Axon Terminals Research Articles

Structural reorganization of medullary dorsal horn astrocytes in a rat model of neuropathic pain.

Multiple studies have shown that astrocytes in the medullary dorsal horn (MDH) play an important role in the development of pathologic pain. However, little is known about the structural reorganization of the peripheral astrocytic processes (PAP), the main functional part of the astrocyte, in MDH in neuropathic state. For this, we investigated the structural relationship between PAP and their adjacent presynaptic axon terminals and postsynaptic dendrites in the superficial laminae of the MDH using electron microscopical immunohistochemistry for ezrin, a marker for PAP, and quantitative analysis in a rat model of neuropathic pain following chronic constriction injury of the infraorbital nerve (CCI-ION). We found that, compared to controls, in rats with CCI-ION, (1) the number, % area, surface density, and volume fraction of ezrin-positive (+) PAP, as well as the fraction of synaptic edge apposed by ezrin + PAP and the degree of its coverage of presynaptic axon terminals and postsynaptic dendrites increased significantly, (2) these effects were abolished by administration of the mGluR5 antagonist 2-methyl-6-(phenylethynyl) pyridine (MPEP). These findings indicate that PAP undergoes structural reorganization around the central synapses of sensory afferents following nerve injury, suggest that it may be mediated by mGluR5, and may represent the structural basis for enhancing astrocyte-neuron interaction in neuropathic pain.

An initial HOPS-mediated fusion event is critical for autophagosome transport initiation from the axon terminal

ABSTRACT In neurons, macroautophagy/autophagy is a frequent and critical process. In the axon, autophagy begins in the axon terminal, where most nascent autophagosomes form. After formation, autophagosomes must initiate transport to exit the axon terminal and move toward the cell body via retrograde transport. During retrograde transport these autophagosomes mature through repetitive fusion events. Complete lysosomal cargo degradation occurs largely in the cell body. The precipitating events to stimulate retrograde autophagosome transport have been debated but their importance is clear: disrupting neuronal autophagy or autophagosome transport is detrimental to neuronal health and function. We have identified the HOPS complex as essential for early autophagosome maturation and consequent initiation of retrograde transport from the axon terminal. In yeast and mammalian cells, HOPS controls fusion between autophagosomes and late endosomes with lysosomes. Using zebrafish strains with loss-of-function mutations in vps18 and vps41, core components of the HOPS complex, we found that disruption of HOPS eliminates autophagosome maturation and disrupts retrograde autophagosome transport initiation from the axon terminal. We confirmed this phenotype was due to loss of HOPS complex formation using an endogenous deletion of the HOPS binding domain in Vps18. Finally, using pharmacological inhibition of lysosomal proteases, we show that initiation of autophagosome retrograde transport requires autophagosome maturation. Together, our data demonstrate that HOPS-mediated fusion events are critical for retrograde autophagosome transport initiation through promoting autophagosome maturation. This reveals critical roles for the HOPS complex in neuronal autophagy which deepens our understanding of the cellular pathology of HOPS-complex linked neurodegenerative diseases. Abbreviations: CORVET: Class C core vacuole/endosome tethering; gRNA: guide RNA; HOPS: homotypic fusion and protein sorting; pLL: posterior lateral line; Vps18: VPS18 core subunit of CORVET and HOPS complexes; Vps41: VPS41 subunit of HOPS complex.

Local protein synthesis at neuromuscular synapses is required for motor functions

Motor neurons are highly polarized, and their axons extend over great distances to form connections with myofibers via neuromuscular junctions (NMJs). Local translation at the NMJs in vivo has not been identified. Here, we utilized motor neuron-labeled RiboTag mice and the TRAP (translating ribosome affinity purification) technique to spatiotemporally profile the translatome at NMJs. We found that mRNAs associated with glucose catabolism, synaptic connection, and protein homeostasis are enriched at presynapses. Local translation at the synapse shifts from the assembly of cytoskeletal components during early developmental stages to energy production in adulthood. The mRNA of neuronal Agrin (Agrn), the key molecule for NMJ assembly, is present at motor axon terminals and locally translated. Disrupting the axonal location of Agrn mRNA causes impairment of synaptic transmission and motor functions in adult mice. Our findings indicate that spatiotemporal regulation of mRNA local translation at NMJs plays critical roles in synaptic transmission and motor functions invivo.

