bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage/Microglia Regulation of Astrocytic Tenascin: Synergistic Action of Transforming Growth Factor-β and Basic Fibroblast Growth Factor
Authors: Smith George M. , Hale Jason H.
Published: April 2018

After injury to the CNS, extracellular matrix molecules such as tenascin are upregulated around the injury site and may be involved in inhibition of axon growth. In the present study, astrocytes were investigated to determine which cell types, growth factors, or cytokines are responsible for the injury-induced regulation of tenascin. The addition of activated macrophage- or microglial-conditioned medium increased astrocytic expression of tenascin 2.5-fold, as determined by Northern and Western blot analysis and ELISA. Of the cytokines and growth factors examined, only transforming growth factor-β1 (TGF-β1) and basic fibroblast growth factor (bFGF) significantly induced an increase in the production of astrocytic tenascin. Examination of macrophage and microglial supernatants showed the presence of TGF-β1 but not bFGF; however, the TGF-β1 concentration in supernatants was lower than that expected to induce an increase in astrocytic tenascin similar to that seen with recombinant TGF-β1. Western blot analysis of astrocytes showed only the presence of bFGF. Compared with the responses of the individual growth factors, tenascin production by astrocytes was dramatically potentiated when grown in the presence of a combination of both TGF-β1 and bFGF. A similar synergistic effect was observed after the addition of either TGF-β1 or bFGF to macrophage-conditioned medium. Northern analysis also showed concomitant increases in TGF-β1, bFGF, and tenascin after CNS injury to animals 14 d of age or older. These results show that the regulation of astrocytic tenascin is mediated by the synergistic action of TGF-β1 and bFGFin vitroand after injuryin vivo.
Role of the Supplementary Motor Area and the Right Premotor Cortex in the Coordination of Bimanual Finger Movements
Authors: Sadato Norihiro , Yonekura Yoshiharu , Waki Atsuo , Yamada Hiroki , Ishii Yasushi
Published: April 2018

To obtain a better understanding of the cortical representation of bimanual coordination, we measured regional cerebral blood flow (rCBF) with15O-labeled water and positron emission tomography (PET). To detect areas with changes of rCBF during bimanual finger movements of different characteristics, we studied 12 right-handed normal volunteers. A complete session consisted of three rest scans and six scans with acoustically paced (1 Hz) bimanual, mirror, or parallel sequential finger movements. Activation of the right dorsal premotor area (PMd) extending to the posterior supplementary motor area (SMA) was significantly stronger during the parallel movements than during the mirror sequential movements (p< 0.05, at cluster level with correction for multiple comparisons). To determine whether these cortical areas truly represented bimanual coordination, a different group of nine normal volunteers was studied with a different task. Subjects performed acoustically paced (2 Hz) abduction–adduction movements of the index finger, making right only, left only, and bimanual mirror and parallel movements. Activation of the posterior SMA and right PMd was significantly greater during the parallel movements than during the bimanual mirror movements or the unimanual movements of either hand (p< 0.01, with anatomical constraint). Thus, the posterior SMA and right PMd appear to be related to the bimanual coordination of finger movements.
Input Summation by Cultured Pyramidal Neurons Is Linear and Position-Independent
Authors: Cash Sydney , Yuste Rafael
Published: April 2018

The role of dendritic morphology in integration and processing of neuronal inputs is still unknown. Models based on passive cable theory suggest that dendrites serve to isolate synapses from one another. Because of decreases in driving force or resistance, two inputs onto the same dendrite would diminish their joint effect, resulting in sublinear summation. When on different dendrites, however, inputs would not interact and therefore would sum linearly. These predictions have not been rigorously tested experimentally. In addition, recent results indicate that dendrites have voltage-sensitive conductances and are not passive cables. To investigate input integration, we characterized the effects of dendritic morphology on the summation of subthreshold excitatory inputs on cultured hippocampal neurons with pyramidal morphologies. We used microiontophoresis of glutamate to systematically position inputs throughout the dendritic tree and tested the summation of two inputs by measuring their individual and joint effects. We find that summation was surprisingly linear regardless of input position. For small inputs, this linearity arose because no significant shunts or changes in driving force occurred and no voltage-dependent channels were opened. Larger inputs also added linearly, but this linearity was caused by balanced action of NMDA and IApotassium conductances. Therefore, active conductances can maintain, paradoxically, a linear input arithmetic. Furthermore, dendritic morphology does not interfere with this linearity, which may be essential for particular neuronal computations.
Differential Intracellular Sorting of Immediate Early Gene mRNAs Depends on Signals in the mRNA Sequence
Authors: Wallace Christopher S. , Lyford Gregory L. , Worley Paul F. , Steward Oswald
Published: April 2018

This study characterizes the differential targeting of recently synthesized immediate early gene (IEG) mRNAs to neuronal cell bodies versus dendrites and tests the hypothesis that this targeting is based on signals in the encoded proteins. A single electroconvulsive seizure induces the expression of a number of IEG mRNAs in granule cells of the dentate gyrus. Most of these IEG mRNAs remain in the cell body, including two that are characterized in the present study (the mRNAs for NGFI-A and COX-2). In contrast, the mRNA forArcmoved rapidly into dendrites at an apparent rate of ∼300 μm/hr. Inhibiting protein synthesis with cycloheximide did not disrupt the differential mRNA sorting, demonstrating that the differential targeting of mRNAs is not dependent on translation.
Sorting of β-Actin mRNA and Protein to Neurites and Growth Cones in Culture
Authors: Bassell Gary J. , Zhang Honglai , Byrd Anne L. , Femino Andrea M. , Singer Robert H. , Taneja Krishan L. , Lifshitz Lawrence M. , Herman Ira M. , Kosik Kenneth S.
Published: April 2018

