Search

Superresolution microscopy (SM) techniques are among the revolutionary methods for molecular and cellular observations in the 21 st century. SM techniques overcome optical limitations, and several new observations using SM lead us to expect these techniques to have a large impact on neuroscience in the near future. Several types of SM have been developed, including structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), and photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM), each with special features. In this Minisymposium, experts in these different types of SM discuss the new structural and functional information about specific important molecules in neuroscience that has been gained with SM. Using these techniques, we have revealed novel mechanisms of endocytosis in nerve growth, fusion pore dynamics, and described quantitative new properties of excitatory and inhibitory synapses. Additional powerful techniques, including single molecule-guided Bayesian localization SM (SIMBA) and expansion microscopy (ExM), alone or combined with super-resolution observation, are also introduced in this session., (Copyright © 2018 the authors 0270-6474/18/389459-09$15.00/0.)

Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes a complex array of pathological events, through toxic gain of function mechanisms, leading to selective motor neuron degeneration. Mitochondrial dysfunction is among the well established toxic effects of mutant SOD1, but its mechanisms are just starting to be elucidated. A portion of mutant SOD1 is localized in mitochondria, where it accumulates mostly on the outer membrane and inside the intermembrane space (IMS). Evidence in cultured cells suggests that mutant SOD1 in the IMS causes mitochondrial dysfunction and compromises cell viability. Therefore, to test its pathogenic role in vivo we generated transgenic mice expressing G93A mutant or wild-type (WT) human SOD1 targeted selectively to the mitochondrial IMS (mito-SOD1). We show that mito-SOD1 is correctly localized in the IMS, where it oligomerizes and acquires enzymatic activity. Mito-G93ASOD1 mice, but not mito-WTSOD1 mice, develop a progressive disease characterized by body weight loss, muscle weakness, brain atrophy, and motor impairment, which is more severe in females. These symptoms are associated with reduced spinal motor neuron counts and impaired mitochondrial bioenergetics, characterized by decreased cytochrome oxidase activity and defective calcium handling. However, there is no evidence of muscle denervation, a cardinal pathological feature of ALS. Together, our findings indicate that mutant SOD1 in the mitochondrial IMS causes mitochondrial dysfunction and neurodegeneration, but per se it is not sufficient to cause a full-fledged ALS phenotype, which requires the participation of mutant SOD1 localized in other cellular compartments.

The frontal eye field (FEF) of the primate neocortex occupies a pivotal position in the matrix of inter-areal projections. In addition to its role in directing saccadic eye movements, it is the source of an attentional signal that modulates the activity of neurons in extrastriate and parietal cortex. Here, we tested the prediction that FEF preferentially excites inhibitory neurons in target areas during attentional modulation. Using the anterograde tracer biotinylated dextran amine, we found that the projections from FEF terminate in all cortical layers of area 46, lateral intraparietal area (LIP), and visual area V4. Axons in layer 1 spread extensively, those in layer 2/3 were most numerous, individual axons in layer 4 formed sprays of collaterals, and those of the deep layers were the finest caliber and irregular. All labeled synapses were the typical asymmetric morphology of excitatory synapses of pyramidal neurons. Dendritic spines were the most frequent synaptic target in all areas (95% in area 46, 89% in V4, 84% in LIP, 78% intrinsic local FEF). The remaining targets were one soma and dendritic shafts, most of which showed characteristics of inhibitory neurons with smooth dendrites (5% of all targets in area 46, 2% in V4, 9% in LIP, and 13% in FEF).

The cerebellum influences behavior and cognition exclusively via Purkinje cell synapses onto neurons in the deep cerebellar and vestibular nuclei. In contrast with the rich information available about the organization of the cerebellar cortex and its synaptic inputs, relatively little is known about microcircuitry postsynaptic to Purkinje cells. Here we examined the cell types and microcircuits through which Purkinje cells influence an oculomotor behavior controlled by the cerebellum, the horizontal vestibulo-ocular reflex, which involves only two eye muscles. Using a combination of anatomical tracing and electrophysiological recordings in transgenic mouse lines, we identified several classes of neurons in the medial vestibular nucleus that receive Purkinje cell synapses from the cerebellar flocculus. Glycinergic and glutamatergic flocculus target neurons (FTNs) with somata densely surrounded by Purkinje cell terminals projected axons to the ipsilateral abducens and oculomotor nuclei, respectively. Of three additional types of FTNs that were sparsely innervated by Purkinje cells, glutamatergic and glycinergic neurons projected to the contralateral and ipsilateral abducens, respectively, and GABAergic neurons projected to contralateral vestibular nuclei. Densely innervated FTNs had high spontaneous firing rates and pronounced postinhibitory rebound firing, and were physiologically homogeneous, whereas the intrinsic excitability of sparsely innervated FTNs varied widely. Heterogeneity in the molecular expression, physiological properties, and postsynaptic targets of FTNs implies that Purkinje cell activity influences the neural control of eye movements in several distinct ways. These results indicate that the cerebellum regulates a simple reflex behavior via at least five different cell types that are postsynaptic to Purkinje cells.

The ability of the olfactory epithelium (OE) to regenerate after injury is mediated by at least two populations of presumed stem cells-globose basal cells (GBCs) and horizontal basal cells (HBCs). Of the two, GBCs are molecularly and phenotypically analogous to the olfactory progenitors of the embryonic placode (OPPs). In contrast, HBCs are a reserve stem cell population that appears later in development and requires activation by severe epithelial damage before contributing to epithelial reconstitution. Neither HBC emergence nor the mechanism of activation after injury is understood. Here we show that the transcription factor p63 (Trp63), which is expressed selectively by adult HBCs, is required for HBC differentiation. The first evidence of HBC differentiation is the expression of p63 by cells that closely resemble embryonic OPPs and adult GBCs by morphology and expression of the transcription factors Sox2, Ascl1, and Hes1. HBC formation is delayed in Ascl1 knock-out OE and is completely abrogated in p63-null mice. Strikingly, other cell types of the OE form normally in the p63 knock-out OE. The role of p63 in HBC differentiation appears to be conserved in the regenerating rat OE, where HBCs disappear and then reappear after tissue lesion. Finally, p63 protein is downregulated in HBCs activated by lesion to become multipotent progenitor cells. Together, our data identify a novel mechanism for the generation of a reserve stem cell population and suggest that a p63-dependent molecular switch is responsible for activating reserve stem cells when they are needed.

PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNA interference knockdown of PSD-95 leads to loss of entire patches of PSD material, and electron microscopy tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA receptor-type, but not NMDA receptor-type, structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three-dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.

Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Such degeneration is not a passive event but rather an active process mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigrostriatal projection that a constitutively active form of the kinase, myristoylated Akt (Myr-Akt), demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here, we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve dopaminergic axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mammalian target of rapamycin (mTor) activity and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.

Acyl-CoA synthetase long-chain family member 4 (ACSL4) converts long-chain fatty acids to acyl-CoAs that are indispensable for lipid metabolism and cell signaling. Mutations in ACSL4 cause nonsyndromic X-linked mental retardation. We previously demonstrated that Drosophila dAcsl is functionally homologous to human ACSL4, and is required for axonal targeting in the brain. Here, we report that Drosophila dAcsl mutants exhibited distally biased axonal aggregates that were immunopositive for the synaptic-vesicle proteins synaptotagmin (Syt) and cysteine-string protein, the late endosome/lysosome marker lysosome-associated membrane protein 1, the autophagosomal marker Atg8, and the multivesicular body marker Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate). In contrast, the axonal distribution of mitochondria and the cell adhesion molecule Fas II (fasciclin II) was normal. Electron microscopy revealed accumulation of prelysomes and multivesicle bodies. These aggregates appear as retrograde instead of anterograde cargos. Live imaging analysis revealed that dAcsl mutations increased the velocity of anterograde transport but reduced the flux, velocity, and processivity of retrograde transport of Syt-enhanced green fluorescent protein-labeled vesicles. Immunohistochemical and electrophysiological analyses showed significantly reduced growth and stability of neuromuscular synapses, and impaired glutamatergic neurotransmission in dAcsl mutants. The axonal aggregates and synaptic defects in dAcsl mutants were fully rescued by neuronal expression of human ACSL4, supporting a functional conservation of ACSL4 across species in the nervous system. Together, our findings demonstrate that dAcsl regulates axonal transport of synaptic vesicles and is required for synaptic development and function. Defects in axonal transport and synaptic function may account, at least in part, for the pathogenesis of ACSL4-related mental retardation.

