Terminal Boutons Research Articles

Infrared inhibition impacts on locally initiated and propagating action potentials and the downstream synaptic transmission.

.Significance: Systematic studies of the physiological outputs induced by infrared (IR)-mediated inhibition of motor nerves can provide guidance for therapeutic applications and offer critical insights into IR light modulation of complex neural networks.Aim: We explore the IR-mediated inhibition of action potentials (APs) that either propagate along single axons or are initiated locally and their downstream synaptic transmission responses.Approach: APs were evoked locally by two-electrode current clamp or at a distance for propagating APs. The neuromuscular transmission was recorded with intracellular electrodes in muscle cells or macro-patch pipettes on terminal bouton clusters.Results: IR light pulses completely and reversibly terminate the locally initiated APs firing at low frequencies, which leads to blocking of the synaptic transmission. However, IR light pulses only suppress but do not block the amplitude and duration of propagating APs nor locally initiated APs firing at high frequencies. Such suppressed APs do not influence the postsynaptic responses at a distance. While the suppression of AP amplitude and duration is similar for propagating and locally evoked APs, only the former exhibits a 7% to 21% increase in the maximum time derivative of the AP rising phase.Conclusions: The suppressed APs of motor axons can resume their waveforms after passing the localized IR light illumination site, leaving the muscular and synaptic responses unchanged. IR-mediated modulation on propagating and locally evoked APs should be considered as two separate models for axonal and somatic modulations.

A Conserved Role for Vezatin Proteins in Cargo-Specific Regulation of Retrograde Axonal Transport.

Active transport of organelles within axons is critical for neuronal health. Retrograde axonal transport, in particular, relays neurotrophic signals received by axon terminals to the nucleus and circulates new material among enpassant synapses. A single motor protein complex, cytoplasmic dynein, is responsible for nearly all retrograde transport within axons: its linkage to and transport of diverse cargos is achieved by cargo-specific regulators. Here, we identify Vezatin as a conserved regulator of retrograde axonal transport. Vertebrate Vezatin (Vezt) is required for the maturation and maintenance of cell-cell junctions and has not previously been implicated in axonal transport. However, a related fungal protein, VezA, has been shown to regulate retrograde transport of endosomes in hyphae. In a forward genetic screen, we identified a loss-of-function mutation in the Drosophila vezatin-like (vezl) gene. We here show that vezl loss prevents a subset of endosomes, including signaling endosomes containing activated BMP receptors, from initiating transport out of motor neuron terminal boutons. vezl loss also decreases the transport of endosomes and dense core vesicles, but not mitochondria, within axon shafts. We disrupted vezt in zebrafish and found that vezt loss specifically impairs the retrograde axonal transport of late endosomes, causing their accumulation in axon terminals. Our work establishes a conserved, cargo-specific role for Vezatin proteins in retrograde axonal transport.

Drosophila enabled promotes synapse morphogenesis and regulates active zone form and function

BackgroundRecent studies of synapse form and function highlight the importance of the actin cytoskeleton in regulating multiple aspects of morphogenesis, neurotransmission, and neural plasticity. The conserved actin-associated protein Enabled (Ena) is known to regulate development of the Drosophila larval neuromuscular junction through a postsynaptic mechanism. However, the functions and regulation of Ena within the presynaptic terminal has not been determined.MethodsHere, we use a conditional genetic approach to address a presynaptic role for Ena on presynaptic morphology and ultrastructure, and also examine the pathway in which Ena functions through epistasis experiments.ResultsWe find that Ena is required to promote the morphogenesis of presynaptic boutons and branches, in contrast to its inhibitory role in muscle. Moreover, while postsynaptic Ena is regulated by microRNA-mediated mechanisms, presynaptic Ena relays the output of the highly conserved receptor protein tyrosine phosphatase Dlar and associated proteins including the heparan sulfate proteoglycan Syndecan, and the non-receptor Abelson tyrosine kinase to regulate addition of presynaptic varicosities. Interestingly, Ena also influences active zones, where it restricts active zone size, regulates the recruitment of synaptic vesicles, and controls the amplitude and frequency of spontaneous glutamate release.ConclusionWe thus show that Ena, under control of the Dlar pathway, is required for presynaptic terminal morphogenesis and bouton addition and that Ena has active zone and neurotransmission phenotypes. Notably, in contrast to Dlar, Ena appears to integrate multiple pathways that regulate synapse form and function.