Genetic labeling of the nucleus of tractus solitarius neurons associated with electrical stimulation of the cervical or auricular vagus nerve in mice

IntroductionVagus nerve stimulation (VNS) is clinically useful for treating epilepsy, depression, and chronic pain. Currently, cervical VNS (cVNS) treatment is well-established, while auricular VNS (aVNS) is under development. Vagal stimulation regulates functions in diverse brain regions; therefore, it is critical to better understand how electrically-evoked vagal inputs following cVNS and aVNS engage with different brain regions. ObjectiveAs vagus inputs are predominantly transmitted to the nucleus of tractus solitarius (NTS), we directly compared the activation of NTS neurons by cVNS or aVNS and the brain regions directly projected by the activated NTS neurons in mice. MethodsWe adopted the targeted recombination in active populations method, which allows for the activity-dependent, tamoxifen-inducible expression of mCherry—a reporter protein—in neurons specifically associated with cVNS or aVNS. ResultscVNS and aVNS induced comparable bilateral mCherry expressions in neurons within the NTS, especially in its caudal section (cNTS). However, the numbers of mCherry-expressing neurons within different subdivisions of cNTS was distinctive. In both cVNS and aVNS, anterogradely labeled mCherry-expressing axonal terminals were similarly observed across different areas of the forebrain, midbrain, and hindbrain. These terminals were enriched in the rostral ventromedial medulla, parabrachial nucleus, periaqueductal gray, thalamic nuclei, central amygdala, and the hypothalamus. Sex difference of cVNS- and aVNS-induced labeling of NTS neurons was modest. ConclusionThe central projections of mCherry-expressing cNTS terminals are comparable between aVNS and cVNS, suggesting that cVNS and aVNS activate distinct but largely overlapping projections into the brain through the cNTS.

Ototoxicity-related changes in GABA immunolabeling within the rat inferior colliculus

Several studies suggest that hearing loss results in changes in the balance between inhibition and excitation in the inferior colliculus (IC). The IC is an integral nucleus within the auditory brainstem. The majority of ascending pathways from the lateral lemniscus (LL), superior olivary complex (SOC), and cochlear nucleus (CN) synapse in the IC before projecting to the thalamus and cortex. Many of these ascending projections provide inhibitory innervation to neurons within the IC. However, the nature and the distribution of this inhibitory input have only been partially elucidated in the rat. The inhibitory neurotransmitter, gamma aminobutyric acid (GABA), from the ventral nucleus of the lateral lemniscus (VNLL), provides the primary inhibitory input to the IC of the rat with GABA from other lemniscal and SOC nuclei providing lesser, but prominent innervation.There is evidence that hearing related conditions can result in dysfunction of IC neurons. These changes may be mediated in part by changes in GABA inputs to IC neurons. We have previously used gene micro-arrays in a study of deafness-related changes in gene expression in the IC and found significant changes in GAD as well as the GABA transporters and GABA receptors (Holt 2005). This is consistent with reports of age and trauma related changes in GABA (Bledsoe et al., 1995; Mossop et al., 2000; Salvi et al., 2000). Ototoxic lesions of the cochlea produced a permanent threshold shift. The number, intensity, and density of GABA positive axon terminals in the IC were compared in normal hearing and deafened rats. While the number of GABA immunolabeled puncta was only minimally different between groups, the intensity of labeling was significantly reduced. The ultrastructural localization and distribution of labeling was also examined. In deafened animals, the number of immuno gold particles was reduced by 78 % in axodendritic and 82 % in axosomatic GABAergic puncta. The affected puncta were primarily associated with small IC neurons. These results suggest that reduced inhibition to IC neurons contribute to the increased neuronal excitability observed in the IC following noise or drug induced hearing loss. Whether these deafness diminished inhibitory inputs originate from intrinsic or extrinsic CNIC sources awaits further study.

