bluuebunny/crossref_metadata_2025 huggingface dataset:
Patterns of Chondroitin Sulfate Immunoreactivity in the Developing Tectum Reflect Regional Differences in Glycosaminoglycan Biosynthesis
Authors: Hoffman-Kim Diane , Lander Arthur D. , Jhaveri Sonal
Published: April 2018

The glycosaminoglycan chondroitin sulfate (CS) is expressed in many parts of the developing brain, both in regions where axons preferentially grow and in areas that axons distinctly avoid. Somein vitrostudies suggest that CS and proteoglycans (PGs) that carry CS enhance axon growth, whereas others suggest that CS and CSPGs inhibit it. In the developing hamster, there is evidence that midbrain raphe cells act as a barrier to prevent growth of optic axons across the tectal midline. Here we show that in the newborn hamster, CS immunoreactivity is substantially higher in midline than in lateral tectum, raising the possibility that CSPGs play a role in the unilateral containment of optic axons. However, analysis of tectal PGs by anion exchange chromatography and denaturing gel electrophoresis failed to detect substantial differences between midline and lateral tectum in either the types or relative amounts of CSPG and heparan sulfate PG protein cores. In contrast, metabolic labeling of tectal slicesin vitrodocumented that incorporation of35S-sulfate into macromolecules is significantly increased at the tectal midline, in a pattern resembling chondroitin sulfate immunoreactivity. This difference was evident whether slices were labeled for 1 hr or overnight and was not paralleled by a difference in overall protein synthesis, suggesting that the rate of synthesis of sulfated macromolecules is specifically elevated in midline tectum. We propose that the concentration of CS at the midline of the developing tectum is a reflection of a higher rate of synthesis or sulfation of glycosaminoglycans by midline cells, rather than a higher level of production of any particular CSPG. These results suggest that the distribution of some axon guidance signals in development may be controlled by differential regulation of glycosaminoglycan biosynthetic enzymes.
Optimal Effectiveness of BDNF for Fetal Nigral Transplants Coincides with the Ontogenic Appearance of BDNF in the Striatum
Authors: Yurek David M. , Hipkens Susan B. , Wiegand Stanley J. , Altar C. Anthony
Published: April 2018

Transplantation of fetal nigral dopamine neurons into the caudate and putamen of Parkinson’s disease patients produces limited symptomatic relief. One approach to augment the outgrowth and function of nigral grafts includes exposure of the graphs to neurotrophic factors; however, the temporal requirements for optimizing these actions are unknown. The present study characterized the ontogeny of brain-derived neurotrophic factor (BDNF) in the rat striatum and used this information to define and evaluate three distinct periods of BDNF infusion into fetal nigral grafts transplanted into the striatum of rats with experimental Parkinson’s disease. At postnatal day 1 (P1), BDNF and dopamine were measured at 17 and 27% of peak levels, respectively, that occurred at P27 for both. Both compounds showed their greatest surge between P7 and P20, increasing from 40% to ∼95% of peak levels. Exogenous BDNF infused into transplants during weeks 1 and 2 after the transplantation, which coincide with the developmental period embryonic day 14 (E14)–P7 for transplanted tissue, did not improve rotational behavior or enhance fiber outgrowth of transplanted dopamine neurons. Delaying the BDNF infusion until transplanted tissue was approximately P8–P21 greatly enhanced the effect on rotational behavior and doubled the area of dopamine fiber outgrowth from the transplants. Delaying the infusion until transplanted tissue was approximately P36–P49 failed to augment fiber outgrowth and decreased the behavioral function of transplants. Thus, the optimal effect of exogenous BDNF on the development of dopamine neurons in fetal nigral transplants occurs at a postnatal age when endogenous dopamine and BDNF show the greatest increases during the normal development of the striatum.
Opioid Inhibition of Hippocampal Interneurons via Modulation of Potassium and Hyperpolarization-Activated Cation (Ih) Currents
Authors: Svoboda Kurt R. , Lupica Carl R.
Published: April 2018

The actions of mu- and delta-opioid agonists (DAMGO and DPDPE, respectively) on GABAergic interneurons in stratum oriens of area CA1 of the hippocampus were examined by using whole-cell voltage-clamp recordings in brain slices. Both agonists consistently generated outward currents of similar magnitude (15–20 pA) in the majority of cells. However, under control conditions, current–voltage (I/V) relationships revealed that only a small number of these cells (3 of 77) demonstrated clear increases in membrane conductance, associated with the activation of the potassium current known asGirk. These interneurons also exhibited a slowly activating, inwardly rectifying current known asIhon hyperpolarizing step commands.Ihwas blocked by the extracellular application of cesium (3–9 mm) or ZD 7288 (10–100 μm) but was insensitive to barium (1–2 mm). In an effort to determine whether the holding current changes were attributable to the modulation ofGirkand/orIh, we used known blockers of these ion channels (barium or cesium and ZD 7288, respectively). Extracellular application of cesium (3–9 mm) or ZD 7288 (25–100 μm) blockedIhand significantly reduced the opioid-induced outward currents by 58%. Under these conditions the opioid agonists activated a potassium current with characteristics similar toGirk. Similarly, during barium (1–2 mm) application the opioid-induced outward currents were reduced by 46%, and a clear reduction inIhand the whole-cell conductance was revealed. These data suggest that the opioids can modulate bothIhandGirkin the same population of stratum oriens interneurons and that the modulation of these ion channels can contribute to the inhibition of interneuron activity in the hippocampus.
TrkB and Neurotrophin-4 Are Important for Development and Maintenance of Sympathetic Preganglionic Neurons Innervating the Adrenal Medulla
Authors: Schober Andreas , Wolf Nicole , Huber Katrin , Hertel Richard , Krieglstein Kerstin , Minichiello Liliana , Kahane Nitza , Widenfalk Johan , Kalcheim Chaya , Olson Lars , Klein Rüdiger , Lewin Gary R. , Unsicker Klaus
Published: April 2018