The transport of mRNAs into developing dendrites and axons may be a basic mechanism to localize cytoskeletal proteins to growth cones and influence microfilament organization. Using isoform-specific antibodies and probes forin situhybridization, we observed distinct localization patterns for β- and γ-actin within cultured cerebrocortical neurons. β-Actin protein was highly enriched within growth cones and filopodia, in contrast to γ-actin protein, which was distributed uniformly throughout the cell. β-Actin protein also was shown to be peripherally localized after transfection of β-actin cDNA bearing an epitope tag. β-Actin mRNAs were localized more frequently to neuronal processes and growth cones, unlike γ-actin mRNAs, which were restricted to the cell body. The rapid localization of β-actin mRNA, but not γ-actin mRNA, into processes and growth cones could be induced by dibutyryl cAMP treatment. Using high-resolutionin situhybridization and image-processing methods, we showed that the distribution of β-actin mRNA within growth cones was statistically nonrandom and demonstrated an association with microtubules. β-Actin mRNAs were detected within minor neurites, axonal processes, and growth cones in the form of spatially distinct granules that colocalized with translational components. Ultrastructural analysis revealed polyribosomes within growth cones that colocalized with cytoskeletal filaments. The transport of β-actin mRNA into developing neurites may be a sequence-specific mechanism to synthesize cytoskeletal proteins directly within processes and growth cones and would provide an additional means to deliver cytoskeletal proteins over long distances.
Up and Down States in Striatal Medium Spiny Neurons Simultaneously Recorded with Spontaneous Activity in Fast-Spiking Interneurons Studied in Cortex–Striatum–Substantia Nigra Organotypic Cultures
Authors: Plenz Dietmar , Kitai Stephen T.
Published: April 2018

In vivointracellular spontaneous activity in striatal medium spiny (MS) projection neurons is characterized by “up” and “down” states. How this type of activity relates to the neuronal activity of striatal fast-spiking (FS) interneurons was examined in the presence of nigral and cortical inputs using cortex–striatum–substantia nigra organotypic cultures grown for 45 ± 4 d. The nigrostriatal projection was confirmed by tyrosine hydroxylase immunoreactivity. Corticostriatal (CS) projection neurons, striatal MS neurons, and FS neurons were intracellularly recorded and morphologically and electrophysiologically characterized. Intracellular spontaneous activity in the cultures consisted of intermittent depolarized periods of 0.5–1 sec duration. Spontaneous depolarizations in MS neurons were restricted to a narrow membrane potential range (up state) during which they occasionally fired single spikes. These up states were completely blocked by the glutamate antagonist CNQX. In FS interneurons, depolarized periods were characterized by large membrane potential fluctuations that occupied a wide range between rest and spike threshold. Also, FS interneurons spontaneously fired at much higher rates than did MS neurons. Simultaneous intracellular recordings established that during spontaneous depolarizations MS neurons and FS interneurons displayed correlated subthreshold neuronal activity in the low frequency range. These results indicate that (1) the CS projection neurons, striatal MS neurons, and FS interneurons grown in cortex–striatum–substantia nigra organotypic cultures show morphological and electrophysiological characteristics similar to those seenin vivo; (2) striatal MS neurons but not FS interneurons show an up state; (3) striatal MS neurons and FS interneurons receive common, presumably cortical inputs in the low frequency range. Our results support the view that the cortex provides a feedforward inhibition of MS neuron activity during the up state via FS interneurons.
MouseZic1Is Involved in Cerebellar Development
Authors: Aruga Jun , Minowa Osamu , Yaginuma Hiroyuki , Kuno Junko , Nagai Takeharu , Noda Tetsuo , Mikoshiba Katsuhiko
Published: April 2018

Zicgenes encode zinc finger proteins, the expression of which is highly restricted to cerebellar granule cells and their precursors. These genes are homologs of theDrosophilapair-rule geneodd-paired. To clarify the role of theZic1gene, we have generated mice deficient inZic1. Homozygous mice showed remarkable ataxia during postnatal development. Nearly all of the mice died within 1 month. Their cerebella were hypoplastic and missing a lobule in the anterior lobe. A bromodeoxyuridine labeling study indicated a reduction both in the proliferating cell fraction in the external germinal layer (EGL), from 14 d postcoitum, and in forward movement of the EGL. These findings suggest thatZic1may determine the cerebellar folial pattern principally via regulation of cell proliferation in the EGL.
Postnatal Expression ofHu-Bcl-2Gene inLurcherMutant Mice Fails to Rescue Purkinje Cells but Protects Inferior Olivary Neurons from Target-Related Cell Death
Authors: Zanjani H. S. , Vogel M. W. , Martinou J. C. , Delhaye-Bouchaud N. , Mariani J.
Published: April 2018