The neurobiological underpinnings of mood and anxiety disorders have been linked to the nucleus accumbens (NAc), a region important in processing the rewarding and emotional salience of stimuli. Using chronic social defeat stress, an animal model of mood and anxiety disorders, we investigated whether alterations in synaptic plasticity are responsible for the long-lasting behavioral symptoms induced by this form of stress. We hypothesized that chronic social defeat stress alters synaptic strength or connectivity of medium spiny neurons (MSNs) in the NAc to induce social avoidance. To test this, we analyzed the synaptic profile of MSNs via confocal imaging of Lucifer-yellow-filled cells, ultrastructural analysis of the postsynaptic density, and electrophysiological recordings of miniature EPSCs (mEPSCs) in mice after social defeat. We found that NAc MSNs have more stubby spine structures with smaller postsynaptic densities and an increase in the frequency of mEPSCs after social defeat. In parallel to these structural changes, we observed significant increases in IκB kinase (IKK) in the NAc after social defeat, a molecular pathway that has been shown to regulate neuronal morphology. Indeed, we find using viral-mediated gene transfer of dominant-negative and constitutively active IKK mutants that activation of IKK signaling pathways during social defeat is both necessary and sufficient to induce synaptic alterations and behavioral effects of the stress. These studies establish a causal role for IKK in regulating stress-induced adaptive plasticity and may present a novel target for drug development in the treatment of mood and anxiety disorders in humans.

Formation and stabilization of postsynaptic glycine receptor (GlyR) clusters result from their association with the polymerized scaffold protein gephyrin. At the cell surface, lateral diffusion and local trapping of GlyR by synaptic gephyrin clusters is one of the main factors controlling their number. However, the mechanisms regulating gephyrin/GlyR cluster sizes are not fully understood. To identify molecular binding partners able to control gephyrin cluster stability, we performed pull-down assays with full-length or truncated gephyrin forms incubated in a rat spinal cord extract, combined with mass spectrometric analysis. We found that heat shock cognate protein 70 (Hsc70), a constitutive member of the heat shock protein 70 (Hsp70) family, selectively binds to the gephyrin G-domain. Immunoelectron microscopy of mouse spinal cord sections showed that Hsc70 could be colocalized with gephyrin at inhibitory synapses. Furthermore, ternary Hsc70-gephyrin-GlyR coclusters were formed following transfection of COS-7 cells. Upon overexpression of Hsc70 in mouse spinal cord neurons, synaptic accumulation of gephyrin was significantly decreased, but GlyR amounts were unaffected. In the same way, Hsc70 inhibition increased gephyrin accumulation at inhibitory synapses without modifying GlyR clustering. Single particle tracking experiments revealed that the increase of gephyrin molecules reduced GlyR diffusion rates without altering GlyR residency at synapses. Our findings demonstrate that Hsc70 regulates gephyrin polymerization independently of its interaction with GlyR. Therefore, gephyrin polymerization and synaptic clustering of GlyR are uncoupled events.

Spinal cord injuries lead to impairments, which are accompanied by extensive reorganization of neuronal circuits caudal to the injury. Locomotor training can aid in the functional recovery after injury, but the neuronal mechanisms associated with such plasticity are only sparsely known. We investigated ultrastructurally the synaptic inputs to tibialis anterior motoneurons (MNs) retrogradely labeled in adult rats that had received a complete midthoracic spinal cord transection at postnatal day 5. A subset of the injured rats received locomotor training. Both γ- and α-MNs were studied. The total number of boutons apposing γ-MNs, but not α-MNs, was reduced after neonatal spinal cord transection. The proportion of inhibitory to excitatory boutons, however, was increased significantly in both α-MNs and γ-MNs in spinally transected rats, but with locomotor training returned to levels observed in intact rats. The specific densities and compositions of synaptic boutons were, however, different between all three groups. Surprisingly, we observed the atypical presence of both C- and M-type boutons apposing the somata of γ-MNs in the spinal rats, regardless of training status. We conclude that a neonatal spinal cord transection induces significant reorganization of synaptic inputs to spinal motoneurons caudal to the site of injury with a net increase in inhibitory influence, which is associated with poor stepping. Spinal cord injury followed by successful locomotor training, however, results in improved bipedal stepping and further synaptic changes with the proportion of inhibitory and excitatory inputs to the motoneurons being similar to that observed in intact rats.

Aggregation of amyloid-beta (Abeta) and Tau protein are hallmarks of Alzheimer's disease (AD), and according to the Abeta-cascade hypothesis, Abeta is considered toxic for neurons and Tau a downstream target of Abeta. We have investigated differentiated primary hippocampal neurons for early localized changes following exposure to Abeta oligomers. Initial events become evident by missorting of endogenous Tau into the somatodendritic compartment, in contrast to axonal sorting in normal neurons. In missorted dendritic regions there is a depletion of spines and local increase in Ca(2+), and breakdown of microtubules. Tau in these regions shows elevated phosphorylation at certain sites diagnostic of AD-Tau (e.g., epitope of antibody 12E8, whose phosphorylation causes detachment of Tau from microtubules, and AT8 epitope), and local elevation of certain kinase activities (e.g., MARK/par-1, BRSK/SADK, p70S6K, cdk5, but not GSK3beta, JNK, MAPK). These local effects occur without global changes in Tau, tubulin, or kinase levels. Somatodendritic missorting occurs not only with Tau, but also with other axonal proteins such as neurofilaments, and correlates with pronounced depletion of microtubules and mitochondria. The Abeta-induced effects on microtubule and mitochondria depletion, Tau missorting, and loss of spines are prevented by taxol, indicating that Abeta-induced microtubule destabilization and corresponding traffic defects are key factors in incipient degeneration. By contrast, the rise in Ca(2+) levels, kinase activities, and Tau phosphorylation cannot be prevented by taxol. Incipient and local changes similar to those of Abeta oligomers can be evoked by cell stressors (e.g., H(2)O(2), glutamate, serum deprivation), suggesting some common mechanism of signaling.

Chronic intermittent hypoxia (CIH) is a concomitant of sleep apnea that produces a slowly developing chemosensory-dependent blood pressure elevation ascribed in part to NMDA receptor-dependent plasticity and reduced nitric oxide (NO) signaling in the carotid body. The hypothalamic paraventricular nucleus (PVN) is responsive to hypoxic stress and also contains neurons that express NMDA receptors and neuronal nitric oxide synthase (nNOS). We tested the hypothesis that extended (35 d) CIH results in a decrease in the surface/synaptic availability of the essential NMDA NR1 subunit in nNOS-containing neurons and NMDA-induced NO production in the PVN of mice. As compared with controls, the 35 d CIH-exposed mice showed a significant increase in blood pressure and an increased density of NR1 immunogold particles located in the cytoplasm of nNOS-containing dendrites. Neither of these between-group differences was seen after 14 d, even though there was already a reduction in the NR1 plasmalemmal density at this time point. Patch-clamp recording of PVN neurons in slices showed a significant reduction in NMDA currents after either 14 or 35 d exposure to CIH compared with sham controls. In contrast, NO production, as measured by the NO-sensitive fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein, was suppressed only in the 35 d CIH group. We conclude that CIH produces a reduction in the surface/synaptic targeting of NR1 in nNOS neurons and decreases NMDA receptor-mediated currents in the PVN before the emergence of hypertension, the development of which may be enabled by suppression of NO signaling in this brain region.

