RESUMO
A better understanding of neuromodulation in a behavioral system requires identification of active modulatory transmitters. Here, we used identifiable neurons in a neurobiological model system, the mollusc Aplysia, to study neuropeptides, a diverse class of neuromodulators. We took advantage of two types of feeding neurons, B48 and B1/B2, in the Aplysia buccal ganglion that might contain different neuropeptides. We performed a representational difference analysis (RDA) by subtraction of mRNAs in B48 versus mRNAs in B1/B2. The RDA identified an unusually long (2025 amino acids) peptide precursor encoding Aplysia leucokinin-like peptides (ALKs; e.g. ALK-1 and ALK-2). Northern blot analysis revealed that, compared with other ganglia (e.g. the pedal-pleural ganglion), ALK mRNA is predominantly present in the buccal ganglion, which controls feeding behavior. We then used in situ hybridization and immunohistochemistry to localize ALKs to specific neurons, including B48. MALDI-TOF MS on single buccal neurons revealed expression of 40 ALK precursor-derived peptides. Among these, ALK-1 and ALK-2 are active in the feeding network; they shortened the radula protraction phase of feeding motor programs triggered by a command-like neuron. We also found that this effect may be mediated by the ALK-stimulated enhancement of activity of an interneuron, which has previously been shown to terminate protraction. We conclude that our multipronged approach is effective for determining the structure and defining the diverse functions of leucokinin-like peptides. Notably, the ALK precursor is the first verified nonarthropod precursor for leucokinin-like peptides with a novel, marked modulatory effect on a specific parameter (protraction duration) of feeding motor programs.
Assuntos
Aplysia/fisiologia , Gânglios dos Invertebrados/fisiologia , Neuropeptídeos/metabolismo , Animais , Aplysia/química , Aplysia/citologia , Aplysia/genética , Comportamento Alimentar , Gânglios dos Invertebrados/química , Gânglios dos Invertebrados/metabolismo , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/análise , Neuropeptídeos/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/análise , RNA Mensageiro/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Improvements in the imaging of neural circuits are essential for studies of network function in both invertebrates and vertebrates. Therefore, CLARITY, a new imaging enhancement technique developed for mouse brains has attracted broad interest from researchers working on other species. We studied the potential of a modified version of CLARITY to enhance the imaging of ganglia in an invertebrate Aplysia. For example, we have modified the hydrogel solution and designed a small container for the Aplysia ganglia. The ganglia were first processed for immunohistochemistry, and then for CLARITY. We examined the compatibility of these techniques and the extent to which the imaging of fluorescence improved using confocal microscopy. We found that CLARITY did indeed enhance the imaging of CP2 immunopositive neurons in Aplysia ganglia. For example, it improved visualization of small, weak immunoreactive neurons deep in the ganglia. Our modifications of CLARITY make this new method suitable for future use in Aplysia experiments. Furthermore, our techniques are likely to facilitate imaging in other invertebrate ganglia.
Assuntos
Aplysia/anatomia & histologia , Gânglios dos Invertebrados/diagnóstico por imagem , Aumento da Imagem/métodos , Animais , Imuno-Histoquímica , Neurônios/citologiaRESUMO
Understanding circuit function requires the characterization of component neurons and their neurotransmitters. Previous work on radula protraction in the Aplysia feeding circuit demonstrated that critical neurons initiate feeding via cholinergic excitation. In contrast, it is less clear how retraction is mediated at the interneuronal level. In particular, glutamate involvement was suggested, but was not directly confirmed. Here we study a suspected glutamatergic retraction interneuron, B64. We used the representational difference analysis (RDA) method to successfully clone an Aplysia vesicular glutamate transporter (ApVGLUT) from B64 and from a glutamatergic motor neuron B38. Previously, RDA was used to characterize novel neuropeptides. Here we demonstrate its utility for characterizing other types of molecules. Bioinformatics suggests that ApVGLUT is more closely related to mammalian VGLUTs than to Drosophila and Caenorhabditis elegans VGLUTs. We expressed ApVGLUT in a cell line, and demonstrated that it indeed transports glutamate in an ATP and proton gradient-dependent manner. We mapped the ApVGLUT distribution in the CNS using in situ hybridization and immunocytochemistry. Further, we demonstrated that B64 is ApVGLUT positive, supporting the idea that it is glutamatergic. Although glutamate is primarily an excitatory transmitter in the mammalian CNS, B64 elicits inhibitory PSPs in protraction neurons to terminate protraction and excitatory PSPs in retraction neurons to maintain retraction. Pharmacological data indicated that both types of PSPs are mediated by glutamate. Thus, glutamate mediates the dual function of B64 in Aplysia. More generally, our systematic approaches based on RDA may facilitate analyses of transmitter actions in small circuits with identifiable neurons.
Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Interneurônios/fisiologia , Rede Nervosa/fisiologia , Proteínas Vesiculares de Transporte de Glutamato/fisiologia , Animais , Aplysia , Células COS , Chlorocebus aethiops , Feminino , Masculino , Rede Nervosa/química , Filogenia , Proteínas Vesiculares de Transporte de Glutamato/análiseRESUMO
Motor activity is often initiated by a population of command-like interneurons. Command-like interneurons that reliably drive programs have received the most attention, so little is known about how less reliable command-like interneurons may contribute to program generation. We study two electrically coupled interneurons, cerebral-buccal interneuron-2 (CBI-2) and CBI-11, which activate feeding motor programs in the mollusk Aplysia californica. Earlier work indicated that, in rested preparations, CBI-2, a powerful activator of programs, can trigger ingestive and egestive programs. CBI-2 reliably generated ingestive patterns only when it was repeatedly stimulated. The ability of CBI-2 to trigger motor activity has been attributed to the two program-promoting peptides it contains, FCAP and CP2. Here, we show that CBI-11 differs from CBI-2 in that it contains FCAP but not CP2. Furthermore, it is weak in its ability to drive programs. On its own, CBI-11 is therefore less effective as a program activator. When it is successful, however, CBI-11 is an effective specifier of motor activity; that is, it drives mostly ingestive programs. Importantly, we found that CBI-2 and CBI-11 complement each other's actions. First, prestimulation of CBI-2 enhanced the ability of CBI-11 to drive programs. This effect appears to be partly mediated by CP2. Second, coactivation of CBI-11 with CBI-2 makes CBI-2 programs immediately ingestive. This effect may be mediated by specific actions that CBI-11 exerts on pattern-generating interneurons. Therefore, different classes of command-like neurons in a motor network may make distinct, but potentially complementary, contributions as either activators or specifiers of motor activity.
Assuntos
Aplysia/fisiologia , Vias Eferentes/fisiologia , Interneurônios/fisiologia , Atividade Motora/fisiologia , Animais , Bochecha/inervação , Bochecha/fisiologia , Interpretação Estatística de Dados , Ingestão de Alimentos/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Comportamento Alimentar/fisiologia , Alimentos , Imuno-Histoquímica , Neuropeptídeos/fisiologia , Técnicas de Patch-ClampRESUMO
During eukaryotic translation, peptides/proteins are created using L-amino acids. However, a D-amino acid-containing peptide (DAACP) can be produced through post-translational modification via an isomerase enzyme. General approaches to identify novel DAACPs and investigate their function, particularly in specific neural circuits, are lacking. This is primarily due to the difficulty in characterizing this modification and due to the limited information on neural circuits in most species. We describe a multipronged approach to overcome these limitations using the sea slug Aplysia californica. Based on bioinformatics and homology to known DAACPs in the land snail Achatina fulica, we targeted two predicted peptides in Aplysia, GFFD, similar to achatin-I (GdFAD versus GFAD, where dF stands for D-phenylalanine), and YAEFLa, identical to fulyal (YdAEFLa versus YAEFLa), using stereoselective analytical methods, i.e. MALDI MS fragmentation analysis and LC-MS/MS. Although YAEFLa in Aplysia was detected only in an all L-form, we found that both GFFD and GdFFD were present in the Aplysia CNS. In situ hybridization and immunolabeling of GFFD/GdFFD-positive neurons and fibers suggested that GFFD/GdFFD might act as an extrinsic modulator of the feeding circuit. Consistent with this hypothesis, we found that GdFFD induced robust activity in the feeding circuit and elicited egestive motor patterns. In contrast, the peptide consisting of all L-amino acids, GFFD, was not bioactive. Our data indicate that the modification of an L-amino acid-containing neuropeptide to a DAACP is essential for peptide bioactivity in a motor circuit, and thus it provides a functional significance to this modification.