Reorganization of the Primate Dorsal Horn in Response to a Deafferentation Lesion Affecting Hand Function.

The loss of sensory input following a spinal deafferentation injury can be debilitating, and this is especially true in primates when the hand is involved. Although significant recovery of function occurs, little is currently understood about the reorganization of the neuronal circuitry, particularly within the dorsal horn. This region receives primary afferent input from the periphery, and cortical input via the somatosensory subcomponent of the corticospinal tract (S1 CST), and is critically important in modulating sensory transmission, both in normal and lesioned states. To determine how dorsal horn circuitry alters to facilitate recovery post-injury, we used an established deafferentation lesion model (dorsal root/dorsal column) in male monkeys to remove sensory input from just the opposing digits (digits 1-3) of one hand. This results in a deficit in fine dexterity that recovers over several months. Electrophysiological mapping, tract tracing, and immunolabeling techniques were combined to delineate specific changes to dorsal horn input circuitry. Our main findings show that (1) there is complementary sprouting of the primary afferent and S1 CST populations into an overlapping region of the reorganizing dorsal horn; (2) S1 CST and primary afferent inputs connect in different ways within this region to facilitate sensory integration; and (3) there is a loss of larger S1 CST terminal boutons in the affected dorsal horn, but no change in the size profile of the spared/sprouted primary afferent terminal boutons post-lesion. Understanding such changes helps to inform new and targeted therapies that best promote recovery.SIGNIFICANCE STATEMENT Spinal injuries that remove sensation from the hand, can be debilitating, though functional recovery does occur. We examined changes to the neuronal circuitry of the dorsal horn in monkeys following a lesion that deafferented three digits of one hand. Little is understood about dorsal horn circuitry, despite the fact that this region loses most of its normal input after such an injury, and is clearly a major focus of reorganization. We found that both the spared primary afferents and somatosensory corticospinal efferents sprouted in an overlapping region of the dorsal horn after injury, and that larger (presumably faster) corticospinal terminals are lost, suggesting a significantly altered cortical modulation of primary afferents. Understanding this changing circuitry is important for designing targeted therapies.

Pupillary light reflex circuits in the macaque monkey: the preganglionic Edinger\u2013Westphal nucleus

The motor outflow for the pupillary light reflex originates in the preganglionic motoneuron subdivision of the Edinger–Westphal nucleus (EWpg), which also mediates lens accommodation. Despite their importance for vision, the morphology, ultrastructure and luminance-related inputs of these motoneurons have not been fully described in primates. In macaque monkeys, we labeled EWpg motoneurons from ciliary ganglion and orbital injections. Both approaches indicated preganglionic motoneurons occupy an EWpg organized as a unitary, ipsilateral cell column. When tracers were placed in the pretectal complex, labeled terminals targeted the ipsilateral EWpg and reached contralateral EWpg by crossing both above and below the cerebral aqueduct. They also terminated in the lateral visceral column, a ventrolateral periaqueductal gray region containing neurons projecting to the contralateral pretectum. Combining olivary pretectal and ciliary ganglion injections to determine whether a direct pupillary light reflex projection is present revealed a labeled motoneuron subpopulation that displayed close associations with labeled pretectal terminal boutons. Ultrastructurally, this subpopulation received synaptic contacts from labeled pretectal terminals that contained numerous clear spherical vesicles, suggesting excitation, and scattered dense-core vesicles, suggesting peptidergic co-transmitters. A variety of axon terminal classes, some of which may serve the near response, synapsed on preganglionic motoneurons. Quantitative analysis indicated that pupillary motoneurons receive more inhibitory inputs than lens motoneurons. To summarize, the pupillary light reflex circuit utilizes a monosynaptic, excitatory, bilateral pretectal projection to a distinct subpopulation of EWpg motoneurons. Furthermore, the interconnections between the lateral visceral column and olivary pretectal nucleus may provide pretectal cells with bilateral retinal fields.