TheLurchermutant has been extensively studied as a model for cell-autonomous and target-related cell death, yet there are still many unknowns concerning the mechanisms of neuronal degeneration in this mutant. As a key regulator of apoptosis, abcl-2transgene has been overexpressed in the heterozygousLurchermutant to investigate the effects of BCL-2 on two types ofin vivoneuronal cell loss inLurcher: cell-autonomous Purkinje cell degeneration and target-related olivary neuron death. Six adult +/Lcmutants expressing a humanbcl-2transgene (Hu-bcl-2) were generated by crossing +/Lcmutants with NSE71Hu-bcl-2transgenic mice. Analysis of these brains showed thatbcl-2overexpression did not prevent +/LcPurkinje cell degeneration, but it did rescue most olivary neurons from target-related cell death. Although the number of olivary neurons was equivalent to wild-type numbers, the inferior olive nucleus was significantly shorter in its rostrocaudal extent, suggesting that olivary neurons are atrophied. We propose thatLurchergene action causes Purkinje cell degeneration independently of a BCL-2-mediated pathway. Furthermore, althoughbcl-2overexpression rescues olivary neurons from target-related cell death, it does not prevent the atrophy associated with the loss of target-related trophic support.
Development of Walking, Swimming and Neuronal Connections after Complete Spinal Cord Transection in the Neonatal Opossum,Monodelphis domestica
Authors: Saunders N. R. , Kitchener P. , Knott G. W. , Nicholls J. G. , Potter A. , Smith T. J.
Published: April 2018

Development of coordinated movements was quantitatively assessed in adult opossums (Monodelphis domestica) with thoracic spinal cords transected by (1) crushing 7–8 d after birth [postnatal days 7–8 (P7–P8)]; at 2–3 years of age, systematic behavioral tests (e.g., climbing, footprint analysis, and swimming) showed only minor differences between control (n= 5) and operated (n= 10) animals; and (2) cutting on P4–P6; at 1 month these opossums exhibited coordinated walking movements but were unable to right themselves from a supine position, unlike controls (n= 6). When tested at 2 or 6 months, they could right themselves and showed remarkable coordination, albeit with more differences from controls than after a crush. No animals with spinal cords that were crushed at P14–18 survived because of cannibalism by the mother. Morphological studies (n= 10) 3 months–3 years after crush at 1 week showed restoration of structural continuity and normal appearance at the lesion site. Animals with cut rather than crushed cords showed continuity but greater morphological deficits. That lesions were complete was demonstrated by examining morphology and nerve impulse conduction immediately after crushing or cutting the spinal cord in controls. After lumbar spinal cord injection of 10 kDa dextran amine, retrogradely labeled cells were found rostral to the lesion in hindbrain and midbrain nuclei. Conduction was restored across the site of the lesion. Thus complete spinal cord transection in neonatalMonodelphiswas followed by development of coordinated movements and repair of the spinal cord, a process that included development of functional connections by axons that crossed the lesion.
Spatial Distribution of Potentiated Synapses in Hippocampus: Dependence on Cellular Mechanisms and Network Properties
Authors: Yeckel M. F. , Berger T. W.
Published: April 2018

Long-term potentiation (LTP) of synaptic transmission, studied intensively in reduced brain preparations such as hippocampal brain slices, is the leading candidate for the cellular/molecular basis of learning and memory. Serious consideration of LTP as underlying information storage in the intact brain, however, requires understanding how LTP can be induced selectively at specific synaptic sites in a neural system when the mechanisms underlying LTP are regulated by other structural and functional properties of the same neural system. In the studies reported here, we tested the hypothesis that different patterns of activity within the same population of entorhinal cortical afferents could lead to a selective potentiation of spatially distinct populations of synapses across different regions of the hippocampus, including those activated multisynaptically. We focused specifically on potentiation of direct, monosynaptic entorhinal input to dentate granule cells, which expresses an NMDA receptor-dependent LTP, and on potentiation of indirect, disynaptic entorhinal input to CA3 pyramidal cells, which is transmitted by the mossy fiber projection of dentate granule cells and expresses an NMDA receptor-independent LTP. The principal findings of these experiments show that lower stimulation frequencies (10–20 Hz) of entorhinal cortical axons selectively induce LTP of mossy fiber input to CA3 transsynaptically via excitation of dentate granule cells, and that patterns of stimulation of that mimic neuronal firing in the entorhinal cortex during endogenous theta rhythm (five-impulse bursts at 200 Hz, interburst intervals of 200 msec) induce LTP both monosynaptically for input to dentate granule cells and transsynaptically for mossy fiber input to CA3.
Hypoactivity of the Spinal Cannabinoid System Results in NMDA-Dependent Hyperalgesia
Authors: Richardson Jennelle Durnett , Aanonsen Lin , Hargreaves Kenneth M.
Published: April 2018

Cannabinoids, such as Δ9-THC, are capable of inhibiting nociception, i.e., pain transmission, at least in part, by interacting with spinal Gi/Go-coupled cannabinoid receptors. What is not known, however, is the antinociceptive role ofendogenousspinal cannabinoids. If endogenous cannabinoids modulate basal nociceptive thresholds, then alterations in this system could be involved in the etiology of certain pain states. In this report we provide evidence for tonic modulation of basal thermal nociceptive thresholds by the spinal cannabinoid system. Administration of oligonucleotides directed against CB1cannabinoid receptor mRNA significantly reduced spinal cannabinoid binding sites and produced significant hyperalgesia when compared with a randomer oligonucleotide control. A second method used to reduce activity of the spinal cannabinoid receptor was intrathecal administration of the cannabinoid receptor antagonist SR 141716A. SR 141716A evoked thermal hyperalgesia with an ED50of 0.0012 fmol. The SR 141716A-induced hyperalgesia was dose-dependently blocked by the administration ofd-AP-5 or MK-801, two antagonists to the NMDA receptor. These results indicate that there is tonic activation of the spinal cannabinoid system under normal conditions. Furthermore, hypoactivity of the spinal cannabinoid system results in an NMDA-dependent hyperalgesia and thus may participate in the etiology of certain chronic pain states.
Reconstruction of Flexor/Extensor Alternation during Fictive Rostral Scratching by Two-Site Stimulation in the Spinal Turtle with a Transverse Spinal Hemisection
Authors: Stein Paul S. G. , McCullough Margaret L. , Currie Scott N.
Published: April 2018

