bluuebunny/crossref_metadata_2025 huggingface dataset:
A distinct subset of tenascin/CS-6-PG-rich astrocytes restricts neuronal growth in vitro
Authors: Meiners S , Powell EM , Geller HM
Published: April 2018

Astrocytes provide an optimal surface for attachment, migration, and growth of CNS neurons. Nonetheless, not all astrocytes are alike: our previous work demonstrated a heterogeneity in the ability of cultured astrocyte monolayers to support neuronal growth. Areas displaying a fibrous, uneven surface ( “rocky” astrocytes) were shown to be restrictive substrates, whereas surrounding, flat areas were permissive substrates. However, whether these cell types are in fact different cannot be addressed using mixed cultures. Therefore, in the current study we used morphological criteria to isolate the two subpopulations from mixed astrocyte cultures established from the cerebral cortex of neonatal rats. Following isolation, the purified populations only produced progeny with the same phenotype as the parent cells. We then measured production of several extracellular matrix molecules putatively involved in neuronal guidance during development and quantitatively assessed neuronal behavior on the purified populations. Immunocytochemistry and immunoblotting showed that rocky astrocytes were enriched in tenascin and chondroitin-6- sulfate-containing proteoglycans, but not in laminin or fibronectin. In addition, these astrocytes, as well as their isolated matrix, were a less permissive substrate for neuronal growth than flat astrocytes/matrix. Neurite outgrowth was significantly increased on rocky astrocytes following treatment with chondroitinase ABC or AC, but not heparitinase or hyaluronidase. These data support a critical role for matrix-bound chondroitin-6-sulfate-containing proteoglycans. We hypothesize that rocky astrocytes represent a subtype of cells which form barriers to neuronal growth during cortical development.
At least two mechanisms are involved in the death of retinal ganglion cells following target ablation in neonatal rats
Authors: Cui Q , Harvey AR
Published: April 2018

Removal of the superior colliculus (SC) in neonatal Wistar rats results in a rapid loss of retinal ganglion cells (RGCs). There is an early twofold increase in RGC death 4–8 hr postlesion (PL) followed by a later 10–11-fold increase in pyknosis about 24 hr PL. We have now used neurotrophic factors (BDNF, NT-4/5, NT-3, NGF, LIF), glutamate receptor antagonists (MK-801, DNQX, CNQX), an antioxidant (N-ace-tyl-L- cysteine), and an NOS inhibitor (L-NAME) to determine whether the early and late phases of lesion-induced RGC death involved similar or different mechanisms. Normal and pyknotic nuclei of tectally projecting RGCs were visualized by injecting the left s.c. of 2 d old rats with diamidino yellow (DY). Two days later the injection site was removed. In most rats, right eyes were injected with factors immediately after the s.c. ablation. Rats were perfused either 6 or 24 hr PL. In the latter group a second intravitreal injection of the appropriate factor was sometimes made 12 hr PL. NT- 4/5 and BDNF significantly decreased RGC pyknosis 6 and 24 hr PL, whereas NT-3 was only protective 6 hr PL. LIF slightly reduced RGC death 24 hr PL, but NGF had no influence on RGC survival at either time point. NT-4/5 also reduced the rate of naturally occurring RGC death. MK-801, DNQX, CNQX, N-acetylcysteine, and L-NAME all prevented the early lesion-induced increase in RGC death but had no significant effect on RGC death measured 24 hr PL; none of these factors significantly reduced the rate of naturally occuring RGC death. Cycloheximide, shown previously to reduce RGC pyknosis 24 hr PL, did not prevent RGC death 6 hr PL. The data indicate that there are at least two mechanisms involved in RGC death after neonatal target ablation. The early increase is related to excitotoxic effects mediated by glutamate receptors and involves NOS and the production of free radicals. We found no evidence that RGC death measured 24 hr PL is dependent on these processes, but the later death does require protein synthesis and, most likely, the activation of an endogenous suicide program. NT-4/5 and BDNF protected RGCs from both types of lesion- induced death.
Differential paired pulse depression of non-NMDA and NMDA currents in pyramidal cells of the rat frontal cortex
Authors: Kang Y
Published: April 2018