Synaptic Specializations of Melanopsin-Retinal Ganglion Cells in Multiple Brain Regions Revealed by Genetic Label for Light and Electron Microscopy

The form and synaptic fine structure of melanopsin-expressing retinal ganglion cells, also called intrinsically photosensitive retinal ganglion cells (ipRGCs), were determined using a new membrane-targeted version of a genetic probe for correlated light and electron microscopy (CLEM). ipRGCs project to multiple brain regions, and because the method labels the entire neuron, it was possible to analyze nerve terminals in multiple retinorecipient brain regions, including the suprachiasmatic nucleus (SCN), olivary pretectal nucleus (OPN), and subregions of the lateral geniculate. Although ipRGCs provide the only direct retinal input to the OPN and SCN, ipRGC terminal arbors and boutons were found to be remarkably different in each target region. A network of dendro-dendritic chemical synapses (DDCSs) was also revealed in the SCN, with ipRGC axon terminals preferentially synapsing on the DDCS-linked cells. The methods developed to enable this analysis should propel other CLEM studies of long-distance brain circuits at high resolution.

Deletion of the \u03b1 subunit of the heterotrimeric Go protein impairs cerebellar cortical development in mice

Go is a member of the pertussis toxin-sensitive Gi/o family. Despite its abundance in the central nervous system, the precise role of Go remains largely unknown compared to other G proteins. In the present study, we explored the functions of Go in the developing cerebellar cortex by deleting its gene, Gnao. We performed a histological analysis with cerebellar sections of adult mice by cresyl violet- and immunostaining. Global deletion of Gnao induced cerebellar hypoplasia, reduced arborization of Purkinje cell dendrites, and atrophied Purkinje cell dendritic spines and the terminal boutons of climbing fibers from the inferior olivary nucleus. These results indicate that Go-mediated signaling pathway regulates maturation of presynaptic parallel fibers from granule cells and climbing fibers during the cerebellar cortical development.

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals.

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

Ultrastructure of the dorsal claustrum in cat. II. Synaptic organization

The claustrum is a bilateral subcortical nucleus situated between the insular cortex and the striatum in the brain of all mammals. It consists of two embryologically distinct subdivisions - dorsal and ventral claustrum. The claustrum has high connectivity with various areas of the cortex, subcortical and allocortical structures. It has long been suggested that the various claustral connections have different types of synaptic contacts at the claustral neurons. However, to the best of our knowledge, the literature data on the ultrastructural organization of the different types of synaptic contacts in the dorsal claustrum are very few. Therefore, the aim of our study was to observe and describe the synaptic organization of the dorsal claustrum in the cat. We used a total of 10 adult male cats and conducted an ultrastructural study under a transmission electron microscope as per established protocol. We described a multitude of dendritic spines, which were subdivided into two types - with and without foot processes. Based on the size and shape of the terminal boutons, the quantity and distribution of vesicles and the characteristic features of the active synaptic zone, we described six types of synaptic boutons, most of which formed asymmetrical synaptic contacts. Furthermore, we reported the presence of axo-dendritic, axo-somatic, dendro-dendritic and axo-axonal synapses. The former two likely represent the morphological substrate of the corticoclaustral pathway, while the remaining two types have the ultrastructural features of inhibitory synapses, likely forming a local inhibitory circuit in the claustrum. In conclusion, the present study shares new information about the neuropil of the claustrum and proposes a systematic classification of the types of synaptic boutons and contacts observed in the dorsal claustrum of the cat, thus supporting its key and complex role as a structure integrating various information within the brain.

Cocaine Directly Inhibits α6-Containing Nicotinic Acetylcholine Receptors in Human SH-EP1 Cells and Mouse VTA DA Neurons.

Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits α6N/α3Cβ2β3-nAChR (α6*-nAChRs) function. Human α6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (VTA) were used as a model of presynaptic α6*-nAChR activation since this method preserves terminal boutons. Patch-clamp recordings in whole-cell configuration were used to measure α6*-nAChR function as well as evaluate the effects of cocaine. In SH-EP1 cells containing heterologously expressed human α6*-nAChRs, cocaine inhibits nicotine-induced inward currents in a concentration-dependent manner with an IC50 value of 30 μM. Interestingly, in the presence of 30 μM cocaine, the maximal current response of the nicotine concentration-response curve is reduced without changing nicotine’s EC50 value, suggesting a noncompetitive mechanism. Furthermore, analysis of whole-cell current kinetics demonstrated that cocaine slows nAChR channel activation but accelerates whole-cell current decay time. Our findings demonstrate that cocaine-induced inhibition occurs solely with bath application, but not during intracellular administration, and this inhibition is not use-dependent. Additionally, in Xenopus oocytes, cocaine inhibits both α6N/α3Cβ2β3-nAChRs and α6M211L/α3ICβ2β3-nCAhRs similarly, suggesting that cocaine may not act on the α3 transmembrane domain of chimeric α6N/α3Cβ2β3-nAChR. In mechanically isolated VTA DA neurons, cocaine abolishes α6*-nAChR-mediated enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs). Collectively, these studies provide the first evidence that cocaine directly inhibits the function of both heterologously and naturally expressed α6*-nAChRs. These findings suggest that α6*-nAChRs may provide a novel pharmacological target mediating the effects of cocaine and may underlie a novel mechanism of cocaine reward and dependence.

Electron microscopic study of Golgi-impregnated and gold-toned neurons and fibers in the claustrum of the cat.

The claustrum is a subcortical nucleus found in the telencephalon of all placental mammals. It is a symmetrical, thin and irregular sheet of grey matter which lies between the inner surface of the insular cortex and the outer surface of the putamen. The claustrum has extensive connections with the visual, auditory, somatosensory and motor regions of the cortex, as well as with subcortical and allocortical regions. The aim of this study was to provide a detailed description of the morphology of different types of Golgi-impregnated and gold-toned neurons and fibers in the dorsal claustrum of the cat employing the combined Golgi-electron microscope Fairén method. We were able to distinguish two major types of neurons: those with dendritic spines (spiny) and those without dendritic spines (aspiny). In both groups we observed large (21-40µm in diameter), medium-sized (16-21µm in diameter) and small cells (10-16µm in diameter), describing their ultrastructural organization and characteristic features, including the presence of terminal boutons. These ultrastructural findings allow us to conclude that large and medium-sized spiny claustral neurons are indeed efferent neurons, projecting to the cortex, while the small spiny and the different types of aspiny neurons are most likely inhibitory local circuit interneurons. The findings in the present study will hopefully contribute to a better understanding of the role of the claustrum.

Nanotopography-Promoted Formation of Axon Collateral Branches of Hippocampal Neurons.

Axon collateral branches, as a key structural motif of neurons, allow neurons to integrate information from highly interconnected, divergent networks by establishing terminal boutons. Although physical cues are generally known to have a comprehensive range of effects on neuronal development, their involvement in axonal branching remains elusive. Herein, it is demonstrated that the nanopillar arrays significantly increase the number of axon collateral branches and also promote their growth. Immunostaining and biochemical analyses indicate that the physical interactions between the nanopillars and the neurons give rise to lateral filopodia at the axon shaft via cytoskeletal changes, leading to the formation of axonal branches. This report, demonstrates that nanotopography regulates axonal branching, and provides a guideline for the design of sophisticated neuron-based devices and scaffolds for neuro-engineering.

Terminal Arbors of Callosal Axons Undergo Plastic Changes in Early-Amputated Rats

Sensory information is processed in specific brain regions, and shared between the cerebral hemispheres by axons that cross the midline through the corpus callosum. However, sensory deprivation usually causes sensory losses and/or functional changes. This is the case of people who suffered limb amputation and show changes of body map organization within the somatosensory cortex (S1) of the deafferented cerebral hemisphere (contralateral to the amputated limb), as well as in the afferented hemisphere (ipsilateral to the amputated limb). Although several studies have approached these functional changes, the possible finer morphological alterations, such as those occurring in callosal axons, still remain unknown. The present work combined histochemistry, single-axon tracing and 3D microscopy to analyze the fine morphological changes that occur in callosal axons of the forepaw representation in early amputated rats. We showed that the forepaw representation in S1 was reduced in the deafferented hemisphere and expanded in the afferented side. Accordingly, after amputation, callosal axons originating from the deafferented cortex undergo an expansion of their terminal arbors with increased number of terminal boutons within the homotopic representation at the afferented cerebral hemisphere. Similar microscale structural changes may underpin the macroscale morphological and functional phenomena that characterize limb amputation in humans.