Astroglia secrete factors that promote synapse formation and maintenance. In culture, glial contact has also been shown to facilitate synaptogenesis. Here, we examined whether glial contact is important for establishing circuits in vivo by simultaneously monitoring differentiation of glial cells and local synaptogenesis over time. Photoreceptor circuits of the vertebrate retina are particularly suitable for this study because of the relatively simple, laminar organization of their connectivity with their target neurons, horizontal cells and bipolar cells. Also, individual photoreceptor terminals are ensheathed within the outer plexiform layer (OPL) by the processes of one type of glia, Müller glia cells (MGs). We conducted in vivo time-lapse multiphoton imaging of the rapidly developing and relatively transparent zebrafish retina to ascertain the time course of MG development relative to OPL synaptogenesis. The emergence of synaptic triads, indicative of functional photoreceptor circuits, and structural association with glial processes were also examined across ages by electron microscopy. We first show that MG processes form territories that tile within the inner and outer synaptic layers. We then demonstrate that cone photoreceptor synapses are assembled before the elaboration of MG processes in the OPL. Using a targeted cell ablation approach, we also determined whether the maintenance of photoreceptor synapses is perturbed when local MGs are absent. We found that removal of MGs had no appreciable effect on the stability of newly formed cone synapses. Thus, in contrast to other CNS circuits, contact from glia is not necessary for the formation or immediate stabilization of outer retinal synapses.

Recent studies have demonstrated protective roles for autophagy in various neurodegenerative disorders, including the polyglutamine diseases; however, the role of autophagy in retinal degeneration has remained unclear. Accumulation of activated rhodopsin in some Drosophila mutants leads to retinal degeneration, and although it is known that activated rhodopsin is degraded in endosomal pathways in normal photoreceptor cells, the contribution of autophagy to rhodopsin regulation has remained elusive. This study reveals that activated rhodopsin is degraded by autophagy in collaboration with endosomal pathways to prevent retinal degeneration. Light-dependent retinal degeneration in the Drosophila visual system is caused by the knockdown or mutation of autophagy-essential components, such as autophagy-related protein 7 and 8 (atg-7/atg-8), or genes essential for PE (phosphatidylethanolamine) biogenesis and autophagosome formation, including Phosphatidylserine decarboxylase (Psd) and CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (Ept). The knockdown of atg-7/8 or Psd/Ept produced an increase in the amount of rhodopsin localized to Rab7-positive late endosomes. This rhodopsin accumulation, followed by retinal degeneration, was suppressed by overexpression of Rab7, which accelerated the endosomal degradation pathway. These results indicate a degree of cross talk between the autophagic and endosomal/lysosomal pathways. Importantly, a reduction in rhodopsin levels rescued Psd knockdown-induced retinal degeneration. Additionally, the Psd knockdown-induced retinal degeneration phenotype was enhanced by Ppt1 inactivation, which causes infantile neuronal ceroid lipofuscinosis, implying that autophagy plays a significant role in its pathogenesis. Collectively, the current data reveal that autophagy suppresses light-dependent retinal degeneration in collaboration with the endosomal degradation pathway and that rhodopsin is a key substrate for autophagic degradation in this context.

Transactivation response DNA-binding protein 43 (TDP-43) is a principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). Mutations in TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, yet multiple neurodegenerative diseases exhibit TDP-43 pathology without known TARDBP mutations. While TDP-43 has been ascribed a number of roles in normal biology, including mRNA splicing and transcription regulation, elucidating disease mechanisms associated with this protein is hindered by the lack of models to dissect such functions. We have generated transgenic (TDP-43PrP) mice expressing full-length human TDP-43 (hTDP-43) driven by the mouse prion promoter to provide a tool to analyze the role of wild-type hTDP-43 in the brain and spinal cord. Expression of hTDP-43 caused a dose-dependent downregulation of mouse TDP-43 RNA and protein. Moderate overexpression of hTDP-43 resulted in TDP-43 truncation, increased cytoplasmic and nuclear ubiquitin levels, and intranuclear and cytoplasmic aggregates that were immunopositive for phosphorylated TDP-43. Of note, abnormal juxtanuclear aggregates of mitochondria were observed, accompanied by enhanced levels of Fis1 and phosphorylated DLP1, key components of the mitochondrial fission machinery. Conversely, a marked reduction in mitofusin 1 expression, which plays an essential role in mitochondrial fusion, was observed in TDP-43PrP mice. Finally, TDP-43PrP mice showed reactive gliosis, axonal and myelin degeneration, gait abnormalities, and early lethality. This TDP-43 transgenic line provides a valuable tool for identifying potential roles of wild-type TDP-43 within the CNS and for studying TDP-43-associated neurotoxicity.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS and remyelination in MS ultimately fails. Although strategies to promote myelin repair are eagerly sought, mechanisms underlying remyelination in vivo have been elusive. CXCR2 is expressed on neutrophils and oligodendrocyte lineage cells in the CNS. CXCR2-positive neutrophils facilitate inflammatory demyelination in demyelination models such as experimental autoimmune encephalomyelitis (EAE) and cuprizone intoxication. Systemic injection of a small molecule CXCR2 antagonist at the onset of EAE decreased demyelinated lesions. These results left the cellular target of the CXCR2 antagonist uncertain and did not clarify whether CXCR2 blockade prevented demyelination or promoted remyelination. Here, we show that the actions of CXCR2 on nonhematopoietic cells unexpectedly delay myelin repair. Bone marrow chimeric mice (Cxcr2(+/-)-->Cxcr2(-/-) and Cxcr2(+/-)-->Cxcr2(+/+)) were subjected to two distinct models of myelin injury. In all cases, myelin repair was more efficient in Cxcr2(+/-)-->Cxcr2(-/-) animals. Oligodendrocyte progenitor cells (OPCs) in demyelinated lesions of Cxcr2(+/-)-->Cxcr2(-/-) mice proliferated earlier and more vigorously than in tissues from Cxcr2(+/-)--> Cxcr2(+/+) animals. In vitro demyelinated CNS slice cultures also showed better myelin repair when CXCR2 was blocked with neutralizing antibodies or was genetically deleted. Our results suggest that CXCR2 inactivation permits optimal spatiotemporal positioning of OPCs in demyelinating lesions to receive local proliferative and differentiating signals. Given that CXCR2 exerts dual functions that promote demyelination and decrease remyelination by actions toward hematopoietic cells and nonhematopoietic cells, respectively, our findings identify CXCR2 as a promising drug target for clinical demyelinating disorders.

Multiple synaptic vesicle (SV) retrieval modes exist in central nerve terminals to maintain a continual supply of SVs for neurotransmission. Two such modes are clathrin-mediated endocytosis (CME), which is dominant during mild neuronal activity, and activity-dependent bulk endocytosis (ADBE), which is dominant during intense neuronal activity. However, little is known about how activation of these SV retrieval modes impact the replenishment of the total SV recycling pool and the pools that reside within it, the readily releasable pool (RRP) and reserve pool. To address this question, we examined the replenishment of all three SV pools by triggering these SV retrieval modes during both high- and low-intensity stimulation of primary rat neuronal cultures. SVs generated by CME and ADBE were differentially labeled using the dyes FM1-43 and FM2-10, and their replenishment of specific SV pools was quantified using stimulation protocols that selectively depleted each pool. Our studies indicate that while the RRP was replenished by CME-generated SVs, ADBE provided additional SVs to increase the capacity of the reserve pool. Morphological analysis of the uptake of the fluid phase marker horseradish peroxidase corroborated these findings. The differential replenishment of specific SV pools by independent SV retrieval modes illustrates how previously experienced neuronal activity impacts the capability of central nerve terminals to respond to future stimuli.