Analyses of fictive scratching motor patterns in the spinal turtle with transverse hemisection provided support for the concept of bilateral shared spinal cord circuitry among neurons responsible for generating left- and right-side rostral, pocket, and caudal fictive scratching. Rhythmic bursts of hip flexor activity, the hip extensor deletion variation of fictive rostral scratching, were elicited by ipsilateral stimulation in the rostral scratch receptive field of a spinal turtle [transection at the segmental border between the second (D2) and third (D3) postcervical spinal segments] with a contralateral transverse hemisection one segment anterior to the hindlimb enlargement (at the D6–D7 segmental border). In addition, other sites were stimulated in this preparation: (1) contralateral sites in a rostral, pocket, or caudal scratch receptive field or (2) ipsilateral sites in a caudal scratch receptive field. A reconstructed fictive rostral scratch motor pattern of rhythmic alternation between hip flexor and hip extensor activation was produced by simultaneous stimulation of one site in the ipsilateral rostral scratch receptive field and another site in one of the other scratch receptive fields. This reconstructed rostral scratch motor pattern resembled the normal rostral scratch motor pattern produced by one-site rostral scratch stimulation of a spinal turtle (D2–D3 transection) with no additional transections. The observation of a reconstructed rostral scratch motor pattern produced by two-site stimulation in the spinal turtle with transverse hemisection supports the concept that hip extensor circuitry activated by stimulation of other scratch receptive fields is shared with circuitry activated by ipsilateral rostral scratch receptive field stimulation.
Differential Modulation of Changes in Hippocampal–Septal Synaptic Excitability by the Amygdala as a Function of Either Elemental or Contextual Fear Conditioning in Mice
Authors: Desmedt Aline , Garcia René , Jaffard Robert
Published: April 2018

Recent data obtained using a classic fear conditioning paradigm showed a dissociation between the retention of associations relative to contextual information (dependent on the hippocampal formation) and the retention of elemental associations (dependent on the amygdala). Furthermore, it was reported that conditioned emotional responses (CERs) could be dissociated from the recollection of the learning experience (declarative memory) in humans and from modifications of the hippocampal–septal excitability in animals. Our aim was to determine whether these two systems (“behavioral expression” system and “factual memory” system) interact by examining the consequences of amygdalar lesions (1) on the modifications of hippocampal–septal excitability and (2) on the behavioral expression of fear (freezing) resulting from an aversive conditioning during reexposure to conditional stimuli (CSs). During conditioning, to modulate the predictive nature of the context and of a discrete stimulus (tone) on the unconditional stimulus (US) occurrence, the phasic discrete CS was paired with the US or randomly distributed with regard to the US. After the lesion, the CER was dramatically reduced during reexposure to the CSs, whatever the type of acquisition. However, the changes in hippocampal–septal excitability persisted but were altered. For controls, a decrease in septal excitability was observed during reexposure to the conditioning context only for the “unpaired group” (predictive context case). Conversely, among lesioned subjects this decrease was observed in the “paired group” (predictive discrete CS case), whereas this decrease was significantly reduced in the unpaired group with respect to the matched control group. The amplitude and the direction of these modifications suggest a differential modulation of hippocampal–septal excitability by the amygdala to amplify the contribution of the more predictive association signaling the occurrence of the aversive event.
A Focal Zone of Thalamic Plasticity
Authors: Parker Jayson L. , Wood Michael L. , Dostrovsky Jonathan O.
Published: April 2018

In this study, sensory maps in the thalamus were investigated by examining their volume and shape. We determined the forelimb representation in adult rats after the removal of hindlimb input by nucleus gracilis lesions. Three-dimensional reconstructions of thalamic sensory maps were obtained from a grid of electrode penetrations. We found that the volume of the shoulder sensory map contracted >50% at an acute time interval (n= 6), followed by a robust volumetric sensory map expansion of 25% at 1 week (n= 8) and 1 month (n= 8) after lesion relative to controls (n= 8). The topology of the volumetric increase was scrutinized by slicing functional maps in the coronal, sagittal, and horizontal planes. The equivalence of such slices from each animal was established by virtue of their distance from either a functional or neuroanatomical landmark. Surprisingly, all of the volumetric increase unequivocally occurred in a circumscribed coronal slice 300 μm thick. This focal zone was located toward the rostral pole of the thalamic tactile relay, the ventroposterolateral nucleus. Analysis in the sagittal plane revealed that, unexpectedly, the shoulder map volume expanded by superimposing its representation on that of the forepaw, via an advancement of the shoulder representation by 0.6 mm medially. We propose a “hot spot” hypothesis in which focal zones of plasticity may not be specific to the thalamus but may have manifestations elsewhere in the nervous system, such as the cerebral cortex or dorsal column nuclei.
Evidence for a 95 kDa Short Form of the α1ASubunit Associated with the ω-Conotoxin MVIIC Receptor of the P/Q-type Ca2+Channels
Authors: Scott Victoria E. S. , Felix Ricardo , Arikkath Jyothi , Campbell Kevin P.
Published: April 2018

