Lateral magnocellular nucleus of the anterior neostriatum (LMAN) in the zebra finch: neuronal connectivity and the emergence of sex differences in cell morphology.

The song system of birds provides a model system to study basic mechanisms of neuronal plasticity and development underlying learned behavior. Song learning and production involve discrete sets of interconnected nuclei in the avian brain. One of these nuclei, the lateral magnocellular nucleus of the anterior neostriatum (LMAN), is the output of the so-called anterior forebrain pathway known to be essential for learning and maintenance of song, both processes depending on auditory feedback. In zebra finches, only males sing and this sexually dimorphic behavior is mirrored by sexual dimorphism in neuronal structure that develops during ontogeny. Female zebra finches are not able to sing and nuclei of the song system are strongly reduced in size or even lacking, when compared to male brains. Only LMAN can be delineated as easily in females as in males. Since female zebra finches, despite being unable to sing, recognize song just as males do and form a memory for song (model acquisition) early in life, LMAN is a putative candidate for song acquisition in both sexes. Therefore, development of LMAN was studied at the cellular and ultrastructural level in both male and female zebra finches. Regressive development of dendritic spines, enlargement of neuronal cell body and nuclei size, as well as changes at the nucleolar level are events all occurring exclusively in males, when song learning progresses. The decline in synapse number and the augmentation in synaptic contact length at synapses in LMAN in males are indicative for synaptic plasticity, whereas in females synapse number and synaptic contact length remain unchanged.     

Membrane and cytoplasmic structure at synaptic junctions in the mammalian central nervous system

Application of rapid freezing, freeze substitution fixation, and freeze fracture techniques to the study of synaptic junctions in the mammalian central nervous system has revealed new aspects of synaptic structure that are consistent with and partially explicate advances in synaptic biochemistry and physiology. In the axoplasm adjacent to the presynaptic active zone, synaptic vesicles are linked to large spectrin-like filamentous proteins by shorter proteins that resemble synapsin I in morphology. This mesh of presynaptic filamentous proteins serves to concentrate synaptic vesicles in the vicinity of the active zone. The affinity with which the vesicles are bound by the mesh is probably modulated by the extent of phosphorylation at specific sites on the constituent filamentous proteins, and changes in the binding affinity result in changes in transmitter release. The structural organization of the postsynaptic density in Purkinje cell dendritic spines consists of very fine strands with adherent, heterogeneous globular proteins. Some of these globular proteins probably correspond to protein kinases and their substrates. The postsynaptic density, positioned at the site of the maximal depolarization caused by synaptic currents, apparently serves as a supporting framework for a variety of proteins, which respond to and transduce postsynaptic depolarization. At least two classes of filamentous protein fill the cytoplasm of spines with a complex mesh, which presumably contributes to maintenance of the spine shape. Membrane bound cisterns are a ubiquitous feature of Purkinje cell dendritic spines. Studies of rapidly frozen tissue with electron probe microanalysis and elemental imaging reveal that these cisterns take up and sequester calcium, which is derived from the extracellular space, and which probably enters the spine as part of the synaptic current.     

Synaptic connections of neurones identified by Golgi impregnation: Characterization by immunocytochemical,enzyme histochemical,and degeneration methods

For more than a century the Golgi method has been providing structural information about the organization of neuronal networks. Recent developments allow the extension of the method to the electron microscopic analysis of the afferent and efferent synaptic connections of identified, Golgi-impregnated neurones. The introduction of degeneration, autoradiographic, enzyme histochemical, and immunocytochemical methods for the characterization of Golgi-impregnated neurones and their pre-and postsynaptic partners makes it possible to establish the origin and also the chemical composition of pre-and postsynaptic elements. Furthermore, for a direct correlation of structure and function the synaptic interconnections between physiologically characterized, intracellularly HRP-filled neurones and Golgi-impregnated cells can be studied. It is thought that most of the neuronal communication takes place at the synaptic junction. In the enterprise of unravelling the circuits underlying the synaptic interactions, the Golgi technique continues to be a powerful tool of analysis.     

Structural and functional correlates of synaptic transmission in the vertebrate neuromuscular junction

Because vertebrate neuromuscular junctions are readily accessible for experimental manipulation, they have provided a superb model in which to examine and test functional correlates of chemical synaptic transmission. In the neuromuscular synapse, acetylcholine receptors have been localized to the crests of the junctional folds and visualized by a variety of ultrastructural techniques. By using ultrarapid freezing techniques with a temporal resolution of less than 1 msec, quantal transmitter release has been correlated with synaptic vesicle exocytosis at discrete sites called “active zones.” Mechanisms for synaptic vesicle membrane retrieval and recycling have been identified by using immunological approaches and correlated with endocytosis via coated pits and coated vesicles. In this review, available ultrastructural, physiological, immunological, and biochemical data have been used to construct an ultrastructural model of neuromuscular synaptic transmission that correlates structure and function at the molecular level.     