Excitatory postsynaptic currents (EPSCs) were induced in layer II-V pyramidal cells in the frontal cortex of the young rat (postnatal day 14–21) by stimulation of layers II/III in the presence of bicuculline using the whole-cell patch-clamp technique. EPSCs usually consisted of fast and slow components that were sensitive to CNQX and APV, respectively. In response to paired stimuli of identical strength, paired pulse depression (PPD) was seen for these EPSCs. The PPD of fast EPSCs was most pronounced at an interstimulus interval (ISI) of 200–300 msec and ceased to occur at ISIs greater than 3–5 sec, while the PPD of slow EPSCs became most pronounced at an ISI of 500–1000 msec and ceased to occur at ISIs greater than 10 sec. The PPD of fast EPSCs was attenuated by (-)-baclofen (1–5 microM) and removed by 2-hydroxy- saclofen (0.2–0.4 mM). By contrast, the PPD of slow EPSCs consisted of early and late components that were attenuated by (-)-baclofen and muscarine (1–5 microM), respectively. The late PPD of slow EPSCs induced in the presence of baclofen was removed by pirenzepine (1–3 microM). Thus, fast and slow components of glutamatergic EPSCs displayed two distinct PPDs. These results suggest that a part of the glutamatergic afferents likely arising from layer II/III pyramidal cells may terminate predominantly on NMDA receptors in pyramidal cells of the frontal cortex and receive distinct presynaptic inhibition through at least the muscarinic receptors.
Deficits in radial arm maze performance in kindled rats: evidence for long-lasting memory dysfunction induced by repeated brief seizures
Authors: Sutula T , Lauersdorf S , Lynch M , Jurgella C , Woodard A
Published: April 2018

Repeated activation of neural pathways by kindling induces brief seizures, permanent increases in seizure susceptibility, neuronal loss in the hippocampus, and mossy fiber sprouting in the dentate gyrus. Because kindling induces permanent cellular alterations in hippocampal pathways that have been implicated in memory, it was of interest to determine if kindling also induces long-lasting impairments in a spatial memory task in rats. In this study, the effects of kindling on memory were investigated by assessing kindled rats in a radial arm maze behavior that is impaired by hippocampal damage. Kindled rats studied at 1 month after the last of 30–134 evoked generalized tonic-clonic seizures acquired competence in the performance of the radial arm maze task at a rate that was indistinguishable from age-matched normal controls, but demonstrated a deficit in the ability to repeat the task on consecutive days. The repetition deficit was not observed in rats that experienced three afterdischarges or three generalized tonic- clonic seizures, and the severity of the deficit varied directly with the number of evoked kindled seizures. Repeated brief seizures evoked by kindling induced a long-lasting deficit in a radial arm maze task that is a rodent model of memory. The observation of a long-lasting deficit in radial arm maze performance in kindled rats suggests that the cellular alterations induced in the hippocampus by seizures could contribute to the memory dysfunction in human epilepsy.
Regulation of GABA transporter activity and mRNA expression by estrogen in rat preoptic area
Authors: Herbison AE , Augood SJ , Simonian SX , Chapman C
Published: April 2018

This study has examined whether estrogen regulates GABA transporter synthesis and activity in the female rat brain. In the first experiment in situ hybridization studies examined the effects of ovariectomy on cellular GABA transporter-1 (GAT-1) mRNA content. A 25% decrease in GAT- 1 mRNA expression was detected within the medial preoptic area (MPOA) but not the parietal cortex, magnocellular preoptic nucleus (Mg-POA) or caudate-putamen (C-P). Estrogen replacement for 7 d returned GAT-1 mRNA content of MPOA cells to levels observed in intact rats. In the second experiment, the effect of increased brain GABA concentrations on GAT-1 mRNA expression was investigated by treating rats with gamma-vinyl GABA, a GABA-transaminase inhibitor. Although resulting in a twofold increase in tissue GABA content, in situ hybridization experiments revealed no changes in GAT-1 transcript expression. A third series of experiments examined GABA transporter activity in vitro using a 3H GABA uptake assay in MPOA, cortex, and C-P punches. Nipecotic acid (10 microM) reduced specific 3H GABA uptake in all three brain regions while 100 microM beta-alanine only reduced uptake in the MPOA. Estrogen treatment for 7 d resulted in a significant increase in 3H GABA uptake in the MPOA but not the cortex or C-P. The presence of a putative estrogen response element in the GAT-1 gene and the effects demonstrated here on GAT-1 mRNA content and GABA transporter activity indicate that estrogen may influence GAT-1 gene transcription to alter GABA transporter function within the MPOA but not the C-P or cortex.
Spontaneous CNS remyelination in beta 2 microglobulin-deficient mice following virus-induced demyelination
Authors: Miller DJ , Rivera-Quinones C , Njenga MK , Leibowitz J , Rodriguez M
Published: April 2018