Homeostatic Shrinkage of Dendritic Spines Requires Melatonin Type 3 Receptor Activation During Sleep.

High-frequency oscillatory activity in cognition-related neural circuits during wakefulness consistently induces the growth of dendritic spines and axonal terminals. Although these structural changes are essential for cognitive functions, it is hypothesized that if these newly expanded structures fail to establish functional connections, they may become superfluous. Sleep is believed to facilitate the reduction of such redundant structures to maintain neural homeostasis. However, the mechanisms underlying this pruning process during sleep remain poorly understood. In this study, that melatonin type 3 receptors (MT3Rs) are selectively expressed in the stellate neurons of the medial entorhinal cortex (MEC) is demonstrated, an area where high melatonin levels are detected during sleep. Activation of MT3Rs during sleep initiates the shrinkage of dendritic spines in stellate neurons by downregulating neural network activity and dephosphorylating synaptic proteins in the MEC. This process is disrupted when MT3R expression is knocked down or when MT3Rs are blocked during sleep. Notably, interference with MT3Rs in the MEC during sleep impairs the acquisition of spatial memory but does not affect object memory acquisition following sleep. These findings reveal novel molecular mechanisms involving melatonin and MT3Rs in the regulation of dendritic spine shrinkage during sleep, which is crucial for the acquisition and consolidation of spatial memory.

H7N7 viral infection elicits pronounced, sex-specific neuroinflammatory responses in vitro.

Influenza A virus (IAV) infection can increase the risk of neuroinflammation, and subsequent neurodegenerative diseases. Certain IAV strains, such as avian H7N7 subtype, possess neurotropic properties, enabling them to directly invade the brain parenchyma and infect neurons and glia cells. Host sex significantly influences the severity of IAV infections. Studies indicate that females of the reproductive age exhibit stronger innate and adaptive immune responses to IAVs compared to males. This heightened immune response correlates with increased morbidity and mortality, and potential neuronal damage in females. Understanding the sex-specific neurotropism of IAV and associated mechanisms leading to adverse neurological outcomes is essential. Our study reveals that primary hippocampal cultures from female mice show heightened interferon-β and pro-inflammatory chemokine secretion following neurotropic IAV infection. We observed sex-specific differences in microglia activation: both sexes showed a transition into a hyper-ramified state, but only male-derived microglia exhibited an increase in amoeboid-shaped cells. These disparities extended to alterations in neuronal morphology. Neurons derived from female mice displayed increased spine density within 24 h post-infection, while no significant change was observed in male cultures. This aligns with sex-specific differences in microglial synaptic pruning. Data suggest that amoeboid-shaped microglia preferentially target postsynaptic terminals, potentially reducing neuronal hyperexcitability. Conversely, hyper-ramified microglia may focus on presynaptic terminals, potentially limiting viral spread. In conclusion, our findings underscore the utility of primary hippocampal cultures, incorporating microglia, as an effective model to study sex-specific, virus-induced effects on brain-resident cells.

Translation of monosynaptic circuits underlying amygdala fMRI neurofeedback training.