The adrenal medulla receives its major presynaptic input from sympathetic preganglionic neurons that are located in the intermediolateral (IML) column of the thoracic spinal cord. The neurotrophic factor concept would predict that these IML neurons receive trophic support from chromaffin cells in the adrenal medulla. We show here that adrenal chromaffin cells in the adult rat store neurotrophin (NT)-4, but do not synthesize or store detectable levels of BDNF or NT-3, respectively. Preganglionic neurons to the adrenal medulla identified by retrograde tracing with fast blue or Fluoro-Gold (FG) express TrkB mRNA. After unilateral destruction of the adrenal medulla, 24% of IML neurons, i.e., all neurons that are preganglionic to the adrenal medulla in spinal cord segments T7–T10, disappear. Administration of NT-4 in gelfoams (6 μg) implanted into the medullectomized adrenal gland rescued all preganglionic neurons as evidenced by their presence after 4 weeks. NT-3 and cytochrome C were not effective. The action of NT-4 is accompanied by massive sprouting of axons in the vicinity of the NT-4 source as monitored by staining for acetylcholinesterase and synaptophysin immunoreactivity, suggesting that NT-4 may enlarge the terminal field of preganglionic nerves and enhance their access to trophic factors. Analysis of TrkB-deficient mice revealed degenerative changes in axon terminals on chromaffin cells. Furthermore, numbers of FG-labeled IML neurons in spinal cord segments T7–T10 of NT-4-deficient adult mice were significantly reduced. These data are consistent with the notion that NT-4 from chromaffin cells operates through TrkB receptors to regulate development and maintenance of the preganglionic innervation of the adrenal medulla.
The Survival-Promoting Effect of Glial Cell Line-Derived Neurotrophic Factor on Axotomized Corticospinal NeuronsIn VivoIs Mediated by an Endogenous Brain-Derived Neurotrophic Factor Mechanism
Authors: Giehl Klaus M. , Schütte Andreas , Mestres Pedro , Yan Qiao
Published: April 2018

Autocrine trophic functions of brain-derived neurotrophic factor (BDNF) have been proposed for many central neurons because this neurotrophin displays striking colocalization with its receptor trkB within the CNS. In the cortex, the distribution patterns of BDNF and trkB expression are almost identical. Corticospinal neurons (CSNs) are a major cortical long-distance projecting system. They are localized in layer V of the somatosensory cortex, and their axons project into the spinal cord where they contribute to the innervation of spinal motoneurons. We have shown recently that adult CSNs express trkB mRNA and are rescued from axotomy-induced death by BDNF treatment. Half of the axotomized CSNs survived without BDNF infusions. These findings raise the possibility that endogenous cortical BDNF is involved in the trophic support of this neuronal population. To test the hypothesis that endogenous cortical BDNF promotes survival of adult CSNs, we infused the BDNF-neutralizing affinity-purified antibody RAB to axotomized and unlesioned CSNs for 7 d. This treatment resulted in increased death of axotomized CSNs. Survival of unlesioned CSNs was not affected by RAB treatment.In situhybridizations for BDNF and trkB mRNA revealed that virtually all CSNs express trkB, whereas only half of them express BDNF. Thus, autocrine/paracrine mechanisms are likely to contribute to the endogenous BDNF protection of axotomized CSNs. We have demonstrated previously that, in addition to BDNF, glial cell line-derived neurotrophic factor (GDNF) and neurotrophin 3 (NT-3) also rescue CSNs from axotomy-induced death. We now show that the rescuing by GDNF requires the presence of endogenous cortical BDNF, implicating a central role of this neurotrophin in the trophic support of axotomized CSNs and a trophic cross-talk between BDNF and GDNF regarding the maintenance of lesioned CSNs. In contrast, NT-3 promotes survival of axotomized CSNs even when endogenous cortical BDNF is neutralized by RAB, indicating a potential of compensatory mechanisms for the trophic support of CSNs.
Critical Role of Axonal A-Type K+Channels and Axonal Geometry in the Gating of Action Potential Propagation along CA3 Pyramidal Cell Axons: A Simulation Study
Authors: Kopysova Irina L. , Debanne Dominique
Published: April 2018

A model of CA3 pyramidal cell axons was used to study a new mode of gating of action potential (AP) propagation along the axon that depends on the activation of A-type K+current (Debanne et al., 1997). The axonal membrane contained voltage-dependent Na+channels, K+channels, and A-type K+channels. The density of axonal A-channels was first determined so that (1) at the resting membrane potential an AP elicited by a somatic depolarization was propagated into all axon collaterals and (2) propagation failures occurred when a brief somatic hyperpolarization preceded the AP induction. Both conditions were fulfilled only when A-channels were distributed in clusters but not when they were homogeneously distributed along the axon. Failure occurs in the proximal part of the axon. Conduction failure could be determined by a single cluster of A-channels, local decrease of axon diameter, or axonal elongation. We estimated the amplitude and temporal parameters of the hyperpolarization required for induction of a conduction block. Transient and small somatic hyperpolarizations, such as simulated GABAAinhibitory postsynaptic potentials, were able to block the AP propagation. It was shown that AP induction had to occur with a short delay (<30 msec) after the hyperpolarization. We discuss the possible conditions in which such local variations of the axon geometry and A-channel density may occur and the incidence of AP propagation failures on hippocampal network properties.
Regulation of Hippocampal Glucocorticoid Receptor Gene Transcription and Protein ExpressionIn Vivo
Authors: Herman James P. , Spencer Robert
Published: April 2018

