Immunolocalization of parvalbumin in two glutamatergic cell types of the guinea pig cochlea: inner hair cells and spinal ganglion neurons

Immunoreactions to a monoclonal antibody raised against parvalbumin, a calcium-binding protein, have been detected in the inner hair cells of the organ of Corti and in the spiral ganglion neurons connected to them (type I neurons). Both cell types probably use an excitatory amino acid as a neurotransmitter (glutamate and/or aspartate). No immunoreactivity was found within the second sensory cell type (outer hair cells) nor in the olivocochlear (efferent) fibers or endings in the cochlea. In the central nervous system, parvalbumin may be involved in calcium-dependent mechanisms leading to neurotransmitter release. It could thus be hypothesized that parvalbumin also have similar implications at the level of the inner hair cell and type I neuron synapses. Additional functions could also be hypothesized for this protein in the cochlea. Within the inner hair cells, parvalbumin may be involved in the ionic regulation following potassium entry during the transduction process. Within type I neurons, by buffering sudden increases in the intracellular calcium concentration, it may allow an adaptation of the firing rate to variations in the intensity of sound stimuli.     

Relais cells and afferent axons in the dorsal part of the lateral geniculate body in the albino rat under geometrical aspects]

1. The average volumes of dendritic domaines of relay neurons (P-neurons) were calculated and the quantitative relations to the neuronal elements situated in this area were investigated. Likewise we carried out measurements and calculations at the terminal parts of afferent axons, to find a conception concerning possible contacts between axons and P-neurons considering quantitative aspects. 2. The dendrites of one P-neuron are distributed in an area of about 0,008 mum3. In this area there are located somata of at least 120 other P-neurons and dendrites of altogether about 900 P-neurons. 3. The type-1-axons (cortical afferents) run almost linearly in the longitudinal system of the CGLd. Traversing a distance adequate to the diameter of a P-neuron (250 mum) the dendrites of 150 to 170 P-neurons may cross the course of one axon. At this distance the axon, however, has just set up about 50 boutons, thus synaptic contacts may be established with one third at most of the existing cells. A type-1-axon that is bifurcating in the entrance area into the CGLd is altogether of about 2000 mum in length and is able to develop about 420 presynaptic profiles. 4. The type-2-axons (retinal afferents) show a distinct terminal branching zone. The Golgi-Kopsch impregnated terminals of type-2a-axons are distributed in a space of 147000 mum3 capacity, the corresponding terminals of type-2b-axons in a space of 443000 mum3. The type-2a-axons having an average number of 23 boutons, may contact the dendritic branching zones of 25 P-neurons. There is a good reason to assume that type-2b-axons are in contact also with terminal dendritic parts of P-neurons. Thus the number of P-cells, which spread their dendrites into the terminal branching zone of one type-2b-axon may amount to 540. The average number of boutons of one type-2b-terminal, however, is only about 160. This means that synaptic contacts may be developed to the P-neurons-dendrites not exceeding 30% of them. 5. Various aspects of divergence of axon terminals in the albino rat's CGLd are discussed.     

Calcium-binding parvalbumin inDrosophila Testis in connection with in vivo irradiation

Summary Assuming a possible role of calcium in the function of the germinal proliferation centre in the testis ofDrosophila melanogaster, the distribution of Ca²⁺-binding protein parvalbumin and structural related proteins in male gonads was tested by several biochemical and immunohistochemical methods. The two dimensional PAGE analysis on 3000 gonads of pupal stages suggests the presence of parvalbumin in this tissue. Immunohistochemical studies confirmed this finding. Parvalbumin-immunoreactivity was located in (or near) membraneous systems, like mitochondria and (or) microtubuli. The immunohistochemical analysis of irradiated gonads showed no radiation damage effect on the distribution of parvalbumin in certain cells and cell components.Abbreviations EDTA ethylenediaminetetraacetate - PAGE polyacrylamid-gelelectrophoresis - pI isolectric point - M_r relative molecular mass     

Song, sexual selection, and a song control nucleus (HVc) in the brains of European sedge warblers

Female sedge warblers select males that have more complex songs as mates. This study tests two predictions concerning HVc, a telencephalic nucleus that is essential for song learning and production: first, that males with more complex songs will have a larger HVc, and second that males who pair successfully will have a larger HVc than unpaired males. Data on song composition and pairing status were collected from wild sedge warblers breeding in Hungary. We found significant positive correlations between three song attributes (repertoire size, song complexity, and song length) and the size of HVc. Males that paired successfully also had more complex songs (repertoire size and song complexity, though not song length) than males that did not. However, we find no direct evidence that males who paired successfully had a larger HVc than unpaired males. These findings are discussed in relation to the possible functions of HVc and also to current views on sexual selection and the evolution of the song control pathway.     