Animal models with selective genetic immunodeficiencies are useful tools to identify pathogenic mechanisms of disease. Resistant (C57BL/6F 129/J) (H-2b) mice are rendered susceptible to Theiler's murine encephalomyelitis virus-induced demyelination by genetic disruption of the beta 2 microglobulin gene [beta 2 m(-l-)]. The absence of beta 2 m prevents the expression of major histocompatibility complex class I molecules and normal levels of functional CD8+ T cells. We tested whether genetic depletion of beta 2 m would permit CNS remyelination after chronic demyelination induced by the Daniel's strain of Theiler's virus. In contrast to the minimal spontaneous remyelination observed in SJL/J mice after infection with the Daniel's strain of Theiler's virus, chronically infected beta 2 m(-I-) mice showed extensive and progressive spontaneous CNS remyelination at 6, 12, and 18 months after infection. Spontaneous remyelination by both oligodendrocytes and Schwann cells occurred despite the presence of persistent virus antigen and RNA, but was associated with diminished virus-specific humoral and delayed-type hypersensitivity responses. These experiments support the hypothesis that the immune response inhibits myelin regeneration after virus-induced CNS demyelination.
Glutaminase-positive and glutaminase-negative pyramidal cells in layer VI of the primary motor and somatosensory cortices: a combined analysis by intracellular staining and immunocytochemistry in the rat
Authors: Kaneko T , Kang Y , Mizuno N
Published: April 2018

Pyramidal neurons in layer VI of the primary motor and somatosensory cortices were examined by a combined method of intracellular recording, biocytin injection, and immunocytochemistry using in vitro slice preparations of rat brain immunofluorescence staining revealed that biocytin-injected pyramidal cells in layer VI were separated into glutaminase (PAG)-immunopositive and PAG-immunonegative cells. Although the two groups of pyramidal cells showed no statistically significant differences in passive membrane properties and spike characteristics, a clear difference was found in spike afterpotentials. Ten of 12 PAG- positive pyramidal cells showed no or a small fast afterhyperpolarization (fAHP), whereas 10 of 11 PAG-negative pyramidal cells displayed a large fAHP. Depolarizing afterpotentials were observed only in PAG-positive pyramidal cells than in PAG-negative cells. In contrast, the arborization of basal dendrites was more developed in PAG-positive pyramidal cells than in PAG-negative cells. The main axons of all the pyramidal cells entered the subcortical axons of all the pyramidal cells entered the subcortical white matter. The local axon collaterals of PAG-positive pyramidal cells were widely spread in the horizontal direction, whereas those of PAG-negative cells were distributed vertically along the dendritic tree. Since PAG is considered to be a marker of glutamatergic neurons in the cerebral cortex, the present results indicate that layer VI pyramidal cells are separated into glutamatergic and nonglutamatergic neurons that have different electrical properties and input-output organizations. Thus, cortical outputs from layer VI are suggested to use at least two distinct systems.
Spinohypothalamic tract neurons in the cervical enlargement of rats: descending axons in the ipsilateral brain
Authors: Zhang X , Kostarczyk E , Giesler GJ
Published: April 2018