Corticobulbar projections from distinct motor cortical areas to the reticular formation in macaque monkeys.

Corticospinal and corticobulbar descending pathways act in parallel with brainstem systems, such as the reticulospinal tract, to ensure the control of voluntary movements via direct or indirect influences onto spinal motoneurons. The aim of this study was to investigate the corticobulbar projections from distinct motor cortical areas onto different nuclei of the reticular formation. Seven adult macaque monkeys were analysed for the location of corticobulbar axonal boutons, and one monkey for reticulospinal neurons' location. The anterograde tracer BDA was injected in the premotor cortex (PM), in the primary motor cortex (M1) or in the supplementary motor area (SMA), in 3, 3 and 1 monkeys respectively. BDA anterograde labelling of corticobulbar axons were analysed on brainstem histological sections and overlapped with adjacent Nissl-stained sections for cytoarchitecture. One adult monkey was analysed for retrograde CB tracer injected in C5-C8 hemispinal cord to visualise reticulospinal neurons. The corticobulbar axons formed bilateral terminal fields with boutons terminaux and en passant, which were quantified in various nuclei belonging to the Ponto-Medullary Reticular Formation (PMRF). The corticobulbar projections from both PM and SMA tended to end mainly ipsilaterally in PMRF, but contralaterally when originating from M1. Furthermore, the corticobulbar projection was less dense when originating from M1 than from non-primary motor areas (PM, SMA). The main nuclei of bouton terminals corresponded to the regions where reticulospinal neurons were located with CB retrograde tracing. In conclusion, the corticobulbar projection differs according to the motor cortical area of origin in density and laterality.

Neurofilaments and Neurofilament Proteins in Health and Disease.

SUMMARYNeurofilaments (NFs) are unique among tissue-specific classes of intermediate filaments (IFs) in being heteropolymers composed of four subunits (NF-L [neurofilament light]; NF-M [neurofilament middle]; NF-H [neurofilament heavy]; and α-internexin or peripherin), each having different domain structures and functions. Here, we review how NFs provide structural support for the highly asymmetric geometries of neurons and, especially, for the marked radial expansion of myelinated axons crucial for effective nerve conduction velocity. NFs in axons extensively cross-bridge and interconnect with other non-IF components of the cytoskeleton, including microtubules, actin filaments, and other fibrous cytoskeletal elements, to establish a regionally specialized network that undergoes exceptionally slow local turnover and serves as a docking platform to organize other organelles and proteins. We also discuss how a small pool of oligomeric and short filamentous precursors in the slow phase of axonal transport maintains this network. A complex pattern of phosphorylation and dephosphorylation events on each subunit modulates filament assembly, turnover, and organization within the axonal cytoskeleton. Multiple factors, and especially turnover rate, determine the size of the network, which can vary substantially along the axon. NF gene mutations cause several neuroaxonal disorders characterized by disrupted subunit assembly and NF aggregation. Additional NF alterations are associated with varied neuropsychiatric disorders. New evidence that subunits of NFs exist within postsynaptic terminal boutons and influence neurotransmission suggests how NF proteins might contribute to normal synaptic function and neuropsychiatric disease states.