Assuntos
Aplysia/fisiologia , Comportamento Animal/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Neuropeptídeos , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Comportamento Animal/fisiologia , Comportamento Alimentar/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologiaRESUMO
Compensatory mechanisms are often used to achieve stability by reducing variance, which can be accomplished via negative feedback during homeostatic regulation. In principle, compensation can also be implemented through feedforward mechanisms where a regulator acts to offset the anticipated output variation; however, few such neural mechanisms have been demonstrated. We provide evidence that an Aplysia neuropeptide, identified using an enhanced representational difference analysis procedure, implements feedforward compensation within the feeding network. We named the novel peptide "allatotropin-related peptide" (ATRP) because of its similarity to insect allatotropin. Mass spectrometry confirmed the peptide's identity, and in situ hybridization and immunostaining mapped its distribution in the Aplysia CNS. ATRP is present in the higher-order cerebral-buccal interneuron (CBI) CBI-4, but not in CBI-2. Previous work showed that CBI-4-elicited motor programs have a shorter protraction duration than those elicited by CBI-2. Here we show that ATRP shortens protraction duration of CBI-2-elicited ingestive programs, suggesting a contribution of ATRP to the parametric differences between CBI-4-evoked and CBI-2-evoked programs. Importantly, because Aplysia muscle contractions are a graded function of motoneuronal activity, one consequence of the shortening of protraction is that it can weaken protraction movements. However, this potential weakening is offset by feedforward compensatory actions exerted by ATRP. Centrally, ATRP increases the activity of protraction motoneurons. Moreover, ATRP is present in peripheral varicosities of protraction motoneurons and enhances peripheral motoneuron-elicited protraction muscle contractions. Therefore, feedforward compensatory mechanisms mediated by ATRP make it possible to generate a faster movement with an amplitude that is not greatly reduced, thereby producing stability.
Assuntos
Retroalimentação Fisiológica/efeitos dos fármacos , Hormônios de Inseto/farmacologia , Interneurônios/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Movimento/efeitos dos fármacos , Neuropeptídeos/farmacologia , Análise de Variância , Animais , Aplysia/fisiologia , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Cromatografia Líquida/métodos , Clonagem Molecular/métodos , Ingestão de Alimentos/efeitos dos fármacos , Hormônios de Inseto/química , Hormônios de Inseto/genética , Interneurônios/classificação , Interneurônios/fisiologia , Modelos Biológicos , Neurônios Motores/fisiologia , Contração Muscular/efeitos dos fármacos , Neuropeptídeos/química , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Radioimunoensaio/métodos , Espectrometria de Massas em TandemRESUMO
Many bioactive neuropeptides containing RFamide at their C terminus have been described in both invertebrates and vertebrates. To obtain insight into the functional logic of RFamide signaling, we investigate it here in the feeding system of Aplysia. We focus on the expression, localization, and actions of two families of RFamide peptides, the FRFamides and FMRFamide, in the central neuronal circuitry and the peripheral musculature that generate the feeding movements. We describe the cloning of the FRFamide precursor protein and show that the FRFamides and FMRFamide are derived from different precursors. We map the expression of the FRFamide and FMRFamide precursors in the feeding circuitry using in situ hybridization and immunostaining and confirm proteolytic processing of the FRFamide precursor by mass spectrometry. We show that the two precursors are expressed in different populations of sensory neurons in the feeding system. In a representative feeding muscle, we demonstrate the presence of both FRFamides and FMRFamide and their release, probably from the processes of the sensory neurons in the muscle. Both centrally and in the periphery, the FRFamides and FMRFamide act in distinct ways, apparently through distinct mechanisms, and nevertheless, from an overall functional perspective, their actions are complementary. Together, the FRFamides and FMRFamide convert feeding motor programs from ingestive to egestive and depress feeding muscle contractions. We conclude that these structurally related peptides, although derived from different precursors, expressed in different neurons, and acting through different mechanisms, remain related to each other in the functional roles that they play in the system.
