bluuebunny/crossref_metadata_2025 huggingface dataset:
Staurosporine-Induced Apoptosis of Cultured Rat Hippocampal Neurons Involves Caspase-1-Like Proteases as Upstream Initiators and Increased Production of Superoxide as a Main Downstream Effector
Authors: Krohn Aaron J. , Preis Elke , Prehn Jochen H. M.
Published: April 2018

We induced apoptosis in cultured rat hippocampal neurons by exposure to the protein kinase inhibitor staurosporine (30 nm, 24 hr). Treatment with the antioxidant (±)-α-tocopherol (100 μm) or the superoxide dismutase-mimetic manganese tetrakis (4-benzoyl acid) porphyrin (1 μm) significantly reduced staurosporine-induced cell death. Using hydroethidine-based digital videomicroscopy, we observed a significant increase in intracellular superoxide production that peaked 6–8 hr into the staurosporine exposure. This increase occurred in the absence of gross mitochondrial depolarization monitored with the voltage-sensitive probe tetramethylrhodamine ethyl ester. We then prepared extracts from staurosporine-treated hippocampal neurons and monitored cleavage of acetyl-Tyr-Val-Ala-Asp-aminomethyl-coumarin and acetyl-Asp-Glu-Val-Asp-AMC, fluorogenic substrates for caspase-1-like and caspase-3-like proteases, respectively. Staurosporine caused a significant increase in caspase-1-like activity that preceded intracellular superoxide production and reached a maximum after 30 min. Caspase-3-like activity paralleled intracellular superoxide production, with peak activity seen after 8 hr. Treatment with the corresponding caspase-3-like protease inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (10 μm) prevented the increase in caspase-3-like activity and staurosporine-induced nuclear fragmentation, but failed to prevent the rise in superoxide production and subsequent cell death. In contrast, treatment with caspase-1-like protease inhibitors reduced both superoxide production and cell death. Of note, antioxidants prevented superoxide production, caspase-3-like protease activity, and cell death even when added 4 hr after the onset of the staurosporine exposure. These results suggest a scenario of an early, caspase-1-like activity followed by a delayed intracellular superoxide production that mediates staurosporine-induced cell death of cultured rat hippocampal neurons.
Protease Inhibitor Coinfusion with Amyloid β-Protein Results in Enhanced Deposition and Toxicity in Rat Brain
Authors: Frautschy Sally A. , Horn David L. , Sigel Jason J. , Harris-White Marni E. , Mendoza John J. , Yang Fusheng , Saido T. C. , Cole Gregory M.
Published: April 2018

Amyloid β-protein, Aβ, is normally produced in brain and is cleared by unknown mechanisms. In Alzheimer’s disease (AD), Aβ accumulates in plaque-like deposits and is implicated genetically in neurodegeneration. Here we investigate mechanisms for Aβ degradation and Aβ toxicityin vivo, focusing on the effects of Aβ40, which is the peptide that accumulates in apolipoprotein E4-associated AD. Chronic intraventricular infusion of Aβ40 into rat brain resulted in limited deposition and toxicity. Coinfusion of Aβ40 with the cysteine protease inhibitor leupeptin resulted in increased extracellular and intracellular Aβ immunoreactivity. Analysis of gliosis and TUNEL in neuron layers of the frontal and entorhinal cortex suggested that leupeptin exacerbated Aβ40 toxicity. This was supported further by the neuronal staining of cathepsin B in endosomes or lysosomes, colocalizing with intracellular Aβ immunoreactivity in pyknotic cells. Leupeptin plus Aβ40 caused limited but significant neuronal phospho-tau immunostaining in the entorhinal cortex. Intriguingly, Aβ40 plus leupeptin induced intracellular accumulation of the more toxic Aβ, Aβ42, in a small group of septal neurons. Leupeptin infusion previously has been reported to interfere with lysosomal proteolysis and to result in the accumulation of lipofuscin, dystrophic neurites, tau- and ubiquitin-positive inclusions, and structures resembling paired helical filaments. Coinfusion of Aβ40 with the serine protease inhibitor aprotinin also increased diffuse extracellular deposition but reduced astrocytosis and TUNEL and was not associated with intracellular Aβ staining. Collectively, these data suggest that an age or Alzheimer’s-related defect in lysosomal/endosomal function could promote Aβ deposition and DNA fragmentation in neurons and glia similar to that found in Alzheimer’s disease.
Selective Activation of Gαoby D2LDopamine Receptors in NS20Y Neuroblastoma Cells
Authors: Watts Val J. , Wiens Brenda L. , Cumbay Medhane G. , Vu Minh N. , Neve Rachael L. , Neve Kim A.
Published: April 2018

D2Ldopamine receptor activation results in rapid inhibition and delayed heterologous sensitization of adenylate cyclase in several host cell types. The D2Ldopamine receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibition and sensitization in a neuronal cell environment and to identify the particular G-proteins involved. Acute activation of D2Lreceptors with the selective D2agonist quinpirole inhibited forskolin-stimulated cAMP accumulation, whereas prolonged incubation (2 hr) with quinpirole resulted in heterologous sensitization (more than twofold) of forskolin-stimulated cAMP accumulation in NS20Y-D2Lcells. To unambiguously identify the pertussis toxin (PTX)-sensitive G-proteins responsible for inhibition and sensitization, we used viral-mediated gene delivery to assess the ability of genetically engineered PTX-resistant G-proteins (Gαi1*, Gαi2*, Gαi3*, and Gαo*) to rescue both responses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibition of GTPγS-stimulated adenylate cyclase, respectively. To assess the specificity of D2L-Gα coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-resistant G-protein α subunits and treated with PTX, and quinpirole-induced responses were measured. Infection of NS20Y-D2Lcells with HSV-Gαo* rescued both inhibition and sensitization in PTX-treated cells, whereas infection with HSV-Gαi1*, HSV-Gαi2*, or HSV-Gαi3* failed to rescue either response. In summary, the current study provides strong evidence that the D2Ldopamine receptor couples to Gαoin neuronal cells, and that this coupling is responsible for both the acute and subacute effects of D2receptor activation on adenylate cyclase activity.
Presynaptic Induction and Expression of Homosynaptic Depression atAplysiaSensorimotor Neuron Synapses
Authors: Armitage Beth A. , Siegelbaum Steven A.
Published: April 2018

