their distinct intrinsic electrophysiological properties. Both type II LNs did not posses voltage-activated Na(+) currents and apparently innervated all glomeruli including the macroglomerulus. Type IIa LNs had significant longer and thicker low-order neurites and innervated each glomerulus entirely and homogeneously, whereas type IIb LNs innervated only parts of each glomerulus. All type II LNs were broadly tuned and responded to odorants of many chemical classes with graded changes in the membrane potential. Type IIa LNs responded with odor-specific elaborate patterns of excitation that could also include "spikelets" riding on the depolarizations and periods of inhibition. In contrast, type IIb LNs responded mostly with sustained, relatively smooth depolarizations. Consistent with the strong active membrane properties of type IIa LNs versus type IIb LNs, the voltage-activated Ca(2+) current of type IIa LNs activated at more hyperpolarized membrane potentials and had a larger transient component.
Assuntos
Potenciais de Ação/fisiologia , Fenômenos Biofísicos/fisiologia , Canais de Cálcio/fisiologia , Interneurônios/classificação , Interneurônios/fisiologia , Condutos Olfatórios/citologia , Potenciais de Ação/efeitos dos fármacos , Análise de Variância , Animais , Fenômenos Biofísicos/efeitos dos fármacos , Biofísica/métodos , Mapeamento Encefálico , Cloreto de Cádmio/farmacologia , Cálcio/metabolismo , Estimulação Elétrica/métodos , Técnicas In Vitro , Interneurônios/efeitos dos fármacos , Lisina/análogos & derivados , Lisina/metabolismo , Rede Nervosa/citologia , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Odorantes , Técnicas de Patch-Clamp/métodos , Periplaneta/anatomia & histologiaRESUMO
Single-cell mass spectrometry has become an established technique to study specific molecular properties such as the neuropeptide complement of identified neurons. Here, we describe a strategy to characterize, by MALDI-TOF mass spectrometry, neurochemical composition of neurons that were identified by their electrophysiological and neuroanatomical characteristics. The workflow for the first time combined perforated patch clamp recordings with dye loading by electroporation for electrophysiological and neuroanatomical characterization as well as chemical profiling of somata by MALDI-TOF mass spectrometry with subsequent immunohistochemistry. To develop our protocol, we used identified central olfactory neurons from the American cockroach Periplaneta americana. First, the combined approach was optimized using a relative homogeneous, well-characterized neuron population of uniglomerular projection neurons, which show acetylcholine esterase immunoreactivity. The general applicability of this approach was verified on local interneurons, which are a diverse neuron population expressing highly differentiated neuropeptidomes. Thus, this study shows that the newly established protocol is suitable to comprehensively analyze electrophysiological, neuroanatomical, and molecular properties of single neurons. We consider this approach an important step to foster single-cell analysis in a wide variety of neuron types.
Assuntos
Neurônios/citologia , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Análise de Célula Única/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acebutolol , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Corantes , Eletroporação , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Lisina/análogos & derivados , Potenciais da Membrana/fisiologia , PeriplanetaRESUMO
Molybdenum cofactor deficiency (MoCD) is an autosomal recessive inborn error of metabolism characterized by neurodegeneration and death in early childhood. The rapid and progressive neurodegeneration in MoCD presents a major clinical challenge and may relate to the poor understanding of the molecular mechanisms involved. Recently, we reported that treating patients with cyclic pyranopterin monophosphate (cPMP) is a successful therapy for a subset of infants with MoCD and prevents irreversible brain damage. Here, we studied S-sulfocysteine (SSC), a structural analog of glutamate that accumulates in the plasma and urine of patients with MoCD, and demonstrated that it acts as an N-methyl D-aspartate receptor (NMDA-R) agonist, leading to calcium influx and downstream cell signaling events and neurotoxicity. SSC treatment activated the protease calpain, and calpain-dependent degradation of the inhibitory synaptic protein gephyrin subsequently exacerbated SSC-mediated excitotoxicity and promoted loss of GABAergic synapses. Pharmacological blockade of NMDA-R, calcium influx, or calpain activity abolished SSC and glutamate neurotoxicity in primary murine neurons. Finally, the NMDA-R antagonist memantine was protective against the manifestation of symptoms in a tungstate-induced MoCD mouse model. These findings demonstrate that SSC drives excitotoxic neurodegeneration in MoCD and introduce NMDA-R antagonists as potential therapeutics for this fatal disease.