Glucocorticoid receptors (GRs) are glucocorticoid-activated transcription factors that modulate expression of a variety of neuronal genes. Appropriate control of GR expression is therefore critical for maintenance of cellular and organismic homeostasis. The present study assessed glucocorticoid regulation of the GR at the gene, mRNA, and protein level. Removal of circulating glucocorticoids (adrenalectomy) increased GR mRNA expression in CA1 and dentate gyrus (DG). Corticosterone (CORT) replacement normalized GR mRNA expression, whereas high doses slightly decreased GR mRNA in CA1. Parallel increases were observed using a probe complementary to the distal 3′ untranslated region, indicating that mRNA changes were not attributable to selection of alternative polyadenylation site. Expression of a GR intronic sequence was also increased by adrenalectomy, consistent with increased gene transcription. Analysis of regional GR protein expression by immunoautoradiography did not reveal changes in GR protein in pyramidal cell layers; however, increased GR signal was seen in the stratum radiatum, indicating redistribution of GR to the cytosol. Western blot analysis confirmed adrenalectomy-induced increases in hippocampal GR levels. Administration of the mineralocorticoid receptor (MR) antagonist spironolactone increased both GR mRNA and protein in CA1 and DG, consistent with MR-mediated inhibition of GR transcription. However, high-dose CORT treatment did not decrease GR mRNA or protein levels. Chronic stress exposure did not downregulate GR mRNA or protein in hippocampus. The results suggest that the hippocampal GR is subject to heterologous regulation by the MR. In contrast, GR autoregulation is only evident during prolonged exposure to high-circulating glucocorticoid levels.
Expression and Endocytosis of Lysosomal Aspartylglucosaminidase in Mouse Primary Neurons
Authors: Kyttälä Aija , Heinonen Outi , Peltonen Leena , Jalanko Anu
Published: April 2018

Aspartylglucosaminuria (AGU) is a neurodegenerative lysosomal storage disease that is caused by mutations in the gene encoding for a soluble hydrolase, aspartylglucosaminidase (AGA). In this study, we have used our recently developed mouse model for AGU and analyzed processing, intracellular localization, and endocytosis of recombinant AGA in telencephalic AGU mouse neuronsin vitro. The processing steps of AGA were found to be similar to the peripheral cells, but both the accumulation of the inactive precursor molecule and delayed lysosomal processing of the enzyme were detected. AGA was distributed to the cell soma and neuronal processes but was not found in the nerve terminals. Endocytotic capability of cultured telencephalic neurons was comparable to that of fibroblasts, and endocytosis of AGA was blocked by free mannose-6-phosphate (M6P), indicating that uptake of the enzyme was mediated by M6P receptors (M6PRs). Uptake of extracellular AGA was also studied in the tumor-derived cell lines rat pheochromocytoma (PC12) and mouse neuroblastoma cells (N18), which both endocytosed AGA poorly as compared with cultured primary neurons. Expression of cation-independent M6PRs (CI-M6PRs) in different cell lines correlated well with the endocytotic capability of these cells. Although a punctate expression pattern of CI-M6PRs was found in fibroblasts and cultured primary neurons, the expression was beyond the detection limit in PC12 and N18 cells. This indicates that PC12 and N18 are not feasible cell lines to describe neuronal uptake of mannose-6-phosphate-tagged proteins. Thisin vitrodata will form an important basis for the brain-targeted therapy of AGU.
Hair Cells and Supporting Cells Share a Common Progenitor in the Avian Inner Ear
Authors: Fekete Donna M. , Muthukumar Shanthini , Karagogeos Domna
Published: April 2018

Sensory organs of the vertebrate inner ear contain two major cell types: hair cells (HCs) and supporting cells (SCs). To study the lineage relationships between these two populations, replication-defective retroviral vectors encoding marker genes were delivered to the otic vesicle of the chicken embryo. The resulting labeled clones were analyzed in the hearing organ of the chicken, called the basilar papilla (BP), after cellular differentiation. BPs were allowed to develop for 2 weeks after delivery of the retrovirus, were removed, and were processed histochemically as whole mounts to identify clones of cells. Clusters of labeled cells were evident in the sensory epithelium, the nonsensory epithelium, and in adjacent tissues. Labeled cell types included HCs, two morphologically distinct types of SCs, homogene cells, border cells, hyaline cells, ganglion cells, and connective tissue cells. Each clone was sectioned and cell-type identification was performed on sensory clones expressing retrovirally transduced β-galactosidase. Cell composition was determined for 41 sensory clones, most of which contained both HCs and SCs. Clones containing one HC and one SC were observed, suggesting that a common progenitor exists that can remain bipotential up to its final mitotic division. The possibility that these two cell types may also arise from a mitotic precursor during HC regeneration in the mature basilar papilla is consistent with their developmental history.
Expression of the Mitotic Motor Protein Eg5 in Postmitotic Neurons: Implications for Neuronal Development
Authors: Ferhat Lotfi , Cook Crist , Chauviere Muriel , Harper Maryannick , Kress Michel , Lyons Gary E. , Baas Peter W.
Published: April 2018