Several neurodegenerative diseases are typified by intraneuronal alpha-synuclein deposits, synaptic dysfunction, and dementia. While even modest alpha-synuclein elevations can be pathologic, the precise cascade of events induced by excessive alpha-synuclein and eventually culminating in synaptotoxicity is unclear. To elucidate this, we developed a quantitative model system to evaluate evolving alpha-synuclein-induced pathologic events with high spatial and temporal resolution, using cultured neurons from brains of transgenic mice overexpressing fluorescent-human-alpha-synuclein. Transgenic alpha-synuclein was pathologically altered over time and overexpressing neurons showed striking neurotransmitter release deficits and enlarged synaptic vesicles; a phenotype reminiscent of previous animal models lacking critical presynaptic proteins. Indeed, several endogenous presynaptic proteins involved in exocytosis and endocytosis were undetectable in a subset of transgenic boutons ("vacant synapses") with diminished levels in the remainder, suggesting that such diminutions were triggering the overall synaptic pathology. Similar synaptic protein alterations were also retrospectively seen in human pathologic brains, highlighting potential relevance to human disease. Collectively the data suggest a previously unknown cascade of events where pathologic alpha-synuclein leads to a loss of a number of critical presynaptic proteins, thereby inducing functional synaptic deficits.

A fundamental property of neuronal networks in Ammon's horn is that each area comprises a single glutamatergic cell population and various types of GABAergic neurons. Here we describe an exception to this rule, in the form of granule cells that reside within the CA3 area and function as glutamatergic nonprincipal cells with distinct properties. CA3 granule cells in normal, healthy rats, similarly to dentate gyrus granule cells, coexpressed calbindin and the homeobox protein Prox1. However, CA3 granule cells were located outside of the dentate gyrus, often hundreds of micrometers from the hilar border, in the lucidum and radiatum layers. CA3 granule cells were present in numbers that were comparable to the rarer GABAergic neuronal subtypes, and their somato-dendritic morphology, intrinsic properties, and perforant path inputs were similar to those of dentate gyrus granule cells. CA3 granule cell axons displayed giant mossy fiber terminals with filopodial extensions, demonstrating that not all mossy fibers originate from the dentate gyrus. Somatic paired recordings revealed that CA3 granule cells innervated CA3 pyramidal and GABAergic cells similarly to conventional mossy fiber synapses. However, CA3 granule cells were distinct in the specific organization of their GABAergic inputs. They received GABAergic synapses from cholecystokinin-expressing mossy fiber-associated cells that did not innervate the dentate granule cell layer, and these synapses demonstrated unusually strong activity-dependent endocannabinoid-mediated inhibition of GABA release. These results indicate that granule cells in the CA3 constitute a glutamatergic, nonprincipal neuronal subtype that is integrated into the CA3 synaptic network.

MicroRNAs, by modulating gene expression, have been implicated as regulators of various cellular and physiological processes, including differentiation, proliferation, and cancer. Here, we study the role of microRNAs in Schwann cell (SC) differentiation by conditional removal of the microRNA processing enzyme Dicer1. We reveal that both male and female mice lacking Dicer1 in SC (Dicer1 conditional knock-outs) display a severe neurological phenotype resembling congenital hypomyelination. Ultrastructural analyses show that many SC lacking Dicer1 are stalled in differentiation at the promyelinating state and fail to myelinate axons. Gene expression analyses reveal a failure to extinguish genes characteristic of the undifferentiated state such as Sox2, Jun, and Ccnd1. Sox2 and Jun are well characterized negative regulators of SC differentiation. Consistent with Sox2/Jun maintenance, Egr2, a master regulator of the myelinating program, is drastically downregulated and likely accounts for the myelination defect. We posit a model wherein microRNAs are critical for downregulation of antecedent programs of gene expression. In SC differentiation, this is particularly relevant in the key developmental transition from a promyelinating to myelinating SC.

The segregation between vesicular glutamate and GABA storage and release forms the molecular foundation between excitatory and inhibitory neurons and guarantees the precise function of neuronal networks. Using immunoisolation of synaptic vesicles, we now show that VGLUT2 and VGAT, and also VGLUT1 and VGLUT2, coexist in a sizeable pool of vesicles. VGAT immunoisolates transport glutamate in addition to GABA. Furthermore, VGLUT activity enhances uptake of GABA and monoamines. Postembedding immunogold double labeling revealed that VGLUT1, VGLUT2, and VGAT coexist in mossy fiber terminals of the hippocampal CA3 area. Similarly, cerebellar mossy fiber terminals harbor VGLUT1, VGLUT2, and VGAT, while parallel and climbing fiber terminals exclusively contain VGLUT1 or VGLUT2, respectively. VGLUT2 was also observed in cerebellar GABAergic basket cells terminals. We conclude that the synaptic coexistence of vesicular glutamate and GABA transporters allows for corelease of both glutamate and GABA from selected nerve terminals, which may prevent systemic overexcitability by downregulating synaptic activity. Furthermore, our data suggest that VGLUT enhances transmitter storage in nonglutamatergic neurons. Thus, synaptic and vesicular coexistence of VGLUT and VGAT is more widespread than previously anticipated, putatively influencing fine-tuning and control of synaptic plasticity.

Age-associated memory impairment (AAMI) occurs in many mammalian species, including humans. In contrast to Alzheimer's disease (AD), in which circuit disruption occurs through neuron death, AAMI is due to circuit and synapse disruption in the absence of significant neuron loss and thus may be more amenable to prevention or treatment. We have investigated the effects of aging on pyramidal neurons and synapse density in layer III of area 46 in dorsolateral prefrontal cortex of young and aged, male and female rhesus monkeys (Macaca mulatta) that were tested for cognitive status through the delayed non-matching-to-sample (DNMS) and delayed response tasks. Cognitive tests revealed an age-related decrement in both acquisition and performance on DNMS. Our morphometric analyses revealed both an age-related loss of spines (33%, p < 0.05) on pyramidal cells and decreased density of axospinous synapses (32%, p < 0.01) in layer III of area 46. In addition, there was an age-related shift in the distribution of spine types reflecting a selective vulnerability of small, thin spines, thought to be particularly plastic and linked to learning. While both synapse density and the overall spine size average of an animal were predictive of number of trials required for acquisition of DNMS (i.e., learning the task), the strongest correlate of behavior was found to be the head volume of thin spines, with no correlation between behavior and mushroom spine size or density. No synaptic index correlated with memory performance once the task was learned.

The continuous release of neurotransmitter could be seen to place a persistent burden on presynaptic proteins, one that could compromise nerve terminal function. This supposition and the molecular mechanisms that might protect highly active synapses merit investigation. In hippocampal cultures from knock-out mice lacking the presynaptic cochaperone cysteine string protein-alpha (CSP-alpha), we observe progressive degeneration of highly active synaptotagmin 2 (Syt2)-expressing GABAergic synapses, but surprisingly not of glutamatergic terminals. In CSP-alpha knock-out mice, synaptic degeneration of basket cell terminals occurs in vivo in the presence of normal glutamatergic synapses onto dentate gyrus granule cells. Consistent with this, in hippocampal cultures from these mice, the frequency of miniature IPSCs, caused by spontaneous GABA release, progressively declines, whereas the frequency of miniature excitatory AMPA receptor-mediated currents (mEPSCs), caused by spontaneous release of glutamate, is normal. However, the mEPSC amplitude progressively decreases. Remarkably, long-term block of glutamatergic transmission in cultures lacking CSP-alpha substantially rescues Syt2-expressing GABAergic synapses from neurodegeneration. These findings demonstrate that elevated neural activity increases synapse vulnerability and that CSP-alpha is essential to maintain presynaptic function under a physiologically high-activity regimen.