Spinohypothalamic tract (SHT) cells are spinal cord neurons with axons that project directly to or through the contralateral hypothalamus. Frequently, SHT axons decussate in the posterior optic chiasm, turn posteriorly and descend to unknown locations in the ipsilateral brain. We attempted to determine the course and the termination of these descending axons. Sixty neurons in the cervical enlargement of rats were antidromically activated initially from the contralateral hypothalamus and then from multiple anterior-posterior levels in the ipsilateral brain. Fifty-three (88%) were backfired with low currents at increased latencies from the ipsilateral brain. The axons of 35 neurons were surrounded with electrode penetrations from which high currents could not activate the neuron antidromically, suggesting the examined axons terminated in the surrounded areas. Seven SHT axons that were surrounded (20%) appeared to terminate in the contralateral hypothalamus, 5 (14%) in the ipsilateral hypothalamus, and 9 (26%) in the ipsilateral thalamus. Fourteen SHT axons (40%) ended in the ipsilateral midbrain mainly in the superior colliculus, cuneiform nucleus or nucleus brachium inferior colliculus. An additional 11 axons were followed even further posteriorly into the ventral pons or rostral medulla. Each of the 26 neurons that could be physiologically classified responded either preferentially or specifically to noxious mechanical stimuli. These results indicate that SHT axons course through a surprisingly long and complex path. After decussating in the hypothalamus, the axons of many SHT neurons descend into the ipsilateral posterior thalamus, midbrain, pons, or even rostral medulla. These axons may provide nociceptive information to a variety of nuclei throughout the diencephalon and brainstem bilaterally.
Mutations in the Caenorhabditis elegans Na,K-ATPase alpha-subunit gene, eat-6, disrupt excitable cell function
Authors: Davis MW , Somerville D , Lee RY , Lockery S , Avery L , Fambrough DM
Published: April 2018

We have cloned a Na,K-ATPase alpha-subunit gene from Caenorhabditis elegans and discovered that it is identical to the gene eat-6, eat-6 mutations cause feeble contractions and slow, delayed relaxations of pharyngeal muscle. The resting membrane potential of eat-6 mutant pharynxes is consistently depolarized compared to wild-type. The action potentials are smaller, and the return to resting potential is slower. To explain these abnormalities, we propose that a reduction of Na,K- ATPase activity in eat-6 mutants leads to a reduction of the ion concentration gradients that power membrane potential changes.
Blockade of neuronal nitric oxide synthase protects against excitotoxicity in vivo
Authors: Schulz JB , Matthews RT , Jenkins BG , Ferrante RJ , Siwek D , Henshaw DR , Cipolloni PB , Mecocci P , Kowall NW , Rosen BR
Published: April 2018

Nitric oxide may be a key mediator of excitotoxic neuronal injury in the central nervous system. We examined the effects of the neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI) on excitotoxic striatal lesions. 7-NI significantly attenuated lesions produced by intrastriatal injections of NMDA, but not kainic acid or alpha-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) 7-NI attenuated secondary striatal excitotoxic lesions produced by the succinate dehydrogenase inhibitor malonate, and the protection was reversed by L- arginine but not by D-arginine, 7-NI produced nearly complete protection against striatal lesions produced by systemic administration of 3-nitropropionic acid (3-NP), another succinate dehydrogenase inhibitor, 7-NI protected against malonate induced decreases in ATP, and increases in lactate, as assessed by 1H magnetic resonance spectroscopy. 7-NI had no effects on spontaneous electrophysiologic activity in the striatum in vivo, suggesting that its effects were not mediated by an interaction with excitatory amino acid receptors. 7-NI attenuated increases in hydroxyl radical, 8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine generation in vivo, which may be a consequence of peroxynitrite formation. The present results implicate neuronal nitric oxide generation in the pathogenesis of both direct and secondary excitotoxic neuronal injury in vivo. As such they suggest that neuronal nitric oxide synthase inhibitors may be useful in the treatment of neurologic diseases in which excitotoxic mechanisms play a role.
A strongly inwardly rectifying K+ channel that is sensitive to ATP
Authors: Collins A , German MS , Jan YN , Jan LY , Zhao B
Published: April 2018