fMRI neurofeedback using autobiographical memory recall to upregulate the amygdala is associated with resting-state functional connectivity (rsFC) changes between the amygdala and the salience and default mode networks (SN and DMN, respectively). We hypothesize the existence of anatomical circuits underlying these rsFC changes. Using a cross-species brain parcellation, we identified in non-human primates locations homologous to the regions of interest (ROIs) from studies showing pre-to-post-neurofeedback changes in rsFC with the left amygdala. We injected bidirectional tracers in the basolateral, lateral, and central amygdala nuclei of adult macaques and used bright- and dark-field microscopy to identify cells and axon terminals in each ROI (SN: anterior cingulate, ventrolateral, and insular cortices; DMN: temporal pole, middle frontal gyrus, angular gyrus, precuneus, posterior cingulate cortex, parahippocampal gyrus, hippocampus, and thalamus). We also performed additional injections in specific ROIs to validate the results following amygdala injections and delineate potential disynaptic pathways. Finally, we used high-resolution diffusion MRI data from four post-mortem macaque brains and one in vivo human brain to translate our findings to the neuroimaging domain. Different amygdala nuclei had significant monosynaptic connections with all the SN and DMN ipsilateral ROIs. Amygdala connections with the DMN contralateral ROIs are disynaptic through the hippocampus and parahippocampal gyrus. Diffusion MRI in both species benefitted from using the ground-truth tracer data to validate its findings, as we identified false-negative ipsilateral and false-positive contralateral connectivity results. This study provides the foundation for future causal investigations of amygdala neurofeedback modulation of the SN and DMN through these anatomic connections.

Transsynaptic BMP Signaling Regulates Fine-Scale Topography between Adjacent Sensory Neurons.

Sensory axons projecting to the central nervous system are organized into topographic maps that represent the locations of sensory stimuli. In some sensory systems, even adjacent sensory axons are arranged topographically, forming "fine-scale" topographic maps. Although several broad molecular gradients are known to instruct coarse topography, we know little about the molecular signaling that regulates fine-scale topography at the level of two adjacent axons. Here, we provide evidence that transsynaptic bone morphogenetic protein (BMP) signaling mediates local interneuronal communication to regulate fine-scale topography in the nociceptive system of Drosophila larvae. We first show that the topographic separation of the axon terminals of adjacent nociceptors requires their common postsynaptic target, the A08n neurons. This phenotype is recapitulated by knockdown of the BMP ligand, Decapentaplegic (Dpp), in these neurons. In addition, removing the Type 2 BMP receptors or their effector (Mad transcription factor) in single nociceptors impairs the fine-scale topography, suggesting the contribution of BMP signaling originated from A08n. This signaling is likely mediated by phospho-Mad in the presynaptic terminals of nociceptors to ensure local interneuronal communication. Finally, reducing Dpp levels in A08n reduces the nociceptor-A08n synaptic contacts. Our data support that transsynaptic BMP signaling establishes the fine-scale topography by facilitating the formation of topographically correct synapses. Local BMP signaling for synapse formation may be a developmental strategy that independently regulates neighboring axon terminals for fine-scale topography.

Graded control of Purkinje cell outputs by cAMP through opposing actions on axonal action potential and transmitter release.

All-or-none signalling by action potentials (APs) in neuronal axons is pivotal for the precisely timed and identical size of outputs to multiple distant targets. However, technical limitations with respect to measuring the signalling in small intact axons have hindered the evaluation of high-fidelity signal propagation. Here, using direct recordings from axonal trunks and/or terminals of cerebellar Purkinje cells in slice and culture, we demonstrate that the timing and amplitude of axonal outputs are gradually modulated by cAMP depending on the length of axon. During the propagation in long axon, APs were attenuated and slowed in conduction by cAMP via specifically decreasing axonal Na+ currents. Consequently, the Ca2+ influx and transmitter release at distal boutons are reduced by cAMP, counteracting its direct facilitating effect on release machinery as observed at various CNS synapses. Together, our tour de force functional dissection has unveiled the axonal distance-dependent graded control of output timing and strength by intracellular signalling. KEY POINTS: The information processing in the nervous system has been classically thought to rely on the axonal faithful and high-speed conduction of action potentials (APs). We demonstrate that the strength and timing of axonal outputs are weakened and delayed, respectively, by cytoplasmic cAMP depending on the axonal length in cerebellar Purkinje cells (PCs). Direct axonal patch clamp recordings uncovered axon-specific attenuation of APs by cAMP through reduction of axonal Na+ currents. cAMP directly augments transmitter release at PC terminals without changing presynaptic Ca2+ influx or readily releasable pool of vesicles, although the extent is weaker compared to other CNS synapses. Two opposite actions of cAMP on PC axons, AP attenuation and release augmentation, together give rise to graded control of synaptic outputs in a manner dependent on the axonal length.