The cellular mechanisms underlying the induction and expression of homosynaptic depression at the glutamatergic synapse betweenAplysiasensory and motor neurons were studied in dissociated cell culture. Intracellular microelectrodes were used to stimulate action potentials in the presynaptic sensory neuron and record the depolarizing EPSP from the motor neuron. Homosynaptic depression (HSD) was induced by repeatedly stimulating the sensory neuron at rates as low as one action potential per minute. Activation of postsynaptic Glu receptors was neither sufficient nor necessary to induce HSD. Thus, repeated applications of exogenous Glu did not depress the synaptically evoked EPSP. Moreover, normal HSD was observed when the sensory neuron was stimulated during a period when the Glu receptors were blocked with the antagonist DNQX. The induction of HSD is thus likely to occur within the presynaptic terminal. We explored the role of presynaptic calcium in the induction of HSD by injecting the sensory neuron with EGTA, a relatively slow calcium chelator that does not alter rapid release but effectively buffers the slow residual calcium transient thought to be important for plasticity. EGTA had little effect on HSD, indicating that residual Caiis not involved. HSD does not appear to involve a decrease in presynaptic calcium influx, because there was no change in the presynaptic calcium transient, measured by calcium indicator dyes, during HSD. We conclude that HSD is induced and expressed in the presynaptic terminal, possibly by a mechanism directly coupled to the release process.
Functional Consequences of Central Serotonin Depletion Produced by Repeated Fenfluramine Administration in Rats
Authors: Baumann Michael H. , Ayestas Mario A. , Rothman Richard B.
Published: April 2018

Repeated administration ofd,l-fenfluramine (FEN) is known to cause prolonged depletion of forebrain serotonin (5-HT) in animals. Ironically, few studies have evaluated functional consequences of such FEN-induced 5-HT loss. In the present work, we examined neuroendocrine and behavioral responses evoked by acute FEN injection in rats that had previously received a 4 d FEN-dosing regimen known to deplete forebrain 5-HT (d,l-FEN, 20 mg/kg, s.c., b.i.d.). Rats were fitted with indwelling jugular catheters before the study to allow for repeated intravenous challenge injections and stress-free blood sampling. At 1 and 2 weeks after the 4 d dosing regimen, acute FEN (1.5 or 3.0 mg/kg, i.v.) produced dose-related elevations in plasma corticosterone and prolactin; these hormonal responses were markedly attenuated in FEN-pretreated rats. Behavioral effects of acute FEN, namely flat body posture and forepaw treading, were also blunted in FEN-pretreated rats. Interestingly, rats exposed to repeated FEN did not display overt abnormalities in hormonal or behavioral parameters under basal (i.e., unprovoked) conditions, despite dramatic decreases in postmortem tissue levels of 5-HT in numerous brain areas. Our results suggest that FEN-induced 5-HT depletion is accompanied by multiple impairments in 5-HT function. Although the clinical relevance of our data are debatable, the findings clearly show the utility of the FEN challenge test for uncoveringin vivofunctional deficits that might otherwise go undetected. FEN should remain an important pharmacological tool for determining the role of 5-HT neurons in mediating diverse physiological and behavioral processes.
Control of Myelination by Specific Patterns of Neural Impulses
Authors: Stevens Beth , Tanner Sandra , Fields R. Douglas
Published: April 2018

A cell culture preparation equipped with stimulating electrodes was used to investigate whether action potential activity can influence myelination of mouse dorsal root ganglia axons by Schwann cells. Myelination was reduced to one-third of normal by low-frequency impulse activity (0.1 Hz), but higher-frequency stimulation (1 Hz) had no effect. The number of Schwann cells and the ultrastructure of compact myelin were not affected. The frequency of stimulation that inhibited myelination decreased expression of the cell adhesion molecule L1, and stimulation under conditions that prevented the reduction in L1 blocked the effects on myelination. This link between myelination and functional activity in the axon at specific frequencies that change axonal expression of L1 could have important consequences for the structural and functional relationship of myelinating axons.
Novel Domain-Specific Actions of Amyloid Precursor Protein on Developing Synapses
Authors: Morimoto Takako , Ohsawa Ikuroh , Takamura Chizuko , Ishiguro Mariko , Nakamura Yasuko , Kohsaka Shinichi
Published: April 2018