Similar to dopamine (DA), cannabinoids strongly influence prefrontal cortical functions, such as working memory, emotional learning, and sensory perception. Although endogenous cannabinoid receptors (CB(1)Rs) are abundantly expressed in the prefrontal cortex (PFC), very little is known about endocannabinoid (eCB) signaling in this brain region. Recent behavioral and electrophysiological evidence has suggested a functional interplay between the dopamine and cannabinoid receptor systems, although the cellular mechanisms underlying this interaction remain to be elucidated. We examined this issue by combining neuroanatomical and electrophysiological techniques in PFC of rats and mice (both genders). Using immunoelectron microscopy, we show that CB(1)Rs and dopamine type 2 receptors (D(2)Rs) colocalize at terminals of symmetrical, presumably GABAergic, synapses in the PFC. Indeed, activation of either receptor can suppress GABA release onto layer 5 pyramidal cells. Furthermore, coactivation of both receptors via repetitive afferent stimulation triggers eCB-mediated long-term depression of inhibitory transmission (I-LTD). This I-LTD is heterosynaptic in nature, requiring glutamate release to activate group I metabotropic glutamate receptors. D(2)Rs most likely facilitate eCB signaling at the presynaptic site as disrupting postsynaptic D(2)R signaling does not diminish I-LTD. Facilitation of eCB-LTD may be one mechanism by which DA modulates neuronal activity in the PFC and regulates PFC-mediated behavior in vivo.

alpha-Synuclein is central in Parkinson's disease pathogenesis. Although initially alpha-synuclein was considered a purely intracellular protein, recent data suggest that it can be detected in the plasma and CSF of humans and in the culture media of neuronal cells. To address a role of secreted alpha-synuclein in neuronal homeostasis, we have generated wild-type alpha-synuclein and beta-galactosidase inducible SH-SY5Y cells. Soluble oligomeric and monomeric species of alpha-synuclein are readily detected in the conditioned media (CM) of these cells at concentrations similar to those observed in human CSF. We have found that, in this model, alpha-synuclein is secreted by externalized vesicles in a calcium-dependent manner. Electron microscopy and liquid chromatography-mass spectrometry proteomic analysis demonstrate that these vesicles have the characteristic hallmarks of exosomes, secreted intraluminar vesicles of multivesicular bodies. Application of CM containing secreted alpha-synuclein causes cell death of recipient neuronal cells, which can be reversed after alpha-synuclein immunodepletion from the CM. High- and low-molecular-weight alpha-synuclein species, isolated from this CM, significantly decrease cell viability. Importantly, treatment of the CM with oligomer-interfering compounds before application rescues the recipient neuronal cells from the observed toxicity. Our results show for the first time that cell-produced alpha-synuclein is secreted via an exosomal, calcium-dependent mechanism and suggest that alpha-synuclein secretion serves to amplify and propagate Parkinson's disease-related pathology.

The adult cerebral cortex is composed of excitatory and inhibitory neurons that arise from progenitor cells in disparate proliferative regions in the developing brain and follow different migratory paths. Excitatory pyramidal neurons originate near the ventricle and migrate radially to their position in the cortical plate along radial glial fibers. On the other hand, inhibitory interneurons arise in the ventral telencephalon and migrate tangentially to enter the developing cortex before migrating radially to reach their correct laminar position. Gap junction adhesion has been shown to play an important mechanistic role in the radial migration of excitatory neurons. We asked whether a similar mechanism governs the tangential or radial migration of inhibitory interneurons. Using short hairpin RNA knockdown of Connexin 43 (Cx43) and Cx26 together with rescue experiments, we found that gap junctions are dispensable for the tangential migration of interneurons, but that Cx43 plays a role in the switch from tangential to radial migration that allows interneurons to enter the cortical plate and find their correct laminar position. Moreover this action is dependent on the adhesive properties and the C terminus of Cx43 but not the Cx43 channel. Thus, the radial phase of interneuron migration resembles that of excitatory neuron migration in terms of dependence on Cx43 adhesion. Furthermore, gap junctions between migrating interneurons and radial processes were observed by electron microscopy. These findings provide mechanistic and structural support for a gap junction-mediated interaction between migrating interneurons and radial glia during the switch from tangential to radial migration.

Damage-induced neuronal endopeptidase (DINE) is a metalloprotease belonging to the neprilysin family. Expression of DINE mRNA is observed predominantly in subsets of neurons in the CNS and peripheral nervous system during embryonic development, as well as after axonal injury. However, the physiological function of DINE and its substrate remain unknown. We generated DINE-deficient mice to examine the physiological role of DINE. Shortly after birth, these mice died of respiratory failure resulting from a dysfunction of the diaphragm, which showed severe atrophy. As DINE was abundantly expressed in motor neurons and there was atrophy of the diaphragm, we analyzed the interaction between motor nerves and skeletal muscles in the DINE-deficient mice. Although there were no obvious deficiencies in numbers of motor neurons in the spinal cord or in the nerve trajectories from the spinal cord to the skeletal muscle in DINE-deficient mice, detailed histochemical analysis demonstrated a significant decrease of nerve terminal arborization in the diaphragm from embryonic day 12.5. In accordance with the decrease of final branching, the diaphragms from DINE-deficient mice exhibited only a few neuromuscular junctions. Similar changes in nerve terminal morphology were also apparent in other skeletal muscles, including the latissimus dorsi and the intercostal muscles. These data suggest that DINE is a crucial molecule in distal axonal arborization into muscle to establish neuromuscular junctions.

Acetylcholine (ACh) plays important roles for higher brain functions, including arousal, attention, and cognition. These effects are mediated largely by muscarinic acetylcholine receptors (mAChRs). However, it remains inconclusive whether the mode of ACh-mAChR signaling is synaptic, so-called "wired," transmission mediated by ACh released into the synaptic cleft, or nonsynaptic, so-called "volume," transmission by ambient ACh. To address this issue, we examined cellular and subcellular distribution of M(1), the most predominant mAChR subtype in the cerebral cortex and hippocampus, and pursued its anatomical relationship with cholinergic varicosities in these regions of adult mice. M(1) was highly expressed in glutamatergic pyramidal neurons, whereas it was low or undetectable in various GABAergic interneuron subtypes. M(1) was preferentially distributed on the extrasynaptic membrane of pyramidal cell dendrites and spines. Cholinergic varicosities often made direct contact to pyramidal cell dendrites and synapses. At such contact sites, however, synapse-like specialization was infrequent, and no particular accumulation was found at around contact sites for both M(1) and presynpatic active zone protein Bassoon. These features contrasted with those of the glutamatergic system, in which AMPA receptor GluA2 and metabotropic receptor mGluR5 were recruited to the synaptic or perisynaptic membrane, respectively, and Bassoon was highly accumulated in the presynaptic terminals. These results suggest that M(1) is so positioned to sense ambient ACh released from cholinergic varicosities at variable distances, and to enhance the synaptic efficacy and excitability of pyramidal cells. These molecular-anatomical arrangements will provide the evidence for volume transmission, at least in M(1)-mediated cortical cholinergic signaling.

Patients with PMP22 deficiency present with focal sensory and motor deficits when peripheral nerves are stressed by mechanical force. It has been hypothesized that these focal deficits are due to mechanically induced conduction block (CB). To test this hypothesis, we induced 60-70% CB (defined by electrophysiological criteria) by nerve compression in an authentic mouse model of hereditary neuropathy with liability to pressure palsies (HNPP) with an inactivation of one of the two pmp22 alleles (pmp22(+/-)). Induction time for the CB was significantly shorter in pmp22(+/-) mice than that in pmp22(+/+) mice. This shortened induction was also found in myelin-associated glycoprotein knock-out mice, but not in the mice with deficiency of myelin protein zero, a major structural protein of compact myelin. Pmp22(+/-) nerves showed intact tomacula with no segmental demyelination in both noncompressed and compressed conditions, normal molecular architecture, and normal concentration of voltage-gated sodium channels by [(3)H]-saxitoxin binding assay. However, focal constrictions were observed in the axonal segments enclosed by tomacula, a pathological hallmark of HNPP. The constricted axons increase axial resistance to action potential propagation, which may hasten the induction of CB in Pmp22 deficiency. Together, these results demonstrate that a function of Pmp22 is to protect the nerve from mechanical injury.