Different types of multiple‐synapse boutons in the cerebellar cortex between physically enriched and ataxic mutant mice

Experience‐dependent synapse remodeling is associated with information storage in the nervous system. Neuronal synapses show alteration in various neurological and cognitive disorders in their structure and function. At the ultrastructural level, parallel fiber boutons contacting multiple spines of Purkinje cells in the cerebellar cortex are commonly observed in physiologically enriched animals as well as pathological ataxic mutants. However, the dendritic origin of those spines on parallel fiber multiple‐synapse boutons (MSBs) has been poorly understood. Here, we investigated this issue by 3‐dimensional ultrastructural analysis to determine synaptic connectivity of MSBs in both mice housed in physically enriched environment and cerebellar ataxic mutants. Our results demonstrated that environmental enrichment selectively induced MSBs to contact spines from the same parent dendrite, indicating focal strengthening of synapse through the simultaneous activation of two adjacent spines. In contrast, ataxic mutants displaying impaired motor coordination had significantly more MSBs involving spines originating from different neighboring dendrites compared to both wild‐type and environmentally enriched animals, suggesting that compromising multiple synapse formation may lead to abnormal motor behavior in the mutant mice. These findings propose that environmental stimulation in normal animals mainly involves the refinement of preexisting synaptic networks, whereas pathological ataxic conditions may results from less‐selective but compromising multiple synaptic formation. This study underscores that different types of multiple synapse boutons may have disparate effects on cerebellar synaptic transmission.     

Synaptic circuitry identified by intracellular labeling with horseradish peroxidase

During the past two decades new techniques have been developed to directly test the dogma that neuronal structure is correlated with neuronal function. In the earliest experiments, Procion yellow was injected into neurons after they had been characterized physiologically; these neurons were then viewed through the light microscope. Recent advances in the method generally employ horseradish peroxidase as the dye which is injected since it diffuses quite readily throughout the injected neuron and produces a stable reaction product for both light and electron microscopic studies. This review explores the utility of examining synaptic circuitry after physiologically recording from axons or neurons and then injecting horseradish peroxidase into them. As a model system, we studied the cat lateral geniculate nucleus and investigated, at the electron microscopic level, the synaptic contribution to this nucleus from retinogeniculate axons, from interneurons, and from the axon collaterals of neurons that project to visual cortex.     

Technique to study three-dimensional spatial arrangement of synaptic vesicles using data from single sections

Synaptic vesicles are membrane-bound organelles storing neurotransmitters in presynaptic terminals and releasing them into the synaptic cleft. Coordinated movements of synaptic vesicles relate to synaptic function and their spatial arrangement can provide useful information about the activity of a synapse. This article presents a technique to extract quantitative information about three-dimensional (3D) spatial arrangement of synaptic vesicles from measurements performed on single ultrathin random sections of a presynaptic terminal. The technique presumes quantification of a 2D density as well as 2D spatial pattern formed by vesicle profiles using a minimum spanning tree (MST) algorithm, in digitized micrographs of a presynaptic terminal. Further, original software was used to simulate a 3D spatial arrangement of synaptic vesicles and their random sectioning. A 3D density and pattern of synaptic vesicles were used as basic input parameters of the model, while a 2D density and MST quantities for vesicle profiles served as output, model-derived parameters allowing one to compare and fit simulated distributions to experimental ones. Pilot simulations performed to check the validity of the technique have shown that a 2D density and MST quantities of vesicle profiles closely relate to a 3D density and spatial pattern of vesicles. The technique was demonstrated in the analysis of spatial distribution of synaptic vesicles in axonal terminals forming asymmetric synaptic densities in the stratum radiatum of the CA1 subfield of the murine hippocampus.     

Immunolocalization of the cellular prion protein in normal brain

We examined the localization of PrP(c) in normal brain using free-floating section immunohistochemistry and monclonal antibody 3F4. In the mature hamster and baboon brain, PrP(c) is localized to the neuropil with a synaptic distribution and the PrP(c) immunoreactivity is denser in regions known for ongoing plasticity. Cell bodies and major fiber tracts have little or no PrP(c) immunoreactivity. At the electron microscopic level, PrP(c) immunoreactivity decorates synaptic profiles, both pre- and postsynaptically. Results obtained with two additional antibodies, 3B5 and Pri-304, showed similar patterns of PrP(c) bands on Western blots, although Pri-304 was less sensitive. On sections through the adult hamster hippocampus, 3B5 and Pri-304 both stained the synaptic neuropil while cell bodies in the pyramidal and dentate granule cell layers were not immunoreactive. Pri-304 differentiated between synaptic layers in the hippocampus and closely resembled the pattern of staining obtained with 3F4. Preliminary results of developing brain showed that PrP(c) is initially localized along fiber tracts in the neonate brain. These results show that PrP(c) has a synaptic distribution in the adult brain and suggest that there are important changes in its distribution during brain development. These results also characterize two additional reagents for studies of PrP(c) localization.     