Assuntos
Sinalização do Cálcio , Cisteína/análogos & derivados , Neurônios GABAérgicos/metabolismo , Erros Inatos do Metabolismo dos Metais/metabolismo , Doenças Neurodegenerativas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cisteína/metabolismo , Modelos Animais de Doenças , Neurônios GABAérgicos/patologia , Células HEK293 , Humanos , Memantina/farmacologia , Erros Inatos do Metabolismo dos Metais/tratamento farmacológico , Erros Inatos do Metabolismo dos Metais/patologia , Camundongos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Compostos Organofosforados/farmacologia , Pterinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Sinapses/metabolismo , Sinapses/patologia , Compostos de Tungstênio/toxicidadeRESUMO
In the insect antennal lobe different types of local interneurons mediate complex excitatory and inhibitory interactions between the glomerular pathways to structure the spatiotemporal representation of odors. Mass spectrometric and immunohistochemical studies have shown that in local interneurons classical neurotransmitters are likely to colocalize with a variety of substances that can potentially act as cotransmitters or neuromodulators. In the antennal lobe of the cockroach Periplaneta americana, gamma-aminobutyric acid (GABA) has been identified as the potential inhibitory transmitter of spiking type I local interneurons, whereas acetylcholine is most likely the excitatory transmitter of nonspiking type IIa1 local interneurons. This study used whole-cell patch clamp recordings combined with single-cell labeling and immunohistochemistry to test if the GABAergic type I local interneurons and the cholinergic type IIa1 local interneurons express allatotropin and tachykinin-related neuropeptides (TKRPs). These are two of the most abundant types of peptides in the insect antennal lobe. GABA-like and choline acetyltransferase (ChAT)-like immunoreactivity were used as markers for GABAergic and cholinergic neurons, respectively. About 50% of the GABA-like immunoreactive (-lir) spiking type I local interneurons were allatotropin-lir, and â¼ 40% of these neurons were TKRP-lir. About 20% of nonspiking ChAT-lir type IIa1 local interneurons were TKRP-lir. Our results suggest that in subpopulations of GABAergic and cholinergic local interneurons, allatotropin and TKRPs might act as cotransmitters or neuromodulators. To unequivocally assign neurotransmitters, cotransmitters, and neuromodulators to identified classes of antennal lobe neurons is an important step to deepen our understanding of information processing in the insect olfactory system.
Assuntos
Antenas de Artrópodes/citologia , Baratas/anatomia & histologia , Hormônios de Inseto/metabolismo , Interneurônios/classificação , Interneurônios/metabolismo , Neuropeptídeos/metabolismo , Taquicininas/metabolismo , Animais , Colina O-Acetiltransferase/metabolismo , Potenciais da Membrana/fisiologia , Odorantes , Condutos Olfatórios/fisiologia , Técnicas de Patch-Clamp , Receptores de Taquicininas/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Behavioral and physiological studies have shown that local interneurons are pivotal for processing odor information in the insect antennal lobe. They mediate inhibitory and excitatory interactions between the glomerular pathways and ultimately shape the tuning profile of projection neurons. To identify putative cholinergic local interneurons in the antennal lobe of Periplaneta americana, an antibody raised against the biosynthetic enzyme choline acetyltransferase (ChAT) was applied to individual morphologically and electrophysiologically characterized local interneurons. In nonspiking type IIa1 local interneurons, which were classified in this study, we found ChAT-like immunoreactivity suggesting that they are most likely excitatory. This is a well-defined population of neurons that generates Ca(2+) -driven spikelets upon depolarization and stimulation with odorants, but not Na(+) -driven action potentials, because they lack voltage-activated transient Na(+) currents. The nonspiking type IIa2 and type IIb local interneurons, in which Ca(2+) -driven spikelets were absent, had no ChAT-like immunoreactivity. The GABA-like immunoreactive, spiking type I local interneurons had no ChAT-like immunoreactivity. In addition, we showed that uniglomerular projection neurons with cell bodies located in the ventral portion of the ventrolateral somata group and projections along the inner antennocerebral tract exhibited ChAT-like immunoreactivity. Assigning potential transmitters and neuromodulators to distinct morphological and electrophysiological types of antennal lobe neurons is an important prerequisite for a detailed understanding of odor information processing in insects.
Assuntos
Colina O-Acetiltransferase/metabolismo , Interneurônios/enzimologia , Periplaneta/fisiologia , Animais , Western Blotting , Encéfalo/fisiologia , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Interneurônios/imunologia , Microscopia de Fluorescência , Neurônios/imunologia , Neurotransmissores/metabolismo , Neurotransmissores/fisiologia , Odorantes , Técnicas de Patch-Clamp , Olfato/fisiologiaRESUMO
A multitude of potential neurotransmitters and neuromodulators, including peptides, have been detected in the antennal lobe (AL), the first synaptic relay of the central olfactory pathway in the insect brain. However, the functional role of neuropeptides in this system has yet to be revealed. An important prerequisite to understanding the role of neuropeptides is to match the functionally different cell types in the AL with their peptide profiles by using electrophysiological recordings combined with immunocytochemical studies and/or single-cell mass spectrometry. The olfactory system of Periplaneta americana is particularly well suited to accomplish this goal because several physiologically distinct neuron types can be unequivocally identified. With the aim to analyze the neuropeptide inventory of the P. americana AL, this study is an essential step in this direction. First, we systematically analyzed different parts of the AL by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry to obtain the complete set of neuropeptides present. Altogether, 56 ion signals could be assigned to products of 10 neuropeptide genes (allatostatins A, B, C, SIFamide, allatotropin, FMRFamide-related peptides [myosuppressin, short neuropeptides F, extended FMRFamides], crustacean cardioactive peptide, tachykinin-related peptides). In a second step, a combination of immunocytochemistry and mass spectrometric profiling of defined AL compartments was used to reveal the spatial distribution of neuropeptide-containing cells. Finally, we demonstrated the feasibility of MALDI-TOF mass spectrometric profiling of single AL neurons, which is an important precondition for combining electrophysiology with peptide profiling at the single-cell level.