Assuntos
Neuropeptídeos/química , Neuropeptídeos/fisiologia , Precursores de Proteínas/química , Precursores de Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Aplysia , Células Cultivadas , FMRFamida/química , FMRFamida/fisiologia , Dados de Sequência Molecular , Ratos , Ratos Sprague-DawleyRESUMO
Feedforward loops (FFLs) are one of many network motifs identified in a variety of complex networks, but their functional role in neural networks is not well understood. We provide evidence that combinatorial actions of multiple modulators may be organized as FFLs to promote a specific network state in the Aplysia feeding motor network. The Aplysia feeding central pattern generator (CPG) receives two distinct inputs-a higher-order interneuron cerebral-buccal interneuron-2 (CBI-2) and the esophageal nerve (EN)-that promote ingestive and egestive motor programs, respectively. EN stimulation elicits a persistent egestive network state, which enables the network to temporarily express egestive programs following a switch of input from the EN to CBI-2. Previous work showed that a modulatory CPG element, B65, is specifically activated by the EN and participates in establishing the egestive state by enhancing activity of egestion-promoting B20 interneurons while suppressing activity and synaptic outputs of ingestion-promoting B40 interneurons. Here a peptidergic contribution is mediated by small cardioactive peptide (SCP). Immunostaining and mass spectrometry show that SCP is present in the EN and is released on EN stimulation. Importantly, SCP directly enhances activity and synaptic outputs of B20 and suppresses activity and synaptic outputs of B40. Moreover, SCP promotes B65 activity. Thus the direct and indirect (through B65) pathways to B20 and B40 from SCPergic neurons constitute two FFLs with one functioning to promote egestive output and the other to suppress ingestive output. This composite FFL consisting of the two combined FFLs appears to be an effective means to co-regulate activity of two competing elements that do not inhibit each other, thereby contributing to establish specific network states.
Assuntos
Aplysia/fisiologia , Comportamento Alimentar/fisiologia , Interneurônios/fisiologia , Rede Nervosa/fisiologia , Animais , Aplysia/anatomia & histologia , Fenômenos Eletrofisiológicos/fisiologia , Modelos Animais , Rede Nervosa/anatomia & histologiaRESUMO
We investigated how a neural analog of a form of arousal induced by a mildly noxious stimulus can promote two antagonistic responses, locomotion and feeding. Two pairs of cerebral serotonergic interneurons in Aplysia, CC9 and CC10, were persistently activated by transient noxious stimuli. Direct stimulation of CC9-10 activated locomotor activity that outlasted the stimulation and enhanced subsequent nerve-evoked locomotor programs. Thus, CC9-10 function both as initiators and as modulators of the locomotor network. CC9-10 also interacted with the feeding circuit but in a fundamentally different manner. CC9-10 did not directly trigger feeding activity or activate feeding command or pattern generating interneurons. CC9-10 did, however, elicit slow EPSPs in serotonergic cells that modulate feeding responses, the metacerebral cells (MCCs). CC9-10 persistently enhanced MCC excitability, but did not activate the MCCs directly. Previous work has demonstrated that the MCCs are activated during food ingestion via a sensory neuron C2. Interestingly, we found that CC9-10 stimulation converted subthreshold C2 mediated excitation of the MCC into suprathreshold excitation. Transient noxious stimuli also enhanced MCC excitability, and this was largely mediated by CC9-10. To summarize, CC9-10 exert actions on the feeding network, but their functional effects appear to be conditional on the presence of food-related inputs to the MCCs. A potential advantage of this arrangement is that it may prevent conflicting responses from being directly evoked by noxious stimuli while also facilitating the ability of food-related stimuli to generate feeding responses in the aftermath of noxious stimulation.