Neuronal voltage-dependent Ca2+channels have been isolated previously and shown to contain a primary α1pore-forming subunit as well as auxiliary α2δ and β subunits, in addition to an uncharacterized 95 kDa protein. In the present study, using multiple approaches, we have extensively characterized the molecular structure of the 95 kDa protein. Separation of the P/Q- and N-type neuronal Ca2+channels showed that the 95 kDa protein is associated exclusively with the ω-Conotoxin MVIIC receptor of the P/Q-type channels. Analysis of purified synaptic plasma membranes and the isolated P/Q-type channels, using α1A-specific antibodies, suggested a structural relationship between the α1Asubunit and the 95 kDa protein. This finding was supported by protein–protein interaction data, which revealed that the β subunit can associate with the 95 kDa protein in addition to the α1Asubunit. Changes in electrophoretic mobility after enzymatic treatment with Endo F indicated that the 95 kDa protein is glycosylated. Furthermore, microsequencing of the 95 kDa protein yielded 13 peptide sequences, all of which are present in the first half of the α1Asubunit up to amino acid 829 of the cytoplasmic linker between repeats II and III. Taken together, our results strongly suggest that the 95 kDa glycoprotein associated with the P/Q-type Ca2+channels is a short form of the α1Asubunit.
Critical Elements Determining Diversity in Agonist Binding and Desensitization of Neuronal Nicotinic Acetylcholine Receptors
Authors: Corringer Pierre-Jean , Bertrand Sonia , Bohler Sébastien , Edelstein Stuart J. , Changeux Jean-Pierre , Bertrand Daniel
Published: April 2018

To identify the molecular determinants underlying the pharmacological diversity of neuronal nicotinic acetylcholine receptors, we compared the α7 homo-oligomeric and α4β2 hetero-oligomeric receptors. Sets of residues from the regions initially identified within the agonist binding site of the α4 subunit were introduced into the α7 agonist binding site, carried by the homo-oligomeric α7-V201–5HT3chimera. Introduction of the α4 residues 183–191 into α7 subunit sequence (chimera C2) selectively increased the apparent affinities for equilibrium binding and for ion channel activation by acetylcholine, resulting in a receptor that no longer displays differences in the responses to acetylcholine and nicotine. Introduction of the α4 residues 151–155 (chimera B) produced a ∼100-fold increase in the apparent affinity for both acetylcholineandnicotine in equilibrium binding measurements. In both cases electrophysiological recordings revealed a much smaller increase (three- to sevenfold) in the apparent affinity for activation, but the concentrations required to desensitize the mutant chimeras parallel the shifts in apparent binding affinity. The data were fitted by a two-state concerted model, and an alteration of the conformational isomerization constant leading to the desensitized state accounts for the chimera B phenotype, whereas alteration of the ligand binding site accounts for the chimera C2phenotype. Point mutation analysis revealed that several residues in both fragments contribute to the phenotypes, with a critical effect of the G152K and T183N mutations. Transfer of α4 amino acids 151–155 and 183–191 into the α7-V201–5HT3chimera thus confers physiological and pharmacological properties typical of the α4β2 receptor.
Excitatory Amino Acid Transporters of the Salamander Retina: Identification, Localization, and Function
Authors: Eliasof Scott , Arriza Jeffrey L. , Leighton Barbara H. , Kavanaugh Michael P. , Amara Susan G.
Published: April 2018

The rapid re-uptake of extracellular glutamate mediated by a family of high-affinity glutamate transporter proteins is essential to continued glutamatergic signaling and neuronal viability, but the contributions of individual transporter subtypes toward cellular physiology are poorly understood. Because the physiology of glutamate transport in the salamander retina has been well described, we have examined the expression and function of glutamate transporter subtypes in this preparation. cDNAs encoding five distinct salamander excitatory amino acid transporter (sEAAT) subtypes were isolated, and their molecular properties and distributions of expression were compared. We report evidence that at least four distinct sEAAT subtypes are expressed in glial (Müller) cells. In addition, four of the five transporter subtypes are localized in neurons throughout the retina. The brightest immunostaining was seen in the synaptic regions of the inner and outer plexiform layers and in the outer nuclear layer. Using electrophysiological measurements in theXenopusoocyte expression system, we also examined the pharmacology and ionic dependence of the four expressing transporter subtypes that make it possible to distinguish, on the basis of functional behavior, among the various subtypes. Although no simple correlation between transporter subtype and retinal cell physiology can be made, the diverse population of sEAAT transporter subtypes with unique localization and functional properties indicates that glutamate transporters play a wide variety of roles in retinal function and are likely to underlie both the uptake of glutamate by Müller cells and the glutamate-elicited chloride conductance involved in signal transduction by photoreceptors and bipolar cells.
Splice Variant-Specific Interaction of the NMDA Receptor Subunit NR1 with Neuronal Intermediate Filaments
Authors: Ehlers Michael D. , Fung Eric T. , O’Brien Richard J. , Huganir Richard L.
Published: April 2018