We identified Pumilio (Pum), a Drosophila translational repressor, in a computational search for metazoan proteins whose activities might be regulated by assembly into ordered aggregates. The search algorithm was based on evolutionary sequence conservation patterns observed for yeast prion proteins, which contain aggregation-prone glutamine/asparagine (Q/N)-rich domains attached to functional domains of normal amino acid composition. We examined aggregation of Pum and its nematode ortholog PUF-9 by expression in yeast. A domain of Pum containing the Q/N-rich sequence, denoted as NQ1, the entire Pum N terminus, and the complete PUF-9 protein localize to macroscopic aggregates (foci) in yeast. NQ1 and PUF-9 can generate the yeast Pin+ trait, which is transmitted by a heritable aggregate. NQ1 also assembles into amyloid fibrils in vitro. In Drosophila, Pum regulates postsynaptic translation at neuromuscular junctions (NMJs). To assess whether NQ1 affects synaptic Pum activity in vivo, we expressed it in muscles. We found that it negatively regulates endogenous Pum, producing gene dosage-dependent pum loss-of-function NMJ phenotypes. NQ1 coexpression also suppresses lethality and NMJ phenotypes caused by overexpression of Pum in muscles. The Q/N block of NQ1 is required for these phenotypic effects. Negative regulation of Pum by NQ1 might be explained by formation of inactive aggregates, but we have been unable to demonstrate that NQ1 aggregates in Drosophila. NQ1 could also regulate Pum by a "dominant-negative" effect, in which it would block Q/N-mediated interactions of Pum with itself or with cofactors required for translational repression.

Increased sensory input from maternal care attenuates neuroendocrine and behavioral responses to stress long term and results in a lifelong phenotype of resilience to depression and improved cognitive function. Whereas the mechanisms of this clinically important effect remain unclear, the early, persistent suppression of the expression of the stress neurohormone corticotropin-releasing hormone (CRH) in hypothalamic neurons has been implicated as a key aspect of this experience-induced neuroplasticity. Here, we tested whether the innervation of hypothalamic CRH neurons of rat pups that received augmented maternal care was altered in a manner that might promote the suppression of CRH expression and studied the cellular mechanisms underlying this suppression. We found that the number of excitatory synapses and the frequency of miniature excitatory synaptic currents onto CRH neurons were reduced in "care-augmented" rats compared with controls, as were the levels of the glutamate vesicular transporter vGlut2. In contrast, analogous parameters of inhibitory synapses were unchanged. Levels of the transcriptional repressor neuron-restrictive silencer factor (NRSF), which negatively regulates Crh gene transcription, were markedly elevated in care-augmented rats, and chromatin immunoprecipitation demonstrated that this repressor was bound to a cognate element (neuron-restrictive silencing element) on the Crh gene. Whereas the reduced excitatory innervation of CRH-expressing neurons dissipated by adulthood, increased NRSF levels and repression of CRH expression persisted, suggesting that augmented early-life experience reprograms Crh gene expression via mechanisms involving transcriptional repression by NRSF.

Systemic injection of lipopolysaccharide (LPS) is a widely used model of immune/inflammatory challenge, which can invoke a host of CNS responses, including activation of the hypothalamic-pituitary-adrenal (HPA) axis. Inducible vascular prostaglandin E(2) (PGE(2)) synthesis by endothelial (ECs) and/or perivascular cells (PVCs) (a macrophage-derived vascular cell type) is implicated in the engagement of HPA and other CNS responses, by virtue of their capacity to express cyclooxygenase-2 (COX-2) and microsomal PGE(2) synthase-1. Evidence from genetic and pharmacologic studies also supports a role for the constitutively expressed COX-1 in inflammation-induced activation of the HPA axis, although histochemical evidence to support relevant localization(s) and regulation of COX-1 expression is lacking. The present experiments fill this void in showing that COX-1 immunoreactivity (IR) and mRNA are detectable in identified PVCs and parenchymal microglia under basal conditions and is robustly expressed in these and ECs 1-3 h after intravenous injection of LPS (2 microg/kg). Confocal and electron microscopic analyses indicate distinct cellular/subcellular localizations of COX-1-IR in the three cell types. Interestingly, COX-1 expression is enhanced in ECs of brain PVC-depleted rats, supporting an anti-inflammatory role of the latter cell type. Functional involvement of COX-1 is indicated by the observation that central, but not systemic, pretreatment with the selective COX-1 inhibitor SC-560 attenuated the early phase of LPS-induced increases in adrenocorticotropin and corticosterone secretion. These findings support an involvement of COX-1 in bidirectional interplay between ECs and PVCs in initiating vascular PGE(2) and downstream HPA response to proinflammatory challenges.

Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in age-dependent neurodegenerative diseases remain largely unknown. Here we show that the autophagy inhibitor 3-methyladenine delays neuronal cell loss caused by dysfunctional endosomal sorting complex required for transport III (ESCRT-III), either through loss of its essential component mSnf7-2 or ectopic expression of the disease protein CHMP2B(Intron5), which is associated with frontotemporal dementia linked to chromosome 3. Neuronal loss was also delayed by reduced activity of the autophagy genes atg5 and atg7. However, the endosomal accumulation of ubiquitinated proteins induced by dysfunctional ESCRT-III was not significantly affected, further confirming the essential contribution of dysregulated autophagy pathway in neurodegeneration. These findings show that autophagic stress by excess accumulation of autophagosomes is detrimental to neuronal survival under certain neurodegenerative conditions.

Heparan sulfate proteoglycans (HSPGs) are concentrated at neuromuscular synapses in many species, including Drosophila. We have established the physiological and patterning functions of HSPGs at the Drosophila neuromuscular junction by using mutations that block heparan sulfate synthesis or sulfation to compromise HSPG function. The mutant animals showed defects in synaptic physiology and morphology suggesting that HSPGs function both presynaptically and postsynaptically; these defects could be rescued by appropriate transgene expression. Of particular interest were selective disruptions of mitochondrial localization, abnormal distributions of Golgi and endoplasmic reticulum markers in the muscle, and a markedly increased level of stimulus-dependent endocytosis in the motoneuron. Our data support the emerging view that HSPG functions are not limited to the cell surface and matrix environments, but also affect a diverse set of cellular processes including membrane trafficking and organelle distributions.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

Neuregulin-1 has a key role in mediating signaling between axons and Schwann cells during development. A limitation to studying its role in adulthood is the embryonic lethality of global Nrg1 gene deletion. We used the Cre-loxP system to generate transgenic mice in which neuregulin-1 is conditionally ablated in the majority of small-diameter and a proportion of large-diameter sensory neurons that have axons conducting in the C- and Adelta-fiber range, respectively. Sensory neuron-specific neuregulin-1 ablation resulted in abnormally large Remak bundles with axons clustered in "polyaxonal" pockets. The total number of axons in the sural nerve was unchanged, but a greater proportion was unmyelinated. In addition, we observed large-diameter axons that were in a 1:1 relationship with Schwann cells, surrounded by a basal lamina but not myelinated. There was no evidence of DRG or Schwann cell death; the markers of different DRG cell populations and cutaneous innervation were unchanged. These anatomical changes were reflected in a slowing of conduction velocity at the lower end of the A-fiber conduction velocity range and a new population of more rapidly conducting C-fibers that are likely to represent large-diameter axons that have failed to myelinate. Conditional neuregulin-1 ablation resulted in a reduced sensitivity to noxious mechanical stimuli. These findings emphasize the importance of neuregulin-1 in mediating the signaling between axons and both myelinating and nonmyelinating Schwann cells required for normal sensory function. Sensory neuronal survival and axonal maintenance, however, are not dependent on axon-derived neuregulin-1 signaling in adulthood.

Fiber tracts should use space and energy efficiently, because both resources constrain neural computation. We found for a myelinated tract (optic nerve) that astrocytes use nearly 30% of the space and >70% of the mitochondria, establishing the significance of astrocytes for the brain's space and energy budgets. Axons are mostly thin with a skewed distribution peaking at 0.7 microm, near the lower limit set by channel noise. This distribution is matched closely by the distribution of mean firing rates measured under naturalistic conditions, suggesting that firing rate increases proportionally with axon diameter. In axons thicker than 0.7 microm, mitochondria occupy a constant fraction of axonal volume--thus, mitochondrial volumes rise as the diameter squared. These results imply a law of diminishing returns: twice the information rate requires more than twice the space and energy capacity. We conclude that the optic nerve conserves space and energy by sending most information at low rates over fine axons with small terminal arbors and sending some information at higher rates over thicker axons with larger terminal arbors but only where more bits per second are needed for a specific purpose. Thicker axons seem to be needed, not for their greater conduction velocity (nor other intrinsic electrophysiological purpose), but instead to support larger terminal arbors and more active zones that transfer information synaptically at higher rates.