CLEM and Super-Resolution Microscopy Pipeline Demonstrate Single Co-Releasing GABA-Glutamate Axon Terminals Establishing Convergent Synapses for Glutamate or GABA Release in Mouse Brain

CLEM and Super-Resolution Microscopy Pipeline Demonstrate Single Co-Releasing GABA-Glutamate Axon Terminals Establishing Convergent Synapses for Glutamate or GABA Release in Mouse Brain

Astrocytes require perineuronal nets to maintain synaptic homeostasis in mice.

Perineuronal nets (PNNs) are densely packed extracellular matrices that cover the cell body of fast-spiking inhibitory neurons. PNNs stabilize synapses inhibiting synaptic plasticity. Here we show that synaptic terminals of fast-spiking interneurons localize to holes in the PNNs in the adult mouse somatosensory cortex. Approximately 95% of holes in the PNNs contain synapses and astrocytic processes expressing Kir4.1, glutamate and GABA transporters. Hence, holes in the PNNs contain tripartite synapses. In the adult mouse brain, PNN degradation causes an expanded astrocytic coverage of the neuronal somata without altering the axon terminals. The loss of PNNs impairs astrocytic transmitter and potassium uptake, resulting in the spillage of glutamate into the extrasynaptic space. Our data show that PNNs and astrocytes cooperate to contain synaptically released signals in physiological conditions. Their combined action is altered in mouse models of Alzheimer's disease and epilepsy where PNNs are disrupted.

Mistargeted retinal axons form synaptically segregated subcircuits in the visual thalamus of albino mice.

A Hebbian model of circuit remodeling predicts that two sets of inputs with sufficiently distinct activity patterns will synaptically capture separate sets of target cells. Mice in which a subset of retinal ganglion cells (RGCs) target the wrong region of the dorsal lateral geniculate nucleus (dLGN) provide the conditions for testing this prediction. In albino mice, mistargeted RGC axons form an island of terminals that is distinct from the surrounding neuropil. Blocking retinal activity during development prevents the formation of this island. However, the synaptic connectivity of the island was unknown. Here, we combine light and electron microscopy to determine if this activity-dependent island of axon terminals represent a synaptically segregated subcircuit. We reconstructed the microcircuitry of the boundary between the island and non-island RGCs and found a remarkably strong segregation within retinogeniculate connectivity. We conclude that, when sets of retinal input are established in the wrong part of the dLGN, the developing circuitry responds by forming a synaptically isolated subcircuit from the otherwise fully connected network. The fact that there is a developmental starting condition that can induce a synaptically segregated microcircuit has important implications for our understanding of the organization of visual circuits and for our understanding of the implementation of activity dependent development.

Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice.

The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor long form (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determined the role of glutamatergic neurotransmission from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces luteinizing hormone (LH) release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LeprCre (LepRb-Cre) mice. We collected blood sequentially before and for 1 hr after intravenous clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of Fos immunoreactive neurons in the PMv. Next, females with deletion of Slc17a6 (Vglut2) in LepRb neurons (LeprΔVGlut2) showed delayed age of puberty, disrupted estrous cycles, increased gonadotropin-releasing hormone (GnRH) concentration in the axon terminals, and disrupted LH secretion, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LeprloxTB) with concomitant deletion of Slc17a6 (Vglut2flox) mice. Rescue of Lepr and deletion of Slc17a6 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LeprloxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation, and became pregnant, while LeprloxTB;Vglut2flox mice showed no pubertal development. Our results indicate that glutamatergic neurotransmission from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.