It is well established that the microtubules of the mitotic spindle are organized by a variety of motor proteins, and it appears that the same motors or closely related variants organize microtubules in the postmitotic neuron. Specifically, cytoplasmic dynein and the kinesin-related motor known as CHO1/MKLP1 are used within the mitotic spindle, and recent studies suggest that they are also essential for the establishment of the axonal and dendritic microtubule arrays of the neuron. Other motors are required to tightly regulate microtubule behaviors in the mitotic spindle, and it is attractive to speculate that these motors might also help to regulate microtubule behaviors in the neuron. Here we show that a homolog of the mitotic kinesin-related motor known as Eg5 continues to be expressed in rodent neurons well after their terminal mitotic division. In neurons, Eg5 is directly associated with the microtubule array and is enriched within the distal regions of developing processes. This distal enrichment is transient, and typically lost after a process has been clearly defined as an axon or a dendrite. Strong expression can resume later in development, and if so, the protein concentrates within newly forming sprouts at the distal tips of dendrites. We suggest that Eg5 generates forces that help to regulate microtubule behaviors within the distal tips of developing axons and dendrites.
Promyelinating Schwann Cells Express Tst-1/SCIP/Oct-6
Authors: Arroyo Edgardo J. , Bermingham John R. , Rosenfeld Michael G. , Scherer Steven S.
Published: April 2018

Tst-1/SCIP/Oct-6, a POU domain transcription factor, is transiently expressed by developing Schwann cells and is required for their normal development into a myelinating phenotype. Intst-1/scip/oct-6–null sciatic nerves, Schwann cells are transiently arrested at the “promyelinating” stage, when they have a one-to-one relationship with an axon but before they have elaborated a myelin sheath. To determine when Schwann cells express Tst-1/SCIP/Oct-6, we examined β-galactosidase (β-gal) expression in heterozygoustst-1/scip/oct-6mice, in which one copy of thetst-1/scip/oct-6gene has been replaced with the LacZ gene. β-Gal expression from the LacZ gene seems to parallel Tst-1/SCIP/Oct-6 expression from the endogenoustst-1/scip/oct-6gene in developing and regenerating sciatic nerves. Furthermore, electron microscopic examination of 5bromo-4-chloro-3-indolyl-β-d-galactopyranoside– (X-gal) and halogenated indolyl-β-d-galactoside– (Bluo-gal) stained nerves showed that promyelinating Schwann cells express the highest levels of β-gal, both in developing and in regenerating nerves. Thus, the expression of β-gal, a surrogate marker of Tst-1/SCIP/Oct-6, peaks at the same stage of Schwann cell development at which development is arrested intst-1/scip/oct-6–null mice, indicating that Tst-1/SCIP/Oct-6 has a critical role in promyelinating Schwann cells.
Specific Targeting of Ganglion Cell Sprouts Provides an Additional Mechanism for Restoring Peripheral Motor Circuits in Pelvic Ganglia after Spinal Nerve Damage
Authors: Kepper Mark E. , Keast Janet R.
Published: April 2018

The pelvic ganglia contain both sympathetic and parasympathetic neurons and provide an interesting model in which to study the effects of a distributed spinal nerve lesion. Previous animal studies have suggested that after either lumbar or sacral nerve injury, some functional connections are restored between preganglionic and postganglionic neurons. It has been proposed that this is because of intact preganglionic axons sprouting collaterals to supply denervated ganglion cells. However, this has never been demonstrated, and our study has investigated whether the ganglion cells themselves contribute to axogenesis and restoration of peripheral circuitry. We have monitored the growth of axons from pelvic ganglion cells after lumbar or sacral nerve injury (partial decentralization), or a combination of the two (total decentralization). These new processes were distinguished from intact preganglionic terminals by their immunoreactivity for substances present only in pelvic ganglion neurons (vasoactive intestinal peptide, neuropeptide Y, and tyrosine hydroxylase). The proportion of pelvic neurons surrounded by these immunostained fibers was then assessed. Complete removal of preganglionic terminals provides the biggest stimulus for growth of new axon processes (sprouts), which grow profusely within just a few days. These arise from each of the main chemical classes of pelvic neurons but grow at different rates and have different distributions. Importantly, some chemical classes of sprouts preferentially supply neurons of dissimilar histochemistry, suggesting the presence of very specific targeting mechanisms rather than random growth. These sprouts are transient, however, those formed after partial decentralization appear to be maintained. Moreover, after lesion of either lumbar or sacral spinal nerves, many sprouts arise from neurons with intact spinal connections and innervate neurons that have lost their preganglionic inputs. This provides a very different alternative mechanism to reestablish communication between preganglionic and postganglionic neurons. In conclusion, we have demonstrated a rapid and selective axogenesis within the pelvic ganglion after spinal nerve injury. This may allow the development of novel strategies by which autonomic nerve pathways can be experimentally manipulated, to facilitate more rapid return of appropriate peripheral reflex control.
Dopamine Depletion Reorganizes Projections from the Nucleus Accumbens and Ventral Pallidum That Mediate Opioid-Induced Motor Activity
Authors: Churchill Lynn , Klitenick Mark A. , Kalivas Peter W.
Published: April 2018