Long‐term stability of axonal boutons in the mouse barrel cortex

Many lines of evidence indicate that postsynaptic dendritic spines are plastic during development and largely stable in adulthood. It remains unclear to what degree presynaptic axonal terminals undergo changes in the developing and mature cortex. In this study, we examined the formation and elimination of fluorescently‐labeled axonal boutons in the living mouse barrel cortex with transcranial two‐photon microscopy. We found that the turnover of axonal boutons was significantly higher in 3‐week‐old young mice than in adult mice (older than 3 months). There was a slight but significant net loss of axonal boutons in mice from 1 to 2 months of age. In both young and adult barrel cortex, axonal boutons existed for at least 1 week were less likely to be eliminated than those recently‐formed boutons. In adulthood, 80% of axonal boutons persisted over 12 months and enriched sensory experience caused a slight but not significant increase in the turnover of axonal boutons over 2–4 weeks. Thus, similar to postsynaptic dendritic spines, presynaptic axonal boutons show remarkable stability after development ends. This long‐term stability of synaptic connections is likely important for reliable sensory processing in the mature somatosensory cortex. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 252–261, 2016     

A thalamo-cerebral connection in vocal center of canary

Canary song is controlled by two groups of thalamo-cerebral nuclei. One, the hyperstriatum ventrale pars caudale (HVc) and the robust nucleus of the archistriatum (RA), is a motor driving system for vocalization. The other group, which includes the HVc, the nucleus magnocellularis of neostriatum (MAN), Area X and the nucleus dorsointermedius posterior thalami (DIP), modulates the driving system. The HVc receives synaptic projections from the MAN and sends fibers to Area X. Axons of Area X monosynaptically innervate the thalamic nucleus, the DIP, from which neurons extend axons back to the cerebral nucleus, the MAN. DIP neurons relay incoming impulses by way of Area X to the MAN. Double labeling of DIP neurons with HRP and Fast Blue shows that axonal terminals from Area X connect directly with DIP neurons which send fibers to the MAN. The axon formed a bulge from which multiple branches extended to the postsynaptic cell bodies covering most of the surface. The structure of the DIP synapse may be related to a characteristic pattern of discharge of the DIP neuron, which is transmitted over thalamic projection to cerebral vocal nuclei.     

Heterogeneity of receptor immunoreactivity at synapses of glycine-utilizing neurons.

Neurons often contain, and probably release, more than one neuroactive substance that may have diverse or opposite actions on the postsynaptic cell. It remains unexplained how these neurons utilize their multiple neuroactive substances while maintaining appropriate resolution of neurotransmitter functions. Here, we have examined the ultrastructural localization of glycine receptors by using a monoclonal antibody directed to the intracellular domain of the strychnine-sensitive glycine receptor. We have found that glycine receptors are only localized to 56% of the synapses made by presumed 'glycinergic' (more accurately, glycine-utilizing) amacrine cells in the turtle retina. The remaining synapses made by these same boutons show no evidence of glycine receptors. As there is no evidence to suggest the presence of a second type of glycine receptor, these data indicate that only a portion of the postsynaptic sites contacted by the glycine-utilizing neurons can respond to glycine. They also suggest that a neuron containing multiple neuroactive substances can selectively affect postsynaptic elements by means of heterogeneous receptor localization.     

GABAA receptor kinetics in the cerebellar nuclei: evidence for detection of transmitter from distant release sites

Neurons of the cerebellar nuclei receive GABAergic input from Purkinje cells. Purkinje boutons have several closely spaced presynaptic densities without GABA transporters, raising the possibility that neurotransmitter released by one presynaptic site diffuses to multiple postsynaptic sites. To test whether such local spillover may contribute to transmission, we studied gating of GABA(A) receptors at 31-33 degrees C in cerebellar nuclear neurons acutely dissociated from mice. Currents were evoked by rapid application of long steps, brief pulses, and high-frequency trains of GABA to outside-out patches. Receptors desensitized and deactivated rapidly, and dose-response measurements estimated an EC(50) of approximately 30 microM. From these data, a kinetic scheme was developed that replicated the recorded currents. Next, we simulated diffusion of GABA in the synaptic cleft, constrained by previous electron microscopic data, and drove the kinetic GABA(A) receptor model with modeled concentration transients. Simulations predicted receptor occupancies of approximately 100% directly opposite the release site and approximately 50% at distant postsynaptic densities, such that receptors up to 700 nm from a release site opened on the timescale of the inhibitory postsynaptic currents before desensitizing. Further simulations of probabilistic release from multiple-site boutons suggested that local spillover-mediated transmission slows the onset and limits the extent of depression during high-frequency signaling.     

Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment

Time-lapse microscopy, retrospective immunohistochemistry, and cultured hippocampal neurons were used to determine the time frame of individual glutamatergic synapse assembly and the temporal order in which specific molecules accumulate at new synaptic junctions. New presynaptic boutons capable of activity-evoked vesicle recycling were observed to form within 30 min of initial axodendritic contact. Clusters of the presynaptic active zone protein Bassoon were present in all new boutons. Conversely, clusters of the postsynaptic molecule SAP90/PSD-95 and glutamate receptors were found on average only approximately 45 min after such boutons were first detected. AMPA- and NMDA-type glutamate receptors displayed similar clustering kinetics. These findings suggest that glutamatergic synapse assembly can occur within 1-2 hr after initial contact and that presynaptic differentiation may precede postsynaptic differentiation.     

Calcium-dependent proteases: an enzyme system active at cellular membranes?

Proteases having a neutral pH optimum and an absolute requirement for calcium ion are found in virtually all mammalian cells. Association of calcium-dependent proteases and a specific inhibitor protein with biological membranes seems to be an important regulatory feature of this proteolytic system, and it is likely that membranes are preferred sites for calcium-dependent protease action. Several recent hypotheses for the physiological function of calcium-dependent proteolysis are consistent with a membrane-associated protease action. Calcium-dependent proteases may participate in cell membrane fusion: the proteolysis of membrane proteins, which is required for the efficient fusion of erythrocytes, may be catalyzed by these enzymes. There is also evidence for the involvement of calcium-dependent proteolysis in postsynaptic membrane remodeling in the hippocampus after long-term potentiation. Although the relationship of the proteolysis to synaptic function is not known, it could have important physiological or pathophysiological consequences. Finally, it has recently been suggested that calcium-dependent proteolysis may be a physiologically significant mechanism for activating membrane-associated protein kinase C after exposure of some cell types to phorbol esters or other mitogens. Further pursuit of these hypotheses may reveal a novel role for intracellular calcium-regulated proteolysis in membrane-associated cell functions.     

Cytochemical detection of actin in the structure of the synaptic apparatus of hippocampal field CA3

Ultrastructural distribution of actin in dendrites, dendritic spines and presynaptic boutons of the hippocampal area CA3 of the guinea pig was investigated using decoration and immunocytochemical methods. The distribution of actin was non-homogeneous in all the parts of neurons. The highest concentration of this contractile protein was revealed in the spine cytoplasm. Here actin forms a dense cytoskeleton meshwork and is present also in postsynaptic densities. An intimate interaction between the spine actin cytoskeleton and the postsynaptic actin densities has been revealed. This feature may indicate the involvement of actin cytoskeleton in the organization and maintenance of dimensions, location and geometry of active zones.     

燕雀上纹状体腹侧尾核对发声和呼吸的调制效应及其纤维联系

在乌拉坦麻醉的鸣禽燕雀（Ｆｒｉｎｇｉｌｌａｍｏｎｔｉｆｒｉｎｇｉｌｌａ）上,观察电刺激上纹状体腹侧尾核（ＨＶｃ）对发声和呼吸的影响,随后在ＨＶｃ内注入ＣＢ－ＨＲＰ溶液,研究ＨＶｃ的中枢联系。结果如下：（１）电刺激ＨＶｃ的不同区域都引起鸣叫反应。（２）长串电脉冲刺激ＨＶｃ,产生明显的呼吸易化效应,表现为增频增幅的呼吸。（３）吸气期用短串电脉冲刺激ＨＶｃ,产生吸气切断效应;刺激落位于呼气相,可使该呼气时程明显延长,以配合鸣叫,然后转变为增频增幅的呼吸。（４）ＣＢＨＲＰ法表明,ＨＶｃ投射到古纹状体粗核和嗅叶Ｘ区,ＨＶｃ接受新纹状体前部大细胞核内侧部、新纹状体中部界面核、端脑听核－Ｌ区、丘脑葡萄形核及脑桥蓝斑核的传入投射。提示ＨＶｃ除控制发声外,尚参与呼吸易化的调制。ＨＶｃ对发声及呼吸的特异性影响,可能在鸣叫与呼吸的协调机制中起重要作用。     

Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila

The glutamatergic neuromuscular junction (NMJ) in Drosophila adds new boutons and branches during larval development. We generated transgenic fruit flies that express a novel green fluorescent membrane protein at the postsynaptic specialization, allowing for repeated noninvasive confocal imaging of synapses in live, developing larvae. As synapses grow, existing synaptic boutons stretch apart and new boutons insert between them; in addition, new boutons are added at the ends of existing strings of boutons. Some boutons are added de novo, while others bud from existing boutons. New branches form as multiple boutons bud from existing boutons. Nascent boutons contain active zones, T bars, and synaptic vesicles; we observe no specialized growth structures. Some new boutons exhibit a lower level of Fasciclin II, suggesting that the levels of this synaptic cell adhesion molecule vary locally during synaptic growth.     

Computation of action potential propagation and presynaptic bouton activation in terminal arborizations of different geometries.

Action potential propagation in axons with bifurcations involving short collaterals with synaptic boutons has been simulated using SPICE, a general purpose electrical circuit simulation program. The large electrical load of the boutons may lead to propagation failure at otherwise uncritical geometric ratios. Because the action potential gradually fails while approaching the branch point, the electrotonic spread of the failing action potential cannot depolarize the terminal boutons above an assumed threshold of 20 mV (Vrest = 0 mV) for the presynaptic calcium inflow, and therefore fails to evoke transmitter release even for boutons attached at short collaterals. For even shorter collaterals the terminal boutons can again be activated by the spread of passive current reflected at the sealed end of the bouton which increases the membrane potential above firing threshold. The action potential is then propagated in anterograde fashion into the main axon and may activate the terminal bouton on the other collateral. Differential activation of the synaptic boutons can be observed without repetitive activation of the main axon and with the assumption of uniform membrane properties. Axon enlargements above a critical size at branch points can increase the safety factor for propagation significantly and may serve a double function: they can act both as presynaptic boutons and as boosters, facilitating invasion of the action potential into the terminal arborizations. The architecture of the terminal arborizations has a profound effect on the activation pattern of synapses, suggesting that terminal arborizations not only distribute neural information to postsynaptic cells but may also be able to process neural information presynaptically.     

Derailed regulates development of the Drosophila neuromuscular junction

Neural function is dependent upon the proper formation and development of synapses. We show here that Wnt5 regulates the growth of the Drosophila neuromuscular junction (NMJ) by signaling through the Derailed receptor. Mutations in both wnt5 and drl result in a significant reduction in the number of synaptic boutons. Cell-type specific rescue experiments show that wnt5 functions in the presynaptic motor neuron while drl likely functions in the postsynaptic muscle cell. Epistatic analyses indicate that drl acts downstream of wnt5 to promote synaptic growth. Structure-function analyses of the Drl protein indicate that normal synaptic growth requires the extracellular Wnt inhibitory factor domain and the intracellular domain, which includes an atypical kinase. Our findings reveal a novel signaling mechanism that regulates morphology of the Drosophila NMJ.     

Distribution and mobility of lectin receptors on synaptic membranes of identified neurons in the central nervous system

The distribution and mobility of concanavalin A (Con A) and Ricinus communis agglutinin (RCA) receptors (binding sites) on the external surfaces of Purkinje, hippocampal pyramidal, and granule cells and their attached boutons were studied using ferritin-lectin conjugates. Dendritic fields of these cells were isolated by microdissection and gently homogenized. Cell fragments and pre- and postsynaptic membranes were labeled with the ferritin-lectin conjugates at a variety of temperatures, and the distribution of lectin receptors was determined by electron microscopy. Both classes of these lectin receptors were concentrated at nearly all open and partially open postsynaptic junctional membranes of asymmetric-type synapses on all three neuron types. Con A receptors were most concentrated at the junctional membrane region, indicating that the mature neuron has a specialized nonrandom organization of carbohydrates on its outer surface. Lectin receptors located on postsynaptic junctional membranes appeared to be restricted in their mobility compared to similar classes of receptors on extrajunctional membrane regions. Labeling with ferritin-RCA and - Con A at 37 degrees C produced clustering of lectin receptors on nonjunctional surfaces; however, Con A and RCA receptors retained their nonrandom topographic distribution on the postsynaptic junctional surface. The restricted mobility of lectin receptors was an inherent property of the postsynaptic membrane since the presynaptic membrane was absent. It is proposed that structures in the postsynaptic density may be transmembrane-linked to postsynaptic receptors and thereby determine topographic distribution and limit diffusion of specialized synaptic molecules. Speicalized receptor displays may play an important role in the formation and maintenance of specific synaptic contacts.     