Mammalian target of rapamycin (mTOR), a well known Akt substrate, regulates multiple cellular functions including cell growth and protein synthesis. The current study identifies a novel role of the Akt/mTOR pathway as a regulator of CNS myelination. Previously, we showed that overexpressing constitutively active Akt in oligodendrocytes in a transgenic mouse model induces enhanced CNS myelination, with no changes in the proliferation or survival of oligodendrocyte progenitor or mature cells. The present study focused on the signaling mechanisms regulating this hypermyelination induced by Akt. Activation of mTOR and its downstream substrates (p70S6 kinase and S6 ribosomal protein) was observed in Akt-overexpressing oligodendrocytes. When mTOR signaling was inhibited chronically in vivo with rapamycin starting at 6 weeks of age, the observed hypermyelination was reduced to approximately the amount of myelin seen in wild-type mice. mTOR inhibition had little impact on wild-type myelination between 6 and 12 weeks of age, suggesting that, in normal adults, myelination is relatively complete and is no longer regulated by mTOR signaling. However, when mTOR was chronically inhibited in young adult wild-type mice, myelination was reduced. These results suggest that, during active myelination, the major Akt signal regulating CNS myelination is the mTOR pathway.

Neuropathic pain results from damage to the peripheral sensory nervous system, which may have a number of causes. The calcium channel subunit alpha(2)delta-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of neuropathic pain, and this is causally related to the onset of allodynia, in which a non-noxious stimulus becomes painful. The therapeutic drugs gabapentin and pregabalin (PGB), which are both alpha(2)delta ligands, have antiallodynic effects, but their mechanism of action has remained elusive. To investigate this, we used an in vivo rat model of neuropathy, unilateral lumbar spinal nerve ligation (SNL), to characterize the distribution of alpha(2)delta-1 in DRG neurons, both at the light- and electron-microscopic level. We found that, on the side of the ligation, alpha(2)delta-1 was increased in the endoplasmic reticulum of DRG somata, in intracellular vesicular structures within their axons, and in the plasma membrane of their presynaptic terminals in superficial layers of the dorsal horn. Chronic PGB treatment of SNL animals, at a dose that alleviated allodynia, markedly reduced the elevation of alpha(2)delta-1 in the spinal cord and ascending axon tracts. In contrast, it had no effect on the upregulation of alpha(2)delta-1 mRNA and protein in DRGs. In vitro, PGB reduced plasma membrane expression of alpha(2)delta-1 without affecting endocytosis. We conclude that the antiallodynic effect of PGB in vivo is associated with impaired anterograde trafficking of alpha(2)delta-1, resulting in its decrease in presynaptic terminals, which would reduce neurotransmitter release and spinal sensitization, an important factor in the maintenance of neuropathic pain.

Mouse models of human disease are helpful for understanding the pathogenesis of the disorder and ultimately for testing potential therapeutic agents. Here, we describe the engineering and characterization of a mouse carrying the I268N mutation in Egr2, observed in patients with recessively inherited Charcot-Marie-Tooth (CMT) disease type 4E, which is predicted to alter the ability of Egr2 to interact with the Nab transcriptional coregulatory proteins. Mice homozygous for Egr2(I268N) develop a congenital hypomyelinating neuropathy similar to their human counterparts. Egr2(I268N) is expressed at normal levels in developing nerve but is unable to interact with Nab proteins or to properly activate transcription of target genes critical for proper peripheral myelin development. Interestingly, Egr2(I268N/I268N) mutant mice maintain normal weight and have only mild tremor until 2 weeks after birth, at which point they rapidly develop worsening weakness and uniformly die within several days. Nerve electrophysiology revealed conduction block, and neuromuscular junctions showed marked terminal sprouting similar to that seen in animals with pharmacologically induced blockade of action potentials or neuromuscular transmission. These studies describe a unique animal model of CMT, whereby weakness is due to conduction block or neuromuscular junction failure rather than secondary axon loss and demonstrate that the Egr2-Nab complex is critical for proper peripheral nerve myelination.

Cyclophilin D (CypD), a regulator of the mitochondrial membrane permeability transition pore (PTP), enhances Ca(2+)-induced mitochondrial permeabilization and cell death in the brain. However, the role of CypD in hypoxic-ischemic (HI) brain injury at different developmental ages is unknown. At postnatal day (P) 9 or P60, littermates of CypD-deficient [knock-out (KO)], wild-type (WT), and heterozygous mice were subjected to HI, and brain injury was evaluated 7 d after HI. CypD deficiency resulted in a significant reduction of HI brain injury at P60 but worsened injury at P9. After HI, caspase-dependent and -independent cell death pathways were more induced in P9 CypD KO mice than in WT controls, and apoptotic activation was minimal at P60. The PTP had a considerably higher induction threshold and lower sensitivity to cyclosporin A in neonatal versus adult mice. On the contrary, Bax inhibition markedly reduced caspase activation and brain injury in immature mice but was ineffective in the adult brain. Our findings suggest that CypD/PTP is critical for the development of brain injury in the adult, whereas Bax-dependent mechanisms prevail in the immature brain. The role of CypD in HI shifts from a predominantly prosurvival protein in the immature to a cell death mediator in the adult brain.

The absence of thyroid hormone (TH) during late gestation and early infancy can cause irreparable deafness in both humans and rodents. A variety of rodent models have been used in an effort to identify the underlying molecular mechanism. Here, we characterize a mouse model of secondary hypothyroidism, pituitary transcription factor 1 (Pit1(dw)), which has profound, congenital deafness that is rescued by oral TH replacement. These mutants have tectorial membrane abnormalities, including a prominent Hensen's stripe, elevated beta-tectorin composition, and disrupted striated-sheet matrix. They lack distortion product otoacoustic emissions and cochlear microphonic responses, and exhibit reduced endocochlear potentials, suggesting defects in outer hair cell function and potassium recycling. Auditory system and hair cell physiology, histology, and anatomy studies reveal novel defects of hormone deficiency related to deafness: (1) permanently impaired expression of KCNJ10 in the stria vascularis of Pit1(dw) mice, which likely contributes to the reduced endocochlear potential, (2) significant outer hair cell loss in the mutants, which may result from cellular stress induced by the lower KCNQ4 expression and current levels in Pit1(dw) mutant outer hair cells, and (3) sensory and strial cell deterioration, which may have implications for thyroid hormone dysregulation in age-related hearing impairment. In summary, we suggest that these defects in outer hair cell and strial cell function are important contributors to the hearing impairment in Pit1(dw) mice.

The motor neuron disease spinal muscular atrophy (SMA) causes profound muscle weakness that most often leads to early death. At autopsy, SMA is characterized by loss of motor neurons and muscle atrophy, but the initial cellular events that precipitate motor unit dysfunction and loss remain poorly characterized. Here, we examined the function and corresponding structure of neuromuscular junction (NMJ) synapses in a mouse model of severe SMA (hSMN2/delta7SMN/mSmn-/-). Surprisingly, most SMA NMJs remained innervated even late in the disease course; however they showed abnormal synaptic transmission. There was a two-fold reduction in the amplitudes of the evoked endplate currents (EPCs), but normal spontaneous miniature EPC (MEPC) amplitudes. These features in combination indicate reduced quantal content. SMA NMJs also demonstrated increased facilitation suggesting a reduced probability of vesicle release. By electron microscopy, we found a decreased density of synaptic vesicles that is likely to contribute to the reduced release probability. In addition to presynaptic defects, there were postsynaptic abnormalities. EPC and MEPC decay time constants were prolonged because of a slowed switch from the fetal acetylcholine receptor (AChR) gamma-subunit to the adult epsilon-subunit. There was also reduced size of AChR clusters and small myofibers, which expressed an immature pattern of myosin heavy chains. Together these results indicate that impaired synaptic vesicle release at NMJs in severe SMA is likely to contribute to failed postnatal maturation of motor units and muscle weakness.