GABAergic innervation of the ciliary ganglion in macaque monkeys – A light and electron microscopic study

AbstractThe vertebrate ciliary ganglion (CG) is a relay station in the parasympathetic pathway activating the iris sphincter and ciliary muscle to mediate pupillary constriction and lens accommodation, respectively. While the postganglionic motoneurons in the CG are cholinergic, as are their inputs, there is evidence from avian studies that GABA may also be involved. Here, we used light and electron microscopic methods to examine the GABAergic innervation of the CG in Macaca fascicularis monkeys. Immunohistochemistry for the gamma aminobutyric acid synthesizing enzyme glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) revealed that all CG neurons are contacted by ChAT‐positive terminals. A subpopulation of 17.5% of CG neurons was associated with terminal boutons expressing GAD‐immunoreactivity in addition. Double‐labeling for GAD and synaptophysin confirmed that these were synaptic terminals. Electron microscopic analysis in conjunction with GABA‐immunogold staining showed that (1) GAD‐positive terminals mainly target dendrites and spines in the perisomatic neuropil of CG neurons; (2) GABA is restricted to a specific terminal type, which displays intermediate features lying between classically excitatory and inhibitory endings; and (3) if a CG neuron is contacted by GABA‐positive terminals, virtually all perisomatic terminals supplying it show GABA immunoreactivity. The source of this GABAergic input and whether GABA contributes to a specific CG function remains to be investigated. Nevertheless, our data indicate that the innervation of the ciliary ganglion is more complex than previously thought, and that GABA may play a neuromodulatory role in the control of lens or pupil function. J. Comp. Neurol. 525:1–1, 2017. © 2016 Wiley Periodicals, Inc.

GABAergic innervation of the ciliary ganglion in macaque monkeys - A light and electron microscopic study.

The vertebrate ciliary ganglion (CG) is a relay station in the parasympathetic pathway activating the iris sphincter and ciliary muscle to mediate pupillary constriction and lens accommodation, respectively. While the postganglionic motoneurons in the CG are cholinergic, as are their inputs, there is evidence from avian studies that GABA may also be involved. Here, we used light and electron microscopic methods to examine the GABAergic innervation of the CG in Macaca fascicularis monkeys. Immunohistochemistry for the gamma aminobutyric acid synthesizing enzyme glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) revealed that all CG neurons are contacted by ChAT-positive terminals. A subpopulation of 17.5% of CG neurons was associated with terminal boutons expressing GAD-immunoreactivity in addition. Double-labeling for GAD and synaptophysin confirmed that these were synaptic terminals. Electron microscopic analysis in conjunction with GABA-immunogold staining showed that (1) GAD-positive terminals mainly target dendrites and spines in the perisomatic neuropil of CG neurons; (2) GABA is restricted to a specific terminal type, which displays intermediate features lying between classically excitatory and inhibitory endings; and (3) if a CG neuron is contacted by GABA-positive terminals, virtually all perisomatic terminals supplying it show GABA immunoreactivity. The source of this GABAergic input and whether GABA contributes to a specific CG function remains to be investigated. Nevertheless, our data indicate that the innervation of the ciliary ganglion is more complex than previously thought, and that GABA may play a neuromodulatory role in the control of lens or pupil function. J. Comp. Neurol. 525:1517-1531, 2017. © 2016 Wiley Periodicals, Inc.

Loss of Huntingtin stimulates capture of retrograde dense-core vesicles to increase synaptic neuropeptide stores

The Huntington’s disease protein Huntingtin (Htt) regulates axonal transport of dense-core vesicles (DCVs) containing neurotrophins and neuropeptides. DCVs travel down axons to reach nerve terminals where they are either captured in synaptic boutons to support later release or reverse direction to reenter the axon as part of vesicle circulation. Currently, the impact of Htt on DCV dynamics in the terminal is unknown. Here we report that knockout of Drosophila Htt selectively reduces retrograde DCV flux at proximal boutons of motoneuron terminals. However, initiation of retrograde transport at the most distal bouton and transport velocity are unaffected suggesting that synaptic capture rate of these retrograde DCVs could be altered. In fact, tracking DCVs shows that retrograde synaptic capture efficiency is significantly elevated by Htt knockout or knockdown. Furthermore, synaptic boutons contain more neuropeptide in Htt knockout larvae even though bouton size, single DCV fluorescence intensity, neuropeptide release in response to electrical stimulation and subsequent activity-dependent capture are unaffected. Thus, loss of Htt increases synaptic capture as DCVs travel by retrograde transport through boutons resulting in reduced transport toward the axon and increased neuropeptide in the terminal. These results therefore identify native Htt as a regulator of synaptic capture and neuropeptide storage.