Motor activity elicited pharmacologically from the nucleus accumbens by the μ-opioid receptor agonistd-Ala-Tyr-Gly-NMePhe-Gly-OH (DAMGO) is augmented in rats sustaining dopamine depletions. GABAergic projections from the nucleus accumbens to ventral pallidum and ventral tegmental area (VTA) are involved because stimulation of GABABreceptors in the VTA (by baclofen) or GABAAreceptors in the ventral pallidum (by muscimol) inhibit the motor response induced by the microinjection of DAMGO into the nucleus accumbens. The present study was done to determine which of these projections is mediating the augmented DAMGO-induced motor activity that follows 6-hydroxydopamine lesions of the nucleus accumbens. The inhibition of DAMGO-induced activation by pallidal injections of muscimol was markedly attenuated in lesioned animals, whereas the inhibition by VTA injections with baclofen was greatly enhanced. A similar switch in emphasis from pallidal to mesencephalic efferents was not observed for dopamine-induced motor activity, because muscimol microinjections inhibited the response elicited by dopamine microinjection into the nucleus accumbens in all subjects. The stimulation of μ-opioid receptors in the ventral pallidum also elicits motor activation, and this is blocked by baclofen microinjection into the VTA. However, after dopamine depletion in the nucleus accumbens, baclofen in the VTA was ineffective in blocking the motor response by DAMGO in the ventral pallidum. These data reveal that dopamine depletion in the nucleus accumbens produces a lesion-induced plasticity that alters the effect of μ-opioid receptor stimulation on efferent projections from the nucleus accumbens and ventral pallidum.
Electrochemical Analysis of Protein Nitrotyrosine and Dityrosine in the Alzheimer Brain Indicates Region-Specific Accumulation
Authors: Hensley Kenneth , Maidt Michael L. , Yu Zhenqiang , Sang Hong , Markesbery William R. , Floyd Robert A.
Published: April 2018

HPLC with electrochemical array detection (HPLC-ECD) was used to quantify 3,3′-dityrosine (diTyr) and 3-nitrotyrosine (3-NO2-Tyr) in four regions of the human brain that are differentially affected in Alzheimer’s disease (AD). DiTyr and 3-NO2-Tyr levels were elevated consistently in the hippocampus and neocortical regions of the AD brain and in ventricular cerebrospinal fluid (VF), reaching quantities five- to eightfold greater than mean concentrations in brain and VF of cognitively normal subjects. Uric acid, a proposed peroxynitrite scavenger, was decreased globally in the AD brain and VF. The results suggest that AD pathogenesis may involve the activation of oxidant-producing inflammatory enzyme systems, including nitric oxide synthase.
Delayed Release of Neurotransmitter from Cerebellar Granule Cells
Authors: Atluri Pradeep P. , Regehr Wade G.
Published: April 2018

At fast chemical synapses the rapid release of neurotransmitter that occurs within a few milliseconds of an action potential is followed by a more sustained elevation of release probability, known as delayed release. Here we characterize the role of calcium in delayed release and test the hypothesis that facilitation and delayed release share a common mechanism. Synapses between cerebellar granule cells and their postsynaptic targets, stellate cells and Purkinje cells, were studied in rat brain slices. Presynaptic calcium transients were measured with calcium-sensitive fluorophores, and delayed release was detected with whole-cell recordings. Calcium influx, presynaptic calcium dynamics, and the number of stimulus pulses were altered to assess their effect on delayed release and facilitation. Following single stimuli, delayed release can be separated into two components: one lasting for tens of milliseconds that is steeply calcium-dependent, the other lasting for hundreds of milliseconds that is driven by low levels of calcium with a nearly linear calcium dependence. The amplitude, calcium dependence, and magnitude of delayed release do not correspond to those of facilitation, indicating that these processes are not simple reflections of a shared mechanism. The steep calcium dependence of delayed release, combined with the large calcium transients observed in these presynaptic terminals, suggests that for physiological conditions delayed release provides a way for cells to influence their postsynaptic targets long after their own action potential activity has subsided.
Noradrenergic-Specific Transcription of the Dopamine β-Hydroxylase Gene Requires Synergy of MultipleCis-Acting Elements Including at Least Two Phox2a-Binding Sites
Authors: Kim Hee-Sun , Seo Hyemyung , Yang Chunying , Brunet Jean-Francois , Kim Kwang-Soo
Published: April 2018

Dopamine β-hydroxylase (DBH) catalyzes the conversion of dopamine to noradrenaline and is selectively expressed in noradrenergic and adrenergic neurons and neuroendocrine cells. Recent data from this laboratory showed that a paired-like homeodomain (HD) protein, Phox2a, interacts with the HD-binding site residing within a composite promoter of the human DBH gene, designated domain IV, in a cell-specific manner and directly controls noradrenergic-specific DBH promoter activity. In this report, we demonstrate that three additional protein-binding sites (i.e., domains I, II, and III) between domain IV and the TATA box are critical for intact DBH promoter activity in noradrenergic cells and that they activate DBH transcription in a highly concerted manner. Transient transfection assays of mutant DBH reporter constructs indicated that domain I was active in every cell line tested, whereas domain III was preferentially active in DBH-positive cells. Remarkably, mutation of domain II was associated with inactivation of DBH promoter activity exclusively in DBH-positive cell lines, defining it as another noradrenergic-specific promoter element. The cell-specific profile of the promoter function of these sequence motifs was further supported byin vitroDNA-binding studies and Southwestern analysis. Furthermore, competition and antibody supershift assays show that transcription factors Sp1 and AP2 are the cognate nuclear factors interacting with domains I and III, respectively. Parallel evidence indicates that domain II is another Phox2a-binding site, demonstrating at least two binding sites for this factor in the upstream DBH promoter. Strikingly, four tandem copies of domain II increased the promoter activity of a minimal DBH promoter by 100- to 200-fold in DBH-positive cell lines without compromising cell specificity. Cotransfection of Phox2a-expression vector dramatically increased the activity of the multiple domain II promoter only in DBH-negative cell lines, confirming that domain II is responsive to Phox2a. Collectively, this study emphasizes a critical role of Phox2a as well as its functional synergism with other transcription factors (e.g., CREB, AP2, and Sp1) in transcriptional activation of the DBH gene.
An Early Phase of EmbryonicDlx5Expression Defines the Rostral Boundary of the Neural Plate
Authors: Yang Lu , Zhang Hailan , Hu Gezhi , Wang Hongyu , Abate-Shen Cory , Shen Michael M.
Published: April 2018