Drosophila larval neuromuscular junction: molecular components and mechanisms underlying synaptic plasticity

Understanding the mechanisms that mediate synaptic plasticity is a primary goal of molecular neuroscience. The Drosophila larval neuromuscular junction provides a particularly useful model for investigating the roles of synaptic components in both structural and functional plasticity. The powerful molecular genetics of this system makes it possible to uncover new synaptic components and signaling molecules, as well as their function in the intact organism. Together with the mouse hippocampus and Aplysia dissociated cell culture, the Drosophila larval neuromuscular junction has been among the most valuable model systems for examining the molecular and cellular basis of neuronal plasticity.     

A computerized method of determining the number of synaptic contacts in a volume of cerebral cortex

A computerized flying-spot scanning device has been used to count the number of synaptic contacts in neural tissue stained with ethanolic phosphotungstic acid (E-PTA). The ability of the computer to idealize the irregular synaptic contacts as solid disks by modifying the information gleaned from scanning the preparation allows the stereological principle of Weibel & Gomez to be used to calculate the number of E-PTA stained synaptic contacts per unit volume of tissue. This technique represents a significant improvement over the time necessary for making similar quantitative determinations manually.     

Expression and plasticity of glutamate receptors in the supraoptic nucleus of the hypothalamus.

Magnocellular neuroendocrine cells (MNCs) of the supraoptic nucleus of the hypothalamus (SON) produce and release the hormones vasopressin (VP) and oxytocin (OT) in response to a variety of stimuli to regulate body water and salt, parturition and lactation. Hormone release is influenced by the pattern of neuronal firing of these MNCs, which, in turn, is governed by intrinsic conductances and synaptic inputs, including those mediated by the neurotransmitter glutamate. Functional and molecular evidence has confirmed the expression of AMPA-, NMDA-, and metabotropic-type glutamate receptors in the SON, that together may orchestrate the effects of glutamatergic transmission on neuroendocrine function. However, the specific roles of the different subtypes of glutamate receptors is not yet clear. As with other central neurons, the subunit composition of glutamate receptors on MNCs will likely determine their properties and may potentially help define the differential properties of VP- and OT-producing MNCs. Possible functions of glutamate receptors on SON MNCs include altering excitatory synaptic transmission of osmotic information, neuronal firing, hormone production and release, and calcium signaling. Of interest are the anatomical, molecular, and functional changes at glutamatergic synapses in the SON that occur in response to pertinent physiological stimuli or development. These types of plasticity may include changes in glutamatergic synaptic density, glutamate receptor levels, or glutamate receptor subunit expression, all of which can affect the efficiency of synaptic transmission.     

Review: Correlative microscopy of Purkinje cells

The Purkinje cell and their synaptic contacts have been described using (1) light microsocopy, (2) transmission and scanning electron microscopy, and freeze etching technique, (3) conventional and field emission scanning electron microscopy and cryofracture methods, (4) confocal laser scanning microscopy using intravital stain FM64, and (5) immunocytochemical techniques for Synapsin-I, PSD9-5, GluR1 subunit of AMPA receptors, N-cadherin, and CamKII alpha. The outer surface and inner content of plasma membrane, cell organelles, cytoskeleton, nucleus, dendritic and axonal processes have been exposed and analyzed in a three-dimensional view. The intramembrane morphology, in bi- and three-dimensional views, and immunocytochemical labeling of synaptic contacts with parallel and climbing fibers, basket and stellate cell axons have been characterized. Freeze etching technique, field emission scanning microscopy and cryofracture methods, and GluR1 immunohistochemistry showed the morphology and localization of postsynaptic receptors. Purkinje cell shows N-cadherin and CamKII alpha immunoreactivity. The correlative microscopy approach provides a deeper understanding of structure and function of the Purkinje cell, a new three-dimensional outer and inner vision, a more detailed study of afferent and intrinsic synaptic junctions, and of intracortical circuits.     