Assuntos
Nível de Alerta/fisiologia , Comportamento Alimentar/psicologia , Locomoção/fisiologia , Neurônios/fisiologia , Serotonina/metabolismo , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Animais , Aplysia , Comportamento Animal , Córtex Cerebral/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Gânglios dos Invertebrados/citologia , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Potenciais Pós-Sinápticos Inibidores/efeitos da radiação , Masculino , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Técnicas de Patch-Clamp/métodos , Estimulação Física/efeitos adversos , Ratos , Ratos Sprague-DawleyRESUMO
A shift in motivational state often produces behavioral change, but the underlying mechanisms are poorly understood. In the marine mollusc, Aplysia californica, feeding-induced transition from a hunger to satiation state leads to a slowdown and an eventual termination of feeding. Because the multifunctional feeding network generates both ingestion and the competing response, egestion, it is possible that the transition from a hunger to a satiety state is associated with network reconfiguration that results in production of fewer ingestive and more egestive responses. Chronic electrophysiological recordings in free-feeding Aplysia showed that as the meal progressed, food elicited fewer ingestive responses and simultaneously increased the number of egestive responses. Injections of Aplysia neuropeptide Y (apNPY) reduced food intake and slowed down the rate of ingestion. apNPY was localized to buccal-ganglion afferents originating in the gut-innervating esophageal nerve (EN), a nerve involved both in satiation and in the generation of egestive programs. During EN stimulation, apNPY was released in the feeding circuit. Importantly, stimulation of the cerebral-buccal interneuron-2, a command-like interneuron that is activated by food and normally elicits ingestive responses, elicited egestive responses in the presence of apNPY. This was accompanied by increased activity of the egestion-promoting interneuron B20 and decreased activity in the ingestion-promoting interneuron B40. Thus, apNPYergic reconfiguration of the feeding central pattern generator plays a role in the gradual transition from hunger to satiety states. More generally, changes in the motivational states may involve not only simple network inhibition but may also require network reconfiguration.
Assuntos
Aplysia/fisiologia , Fome/fisiologia , Neuropeptídeo Y/metabolismo , Resposta de Saciedade/fisiologia , Animais , Bochecha/fisiologia , Comportamento Alimentar/fisiologia , Gânglios dos Invertebrados/metabolismo , Imuno-Histoquímica , Masculino , Motivação , Neurônios/metabolismo , Neuropeptídeo Y/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Neurons can contain both neuropeptides and "classic" small molecule transmitters. Much progress has been made in studies designed to determine the functional significance of this arrangement in experiments conducted in invertebrates and in the vertebrate autonomic nervous system. In this review article, we describe some of this research. In particular, we review early studies that related peptide release to physiological firing patterns of neurons. Additionally, we discuss more recent experiments informed by this early work that have sought to determine the functional significance of peptide cotransmission in the situation where peptides are released from neurons that are part of (i.e., are intrinsic to) a behavior generating circuit in the CNS. In this situation, peptide release will presumably be tightly coupled to the manner in which a network is activated. For example, data obtained in early studies suggest that peptide release will be potentiated when behavior is executed rapidly and intervals between periods of neural activity are relatively short. Further, early studies demonstrated that when neural activity is maintained, there are progressive changes (e.g., increases) in the amount of peptide that is released (even in the absence of a change in neural activity). This suggests that intrinsic peptidergic modulators in the CNS are likely to exert effects that are manifested dynamically in an activity-dependent manner. This type of modulation is likely to differ markedly from the modulation that occurs when a peptide hormone is present at a relatively fixed concentration in the blood.
Assuntos
Rede Nervosa/metabolismo , Plasticidade Neuronal/fisiologia , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Animais , HumanosRESUMO
Neuropeptides are present in species throughout the animal kingdom and generally exert actions that are distinct from those of small molecule transmitters. It has, therefore, been of interest to define the unique behavioral role of this class of substances. Progress in this regard has been made in experimentally advantageous invertebrate preparations. We focus on one such system, the feeding circuit in the mollusc Aplysia. We review research conducted over several decades that played an important role in establishing that peptide cotransmitters are released under behaviorally relevant conditions. We describe how this was accomplished. For example, we describe techniques developed to purify novel peptides, localize them to identified neurons, and detect endogenous peptide release. We also describe physiological experiments that demonstrated that peptides are bioactive under behaviorally relevant conditions. The feeding system is like others in that peptides exert effects that are both convergent and divergent. Work in the feeding system clearly illustrates how this creates potential for behavioral flexibility. Finally, we discuss experiments that determined physiological consequences of one of the hallmark features of peptidergic modulation, its persistence. Research in the feeding system demonstrated that this persistence can change network state and play an important role in determining network output.