Relatively little is known about the molecular events that specify the rostrocaudal axis of the neural plate. Here we show that a member of theDistal-less(Dlx) homeobox gene family,Dlx5, is one of the earliest known markers for the most rostral ectoderm, before the formation of an overt neural plate. During late gastrulationDlx5expression becomes localized to the anterior neural ridge, which defines the rostral boundary of the neural plate, and also extends caudolaterally, marking the region of the presumptive neural crest. Subsequently,Dlx5is expressed in tissues (olfactory epithelium, ventral cephalic epithelium) that are believed to derive from the anterior neural ridge, based on the avian fate map. The early phase ofDlx5expression in the anterior neural ridge and its derivatives is distinct from a later phase of expression in the ventral telencephalon and diencephalon and also appears to be unique forDlx5among members of theDlxfamily. Another distinctive feature ofDlx5expression is the occurrence of an alternative transcript (δDlx5), which encodes a truncated protein lacking the homeodomain, and represents a significant fraction of totalDlx5transcripts at all embryonic stages that were examined. In contrast with full-length DLX5, the δDLX5 truncated protein is deficient in DNA-binding activity and does not interact with the homeoprotein partner MSX1. Taken together, our findings suggest thatDlx5activity may be regulated via the expression of an alternative transcript and demonstrate thatDlx5marks the anterior boundary of the neural plate.
Multiple Restricted Origin of Oligodendrocytes
Authors: Spassky N. , Goujet-Zalc C. , Parmantier E. , Olivier C. , Martinez S. , Ivanova A. , Ikenaka K. , Macklin W. , Cerruti I. , Zalc B. , Thomas J.-L.
Published: April 2018

Theplpgene encodes the proteolipid protein and its alternatively spliced product DM-20, major proteins of CNS myelin. In the mouse,plp/dm-20transcripts are expressed beginning at embryonic day 9.5 (E9.5) by restricted foci of germinative neuroepithelial cells. To determine the identity of the neural precursors expressingplp/dm- 20, a zeomycin resistance gene fused to thelacZreporter was expressed in transgenic mice under the control of theplpregulatory sequences. In the three different lines generated, the pattern of β-galactosidase expression was similar and superimposable on the expression pattern of endogenousplp/dm-20. Bothin vivoandin vitro, the transgene was expressed by O4+pre-oligodendrocytes, and later by RIP+differentiated oligodendrocytes, but not by neuronal cells, astrocytes, or radial glial cells. After zeomycin selection, a dramatic enrichment in O4+pre-oligodendrocytes was observed in cultures derived from E12.5 transgenic embryos. This enrichment indicates the oligodendroglial specification of neural precursors that continuously expressplp/dm-20. Earlyplp/dm-20-expressing precursors, however, appear to be a separate population from previously describedPDGFRα oligodendrocyte precursors, as shown by the striking differences in their (1) patterns of distribution and (2) responsiveness to PDGF. These data suggest that oligodendrocytes have a plural origin and that earlyplp/dm-20defines one of the neural lineages generating oligodendrocytes.
Cervical Dorsal Rhizotomy Enhances Serotonergic Innervation of Phrenic Motoneurons and Serotonin-Dependent Long-Term Facilitation of Respiratory Motor Output in Rats
Authors: Kinkead Richard , Zhan Wen-Zhi , Prakash Y. S. , Bach Karen B. , Sieck Gary C. , Mitchell Gordon S.
Published: April 2018

We tested the hypothesis that spinal plasticity elicited by chronic bilateral cervical dorsal rhizotomy (C3–C5; CDR) has functional implications for respiratory motor control. Surgery was performed on rats (CDR or sham-operated) 26 d before phrenic motoneurons were retrogradely labeled with cholera toxin. Rats were killed 2 d later, and their spinal cords were harvested and processed to reveal the cholera toxin-labeled phrenic motoneurons and serotonin-immunoreactive terminals. The number of serotonin-immunoreactive terminals within 5 μm of labeled phrenic motoneuron soma and primary dendrites increased 2.1-fold after CDR versus sham-operation. Time-dependent phrenic motor responses to hypoxia were compared among CDR, sham-operated, and control rats. Anesthetized, paralyzed, vagotomized, and artificially ventilated rats were exposed to three, 5 min episodes of isocapnic hypoxia (FiO2= 0.11), separated by 5 min hyperoxic intervals (FiO2= 0.5). One hour after hypoxia, a long-lasting, serotonin-dependent enhancement of phrenic motor output (long-term facilitation) was observed in both sham and control rats. After CDR, long-term facilitation was 108 and 163% greater than control and sham responses, respectively. Pretreatment of CDR rats with a 5-HT2receptor antagonist (ketanserin tartrate, 2 mg/kg, i.v.) before episodic hypoxia prevented long-term facilitation and revealed a modest (−28 ± 13%;p< 0.05) long-lasting depression of phrenic motor output. The results indicate that CDR: (1) increases serotonergic innervation of the phrenic motor nucleus; and (2) augments serotonin-dependent long-term facilitation of phrenic motor output. These results further suggest a form of plasticity based on changes in the capacity for neuromodulation.
Prepulse Inhibition of theTritoniaEscape Swim
Authors: Mongeluzi Donna L. , Hoppe Travis A. , Frost William N.
Published: April 2018