Electrophysiological analysis of synaptic interactions within peg sensilla of scorpion pectines

Pectines are unique, midventral sensory appendages that help direct mating and food-finding behaviors in scorpions. Dense two-dimensional arrays of bimodally sensitive (chemical and mechanical) peg sensilla form the primary sensory structures on pectines. Several qualities of peg sensilla make them well suited to electrophysiological investigation, including accessibility, stability of extracellular recordings, and the ease with which spiking cells can be identified and categorized. Cross-correlations of spontaneous neural activity show signs of synaptic interactions between sensillar neurons in all species examined to date (Paruroctonus mesaensis, Hadrurus arizonensis, Centruroides vittatus) representing three families and two superfamilies. Both excitatory and inhibitory interactions have been observed, as well as possible dyadic synaptic arrangement. Computer simulations of cross-correlograms are consistent with experimental data and may help provide additional insight into functionality of synaptic connections. Intra-sensillar interactions, coupled with the topographic order of peg sensilla and their central nervous system projections, may allow scorpions to precisely resolve microfeatures of chemical stimuli. Future research directions include inter-sensillar recordings to determine whether synaptic interactions extend between adjacent sensilla. Other unresolved questions that can be approached electrophysiologically are whether mechanosensory cells interact with chemosensitive cells and how the synaptic circuits function under specific chemical and mechanical stimulation.     

Freeze-fracture organization of hair cell synapses in the sensory epithelium of guinea pig organ of Corti

The fine structure of both the afferent and efferent hair cell synapses in the sensory epithelium of guinea pig organ of Corti was examined by freeze-fracture electron microscopy. In the afferent synapse, barlike aggregates of intramembrane particles (IMPs) of about 10 nm in diameter were seen on the P-face of the afferent presynaptic membrane directly beneath the presynaptic dense projection which is located in the active zone of the presynaptic membrane. Small and large depressions have been seen on the presynaptic membrane. The former were observed in the proximity of the barlike aggregates, while the latter were observed some distance from the aggregate. In outer hair cells, IMPs of about 10 nm in diameter were seen on the P-face of the afferent postsynaptic membrane at a density of 3,000/μm². In the efferent synapse, many aggregates composed of from several to tens of large IMPs of 13 nm in diameter were observed on the presynaptic membrane. These aggregates were localized to small membrane depressions, which tended to be deeper as particle number per aggregate increased. Dense populations of IMPs of about 9 nm in diameter were observed on the P-face of the efferent postsynaptic membrane at a density of 4,000/μm². A fenestrated subsynaptic cistern completely covers the efferent postsynaptic membrane. Moreover, the subsynaptic cistern spans several efferent postsynaptic membranes when efferent synapses are gathered in a group. In the afferent and efferent synapses of hair cells, specializations of the synaptic membranes were represented by marked aggregates characteristic of IMPs. In the efferent synapse, IMP movement inside the synaptic membrane was proposed in relationship to retrival of synaptic vesicle membrane. Structural relationship between the subsynaptic cistern and efferent postsynaptic membrane was revealed.     

Synapses on the mauthner cell of the goldfish: Thin section,freeze-fracture,and immunocytochemical studies

Large myelinated club ending and small-vesicle bouton synapses on the distal part of the lateral dendrite of the goldfish Mauthner cell were investigated with thin section, freeze-fracture, and immunocytochemical electron microscopic methods. Large myelinated club endings form mixed synapses, having both gap junctions and chemical synaptic junctions. The correlation of the number of gap junction particles (connexons) and the data from electrophysiological studies of single large myelinated club ending synapses suggest that only a small fraction of gap junction channels are open at any given time during electrical synaptic transmission. The chemical synaptic junctions at the large myelinated club ending synapse have large, round synaptic vesicles, indicating that they are excitatory. This result is in agreement with electrophysiological data demonstrating the excitatory nature of this chemical synapse. Freeze-fracture of these excitatory chemical synaptic junctions reveals the presence of the intramembrane particle aggregates in the postsynaptic E face. Small-vesicle boutons form chemical synaptic junctions with small, flat or oval synaptic vesicles. These structural data, in combination with previous electrophysiological studies, suggest that the small-vesicle bouton synapses are inhibitory. In support of this theory, the cytoplasmic side of the postsynaptic membrane of some of these synapses show positive immunocytochemical reaction to monoclonal antibodies against the rat glycine receptor. Freeze-fracture data reveal intramembrane particle aggregates in the postsynaptic P face of some small-vesicle bouton synapses which could possibly represent glycine receptor aggregates.     