NMDA receptors are excitatory neurotransmitter receptors critical for synaptic plasticity and neuronal development in the mammalian brain. These receptors are found highly concentrated in the postsynaptic membrane of glutamatergic synapses. To investigate the molecular mechanisms underlying NMDA receptor localization, we used the yeast two-hybrid system to identify proteins expressed in the brain that interact with the NMDA receptor subunit NR1. Here we report that the 68 kDa neurofilament subunit NF-L directly interacts with the NR1 subunit. This interaction occurs between the cytoplasmic C-terminal domain of NR1 and the rod domain of NF-L. However, NR1 splice variants lacking the first C-terminal exon cassette (C1) failed to associate with NF-L. Immunogold electron microscopy revealed a preferential localization of NR1 at the ends ofin vitro-assembled neurofilaments. Overexpression of C1 cassette-containing NR1 constructs in fibroblast cells disrupted the assembly of recombinant neurofilaments. In addition, NR1 and NF-L cofractionated in detergent-treated rat brain synaptic plasma membranes. Furthermore, NR1 and NF-L colocalize in the dendrites and growth cones of cultured hippocampal neurons. These results demonstrate the splice variant-specific association of NR1 with neurofilaments and suggest a possible mechanism for anchoring or localizing NMDA receptors in the neuronal plasma membrane.
Presynaptic Localization of Kv1.4-Containing A-Type Potassium Channels Near Excitatory Synapses in the Hippocampus
Authors: Cooper Edward C. , Milroy Antonia , Jan Yuh Nung , Jan Lily Yeh , Lowenstein Daniel H.
Published: April 2018

MammalianShakervoltage-gated potassium channels that contain the Kv1.4 subunit exhibit rapid activation and prominent inactivation processes, which enable these channels to integrate brief (approximiately milliseconds) depolarizations over time intervals of up to tens of seconds. In the hippocampus, Kv1.4 immunoreactivity is detected at greatest density in two regions: (1) the middle molecular layer (MML), where perforant path axons synapse with dentate granule cells, and (2) the stratum lucidum (SL) of CA3, where the mossy fibers travel in tight fasciculi and form en passante synapses onto CA3 pyramidal cells. We have studied the localization of Kv1.4 within these regions in detail. First, we compared the distribution of Kv1.4 and synaptophysin (a synaptic vesicle protein primarily localized near termini) under confocal immunofluorescence microscopy. In the MML, Kv1.4 and synaptophysin immunofluorescence appeared to overlap. In the SL, however, Kv1.4 and synaptophysin staining was detected in nonoverlapping, irregular patches (∼5–10 μm in diameter). Ultrastructural studies of these two regions revealed that Kv1.4 immunoreactivity was absent from the surface membranes of cell bodies and dendrites and occurred prominently on axons, including axonal “necks” near termini. Small excitatory synaptic boutons also were labeled in the MML; by contrast, the mossy fiber synaptic expansions in the SL were not stained. These localizations may enable Kv1.4-containing channels to regulate the process of neurotransmitter release at these excitatory synapses.
Cellular Localization of Ephrin-A2, Ephrin-A5, and Other Functional Guidance Cues Underlies Retinotopic Development across Species
Authors: Davenport Roger W. , Thies Edda , Zhou Renping , Nelson Phillip G.
Published: April 2018

Avian retinotectal and rodent retinocollicular systems are general model systems used to examine developmental processes that underpin topographically organized neuronal circuits. The two systems rely on guidance components to establish their precise retinotopic maps, but many cellular events differ during their development. For example, compared with the chick, a generally less restricted outgrowth pattern is observed when retinae innervate their targets in rodents. Cellular or molecular distributions of guidance components may account for such differences in retinotopic development across species. Candidate repellent molecules, such as ephrin-A2 and ephrin-A5, have been cloned in both chick and rodents; however, it has not yet been shown in rodents that living cells express sufficient amounts of any repellent components to deter outgrowth. We used a coculture assay that gives cellular resolution of retinotarget interactions and demonstrate that living, caudal superior colliculus cells selectively prevent extension of axons from temporal regions of the retinae. Time-lapse video microscopy revealed the cellular localization of permissive and repulsive guidance components in rodents, which differed from that in chick. To analyze the potential molecular basis for these differences, we investigated the function and localization of ephrin-A2 and -A5. Cells transfected with ephrin-A2 and -A5 selectively repelled retinal axons. Ephrin-A2 and -A5 RNA expression patterns differed across cell populations and between species, suggesting molecular mechanisms and key cellular interactions that may underlie fundamental differences in the development of retinotectal and retinocollicular maps.
Acetylcholinesterase Enhances Neurite Growth and Synapse Development through Alternative Contributions of Its Hydrolytic Capacity, Core Protein, and Variable C Termini
Authors: Sternfeld Meira , Ming Guo-li , Song Hong-jun , Sela Keren , Timberg Rina , Poo Mu-ming , Soreq Hermona
Published: April 2018