Presenting a weak stimulus just before a strong, startle stimulus reduces the amplitude of the ensuing startle response in humans and other vertebrates. This phenomenon, termed “prepulse inhibition” (PPI), appears to function to reduce distraction while processing sensory input. To date, no detailed neural mechanism has been described for PPI. Here we demonstrate PPI in the marine molluskTritonia diomedea, which has a nervous system highly suitable for cellular analyses. We found that a 100 msec vibrotactile prepulse prevented the animal’s escape swim response to a closely following 1 sec tail shock. This inhibition was highly transient, with a significant effect lasting just 2.5 sec. These findings indicate that theTritoniaescape swim response undergoes a form of PPI phenomenologically similar to that observed in vertebrates. Further tests showed that the vibrotactile stimulus had no inhibitory effect if applied after tail shock, while the animal was preparing to swim, but it acted to terminate swims once they were actively under way. As a first step toward a cellular analysis of PPI, we recorded from neurons of the swim circuit in a semi-intact preparation and found that the vibrotactile stimulus used in the behavioral experiments also prevented the tail shock-elicited swim motor program. These results represent the first explicit demonstration of PPI in an invertebrate and establishTritoniaas a model system for analyzing its physiological basis.
Synaptic Regulation of Action Potential Timing in Neostriatal Cholinergic Interneurons
Authors: Bennett Ben D. , Wilson Charles J.
Published: April 2018

Action potentials in neostriatal cholinergic interneurons recordedin vivoare triggered by summation of two or three discrete synaptic depolarizations (Wilson et al., 1990). The ability and precision with which EPSPs and IPSPs regulate action potential timing was therefore investigatedin vitro. Cholinergic interneurons were identified on the basis of morphological and electrophysiological characteristics in neostriatal slices taken from 2- to 3-week-old postnatal rats recorded at 24–26°C.

During periods of induced regular firing, intrastriatal stimuli were used to evoke pharmacologically isolated monosynaptic AMPA receptor-mediated EPSPs or GABAAreceptor-mediated IPSPs. EPSPs evoked during the interspike interval (ISI) produced a phase-dependent decrease in the ISI, whereas IPSPs produced a phase-independent prolongation of the ISI. Injection of brief depolarizing currents mimicked the action of EPSPs and revealed an alteration in the input resistance during the ISI. In contrast to IPSPs, the ability of brief hyperpolarizing current injections to delay spike generation was phase-dependent. After blockade of GABAergic and glutamatergic synaptic transmission, stimuli failed to produce a detectable conductance change but could still prolong the subsequent ISI primarily through a D1 dopamine receptor-mediated enhancement of the afterhyperpolarization (AHP).

Hence, EPSPs are ideally suited to provide a precise regulation of spike timing in cholinergic cells, whereas IPSPs are more likely to influence the overall level of excitability. The D1-mediated modulation of the AHP may contribute to the prolonged ISI seen in tonically active neuronsin vivoin monkeys trained to respond to a sensory cue.
The Epithelial Inward Rectifier Channel Kir7.1 Displays Unusual K+Permeation Properties
Authors: Döring Frank , Derst Christian , Wischmeyer Erhard , Karschin Christine , Schneggenburger Ralf , Daut Jürgen , Karschin Andreas
Published: April 2018

Rat and human cDNAs were isolated that both encoded a 360 amino acid polypeptide with a tertiary structure typical of inwardly rectifying K+channel (Kir) subunits. The new proteins, termed Kir7.1, were <37% identical to other Kir subunits and showed various unique residues at conserved sites, particularly near the pore region. High levels of Kir7.1 transcripts were detected in rat brain, lung, kidney, and testis.In situhybridization of rat brain sections demonstrated that Kir7.1 mRNA was absent from neurons and glia but strongly expressed in the secretory epithelial cells of the choroid plexus (as confirmed byin situpatch-clamp measurements). In cRNA-injectedXenopusoocytes Kir7.1 generated macroscopic Kir currents that showed a very shallow dependence on external K+([K+]e), which is in marked contrast to all other Kir channels. At a holding potential of −100 mV, the inward current through Kir7.1 averaged −3.8 ± 1.04 μA with 2 mm[K+]eand −4.82 ± 1.87 μA with 96 mm[K+]e. Kir7.1 has a methionine at position 125 in the pore region where other Kir channels have an arginine. When this residue was replaced by the conserved arginine in mutant Kir7.1 channels, the pronounced dependence of K+permeability on [K+]e, characteristic for other Kir channels, was restored and the Ba2+sensitivity was increased by a factor of ∼25 (Ki= 27 μm). These findings support the important role of this site in the regulation of K+permeability in Kir channels by extracellular cations.
Acute Inactivation of Tau Has No Effect on Dynamics of Microtubules in Growing Axons of Cultured Sympathetic Neurons
Authors: Tint Irina , Slaughter Theresa , Fischer Itzhak , Black Mark M.
Published: April 2018