Targeting Corticotropin-Releasing Factor Projections from the Oval Nucleus of the Bed Nucleus of the Stria Terminalis Using Cell-Type Specific Neuronal Tracing Studies in Mouse and Rat Brain.

The bed nucleus of the stria terminalis (BNST) is known to play a critical role in mediating the behavioural and autonomic responses to stressors. The oval nucleus of the BNST (BNSTov) contains cell bodies that synthesise the stress hormone corticotropin-releasing factor (CRF). Although afferent fibres originating from the BNSTov have been shown to innervate several key structures of the neuroendocrine and central autonomic system, the question remains as to whether some of these fibres are CRF-positive. To directly address this question, we injected a 'floxed' anterograde tracer (rAAV5/EF1a-DIO-mCherry) into the BNSTov of CRFp3.0CreGFP transgenic mice, which express a green fluorescent protein (GFP) under the control of the CRF promoter. Serial sections were then analysed for the presence of double-labelled fibres in potential projection sites. To determine whether CRF neurons in the rat BNSTov send comparable projections, we infused rat BNSTov with an adeno-associated viral vector (AAV) in which the human synapsin promoter drives enhanced GFP expression. We then used CRF immunoreactivity to examine double-labelled fluorescent fibres and axon terminals in projection sites from brain sections of the AAV-infused rats. We have observed several terminal fields in the mouse and rat brain with double-labelled fibres in the Dorsal raphe nucleus (DRD), the paraventricular nucleus of the hypothalamus and, to a lesser extent, in the ventral tegmental area. We found double-labelled terminal boutons in the nucleus accumbens shell, prelimbic cortex and posterior basolateral nucleus of the amygdala. The most intense double-labelling was found in midbrain, including substantia nigra pars compacta, red nucleus, periaqueductal grey and pontine nuclei, as well as DRD. The results of the present study indicate that CRF neurons are the output neurons of the BNSTov and they send projections not only to the centres of neuroendocrine and autonomic regulation, but also regions modulating reward and motivation, vigilance and motor function, as well as affective behaviour.

Connexions between the dorsomedial division of the ventromedial hypothalamus and the dorsal periaqueductal grey matter are critical in the elaboration of hypothalamically mediated panic-like behaviour

The electrical and chemical stimulation of the dorsal periaqueductal grey matter (dPAG) elicits panic-like explosive escape behaviour. Although neurons of the ventromedial hypothalamus (VMH) seem to organise oriented escape behaviour, when stimulated with excitatory amino acids at higher doses, non-oriented/explosive escape reactions can also be displayed. The aim of this work was to examine the importance of reciprocal projections between the VMH and the dPAG for the organisation of this panic-like behaviour. The chemical stimulation of the VMH with 9nmol of N-methyl-d-aspartic acid (NMDA) elicited oriented and non-oriented escape behaviours. The pretreatment of the dPAG with a non-selective blocker of synaptic contacts, cobalt chloride (CoCl2), followed by stimulation of the dorsomedial part of the ventromedial hypothalamus (dmVMH) with 9nmol of NMDA, abolished the non-oriented/explosive escape and freezing responses elicited by the stimulation of the dmVMH. Nonetheless, the rats still showed oriented escape to the burrow. On the other hand, when the blockade of the dmVMH with CoCl2 was followed by stimulation of the dPAG with 6nmol of NMDA, no effect was observed either on the non-oriented/explosive escape or on the freezing behaviour organised by the dPAG. Furthermore, Fos protein-labelled neurons were observed in the dPAG after the stimulation of the dmVMH with 9nmol of NMDA. Additionally, when the anterograde neurotracer biotinylated dextran amine (BDA) was deposited in the dmVMH subsequent stimulation of the dmVMH produced BDA-labelled neural fibres with terminal boutons surrounding Fos-labelled neurons in the dPAG, suggesting synaptic contacts between dmVMH and dPAG neurons for eliciting panic-like behavioural responses. The current data suggest that the dPAG is the key structure that organises non-oriented/explosive escape reactions associated with panic attack-like behaviours.