The effect of the secretory form of amyloid precursor protein (sAPP) on synaptic transmission was examined by using developing neuromuscular synapses inXenopuscell cultures. The frequency of spontaneous postsynaptic currents (SSCs) was reduced by the addition of sAPP, whereas the amplitude of impulse-evoked postsynaptic currents (ESCs) was increased by sAPP. These opposing effects on spontaneous versus evoked release were separated by using the specific domain of APP. The C-terminal fragment of sAPP (CAPP) only reduced SSC frequency and did not affect ESCs. By contrast, the N-terminal fragment of sAPP (NAPP) did not affect SSC frequency but did increase ESC amplitude. The reduction of SSC frequency by sAPP appears to be mediated by activation of potassium channels through a cGMP-dependent pathway, whereas the increase of ESC amplitude is mediated by a different pathway involving activation of protein kinase(s). These results suggest the potential role of sAPP as a modulator of synaptic activity by two specific domains.
Attenuated Neurotransmitter Release and Spreading Depression-Like Depolarizations after Focal Ischemia in Mutant Mice with Disrupted Type I Nitric Oxide Synthase Gene
Authors: Shimizu-Sasamata Masao , Bosque-Hamilton Prince , Huang Paul L. , Moskowitz Michael A. , Lo Eng H.
Published: April 2018

Nitric oxide (NO) plays a complex role in the pathophysiology of cerebral ischemia. In this study, mutant mice with disrupted type I (neuronal) NO synthase (nNOS) were compared with wild-type littermates after permanent focal ischemia. Cerebral blood flow in the central and peripheral zones of the ischemic distribution were measured with laser doppler flowmetry. Simultaneously, microdialysis electrodes were used to measure extracellular amino acid concentrations and DC potential in these same locations. Blood flow was reduced to <25 and 60% of baseline levels in the central and peripheral zones, respectively; there were no differences in nNOS mutants versus wild-type mice. Within the central ischemic zone, DC potentials rapidly shifted to −20 mV in all mice. In the ischemic periphery, spreading depression (SD)-like waves of depolarization were observed. SD-like events were significantly fewer in the nNOS mutant mice. Concurrent with these hemodynamic and electrophysiological perturbations, extracellular elevations in amino acids occurred after ischemia. There were no detectable differences between wild-type and mutant mice in the ischemic periphery. However, in the central zone of ischemia, elevations in glutamate and GABA were significantly lower in the nNOS mutants. Twenty-four hour infarct volumes in the nNOS mutant mice were significantly smaller than in their wild-type littermates. Overall, the number of SD-like depolarizations and the integrated efflux of glutamate were significantly correlated with infarct size. These results suggest that NO derived from the nNOS isoform contributes to tissue damage after focal ischemia by amplifying excitotoxic amino acid release in the core and deleterious waves of SD-like depolarizations in the periphery.
Neuronal Receptors Mediating Responses to AntibodyActivated Laminin-1
Authors: Ivins Jonathan K. , Colognato Holly , Kreidberg Jordan A. , Yurchenco Peter D. , Lander Arthur D.
Published: April 2018

Embryonic retinal neurons lose the ability to extend neurites on laminin-1 (LN-1) with increasing developmental age yet still do so on other laminin isoforms. However, after treatment of LN-1 with antibodies to “short-arm” regions or removal of the short arms proteolytically, LN-1 supports attachment and extension of neurites even by late embryonic retinal neurons. We have mapped a domain for antibody-mediated “activation” of LN-1 to the N-terminal end of the α1 chain. Furthermore, we show that the primary receptors used in the retinal neuron response to “activated” LN-1 are integrins α3β1 and α6β1; these are the same receptors used by these neurons for outgrowth on other LN isoforms. Interestingly, α3β1 is preferentially involved in neurite outgrowth, whereas α6β1 preferentially mediates attachment and spreading. However, in cultures from α3 integrin-deficient mice, α6β1 mediates retinal ganglion cell neurite outgrowth and compensates for the absence of α3β1. Finally, we show that key features of the retinal neuron response to LN-1 also characterize neurons of the hippocampus, thalamus, and cerebral cortex; these include poor response to untreated LN-1, extensive neurite outgrowth on antibody-activated LN-1 or on fragment E8, and dependence of this response on integrin α6β1 and at least one other long arm-binding β1 integrin. These data suggest that regulation of LN-1 function via the process of activation could have important consequences for axonal regeneration. Curiously, the data also imply that the mechanism of laminin activation involves enhanced function at sites that cannot be considered cryptic.
Neuronal Activity Induction of the Stathmin-Like Gene RB3 in the Rat Hippocampus: Possible Role in Neuronal Plasticity
Authors: Beilharz Erica J. , Zhukovsky Eugene , Lanahan Anthony A. , Worley Paul F. , Nikolich Karoly , Goodman Laurie J.
Published: April 2018

Synaptic activity induces a rapid transcriptional response that is essential for the establishment of long-term neuronal plasticity. Using a differential cloning technique, we have identified a gene induced by seizure activity in the brain as RB3. RB3 is a recently cloned gene belonging to the stathmin family of phosphoproteins. Like SCG10, RB3 is brain-specific, althoughin situhybridization results show that the expression of RB3 is more ubiquitous than is that of SCG10. Using genomic DNA sequencing, we show that the 27 amino acid sequence unique to the RB3" transcript is encoded by an alternatively spliced exon, exon 2′. Using a peptide antibody raised against exon 2′ to detect RB3" and an anti-Flag antibody to detect an epitope-tagged version of RB3, we show that both proteins are localized to the Golgi apparatus of transfected COS7 cells. Of particular interest, RB3 mRNA, but not SCG10 mRNA, is rapidly induced in the dentate gyrus granule layer of the hippocampus after electrically induced seizure activity as well as stimuli leading to long-term potentiation (LTP). In addition, RB3 mRNA is induced in pheochromocytoma (PC12) cells treated with 250 ng/ml NGF. These results suggest that RB3 may play a role in activity-induced neuronal plasticity and neuronal differentiation.
Regulation of F-Actin Stability in Dendritic Spines by Glutamate Receptors and Calcineurin
Authors: Halpain Shelley , Hipolito Arlene , Saffer Linda
Published: April 2018