Assuntos
Aplysia/metabolismo , Ingestão de Alimentos/fisiologia , Comportamento Alimentar/fisiologia , Neuropeptídeos/metabolismo , Animais , Neurônios/metabolismoRESUMO
When individual neurons in a circuit contain multiple neuropeptides, these peptides can target different sets of follower neurons. This endows the circuit with a certain degree of flexibility. Here we identified a novel family of peptides, the Aplysia SPTR-Gene Family-Derived peptides (apSPTR-GF-DPs). We demonstrated apSPTR-GF-DPs, particularly apSPTR-GF-DP2, are expressed in the Aplysia CNS using immunohistochemistry and MALDI-TOF MS. Furthermore, apSPTR-GF-DP2 is present in single projection neurons, e.g., in the cerebral-buccal interneuron-12 (CBI-12). Previous studies have demonstrated that CBI-12 contains two other peptides, FCAP/CP2. In addition, CBI-12 and CP2 promote shortening of the protraction phase of motor programs. Here, we demonstrate that FCAP shortens protraction. Moreover, we show that apSPTR-GF-DP2 also shortens protraction. Surprisingly, apSPTR-GF-DP2 does not increase the excitability of retraction interneuron B64. B64 terminates protraction and is modulated by FCAP/CP2 and CBI-12. Instead, we show that apSPTR-GF-DP2 and CBI-12 increase B20 excitability and B20 activity can shorten protraction. Taken together, these data indicate that different CBI-12 peptides target different sets of pattern-generating interneurons to exert similar modulatory actions. These findings provide the first definitive evidence for SPTR-GF's role in modulation of feeding, and a form of molecular degeneracy by multiple peptide cotransmitters in single identified neurons.
Assuntos
Aplysia/metabolismo , Atividade Motora/fisiologia , Neuropeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Aplysia/citologia , Biologia Computacional , Ingestão de Alimentos/fisiologia , Gânglios dos Invertebrados/citologia , Gânglios dos Invertebrados/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/genética , Processamento de Proteína Pós-Traducional , Ratos Sprague-Dawley , Alinhamento de SequênciaRESUMO
The Aplysia feeding system is advantageous for investigating the role of neuropeptides in behavioral plasticity. One family of Aplysia neuropeptides is the myomodulins (MMs), originally purified from one of the feeding muscles, the accessory radula closer (ARC). However, two MMs, MMc and MMe, are not encoded on the only known MM gene. Here, we identify MM gene 2 (MMG2), which encodes MMc and MMe and four new neuropeptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to verify that these novel MMG2-derived peptides (MMG2-DPs), as well as MMc and MMe, are synthesized from the precursor. Using antibodies against the MMG2-DPs, we demonstrate that neuronal processes that stain for MMG2-DPs are found in the buccal ganglion, which contains the feeding network, and in the buccal musculature including the ARC muscle. Surprisingly, however, no immunostaining is observed in buccal neurons including the ARC motoneurons. In situ hybridization reveals only few MMG2-expressing neurons that are mostly located in the pedal ganglion. Using immunohistochemical and electrophysiological techniques, we demonstrate that some of these pedal neurons project to the buccal ganglion and are the likely source of the MMG2-DP innervation of the feeding network and musculature. We show that the MMG2-DPs are bioactive both centrally and peripherally: they bias egestive feeding programs toward ingestive ones, and they modulate ARC muscle contractions. The multiple actions of the MMG2-DPs suggest that these peptides play a broad role in behavioral plasticity and that the pedal-buccal projection neurons that express them are a novel source of extrinsic modulation of the feeding system of Aplysia.
Assuntos
Aplysia/genética , Comportamento Alimentar/fisiologia , Neuropeptídeos/genética , Precursores de Proteínas/genética , Sequência de Aminoácidos/genética , Animais , Aplysia/fisiologia , Gânglios dos Invertebrados/fisiologia , Dados de Sequência Molecular , Neuropeptídeos/fisiologia , Precursores de Proteínas/fisiologiaRESUMO
Neuropeptide FF (NPFF) is an RF-amide peptide with pleiotropic functions in the mammalian central nervous system, including pain modulation, opiate interactions, cardiovascular regulation and neuroendocrine effects. To gain insights into the transcriptional mechanisms that regulate NPFF gene expression, we cloned and sequenced 9.8 and 1.5 kb of the mouse and rat NPFF 5'-flanking region, respectively. Regions with high sequence homology between mouse, rat and human were expected to have high probability to interact with regulatory proteins and were studied further. Electromobility shift assays revealed one region that may interact with the homeobox proteins Oct-1, PDX1, Pit-1 and MEIS and two consensus DRE sites that bind a nuclear protein, which was identified as the downstream regulatory element antagonistic modulator DREAM by supershift assays. The distribution of NPFF gene expression was examined in the mouse using in situ hybridization and RT-PCR. NPFF expression was also evident during mouse embryogenesis. A fixed transcription initiation site for the mouse NPFF gene was found. A novel splice variant with a retained intron of the NPFF gene was characterized. Chimeric luciferase reporter gene constructs for the mouse NPFF gene revealed a minimal promoter region and a region with transcriptional suppressor features. An NGF responsive area was found using mouse NPFF reporter gene constructs. We postulate that Oct-1, PDX1, Pit-1, MEIS and DREAM are likely transcriptional regulators of NPFF gene expression.