Accumulated indirect evidence suggests nerve growth-promoting activities for acetylcholinesterase (AChE). To determine unequivocally whether such activities exist, whether they are related to the capacities of this enzyme to hydrolyze acetylcholine and enhance synapse development, and whether they are associated with alternative splicing variants of AChEmRNA, we used four recombinant human AChEDNA vectors. WhenXenopus laevisembryos were injected with a vector expressing the synapse-characteristic human AChE-E6, which contains the exon 6-encoded C terminus, cultured spinal neurons expressing this enzyme grew threefold faster than co-cultured control neurons. Similar enhancement occurred in neurons expressing an insertion-inactivated human AChE-E6-IN protein, containing the same C terminus, and displaying indistinguishable immunochemical and electrophoretic migration properties from AChE-E6, but incapable of hydrolyzing acetylcholine. In contrast, the nonsynaptic secretory human AChE-I4, which contains the pseudointron 4-derived C terminus, did not affect neurite growth. Moreover, no growth promotion occurred in neurons expressing the catalytically active C-terminally truncated human AChE-E4, demonstrating a dominant role for the E6-derived C terminus in neurite extension. Also, AChE-E6 was the only active enzyme variant to be associated withXenopusmembranes. However, postsynaptic length measurements demonstrated that both AChE-E6 and AChE-E4 enhanced the development of neuromuscular junctionsin vivo, unlike the catalytically inert AChE-E6-IN and the nonsynaptic AChE-I4. These findings demonstrate an evolutionarily conserved synaptogenic activity for AChE that depends on its hydrolytic capacity but not on its membrane association. Moreover, this synaptogenic effect differs from the growth-promoting activity of AChE, which is unrelated to its hydrolytic capacity yet depends on its exon 6-mediated membrane association.
The Synaptic Basis of GABAA,slow
Authors: Banks Matthew I. , Li Tong-Bin , Pearce Robert A.
Published: April 2018

Although two kinetically distinct evoked GABAAresponses (GABAA,fastand GABAA,slow) have been observed in CA1 pyramidal neurons, studies of spontaneous IPSCs (sIPSCs) in these neurons have reported only a single population of events that resemble GABAA,fastin their rise and decay kinetics. The absence of slow sIPSCs calls into question the synaptic basis of GABAA,slow. We present evidence here that both evoked responses are synaptic in origin, because two classes of minimally evoked, spontaneous and miniature IPSCs exist that correspond to GABAA,fastand GABAA,slow. Slow sIPSCs occur infrequently, suggesting that the cells underlying these events have a low spontaneous firing rate, unlike the cells giving rise to fast sIPSCs. Like evoked GABAA,fastand GABAA,slow, fast and slow sIPSCs are modulated differentially by furosemide, a subtype-specific GABAAantagonist. Furosemide blocks fast IPSCs by acting directly on the postsynaptic receptors, because it reduces the amplitude of both miniature IPSCs and the responses of excised patches to applied GABA. Thus, in the hippocampus, parallel inhibitory circuits are composed of separate populations of interneurons that contact anatomically segregated and pharmacologically distinct postsynaptic receptors.
Peripheral Axotomy Induces Long-Term c-Jun Amino-Terminal Kinase-1 Activation and Activator Protein-1 Binding Activity by c-Jun and junD in Adult Rat Dorsal Root GangliaIn Vivo
Authors: Kenney Anna Marie , Kocsis Jeffery D.
Published: April 2018

One of the earliest documented molecular events after sciatic nerve injury in adult rats is the rapid, long-term upregulation of the immediate early gene transcription factor c-Jun mRNA and protein in lumbar dorsal root ganglion (DRG) neurons, suggesting that c-Jun may regulate genes that are important both in the early post-injury period and during later peripheral axonal regeneration. However, neither the mechanism through which c-Jun protein is increased nor the level of its post-injury transcriptional activity in axotomized DRGs has been characterized. To determine whether transcriptional activation of c-Jun occurs in response to nerve injuryin vivoand is associated with axonal regeneration, we have assayed axotomized adult rat DRGs for evidence of jun kinase activation, c-Jun phosphorylation, and activator protein-1 (AP-1) binding. We report that sciatic nerve transection resulted in chronic activation of c-Jun amino-terminal kinase-1 (JNK) in L4/L5 DRGs concomitant with c-Jun amino-terminal phosphorylation in neurons, and lasting AP-1 binding activity, with both c-Jun and JunD participating in DNA binding complexes. The timing of JNK activation was dependent on the distance of the axotomy site from the DRGs, suggesting the requirement for a retrograde transport-mediated signal. AP-1 binding and c-Jun protein returned to basal levels in DRGs as peripheral regeneration was completed but remained elevated in the case of chronic sprouting, indicating that c-Jun may regulate target genes that are involved in axonal outgrowth.
Preconditioning with Bright Light Evokes a Protective Response against Light Damage in the Rat Retina
Authors: Liu Changdong , Peng Min , Laties Alan M. , Wen Rong
Published: April 2018

Constant exposure to bright light induces photoreceptor degeneration and at the same time upregulates the expression of several neurotrophic factors in the retina. At issue is whether the induced neurotrophic factors protect photoreceptors. We used a preconditioning paradigm to show that animals preconditioned with bright light became resistant to subsequent light damage. The preconditioning consisted of a 12–48 hr preexposure, followed by a 48 hr “rest phase” of normal cyclic lighting. The greatest protection was achieved by a 12 hr preexposure. Preconditioning induces a prolonged increase in two endogenous neurotrophic factors: basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). It also stimulates the phosphorylation of extracellular signal-regulated protein kinases (Erks) in both photoreceptors and Müller cells. These findings indicate that exposure to bright light initiates two opposing processes: a fast degenerative process that kills photoreceptors and a relatively slower process that leads to the protection of photoreceptors. The extent of light damage, therefore, depends on the interaction of the two processes. These results also suggest a role of endogenous bFGF and CNTF in photoreceptor protection and the importance of Erk activation in photoreceptor survival.
Differential Regional Expression and Ultrastructural Localization of α-Actinin-2, a Putative NMDA Receptor-Anchoring Protein, in Rat Brain
Authors: Wyszynski Michael , Kharazia Viktor , Shanghvi Roopal , Rao Anuradha , Beggs Alan H. , Craig Ann Marie , Weinberg Richard , Sheng Morgan
Published: April 2018