Neuronal degeneration and cell death can result from excessive activation of receptors for the excitatory neurotransmitter glutamate; however, the very earliest changes in cytoskeletal organization have not been well documented. We have used anin vitromodel system to examine the early consequences of intense glutamate receptor activation on dendritic spine synapses. Cultured hippocampal neurons exposed to NMDA for as little as 5 min exhibited a rapid and extensive loss of dendritic spines. Staining for the presynaptic marker synapsin 1 and the postsynaptic density proteins PSD-95 and the NR1 subunit of NMDA receptors remained intact. The disappearance of spines was accompanied by a selective loss of filamentous actin staining at synapses. The NMDA-induced loss of spine actin was time- and concentration-dependent and blocked by NMDA receptor antagonists. The effect was mimicked byl-glutamate, AMPA, and ionomycin but not by agonists of L-type calcium channels or of metabotropic glutamate receptors. The effect of NMDA on local actin assembly was strongly attenuated by pretreatment with an actin stabilizing compound or by an antagonist of the calcium-dependent protein phosphatase calcineurin. Immunoreactivity for calcineurin was enriched at synapses together with F-actin. These results indicate that the actin-mediated stability of synaptic structure is disrupted by intense glutamate receptor activity and that calcineurin blockers may be useful in preventing such destabilization.
Nerve Terminal Currents Induced by Autoreception of Acetylcholine Release
Authors: Fu Wen-Mei , Liou Houng-Chi , Chen Yu-Hwa
Published: April 2018

The activation of autoreceptors is known to be important in the modulation of presynaptic transmitter secretion in peripheral and central neurons. Using whole-cell recordings made from the free growth cone of myocyte-contact motoneurons ofXenopuscell cultures, we have observed spontaneous nerve terminal currents (NTCs). These spontaneous NTCs are blocked by d-tubocurarine (d-TC) and α-bungarotoxin (α-BuTx), indicating that endogenously released acetylcholine (ACh) can produce substantial membrane depolarization in the nerve terminals. Local application of NMDA to the growth cone increased the frequency of spontaneous NTCs. When the electrical stimulations were applied at the soma to initiate evoked-release of ACh, evoked ACh-induced potentials were recorded in the nerve terminals, which were inhibited by d-TC and hexamethonium but not by atropine. Replacement of normal Ringer’s solution with high-Mg2+, low-Ca2+solution also reversibly inhibited evoked ACh-induced potentials. The possible regulatory role of presynaptic nicotinic autoreceptors on the synaptic transmission was also examined. When the innervated myocyte was whole-cell voltage-clamped to record synaptic currents, application of hexamethonium inhibited the amplitude of evoked synaptic currents at a higher degree than that of iontophoretic ACh-induced currents. Furthermore, hexamethonium markedly reduced the frequency of spontaneous synaptic currents at high-activity synapses. Pretreatment of neurons with α-BuTx also inhibited the evoked synaptic currents in manipulated synapses. These results suggest that ACh released spontaneously or by electrical stimulation may act on the presynaptic nicotinic autoreceptors of the same nerve terminals to produce membrane potential change and to regulate synaptic transmission.
Evidence for a Role of the Chemorepellent Semaphorin III and Its Receptor Neuropilin-1 in the Regeneration of Primary Olfactory Axons
Authors: Pasterkamp R. Jeroen , De Winter Fred , Holtmaat Anthony J. G. D. , Verhaagen Joost
Published: April 2018

To explore a role for chemorepulsive axon guidance mechanisms in the regeneration of primary olfactory axons, we examined the expression of the chemorepellent semaphorin III (sema III), its receptor neuropilin-1, and collapsin response mediator protein-2 (CRMP-2) during regeneration of the olfactory system. In the intact olfactory system, neuropilin-1 and CRMP-2 mRNA expression define a distinct population of olfactory receptor neurons, corresponding to immature (B-50/GAP-43-positive) and a subset of mature (olfactory marker protein-positive) neurons located in the lower half of the olfactory epithelium. Sema III mRNA is expressed in pial sheet cells and in second-order olfactory neurons that are the target cells of neuropilin-1-positive primary olfactory axons. These data suggest that in the intact olfactory bulb sema III creates a molecular barrier, which helps restrict ingrowing olfactory axons to the nerve and glomerular layers of the bulb. Both axotomy of the primary olfactory nerve and bulbectomy induce the formation of new olfactory receptor neurons expressing neuropilin-1 and CRMP-2 mRNA. After axotomy, sema III mRNA is transiently induced in cells at the site of the lesion. These cells align regenerating bundles of olfactory axons. In contrast to the transient appearance of sema III-positive cells at the lesion site after axotomy, sema III-positive cells increase progressively after bulbectomy, apparently preventing regenerating neuropilin-1-positive nerve bundles from growing deeper into the lesion area. The presence of sema III in scar tissue and the concomitant expression of its receptor neuropilin-1 on regenerating olfactory axons suggests that semaphorin-mediated chemorepulsive signal transduction may contribute to the regenerative failure of these axons after bulbectomy.
Specific and Differential Activation of Mitogen-Activated Protein Kinase Cascades by Unfamiliar Taste in the Insular Cortex of the Behaving Rat
Authors: Berman Diego E. , Hazvi Shoshi , Rosenblum Kobi , Seger Rony , Dudai Yadin
Published: April 2018