Glutamate neurotransmission from leptin receptor cells is required for typical puberty and reproductive function in female mice

The hypothalamic ventral premammillary nucleus (PMv) is a glutamatergic nucleus essential for the metabolic control of reproduction. However, conditional deletion of leptin receptor long form (LepRb) in vesicular glutamate transporter 2 (Vglut2) expressing neurons results in virtually no reproductive deficits. In this study, we determined the role of glutamatergic neurotransmission from leptin responsive PMv neurons on puberty and fertility. We first assessed if stimulation of PMv neurons induces luteinizing hormone (LH) release in fed adult females. We used the stimulatory form of designer receptor exclusively activated by designer drugs (DREADDs) in LeprCre (LepRb-Cre) mice. We collected blood sequentially before and for 1 hr after intravenous clozapine-N-oxide injection. LH level increased in animals correctly targeted to the PMv, and LH level was correlated to the number of Fos immunoreactive neurons in the PMv. Next, females with deletion of Slc17a6 (Vglut2) in LepRb neurons (LeprΔVGlut2) showed delayed age of puberty, disrupted estrous cycles, increased gonadotropin-releasing hormone (GnRH) concentration in the axon terminals, and disrupted LH secretion, suggesting impaired GnRH release. To assess if glutamate is required for PMv actions in pubertal development, we generated a Cre-induced reexpression of endogenous LepRb (LeprloxTB) with concomitant deletion of Slc17a6 (Vglut2flox) mice. Rescue of Lepr and deletion of Slc17a6 in the PMv was obtained by stereotaxic injection of an adeno-associated virus vector expressing Cre recombinase. Control LeprloxTB mice with PMv LepRb rescue showed vaginal opening, follicle maturation, and became pregnant, while LeprloxTB;Vglut2flox mice showed no pubertal development. Our results indicate that glutamatergic neurotransmission from leptin sensitive neurons regulates the reproductive axis, and that leptin action on pubertal development via PMv neurons requires Vglut2.

Inhibitory Pedunculopontine Neurons Gate Dopamine-Mediated Motor Actions of Unsigned Valence.

The pedunculopontine nucleus (PPN) maintains a bidirectional connectivity with the basal ganglia that supports their shared roles in the selection and execution of motor actions. Previous studies identified a role for PPN neurons in goal-directed behavior, but the cellular substrates underlying this function have not been elucidated. We recently revealed the existence of a monosynaptic GABAergic input from the PPN that inhibits dopamine neurons of the substantia nigra. Activation of this pathway interferes with the execution of learned motor sequences when the actions are rewarded, even though the inhibition of dopamine neurons did not shift the value of the action, hence suggesting executive control over the gating of behavior. To test the attributes of the inhibition of dopamine neurons by the PPN in the context of goal-directed behavior regardless of whether the outcome is positively or negatively reinforced. We delivered optogenetic stimulation to PPN GABAergic axon terminals in the substantia nigra during a battery of behavioral tasks with positive and negative valence. Inhibition of dopamine neurons by PPN optogenetic activation during an appetitive task impaired the initiation and overall execution of the behavioral sequence without affecting the consumption of reward. During an active avoidance task, the same activation impaired the ability of mice to avoid a foot shock, but their escape response was unaffected. In addition, responses to potential threats were significantly attenuated. Our results show that PPN GABAergic neurons modulate learned, goal-directed behavior of unsigned valence without affecting overall motor behavior.

Where Do Core Thalamocortical Axons Terminate in Mammalian Neocortex When There Is No Cytoarchitecturally Distinct Layer 4?