The complex-shaped ‘perforated’ synapse,a problem in quantitative stereology of the brain

Failure to appreciate the consequences for stereological work of the simultaneous presence of complex-shaped perforated and disc-like non-perforated synapses in brain tissue results in underestimation of synaptic profile length and overestimation of synaptic density when measured in randomly selected ultrathin E-PTA slices. This problem can be solved by using serial slices and a calculation method which makes no assumptions about synaptic size and shape. A three-dimensional reconstruction is unnecessary.     

Nidogens-Extracellular matrix linker molecules

Nidogens/entactins are a family of highly conserved, sulfated glycoproteins. Biochemical studies have implicated them as having a major structural role in the basement membrane. However despite being ubiquitous components of this specialized extracellular matrix and having a wide spectrum of binding partners, genetic analysis has shown that they are not required for the overall architecture of the basement membrane. Rather in development they play an important role in its stabilization especially in tissues undergoing rapid growth or turnover. Nidogen breakdown has been implicated as a key event in the basement membrane degradation occurring in mammary gland involution. A number of studies, most compellingly those in C. elegans, demonstrated that nidogens may have other nonstructural roles and be involved in axonal pathfinding and synaptic transmission.     

Presynaptic proteins of ribbon synapses in the retina

The synapses of photoreceptors and bipolar cells in the retina are characterized ultrastructurally by the presence of an electron-dense bar, the synaptic ribbon, lying perpendicular to the plasma membrane at the active zone and extending about 0.5 microm into the cytoplasm. Hence, these synapses are known as ribbon synapses. All neurons that make ribbon synapses release neurotransmitter tonically. That is, neurotransmitter is released continuously from these neurons and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven, phasic release from other neurons. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but several differences that may be important determinants of tonic transmitter release have been identified in the retina by immunohistochemistry. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.     

3D analysis of synaptic vesicle density and distribution after acute foot‐shock stress by using serial section transmission electron microscopy

Behavioural stress has shown to strongly affect neurotransmission within the neocortex. In this study, we analysed the effect of an acute stress model on density and distribution of neurotransmitter‐containing vesicles within medial prefrontal cortex. Serial section transmission electron microscopy was employed to compare two groups of male rats: (1) rats subjected to foot‐shock stress and (2) rats with sham stress as control group. Two‐dimensional (2D) density measures are common in microscopic images and are estimated by following a 2D path in‐section. However, this method ignores the slant of the active zone and thickness of the section. In fact, the active zone is a surface in three‐dimension (3D) and the 2D measures do not accurately reflect the geometric configuration unless the active zone is perpendicular to the sectioning angle. We investigated synaptic vesicle density as a function of distance from the active zone in 3D. We reconstructed a 3D dataset by estimating the thickness of all sections and by registering all the image sections into a common coordinate system. Finally, we estimated the density as the average number of vesicles per area and volume and modelled the synaptic vesicle distribution by fitting a one‐dimensional parametrized distribution that took into account the location uncertainty due to section thickness. Our results showed a clear structural difference in synaptic vesicle density and distribution between stressed and control group with improved separation by 3D measures in comparison to the 2D measures. Our results showed that acute foot‐shock stress exposure significantly affected both the spatial distribution and density of the synaptic vesicles within the presynaptic terminal.     

Synaptic structure, distribution, and circuitry in the central nervous system of the locust and related insects.

The Orthopteran central nervous system has proved a fertile substrate for combined morphological and physiological studies of identified neurons. Electron microscopy reveals two major types of synaptic contacts between nerve fibres: chemical synapses (which predominate) and electrotonic (gap) junctions. The chemical synapses are characterized by a structural asymmetry between the pre- and postsynaptic electron dense paramembranous structures. The postsynaptic paramembranous density defines the extent of a synaptic contact that varies according to synaptic type and location in single identified neurons. Synaptic bars are the most prominent presynaptic element at both monadic and dyadic (divergent) synapses. These are associated with small electron lucent synaptic vesicles in neurons that are cholinergic or glutamatergic (round vesicles) or GABAergic (pleomorphic vesicles). Dense core vesicles of different sizes are indicative of the presence of peptide or amine transmitters. Synapses are mostly found on small-diameter neuropilar branches and the number of synaptic contacts constituting a single physiological synapse ranges from a few tens to several thousand depending on the neurones involved. Some principles of synaptic circuitry can be deduced from the analysis of highly ordered brain neuropiles. With the light microscope, synaptic location can be inferred from the distribution of the presynaptic protein synapsin I. In the ventral nerve cord, identified neurons that are components of circuits subserving known behaviours, have been studied using electrophysiology in combination with light and electron microscopy and immunocytochemistry of neuroactive compounds. This has allowed the synaptic distribution of the major classes of neurone in the ventral nerve cord to be analysed within a functional context.