Fast chemical neurotransmission is dependent on ionotropic receptors that are concentrated and immobilized at specific postsynaptic sites. The mechanisms of receptor clustering and anchoring in neuronal synapses are poorly understood but presumably involve molecular linkage of membrane receptor proteins to the postsynaptic cytoskeleton. Recently the actin-binding protein α-actinin-2 was shown to bind directly to the NMDA receptor subunits NR1 and NR2B (Wyszynski et al., 1997), suggesting that α-actinin-2 may function to attach NMDA receptors to the actin cytoskeleton. Here we show that α-actinin-2 is localized specifically in glutamatergic synapses in cultured hippocampal neurons. By immunogold electron microscopy, α-actinin-2 is concentrated over the postsynaptic density (PSD) of numerous asymmetric synapses where it colocalizes with NR1 immunoreactivity. Thus α-actinin-2 is appropriately positioned at the ultrastructural level to function as a postsynaptic-anchoring protein for NMDA receptors. α-Actinin-2 is not, however, exclusively found at the PSD; immunogold labeling was also associated with filaments and the spine apparatus of dendritic spines and with microtubules in dendritic shafts. α-Actinin-2 showed marked differential regional expression in rat brain. For instance, the protein is expressed at much higher levels in dentate gyrus than in area CA1 of the hippocampus. This differential regional expression implies that glutamatergic synapses in various parts of the brain differ with respect to their α-actinin-2 content and thus, potentially, the extent of possible interaction between α-actinin-2 and the NMDA receptor.
Neuronal Basic Helix-Loop-Helix Proteins (NEX, neuroD, NDRF): Spatiotemporal Expression and Targeted Disruption of the NEX Gene in Transgenic Mice
Authors: Schwab Markus H. , Druffel-Augustin Silke , Gass Peter , Jung Martin , Klugmann Matthias , Bartholomae Angelika , Rossner Moritz J. , Nave Klaus-Armin
Published: April 2018

Basic helix-loop-helix (bHLH) genes have emerged as important regulators of neuronal determination and differentiation in vertebrates. Three putative neuronal differentiation factors [NEX for neuronal helix-loop-helix protein-1 (mammalian atonal homolog-2), neuroD (β-2), and NDRF for neuroD-related factor (neuroD2)] are highly homologous to each other in the bHLH region and comprise a new bHLH subfamily. To study the role of NEX, the first bHLH protein identified in this group, we have disrupted the NEX gene by homologous recombination. NEX-deficient mice have no obvious developmental defect, and CNS neurons appear fully differentiated. To investigate further whether the absence of NEX is compensated for by neuroD and NDRF, we compared the spatiotemporal expression of all three genes. We demonstrate, byin situhybridization, that the transcription patterns of NEX, neuroD, and NDRF genes are highly overlapping in the developing CNS of normal rats between embryonic day 12 and adult stages but are not strictly identical. The most prominent transcription of each gene marks the dorsal neuroepithelium of the telencephalon in early development and is sustained in the adult neocortex, hippocampus, and cerebellum. In general, neuroD provides the earliest marker of neuronal differentiation in any given region compared with NDRF or NEX. Whereas a few CNS regions are specific for neuroD, no region was detected in which solely NEX or NDRF is expressed. This suggests that the function of the mutant NEX gene in neuronal differentiation is compensated for by neuroD and NDRF and that, in analogy with myogenic bHLH proteins, neuronal differentiation factors are at least in part equivalent in function.
Distinct Modes of Neuronal Migration in Different Domains of Developing Cerebellar Cortex
Authors: Komuro Hitoshi , Rakic Pasko
Published: April 2018

As postmitotic neurons migrate to their final destinations, they encounter different cellular microenvironments, but functional responses of migrating neurons to changes in local environmental cues have not been examined. In the present study, we used a confocal microscope on acute cerebellar slice preparations to examine real-time changes in the shape of granule cells, as well as the mode and rate of their migration as they transit different microenvironments. The rate of granule cell movement is fastest in the molecular layer, whereas their elongated somata and long leading processes remain in close contact with Bergmann glial fibers. Cell movement is slowest in the Purkinje cell layer after granule cells detach from the surface of Bergmann glia and the somata become transiently round, whereas the leading processes considerably shorten. Surprisingly, after entering the internal granular layer, granule cells re-extend both their somata and leading processes as they resume rapid movement independent of Bergmann glial fibers. In this last phase of migration, described here for the first time, most granule cells move radially for >100 μm (a distance comparable to that observed in the molecular layer) until they reach the deep strata of the internal granular layer, where they become rounded again and form synaptic contacts with mossy fiber terminals. These observations reveal that migrating neurons alter their shape, rate, and mode of movement in response to local environmental cues and open the possibility for testing the role of signaling molecules in cerebellar neurogenesis.