Although the mammalian cerebral cortex is most often described as a hexalaminar structure, there are cortical areas (primary motor cortex) and species (elephants, cetaceans, and hippopotami), where a cytoarchitecturally indistinct, or absent, layer 4 is noted. Thalamocortical projections from the core, or first order, thalamic system terminate primarily in layers 4/inner 3. We explored the termination sites of core thalamocortical projections in cortical areas and in species where there is no cytoarchitecturally distinct layer 4 using the immunolocalization of vesicular glutamate transporter 2, a known marker of core thalamocortical axon terminals, in 31 mammal species spanning the eutherian radiation. Several variations from the canonical cortical column outline of layer 4 and core thalamocortical inputs were noted. In shrews/microchiropterans, layer 4 was present, but many core thalamocortical projections terminated in layer 1 in addition to layers 4 and inner 3. In primate primary visual cortex, the sublaminated layer 4 was associated with a specialized core thalamocortical projection pattern. In primate primary motor cortex, no cytoarchitecturally distinct layer 4 was evident and the core thalamocortical projections terminated throughout layer 3. In the African elephant, cetaceans, and river hippopotamus, no cytoarchitecturally distinct layer 4 was observed and core thalamocortical projections terminated primarily in inner layer 3 and less densely in outer layer 3. These findings are contextualized in terms of cortical processing, perception, and the evolutionary trajectory leading to an indistinct or absent cortical layer 4.

Isoforms of terminal selector Mamo control axon guidance during adult Drosophila memory center construction via Semaphorin-1a

In animals undergoing metamorphosis, the appearance of the nervous system is coincidently transformed by the morphogenesis of neurons. Such morphogenic alterations are exemplified in three types of intrinsic neurons in the Drosophila memory center. In contrast to the well-characterized remodeling of γ neurons, the morphogenesis of α/β and α’/β′ neurons has not been adequately explored. Here, we show that mamo, a BTB-zinc finger transcription factor that acts as a terminal selector for α’/β′ neurons, controls the formation of the correct axonal pattern of α’/β′ neurons. Intriguingly, specific Mamo isoforms are preferentially expressed in α’/β′ neurons to regulate the expression of axon guidance molecule Semaphorin-1a. This action directs proper axon guidance in α’/β′ neurons, which is also crucial for wiring of α’/β’ neurons with downstream neurons. Taken together, our results provide molecular insights into how neurons establish correct axonal patterns in circuitry assembly during adult memory center construction.

Immunohistochemistry and Morphometric Analysis of Drosophila Larval Body Wall Neuromuscular Junction Preparations.

The Drosophila neuromuscular junction (NMJ) is an excellent model for studying vertebrate glutamatergic synapses. Researchers have uncovered fundamental mechanisms at the fly NMJ that are conserved in higher-order organisms. To gain molecular and structural insight into these and other structures, immunolabeling is invaluable. In this protocol, we describe how to use immunolabeling to visualize embryonic/larval presynaptic and postsynaptic structures at the NMJ. We also include details about amplification of weak immunohistochemistry signals and how to use these signals to quantify synaptic growth via bouton counting. Boutons are bead-like structures at motor axon terminals that house synapses, and the number of boutons reflects the size of the NMJ. We also describe how to identify the different bouton types.

Early and selective localization of tau filaments to glutamatergic subcellular domains within the human anterodorsal thalamus

Widespread cortical accumulation of misfolded pathological tau proteins (ptau) in the form of paired helical filaments is a major hallmark of Alzheimer’s disease. Subcellular localization of ptau at various stages of disease progression is likely to be informative of the cellular mechanisms involving its spread. Here, we found that the density of ptau within several distinct rostral thalamic nuclei in post-mortem human tissue (n = 25 cases) increased with the disease stage, with the anterodorsal nucleus (ADn) consistently being the most affected. In the ADn, ptau-positive elements were present already in the pre-cortical (Braak 0) stage. Tau pathology preferentially affected the calretinin-expressing subpopulation of glutamatergic neurons in the ADn. At the subcellular level, we detected ptau immunoreactivity in ADn cell bodies, dendrites, and in a specialized type of presynaptic terminal that expresses vesicular glutamate transporter 2 (vGLUT2) and likely originates from the mammillary body. The ptau-containing terminals displayed signs of degeneration, including endosomal/lysosomal organelles. In contrast, corticothalamic axon terminals lacked ptau. The data demonstrate the involvement of a specific cell population in ADn at the onset of the disease. The presence of ptau in subcortical glutamatergic presynaptic terminals supports hypotheses about the transsynaptic spread of tau selectively affecting specialized axonal pathways.