Rats were given to drink an unfamiliar taste solution under conditions that result in long-term memory of that taste. The insular cortex, which contains the taste cortex, was then removed and assayed for activation of mitogen-activated protein kinase (MAPK) cascades by using antibodies to the activated forms of various MAPKs. Extracellular responsive kinase 1–2 (ERK1–2) in the cortical homogenate was significantly activated within <30 min of drinking the taste solution, without alteration in the total level of the ERK1–2 proteins. The activity subsided to basal levels within <60 min. In contrast, ERK1–2 was not activated when the taste was made familiar. The effect of the unfamiliar taste was specific to the insular cortex. Jun N-terminal kinase 1–2 (JNK1–2) was activated by drinking the taste but with a delayed time course, whereas the activity of Akt kinase and p38MAPK remained unchanged. Elk-1, a member of the ternary complex factor and an ERK/JNK downstream substrate, was activated with a time course similar to that of ERK1–2. Microinjection of a reversible inhibitor of MAPK/ERK kinase into the insular cortex shortly before exposure to the novel taste in a conditioned taste aversion training paradigm attenuated long-term taste aversion memory without significantly affecting short-term memory or the sensory, motor, and motivational faculties required to express long-term taste aversion memory. It was concluded that ERK and JNK are specifically and differentially activated in the insular cortex after exposure to a novel taste, and that this activation is required for consolidation of long-term taste memory.
Galanin Modulation of Seizures and Seizure Modulation of Hippocampal Galanin in Animal Models of Status Epilepticus
Authors: Mazarati Andrey M. , Liu Hantao , Soomets Ursel , Sankar Raman , Shin Don , Katsumori Hiroshi , Langel Ülo , Wasterlain Claude G.
Published: April 2018

We examined the role of hippocampal galanin in an animal model of status epilepticus (SE). Control rats showed abundant galanin-immunoreactive (Gal-IR) fibers in the dentate hilus, whereas no Gal-IR neurons were observed. Three hours after the onset of self-sustaining SE (SSSE), induced either by intermittent stimulation of the perforant path for 30 min (PPS) or by injection of lithium and pilocarpine, Gal-IR fibers disappeared in the hilus and remained absent for up to 1 week afterward. Twelve hours after the induction of SE by PPS or 3 hr after pilocarpine administration, Gal-IR neurons appeared in the hilus; these neurons increased in number after 1 d and gradually declined 3 and 7 d later. Galanin concentration in the hippocampus, measured by ELISA, significantly decreased on the plateau of SSSE and increased 24 hr after PPS. Galanin (0.05 nmol) injected into the hilus prevented the induction of SSSE, and 0.5 nmol of galanin stopped established SSSE. These effects were attenuated by galanin receptor antagonists (M35 > M40 ≥ M15). 2-Ala-galanin (5 nmol), a putative agonist of galanin type 2 receptors, prevented but was unable to stop SSSE. M35 facilitated the development of SSSE when given before PPS. We suggest that hippocampal galanin acts as an endogenous anticonvulsant via galanin receptors. SE-induced galanin depletion in the hippocampus may contribute to the maintenance of seizure activity, whereas the increase of galanin concentration and the appearance of galanin-immunoreactive neurons may favor the cessation of SSSE. The seizure-protecting action of galanin SSSE opens new perspectives in the treatment of SE.
Experience-Dependent Plasticity of Adult Rat S1 Cortex Requires Local NMDA Receptor Activation
Authors: Rema V. , Armstrong-James M. , Ebner F. F.
Published: April 2018

The effect of blocking NMDA glutamate receptors in adult rat cortex on experience-dependent synaptic plasticity of barrel cortex neurons was studied by infusing D-AP5 with an osmotic minipump over barrel cortex for 5 d of novel sensory experience. In acute pilot studies, 500 μmD-AP5 was shown to specifically suppress NMDA receptor (NMDAR)-dependent responses of single cells in cortical layers I–IV. To induce plasticity, all whiskers except D2 and D1 were cut close to the face 1 d after pump insertion. The animals were housed with 2 cage mates before recording 4 d later. This pairing of two whiskers for several days in awake animals generates highly significant biases in responses from D2 layer IV (barrel) cells to the intact D1 whisker as opposed to the cut D3 whisker. D-AP5 completely prevented the D1/D3 surround whisker bias from occurring in the D2 barrel cells (p> 0.6 for D1 > D3, Wilcoxon). Fast-spike and slow-spike barrel cells were affected equally, suggesting parity for inhibitory and excitatory cell plasticity. D-AP5 only partially suppressed the D1/D3 bias in supragranular layers (layers II–III) in the same penetrations (p< 0.042 for D1 > D3). In control animals, the inactive L-AP5 isomer allowed the bias to develop normally toward the intact surround whisker (p< 0.001 for D1 > D3) for cells in all layers. We conclude that experience-dependent synaptic plasticity of mature barrel cortex is cortically dependent and that modification of local cortical NMDARs is necessary for its expression.
Subcellular Redistribution of m2 Muscarinic Acetylcholine Receptors in Striatal InterneuronsIn Vivoafter Acute Cholinergic Stimulation
Authors: Bernard Véronique , Laribi Ouahiba , Levey Allan I. , Bloch Bertrand
Published: April 2018

The purpose of our work was to investigate how the cholinergic environment influences the targeting and the intracellular trafficking of the muscarinic receptor m2 (m2R)in vivo. To address this question, we have used immunohistochemical approaches at light and electron microscopic levels to detect the m2R in control rats and rats treated with muscarinic receptor agonists.