Center-surround antagonistic receptive field (CSARF) organization is the basic synaptic circuit that serves as elementary building blocks for spatial information processing in the visual system. Cells with such receptive fields converge into higher-order visual neurons to form more complex receptive fields. Retinal bipolar cells (BCs) are the first neurons along the visual pathway that exhibit CSARF organization. BCs have been classified according to their response polarities and rod/cone inputs, and they project signals to target cells at different sublaminae of the inner plexiform layer. On the other hand, CSARFs of various types of BCs have been assumed be organized the same way. Here we examined center and surround responses of over 250 salamander BCs, and demonstrated that different types of BCs exhibit different patterns of dye coupling, receptive field center size, surround response strength, and conductance changes associated with center and surround responses. We show that BC receptive field center sizes varied with the degree of BC-BC coupling, and that surround responses of different BCs are mediated by different combinations of five lateral synaptic pathways mediated by the horizontal cells and amacrine cells. The finding of heterogeneous receptive field circuitry fundamentally challenges the common assumption that CSARFs of different subtypes of visual neurons are mediated by the same synaptic pathways. BCs carrying different visual signals use different synaptic circuits to process spatial information, allowing shape and contrast computation be differentially modulated by various lighting and adaptation conditions.

The adult mammalian brain maintains a prominent stem cell niche in the subventricular zone supplying new neurons to the olfactory bulb. We examined the dynamics of synaptogenesis by imaging the formation and elimination of clusters of a postsynaptic marker (PSD95), genetically targeted to adult-born neurons. We imaged in vivo adult-born periglomerular neurons (PGNs) during two phases of development, immaturity and maturity. Immature PGNs showed high levels of PSD95 puncta dynamics during 12-72 h intervals. Mature PGNs were more stable compared with immature PGNs but still remained dynamic, suggesting that synaptogenesis persists long after these neurons integrated into the network. By combining intrinsic signal and two photon imaging we followed PSD95 puncta in sensory enriched glomeruli. Sensory input upregulated the development of adult-born PGNs only in enriched glomeruli. Our data provide evidence for an activity-based mechanism that enhances synaptogenesis of adult-born PGNs during their initial phases of development.

Axonal dysfunction is the major phenotypic change in many neurodegenerative diseases, but the processes underlying this impairment are not clear. Modifier of cell adhesion (MOCA) is a presenilin binding protein that functions as a guanine nucleotide exchange factor for Rac1. The loss of MOCA in mice leads to axonal degeneration and causes sensorimotor impairments by decreasing cofilin phosphorylation and altering its upstream signaling partners LIM kinase and p21-activated kinase, an enzyme directly downstream of Rac1. The dystrophic axons found in MOCA-deficient mice are associated with abnormal aggregates of neurofilament protein, the disorganization of the axonal cytoskeleton, and the accumulation of autophagic vacuoles and polyubiquitinated proteins. Furthermore, MOCA deficiency causes an alteration in the actin cytoskeleton and the formation of cofilin-containing rod-like structures. The dystrophic axons show functional abnormalities, including impaired axonal transport. These findings demonstrate that MOCA is required for maintaining the functional integrity of axons and define a model for the steps leading to axonal degeneration.

Recently, a mutation in the mitochondrial protease Omi/HtrA2, G399S, was found in sporadic Parkinson's disease (PD) patients, leading to the designation of Omi/HtrA2 as PD locus 13 (PARK13). G399S reportedly results in reduced Omi protease activity. In vitro studies have suggested that Omi/HtrA2 acts downstream of PINK1, mutations in which mediate recessive forms of PD. We, as well as other, have previously shown that the Drosophila homologs of the familial PD genes, PINK1 (PARK6) and PARKIN (PARK2), function in a common genetic pathway to regulate mitochondrial integrity and dynamics. Whether Omi/HtrA2 regulates mitochondrial integrity and whether it acts downstream of PINK1 in vivo remain to be explored. Here, we show that Omi/HtrA2 null mutants in Drosophila, in contrast to pink1 or parkin null mutants, do not show mitochondrial morphological defects. Extensive genetic interaction studies do not provide support for models in which Omi/HtrA2 functions in the same genetic pathway as pink1, or carries out partially redundant functions with pink1, at least with respect to regulation of mitochondrial integrity and dynamics. Furthermore, Omi/HtrA2 G399S retains significant, if not full, function of Omi/HtrA2, compared with expression of protease-compromised versions of the protein. In light of recent findings showing that G399S can be found at comparable frequencies in PD patients and healthy controls, we do not favor a hypothesis in which Omi/HtrA2 plays an essential role in PD pathogenesis, at least with respect to regulation of mitochondrial integrity in the pink1/parkin pathway.

Rho GTPases activated by GDP/GTP exchange factors (GEFs) play key roles in the developing and adult nervous system. Kalirin-7 (Kal7), the predominant adult splice form of the multifunctional Kalirin RhoGEF, includes a PDZ [postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] binding domain and localizes to the postsynaptic side of excitatory synapses. In vitro studies demonstrated that overexpression of Kal7 increased dendritic spine density, whereas reduced expression of endogenous Kal7 decreased spine density. To evaluate the role of Kal7 in vivo, mice lacking the terminal exon unique to Kal7 were created. Mice lacking both copies of the Kal7 exon (Kal7(KO)) grew and reproduced normally. Golgi impregnation and electron microscopy revealed decreased hippocampal spine density in Kal7(KO) mice. Behaviorally, Kal7(KO) mice showed decreased anxiety-like behavior in the elevated zero maze and impaired acquisition of a passive avoidance task, but normal behavior in open field, object recognition, and radial arm maze tasks. Kal7(KO) mice were deficient in hippocampal long-term potentiation. Western blot analysis confirmed the absence of Kal7 and revealed compensatory increases in larger Kalirin isoforms. PSDs purified from the cortices of Kal7(KO) mice showed a deficit in Cdk5, a kinase known to phosphorylate Kal7 and play an essential role in synaptic function. The early stages of excitatory synaptic development proceeded normally in cortical neurons prepared from Kal7(KO) mice, with decreased excitatory synapses apparent only after 21 d in vitro. Expression of exogenous Kal7 in Kal7(KO) neurons rescued this deficit. Kal7 plays an essential role in synaptic structure and function, affecting a subset of cognitive processes.

Cranial visceral afferents travel via the solitary tract (ST) to contact neurons within the ST nucleus (NTS) and activate homeostatic reflexes. Hypothalamic projections from the paraventricular nucleus (PVN) release oxytocin (OT) to modulate visceral afferent communication with NTS neurons. However, the cellular mechanisms through which OT acts are poorly understood. Here, we electrophysiologically identified second-order NTS neurons in horizontal brainstem slices by their low-jitter, ST-evoked glutamatergic EPSCs. OT increased the frequency of miniature EPSCs in half of the NTS second-order neurons (13/24) but did not alter event kinetics or amplitudes. These actions were blocked by a selective OT receptor antagonist. OT increased the amplitude of ST-evoked EPSCs with no effect on event kinetics. Variance-mean analysis of ST-evoked EPSCs indicated OT selectively increased the release probability of glutamate from the ST afferent terminals. In OT-sensitive neurons, OT evoked an inward holding current and increased input resistance. The OT-sensitive current reversed at the K(+) equilibrium potential. In in vivo studies, NTS neurons excited by vagal cardiopulmonary afferents were juxtacellularly labeled with Neurobiotin and sections were stained to show filled neurons and OT-immunoreactive axons. Half of these physiologically characterized neurons (5/10) showed close appositions by OT fibers consistent with synaptic contacts. Electron microscopy of medial NTS found immunoreactive OT within synaptic boutons. Together, these findings suggest that OT released from PVN axons acts on a subset of second-order neurons within medial NTS to enhance visceral afferent transmission via presynaptic and postsynaptic mechanisms.