Assuntos
Regulação da Expressão Gênica , Oligopeptídeos/genética , Fatores de Transcrição/genética , Região 5'-Flanqueadora/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Nervoso Central/metabolismo , Mapeamento Cromossômico/veterinária , Clonagem Molecular , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Homeodomínio/fisiologia , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteína Meis1 , Proteínas de Neoplasias/fisiologia , Fator 1 de Transcrição de Octâmero/fisiologia , Ratos , Alinhamento de Sequência , Transativadores/fisiologia , Fator de Transcrição Pit-1/fisiologiaRESUMO
One emerging principle is that neuromodulators, such as neuropeptides, regulate multiple behaviors, particularly motivated behaviors, e.g., feeding and locomotion. However, how neuromodulators act on multiple neural networks to exert their actions remains poorly understood. These actions depend on the chemical form of the peptide, e.g., an alternation of L- to D-form of an amino acid can endow the peptide with bioactivity, as is the case for the Aplysia peptide GdFFD (where dF indicates D-phenylalanine). GdFFD has been shown to act as an extrinsic neuromodulator in the feeding network, while the all L-amino acid form, GFFD, was not bioactive. Given that both GdFFD/GFFD are also present in pedal neurons that mediate locomotion, we sought to determine whether they impact locomotion. We first examined effects of both peptides on isolated ganglia, and monitored fictive programs using the parapedal commissural nerve (PPCN). Indeed, GdFFD was bioactive and GFFD was not. GdFFD increased the frequency with which neural activity was observed in the PPCN. In part, there was an increase in bursting spiking activity that resembled fictive locomotion. Additionally, there was significant activity between bursts. To determine how the peptide-induced activity in the isolated CNS is translated into behavior, we recorded animal movements, and developed a computer program to automatically track the animal and calculate the path of movement and velocity of locomotion. We found that GdFFD significantly reduced locomotion and induced a foot curl. These data suggest that the increase in PPCN activity observed in the isolated CNS during GdFFD application corresponds to a reduction, rather than an increase, in locomotion. In contrast, GFFD had no effect. Thus, our study suggests that GdFFD may act as an intrinsic neuromodulator in the Aplysia locomotor network. More generally, our study indicates that physiological and behavioral analyses should be combined to evaluate peptide actions.
Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Neuropeptídeos/farmacologia , Animais , Aplysia , EletrofisiologiaRESUMO
GABAergic inhibitory interneurons regulate the activity of diverse types of neural networks, but the specific roles of these interneurons in motor control are poorly understood. In the Aplysia feeding motor network, three interneurons, cerebral-buccal interneuron-3 (CBI-3) and buccal interneurons B40 and B34, are GABA-immunoreactive and evoke fast IPSPs in their postsynaptic followers. Using a combination of pharmacological experiments with GABA antagonists, agonists, and uptake inhibitors, we found that these fast IPSPs are likely mediated by GABA. Functionally, these fast IPSPs specify two parameters for ingestive motor programs elicited by the command-like interneuron CBI-2: (1) the appropriate phasing of activity of the radula closer motor neuron B8 relative to protraction-retraction, and (2) protraction duration. First, in ingestive programs, B8 activity is phased such that it fires minimally during protraction. CBI-3 and B40 exert fast inhibition to minimize B8 activity during protraction, by either acting directly on B8 (B40) or indirectly on B8 (CBI-3). Second, these ingestive programs are characterized by long protraction duration, which is promoted by B40 and B34 because hyperpolarization of either cell shortens protraction. Such effects of B40 and B34 are attributable, at least partly, to their inhibitory effects on the retraction-phase interneuron B64 whose activation terminates protraction. Consistent with a GABAergic contribution to both B8 phasing and protraction duration, blockade of GABAergic inhibition by picrotoxin increases B8 activity during protraction and shortens protraction, without disrupting the integrity of motor programs. Thus, the concerted actions of GABAergic inhibition from three Aplysia feeding interneurons contribute to the specification of multiple features that define the motor program as an ingestive one.