In control animals, m2Rs were located mostly at postsynaptic sites at the plasma membrane of perikarya and dendrites of cholinergic and NPY–somatostatin interneurons as autoreceptors and heteroreceptors, respectively. Presynaptic receptors were also detected in boutons. The m2Rs were usually detected at extrasynaptic sites, but they could be found rarely in association with symmetrical synapses, suggesting that the cholinergic transmission mediated by m2R occurs via synaptic and nonsynaptic mechanisms. The stimulation of muscarinic receptors with oxotremorine provoked a dramatic alteration of m2R compartmentalization, including endocytosis with a decrease of the density of m2R at the membrane (−63%) and an increase of those associated with endosomes (+86%) in perikarya. The very strong increase of m2R associated with multivesicular bodies (+732%) suggests that oxotremorine activated degradation. The slight increase in the Golgi apparatus (+26%) suggests that the m2R stimulation had an effect on the maturation of m2R. The substance P receptor located at the membrane of the same neurons was unaffected by oxotremorine.

Our data demonstrate that cholinergic stimulation dramatically influences the subcellular distribution of m2R in striatal interneuronsin vivo. These events may have key roles in controlling abundance and availability of muscarinic receptors via regulation of receptor endocytosis, degradation, and/or neosynthesis. Further, the control of muscarinic receptor trafficking may influence the activity of striatal interneurons, including neurotransmitter release and/or electric activity.
Randomized Retinal Ganglion Cell Axon Routing at the Optic Chiasm of GAP-43-Deficient Mice: Association with Midline Recrossing and Lack of Normal Ipsilateral Axon Turning
Authors: Sretavan David W. , Kruger Kelly
Published: April 2018

During mammalian development, retinal ganglion cell (RGC) axons from nasal retina cross the optic chiasm midline, whereas temporal retina axons do not and grow ipsilaterally, resulting in a projection of part of the visual world onto one side of the brain while the remaining part is represented on the opposite side. Previous studies have shown that RGC axons in GAP-43-deficient mice initially fail to grow from the optic chiasm to form optic tracts and are delayed temporarily in the midline region. Here we show that this delayed RGC axon exit from the chiasm is characterized by abnormal randomized axon routing into the ipsilateral and contralateral optic tracts, leading to duplicated representations of the visual world in both sides of the brain. Within the chiasm, individual contralaterally projecting axons grow in unusual semicircular trajectories, and the normal ipsilateral turning of ventral temporal axons is absent. These effects on both axon populations suggest that GAP-43 does not mediate pathfinding specifically for one or the other axon population but is more consistent with a model in which the initial pathfinding defect at the chiasm/tract transition zone leads to axons backing up into the chiasm, resulting in circular trajectories and eventual random axon exit into one or the other optic tract. Unusual RGC axon trajectories include chiasm midline recrossing similar to abnormal CNS midline recrossing in invertebrate “roundabout” mutants andDrosophilawith altered calmodulin function. This resemblance and the fact that GAP-43 also has been proposed to regulate calmodulin availability raise the possibility that calmodulin function is involved in CNS midline axon guidance in both vertebrates and invertebrates.
Abnormal Targeting of Developing Hippocampal Mossy Fibers after Epileptiform Activities via L-type Ca2+Channel ActivationIn Vitro
Authors: Ikegaya Yuji
Published: April 2018

The hippocampal mossy fibers, which originate from the dentate granule cells, develop mainly in the early postnatal period and are involved in numerous pathological processes. In this study, hippocampal slices prepared from premature rats were cultivated in the presence of convulsants to evaluate the influences of epileptiform activities on mossy fiber ontogeny. Electrophysiological and histochemical analyses revealed that prolonged hyperexcitability inhibited proper growth of the mossy fibers and caused ectopic innervation to the stratum oriens and the dentate molecular layer. These phenomena were prevented by pharmacological blockade of L-type Ca2+channels, which did not affect convulsant-evoked ictal bursts. After single-pulse stimulation of the stratum granulosum in the slices cultured under paroxysmal conditions, the dentate gyrus displayed excessive excitation, but synaptic transmission to the CA3 region was hypoactive. However, brief repetitive stimulation elicited delayed epileptiform discharges in the CA3 region that were inhibited by an NMDA receptor antagonist. Chronic treatment with an L-type Ca2+channel blocker ameliorated such aberrant neurotransmissions. These results suggest that ictal neuron activities at the developmental stage of the mossy fibers bring about the errant maturation associated with hippocampal dysfunction, which may form a cellular basis for the sequelae of childhood epilepsy, including chronic epilepsy or cognitive deficits. Thus I propose that L-type Ca2+channel blockers can ameliorate the aversive prognosis of childhood epilepsy.
NovelGLRA1Missense Mutation (P250T) in Dominant Hyperekplexia Defines an Intracellular Determinant of Glycine Receptor Channel Gating
Authors: Saul Brigitta , Kuner Thomas , Sobetzko Diana , Brune Wolfram , Hanefeld Folker , Meinck Hans-Michael , Becker Cord-Michael
Published: April 2018

Missense mutations as well as a null allele of the human glycine receptor α1 subunit geneGLRA1result in the neurological disorder hyperekplexia [startle disease, stiff baby syndrome, Mendelian Inheritance in Man (MIM) #149400]. In a pedigree showing dominant transmission of hyperekplexia, we identified a novel point mutation C1128A ofGLRA1. This mutation encodes an amino acid substitution (P250T) in the cytoplasmic loop linking transmembrane regions M1 and M2 of the mature α1 polypeptide. After recombinant expression, homomeric α1P250Tsubunit channels showed a strong reduction of maximum whole-cell chloride currents and an altered desensitization, consistent with a prolonged recovery from desensitization. Apparent glycine binding was less affected, yielding an approximately fivefold increase inKivalues. Topological analysis predicts that the substitution of proline 250 leads to the loss of an angular polypeptide structure, thereby destabilizing open channel conformations. Thus, the novelGLRA1mutant allele P250T defines an intracellular determinant of glycine receptor channel gating.
Identification of Microglial Signal Transduction Pathways Mediating a Neurotoxic Response to Amyloidogenic Fragments of β-Amyloid and Prion Proteins
Authors: Combs Colin K. , Johnson Derrick E. , Cannady Steve B. , Lehman Timothy M. , Landreth Gary E.
Published: April 2018