Tau is a developmentally regulated microtubule (MT)-associated protein in neurons that has been implicated in neuronal morphogenesis. On the basis of test tube studies, tau has been proposed to function in axon growth by stabilizing MTs and thereby promoting MT assembly. We have tested this hypothesis by examining the effects of acute inactivation of tau on axonal MTs. Tau was inactivated by microinjecting purified antibodies against recombinant tau into neurons before they extended axons. The injected antibodies quantitatively precipitated tau into aggregates in the soma. With these conditions the neurons elaborate normal-appearing axons, and MTs extend throughout the axons and into the growth cones, but the axons and their MTs are depleted of tau. The immunodepletion of tau had no detectable effect on several parameters of the dynamics of axonal MTs. Depletion of tau also was not accompanied by a reorganization of other major MT-associated proteins or actin filaments in these neurons. Thus, neurons effectively depleted of tau can extend axons that resemble those of control cells, and the axons contain normal-appearing MT arrays with normal dynamic behavior. These observations are exactly the opposite of those expected on the basis of the hypothesis that the stability of axonal MTs is a direct function of their content of tau, indicating that tau in growing axons of cultured sympathetic neurons is not specialized to promote microtubule assembly and stability.
Ceramide Inhibits Inwardly Rectifying K+Currents via a Ras- and Raf-1-Dependent Pathway in Cultured Oligodendrocytes
Authors: Hida Hideki , Takeda Margaret , Soliven Betty
Published: April 2018

Ceramide is a lipid mediator implicated in apoptosis induced by proinflammatory cytokines in many cell types, including oligodendrocytes (OLGs). To determine whether ceramide modulates transmembrane signaling events in OLGs, we studied its effect on intracellular Ca2+(Cai), resting membrane potential and inwardly rectifying K+currents (IKir) in cultured neonatal rat OLGs. We report here that (1) exposure to C2-ceramide (cer) rarely increases OLG Cai, whereas sphingosine elicits sustained increase in Cai; (2) cer causes OLG depolarization, an effect mimicked by sphingosine-1-phosphate but not by sphingosine; and (3) cer, but not its inactive analog dihydroceramide, inhibits OLGIKir. The cer effect is attenuated by Ras antibody Y13-259, by protein kinase C inhibitory peptide (19–36), and by suppression of c-Raf-1 expression with antisense raf-1 oligonucleotides. We conclude that cer-induced OLG depolarization is mediated via inhibition ofIKirby a Ras- and raf-1-dependent pathway, which results in the phosphorylation of the inward rectifier K+channel protein.
Myelin Gene Expression after Experimental Contusive Spinal Cord Injury
Authors: Wrathall Jean R. , Li Wen , Hudson Lynn D.
Published: April 2018

After incomplete traumatic spinal cord injury (SCI), the spared tissue exhibits abnormal myelination that is associated with reduced or blocked axonal conductance. To examine the molecular basis of the abnormal myelination, we used a standardized rat model of incomplete SCI and compared normal uninjured tissue with that after contusion injury. We evaluated expression of mRNA for myelin proteins usingin situhybridization with oligonucleotide probes to proteolipid protein (PLP), the major protein in central myelin; myelin basic protein (MBP), a major component of central myelin and a minor component of peripheral myelin; and protein zero (P0), the major structural protein of peripheral myelin, as well as myelin transcription factor 1 (MYT1). We found reduced expression of PLP and MBP chronically after SCI in the dorsal, lateral, and ventral white matter both rostral and caudal to the injury epicenter. Detailed studies of PLP at 2 months after injury indicated that the density of expressing cells was normal but mRNA per cell was reduced. In addition, P0, normally restricted to the peripheral nervous system, was expressed both at the epicenter and in lesioned areas at least 4 mm rostral and caudal to it. Thus, after SCI, abnormal myelination of residual axons may be caused, at least in part, by changes in the transcriptional regulation of genes for myelin proteins and by altered distribution of myelin-producing cells. In addition, the expression of MYT1 mRNA and protein seemed to be upregulated after SCI in a pattern suggesting the presence of undifferentiated progenitor cells in the chronically injured cord.
Neuronal Differentiation of Precursors in the Neocortical Ventricular Zone Is Triggered by BMP
Authors: Li Weiwei , Cogswell Catherine A. , LoTurco Joseph J.
Published: April 2018

Neocortical neurons begin to differentiate soon after they are generated by mitoses at the surface of the ventricular zone (VZ). We provide evidence here that bone morphogenetic protein (BMP) triggers neuronal differentiation of neocortical precursors within the VZ. In cultures of dissociated neocortical neuroepithelial cells, BMPs increase the number of MAP-2– and TUJ1-positive cells within 24 hr of treatment. In explant cultures, BMP-4 treatment leads to an increase in the number of TUJ1-positive cells within the ventricular zone. Furthermore, truncated, dominant-negative, BMP type I receptor, introduced into neocortical precursors by retrovirus-mediated gene transfer, blocks neurite elaboration and migration out of the VZ. Finally, immunocytochemistry indicates that BMP protein is present at the VZ surface. Together, these results indicate that BMP protein is present within the VZ, that BMP is capable of promoting neuronal differentiation, and that signaling through BMP receptors triggers neuronal precursors to differentiate and migrate out of the VZ.