Assuntos
Aplysia/fisiologia , Comportamento Alimentar/fisiologia , Interneurônios/fisiologia , Atividade Motora/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Comportamento Alimentar/efeitos dos fármacos , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Moduladores GABAérgicos/farmacologia , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Técnicas In Vitro , Interneurônios/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Atividade Motora/efeitos dos fármacos , Rede Nervosa/citologia , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Picrotoxina/farmacologiaRESUMO
Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan.
Assuntos
Aplysia/metabolismo , Urotensinas/fisiologia , Sequência de Aminoácidos , Animais , Aplysia/genética , Aplysia/fisiologia , Sequência de Bases , Sistema Nervoso Central/metabolismo , Fracionamento Químico , Eletrofisiologia , Comportamento Alimentar/fisiologia , Espectrometria de Massas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Resposta de Saciedade/fisiologia , Análise de Sequência de Proteína , Espectrometria de Massas em Tandem , Urotensinas/química , Urotensinas/genéticaRESUMO
It has been proposed that a choice of specific behaviors can be mediated either by activation of behavior-specific higher order neurons or by distinct combinations of such neurons in different behaviors. We examined the role that two higher order neurons, CBI-2 and CBI-3, play in the selection of motor programs that correspond to ingestion and egestion, two stimulus-dependent behaviors that are generated by a single central pattern generator (CPG) of Aplysia. We found that CBI-2 could evoke either ingestive, egestive, or ambiguous motor programs depending on the regime of stimulation. When CBI-2 recruited CBI-3 firing via electrical coupling, the motor program tended to be ingestive. In the absence of CBI-3 activation, the program was usually egestive. When CBI-2 was stimulated to produce ingestive programs, hyperpolarization of CBI-3 converted the programs to egestive or ambiguous. When CBI-2 was stimulated to produce egestive or ambiguous programs, co-stimulation of CBI-3 converted them into ingestive. These findings are consistent with the idea that combinatorial commands are responsible for the choice of specific behaviors. Additional support for this view comes from the observations that appropriate stimulus conditions exist both for activation of CBI-2 together with CBI-3, and for activation of CBI-2 without a concomitant activation of CBI-3. The ability of CBI-3 to convert egestive and ambiguous programs into ingestive ones was mimicked by application of APGWamide, a neuropeptide that we have detected in CBI-3 by immunostaining. Thus combinatorial actions of higher order neurons that underlie pattern selection may involve the use of modulators released by specific higher order neurons.
Assuntos
Interneurônios/fisiologia , Neurônios Motores/fisiologia , Peptídeos/fisiologia , Transmissão Sináptica/fisiologia , Animais , Aplysia , Análise por Conglomerados , Estimulação Elétrica , Comportamento Alimentar/fisiologia , Gânglios dos Invertebrados/citologia , Gânglios dos Invertebrados/fisiologia , Imuno-Histoquímica , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neuropeptídeos/farmacologia , Neuropeptídeos/fisiologia , Peptídeos/farmacologia , Estimulação Física , Processamento de Sinais Assistido por Computador , Estimulação QuímicaRESUMO
As an initial step to study the function of the gene encoding the human neuropeptide FF (NPFF), we cloned a 4.7-kb sequence from the promoter region. Primer extension and 5'-rapid amplification of cDNA ends revealed multiple transcription initiation sites. Northern blot analysis of the mRNA expression revealed a specific signal only in poly(A) + RNA from medulla and spinal cord. Chimeric luciferase reporter gene constructs were transiently transfected in A549, U-251 MG, SK-N-SH, SK-N-AS and PC12 cells. The promoter activity was directly comparable with the level of endogenous NPFF mRNA as determined by real-time quantitative RT-PCR. The highest promoter activity was measured when a region from - 552 to - 830 bp of the 5'-flanking region was fused to the constructs, and a potential silencer element was localized between nucleotides -220 and -551. A twofold increase in NPFF mRNA was observed after 72 h of nerve growth factor stimulation of PC12 cells and the region between - 61 and - 214 bp of the 5'-flanking region was found to be responsive to this stimulation. We postulate that control of human NPFF gene expression is the result of both positive and negative regulatory elements and the use of multiple transcription initiation sites.