Microglial interaction with amyloid fibrils in the brains of Alzheimer’s and prion disease patients results in the inflammatory activation of these cells. We observed that primary microglial cultures and the THP-1 monocytic cell line are stimulated by fibrillar β-amyloid and prion peptides to activate identical tyrosine kinase-dependent inflammatory signal transduction cascades. The tyrosine kinases Lyn and Syk are activated by the fibrillar peptides and initiate a signaling cascade resulting in a transient release of intracellular calcium that results in the activation of classical PKC and the recently described calcium-sensitive tyrosine kinase PYK2. Activation of the MAP kinases ERK1 and ERK2 follows as a subsequent downstream signaling event. We demonstrate that PYK2 is positioned downstream of Lyn, Syk, and PKC. PKC is a necessary intermediate required for ERK activation. Importantly, the signaling response elicited by β-amyloid and prion fibrils leads to the production of neurotoxic products. We have demonstrated in a tissue culture model that conditioned media from β-amyloid- and prion-stimulated microglia or from THP-1 monocytes are neurotoxic to mouse cortical neurons. This toxicity can be ameliorated by treating THP-1 cells with specific enzyme inhibitors that target various components of the signal transduction pathway linked to the inflammatory responses.
The Kinesin Motor KIF3A Is a Component of the Presynaptic Ribbon in Vertebrate Photoreceptors
Authors: Muresan Virgil , Lyass Asya , Schnapp Bruce J.
Published: April 2018

Kinesin motors are presumed to transport various membrane compartments within neurons, but their specificin vivofunctions, cargoes, and expression patterns in the brain are unclear. We have investigated the distribution of KIF3A, a member of the heteromeric family of kinesins, in the vertebrate retina. We find KIF3A at two distinct sites within photoreceptors: at the basal body of the connecting cilium axoneme and at the synaptic ribbon. Immunoelectron microscopy of the photoreceptor ribbon synapse shows KIF3A to be concentrated both at the ribbon matrix and on vesicles docked at the ribbon, a result that is consistent with the presence of both detergent-extractable and resistant KIF3A fractions at these synapses. KIF3A is also present in the inner plexiform layer, again at presynaptic ribbons. These findings suggest that within a single cell, the photoreceptor, one kinesin polypeptide, KIF3A, can serve two distinct functions, one specific for ribbon synapses.
Developmental Synaptic Changes Increase the Range of Integrative Capabilities of an Identified Excitatory Neocortical Connection
Authors: Angulo Marı́a Cecilia , Staiger Jochen F. , Rossier Jean , Audinat Etienne
Published: April 2018

Excitatory synaptic transmission between pyramidal cells and fast-spiking (FS) interneurons of layer V of the motor cortex was investigated in acute slices by using paired recordings at 30°C combined with morphological analysis. The presynaptic and postsynaptic properties at these identified central synapses were compared between 3- and 5-week-old rats. At these two postnatal developmental stages, unitary EPSCs were mediated by the activation of AMPA receptors with fast kinetics at a holding potential of −72 mV. The amplitude distribution analysis of the EPSCs indicates that, at both stages, pyramidal–FS connections consisted of multiple functional release sites. The apparent quantal size obtained by decreasing the external calcium ([Ca2+]e) varied from 11 to 29 pA near resting membrane potential. In young rats, pairs of presynaptic action potentials elicited unitary synaptic responses that displayed paired-pulse depression at all tested frequencies. In older animals, inputs from different pyramidal cells onto the same FS interneuron had different paired-pulse response characteristics and, at most of these connections, a switch from depression to facilitation occurred when decreasing the rate of presynaptic stimulation. The balance between facilitation and depression endows pyramidal–FS connections from 5-week-old animals with wide integrative capabilities and confers unique functional properties to each synapse.
Ischemic Tolerance in Murine Cortical Cell Culture: Critical Role for NMDA Receptors
Authors: Grabb Margaret C. , Choi Dennis W.
Published: April 2018

Murine cortical cultures containing both neurons and glia (daysin vitro13–15) were exposed to periods of oxygen–glucose deprivation (5–30 min) too brief to induce neuronal death. Cultures “preconditioned” by sublethal oxygen–glucose deprivation exhibited 30–50% less neuronal death than controls when exposed to a 45–55 min period of oxygen–glucose deprivation 24 hr later. This preconditioning-induced neuroprotection was specific in that neuronal death induced by exposure to excitotoxins or to staurosporine was not attenuated. Neuroprotection was lost if the time between the preconditioning and severe insult were decreased to 7 hr or increased to 72 hr and was blocked if the NMDA antagonist 100 μm3-((d)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid was applied during the preconditioning insult. This was true even if the duration of preconditioning was increased as far as possible (while still remaining sublethal). A similar preconditioning effect was also produced by sublethal exposure to high K+, glutamate, or NMDA but not to kainate ortrans-1-aminocyclopentane-1,3-dicarboxylic acid.
Intrapreoptic Microinjection of GHRH or Its Antagonist Alters Sleep in Rats
Authors: Zhang Jianyi , Obál Ferenc , Zheng Tong , Fang Jidong , Taishi Ping , Krueger James M.
Published: April 2018