We have cloned an inwardly rectifying K+ channel from the hamster insulinoma cDNA library and shown that it is inhibited by cytoplasmic ATP. The channel is 90.97% identical to the IRK3 channels cloned from other species, and its mRNA is found primarily in the brain. When expressed in Xenopus oocytes, the channel displays strong inward rectification typical of inward rectifiers. The channel is inhibited reversibly by physiological concentrations of ATP via a mechanism that does not appear to involve ATP hydrolysis, as shown by studies of channels in excised inside-out membrane patches. This effect is antagonized by ADP, again in the physiological range, implying that this channel is sensitive to the index of metabolic state, i.e., the intracellular [ATP]/[ADP] ratio. This channel is different from previously known ATP-sensitive K+ channels, although it may also be stimulated by MgATP, as are other ATP-sensitive K+ channels. The potential physiological significance of these ATP-dependent regulations will be discussed.
Metabotropic glutamate receptor modulation of voltage-gated Ca2+ channels involves multiple receptor subtypes in cortical neurons
Authors: Choi S , Lovinger DM
Published: April 2018

Metabotropic glutamate receptor (mGluR) modulation of voltage-gated Ca2+ channels was examined in isolated deep layer frontoparietal cortical neurons under conditions designed to isolate calcium- independent modulatory pathways. Trans-1-aminocyclopentane-1,3- dicarboxylate (t-ACPD), a nonspecific mGluR agonist, produced rapid and reversible inhibition of Ca2+ channels. This effect was mimicked by agonists for group I and group II, but not group III, mGluRs. Effects of group I and II agonists often were observed in the same neurons, but separate subgroups of neurons were unresponsive to the group I agonist quisqualate or the group II agonist 2-(2,3-dicarboxycyclopropyl) glycine (DCG-IV). Inhibition by quisqualate and DCG-IV was nonocclusive in neurons responding to both agonists. These agonists thus appear to act on different mGluRs. The mGluR antagonist alpha-methyl-4- carboxylphenylglycine attenuated inhibition by t-ACPD, quisqualate, and DCG-IV. Inhibition by quisqualate and DCG-IV was voltage-dependent. Although the effects of both agonists were greatly reduced by N- ethylmaleimide (NEM), inhibition by DCG-IV was more sensitive to NEM than inhibition by quisqualate. t-ACPD-induced inhibition was reduced by omega-conotoxin GVIA (omega-CgTx) and omega-agatoxin IVA (omega- AgTx) but was affected little by nifedipine. Inhibition by DCG-IV and quisqualate also was reduced by omega-CgTx. We conclude that multiple mGluR subtypes inhibit Ca2+ channels in cortical neurons and that N- and possibly P-type channels are inhibited. Modulation is via a rapid- onset, voltage-dependent mechanism that likely involves a pertussis toxin (PTX)-sensitive G-protein. Type I mGluRs may work via additional PTX-insensitive pathways.
Calcium-independent activation of the secretory apparatus by ruthenium red in hippocampal neurons: a new tool to assess modulation of presynaptic function
Authors: Trudeau LE , Doyle RT , Emery DG , Haydon PG
Published: April 2018

The functional plasticity of the nervous system may result in part from the direct modulation of the effectiveness of the release machinery of synaptic terminals. To date, direct modulation of secretion in neurons has proven difficult to study because of the lack of a suitable tool to probe the release machinery independently of calcium influx. We report that the polyvalent cation ruthenium red (RR) directly evokes rapid and reversible calcium-independent quantal secretion in hippocampal neurons by binding to external sites on the presynaptic terminal membrane. This binding can be displaced by heparin and is not associated with ultrastructural damage to the synaptic terminals. The use of RR-evoked release as a tool has allowed us to detect a direct modulation of the secretory apparatus after activation of A1 adenosine receptors on hippocampal neurons.
In vitro ischemia promotes calcium influx and intracellular calcium release in hippocampal astrocytes
Authors: Duffy S , MacVicar BA
Published: April 2018