黄喉鵐高级发声中枢及其壳区的神经投射和P物质在发声听觉中枢的分布

用生物素示踪法和P物质 (SP)免疫组化技术研究表明 :黄喉的高级发声中枢 (HVc)接受端脑听区 (L)、新纹状体中部界面核、新纹状体巨细胞核 (MAN)、丘脑葡萄形核、桥脑蓝斑核的传入 ,并有神经纤维投射到古纹状体栎核 (RA)和嗅叶X区 (X) ;HVc壳投射到RA壳并接受L的传入。听觉控制与学习通路与发声中枢之间有许多神经联系 ,提示黄喉发声学习依赖于听觉反馈。在HVc、RA和MAN有SP阳性细胞体 ,在X、中脑背内侧核和延髓舌下神经核气管鸣管部、丘脑卵圆核壳区、中脑背外侧核壳区及中脑丘间核有SP阳性纤维和终末。SP广泛分布于发声 -听觉中枢 ,可能参与了它们的活动     

黄喉鹀高级发声中枢及其壳区的神经投射和P物质在发声听觉中枢的分布

用生物素示踪法和P物质(SP)免疫组化技术研究表明:黄喉(巫鸟)的高级发声中枢(HVc)接受端脑听区(L)、新纹状体中部界面核、新纹状体巨细胞核(MAN)、丘脑葡萄形核、桥脑蓝斑核的传入,并有神经纤维投射到古纹状体栎核(RA)和嗅叶X区(X);HVc壳投射到RA壳并接受L的传入.听觉控制与学习通路与发声中枢之间有许多神经联系,提示黄喉(巫鸟)发声学习依赖于听觉反馈.在HVc、RA和MAN有SP阳性细胞体,在X、中脑背内侧核和延髓舌下神经核气管鸣管部、丘脑卵圆核壳区、中脑背外侧核壳区及中脑丘间核有SP阳性纤维和终末.SP广泛分布于发声-听觉中枢,可能参与了它们的活动.     

Regulation of neuronal excitability in Drosophila by constitutively active CaMKII

The ability of calcium/calmodulin-dependent protein kinase II (CaMKII) to become calcium independent after autophosphorylation makes this enzyme a temporal marker of neuronal activity. Here we show that the calcium-independent form of CaMKII has unique effects on larval viability, locomotion, and neuronal excitability in Drosophila. Expression of constitutively active T287D, but not calcium-dependent T287A, mutant CaMKII in Drosophila neurons resulted in decreased viability, behavioral defects, and failure of action potential propagation. The actions of T287D may be mediated, at least in part, by increased potassium conductances. Expression of T287D CaMKII also stimulated an increase in the number of boutons at the larval neuromuscular junction, but did not affect the mechanics of release. This study defines a role for autophosphorylation of CaMKII in the regulation of multiple neuronal functions including the intrinsic properties of neurons.     

Basal forebrain cholinergic modulation of auditory activity in the zebra finch song system

The cholinergic basis of auditory "gating" in the sensorimotor nucleus HVc and its efferent target robustus archistriatalis (RA) was investigated in anesthetized zebra finches. Injections of cholinergic agonists carbachol or muscarine into HVc strongly affected discharge rates and diminished auditory responsiveness in both HVc and its target RA, changes toward an awake-like condition. HVc nicotine injections produced similar strong effects in HVc, but weaker and inconsistent effects in RA. Stimulation of basal forebrain (BF) produced an initial transient network shutdown followed by diminished auditory responsiveness in HVc and RA. All stimulation effects were blocked when preceded by HVc injections of nicotinic or muscarinic antagonists. Thus, BF cholinergic modulation of song system auditory activity acting via functionally distinct HVc circuits can contribute to auditory gating. We hypothesize that wakeful BF activity levels block sensory input to motor systems and adaptively change during behavior to allow sensorimotor feedback such as auditory feedback during singing.