Previous reports indicate that growth hormone–releasing hormone (GHRH) is involved in sleep regulation. The site of action mediating the nonrapid eye movement sleep (NREMS)–promoting effects of GHRH is not known, but it is independent from the pituitary. GHRH (0.001, 0.01, and 0.1 nmol/kg) or a competitive antagonist of GHRH (0.003, 0.3, and 14 nmol/kg) was microinjected into the preoptic area, and the sleep–wake activity was recorded for 23 hr after injection in rats. GHRH elicited dose-dependent increases in the duration and in the intensity of NREMS compared with that in control records after intrapreoptic injection of physiological saline. The antagonist decreased the duration and intensity of NREMS and prolonged sleep latency. Consistent alterations in rapid eye movement sleep (REMS) and in brain temperature were not found. The GHRH antagonist also attenuated the enhancements in NREMS elicited by 3 hr of sleep deprivation. Histological verification of the injection sites showed that the majority of the effective injections were in the preoptic area and the diagonal band of Broca. The results indicate that the preoptic area mediates the sleep-promoting activity of GHRH.
A Glial Cell Line-Derived Neurotrophic Factor-Secreting Clone of the Schwann Cell Line SCTM41 Enhances Survival and Fiber Outgrowth from Embryonic Nigral Neurons Grafted to the Striatum and to the Lesioned Substantia Nigra
Authors: Wilby Martin J. , Sinclair Simon R. , Muir Elizabeth M. , Zietlow Rike , Adcock Kathryn H. , Horellou Philippe , Rogers John H. , Dunnett Stephen B. , Fawcett James W.
Published: April 2018

We have developed a novel Schwann cell line, SCTM41, derived from postnatal sciatic nerve cultures and have stably transfected a clone with a rat glial cell line-derived neurotrophic factor (GDNF) construct. Coculture with this GDNF-secreting clone enhancesin vitrosurvival and fiber growth of embryonic dopaminergic neurons. In the rat unilateral 6-OHDA lesion model of Parkinson’s disease, we have therefore made cografts of these cells with embryonic day 14 ventral mesencephalic grafts and assayed for effects on dopaminergic cell survival and process outgrowth. We show that cografts of GDNF-secreting Schwann cell lines improve the survival of intrastriatal embryonic dopaminergic neuronal grafts and improve neurite outgrowth into the host neuropil but have no additional effect on amphetamine-induced rotation. We next looked to see whether bridge grafts of GDNF-secreting SCTM41 cells would promote the growth of axons to their striatal targets from dopaminergic neurons implanted orthotopically into the 6-OHDA-lesioned substantia nigra. We show that such bridge grafts increase the survival of implanted embryonic dopaminergic neurons and promote the growth of axons through the grafts to the striatum.
Early Visual Experience Shapes the Representation of Auditory Space in the Forebrain Gaze Fields of the Barn Owl
Authors: Miller Greg L. , Knudsen Eric I.
Published: April 2018

Auditory spatial information is processed in parallel forebrain and midbrain pathways. Sensory experience early in life has been shown to exert a powerful influence on the representation of auditory space in the midbrain space-processing pathway. The goal of this study was to determine whether early experience also shapes the representation of auditory space in the forebrain.

Owls were raised wearing prismatic spectacles that shifted the visual field in the horizontal plane. This manipulation altered the relationship between interaural time differences (ITDs), the principal cue used for azimuthal localization, and locations of auditory stimuli in the visual field. Extracellular recordings were used to characterize ITD tuning in the auditory archistriatum (AAr), a subdivision of the forebrain gaze fields, in normal and prism-reared owls.

Prism rearing altered the representation of ITD in the AAr. In prism-reared owls, unit tuning for ITD was shifted in the adaptive direction, according to the direction of the optical displacement imposed by the spectacles. Changes in ITD tuning involved the acquisition of unit responses to adaptive ITD values and, to a lesser extent, the elimination of responses to nonadaptive (previously normal) ITD values. Shifts in ITD tuning in the AAr were similar to shifts in ITD tuning observed in the optic tectum of the same owls.

This experience-based adjustment of binaural tuning in the AAr helps to maintain mutual registry between the forebrain and midbrain representations of auditory space and may help to ensure consistent behavioral responses to auditory stimuli.
Cholinergic and GABAergic Inputs Drive Patterned Spontaneous Motoneuron Activity before Target Contact
Authors: Milner Louise D. , Landmesser Lynn T.
Published: April 2018

Patterned spontaneous electrical activity has been demonstrated in a number of developing neural circuits and has been proposed to play a role in refining connectivity once axons reach their targets. Using an isolated spinal cord preparation, we have found that chick lumbosacral motor axons exhibit highly regular bursts of activity from embryonic day 4 (E4) (stage 24–25), shortly after they exit the spinal cord and while still en route toward their target muscles. Similar bursts could be evoked by stimulating descending pathways at cervical or thoracic levels. Unlike older embryonic cord circuits, the major excitatory transmitter driving activity was not glutamate but acetylcholine, acting primarily though nicotinic non-α7 receptors. The circuit driving bursting was surprisingly robust and plastic, because bursting was only transiently blocked by cholinergic antagonists, and following recovery, was now driven by GABAergic inputs. Permanent blockade of spontaneous activity was only achieved by a combination of cholinergic antagonists and bicuculline, a GABAAantagonist. The early occurrence of patterned motor activity suggests that it could be playing a role in either peripheral pathfinding or spinal cord circuit formation and maturation. Finally, the characteristic differences in burst parameters already evident between different motoneuron pools at E4 would require that the combination of transcription factors responsible for specifying pool identity to have acted even earlier.