The intracellular calcium concentration ([Ca2+])i of astrocytes within rat hippocampal slices was measured during simultaneous hypoxia and hypoglycemia to examine the early intracellular signaling events induced by this in vitro model of ischemia. Hypoxia-hypoglycemia for 3.3–7.5 min evoked [Ca2+]i increases in astrocytes iontophoretically loaded with calcium orange (11/14 slices; 2.5 min to peak [Ca2+]i, 5 min to > 60 min duration). Calcium elevations also were observed in the absence of extracellular calcium ([Ca2+]o) (4/4 slices), indicative of Ca2+ release from internal stores. Hypoxia-hypoglycemia depolarized astrocytes (51 +/- 16 mV), suggesting additional contribution from voltage-gated Ca2+ influx. Depolarization of a similar magnitude (51 +/- 4 mV) by 50 mM extracellular potassium ([K+]o triggered [Ca2+]i increases (20/24 slices), which were blocked by removal of [Ca2+]o (8/8 slices) indicating that depolarization promoted Ca2+ influx. Voltage- gated Ca2+ influx and internal release were measured in accurately isolated astrocytes during in vitro ischemia to examine these processes in the absence of surrounding neurons. Hypoxia-hypoglycemia (7.5–34.0 min) induced only modest, slow increases in the basal [Ca2+]i of Fura-2- loaded isolated astrocytes (average 12% increase in Fura-2 ratio R340/380 after 10 min) that were blocked by [Ca2+]o removal. Voltage- gated Ca2+ influx was still functional under ischemia, however, as 50 mM [K+]o evoked [Ca2+]i increases (14/14 cells, delta R340/380 of 48%) approximately equal to preischemic responses. Isolated neurons displayed large irreversible increases in basal [Ca2+]i after 1.5–6.5 min in vitro ischemia (10/12 cells; average delta R 340/380 of 152%). The absence of significant basal [Ca2+]i increases on isolated astrocytes indicates that ischemia-induced Ca2+ influx and internal release in astrocytes within slices depend on signals released from neurons (K+, neurotransmitters). Ischemic [Ca2+]i elevations may constitute a signaling mechanism for postischemic reactive responses.
The alpha 6 subunit of the GABAA receptor is concentrated in both inhibitory and excitatory synapses on cerebellar granule cells
Authors: Nusser Z , Sieghart W , Stephenson FA , Somogyi P
Published: April 2018

Although three distinct subunits seem to be sufficient to form a functional pentameric GABAA receptor channel, cerebellar granule cells express nRNA for nine subunits. They receive GABAergic input from a relatively homogenous population of Golgi cells. It is not known whether all subunits are distributed similarly on the surface of granule cells or whether some of them have differential subcellular distribution resulting in distinct types of synaptic and/or extrasynaptic channels. Antibodies to different parts of the alpha 6 and alpha 1 subunits of the GABAA receptor and electron microscopic immunogold localization were used to determine the precise subcellular distribution of these subunits in relation to specific synaptic inputs. Both subunits were present in the extrasynaptic dendritic and somatic membranes at lower densities than in synaptic junctions. The alpha 6 and alpha 1 subunits were colocalized in many GABAergic Golgi synapses, demonstrating that both subunits are involved in synaptic transmission in the same synapse. Synapses immunopositive for only one of the alpha subunits were also found. The alpha 6, but not the alpha 1, subunit was also concentrated in glutamatergic mossy fiber synapses, indicating that the alpha 6 subunit may have several roles depending on its different locations. The results demonstrate a partially differential synaptic targeting of two distinct GABAA receptor subunits on the surface of the same type of neuron.
Axonal transport and distribution of synaptobrevin I and II in the rat peripheral nervous system
Authors: Li JY , Edelmann L , Jahn R , Dahlstrom A
Published: April 2018

Synaptobrevin, a membrane protein of synaptic vesicles that plays a key role in exocytosis, occurs in two closely related isoforms, synaptobrevin I and II. We have analyzed the axonal transport of both isoforms in sciatic nerve and spinal roots. When fast axonal transport was interrupted by crushing, the proteins accumulated continuously proximal to the crush. Accumulation also was observed distal to the crush, but to a lesser extent (47 and 63% of the proximal accumulation for synaptobrevin I and II, respectively). Immunoelectron microscopy revealed that, proximal to the crush, synaptobrevin I and II were associated with small clear vesicles reminiscent of typical synaptic vesicles. Distal to the crush, membranes positive for synaptobrevin I or II were more heterogeneous, including larger membrane profiles that may represent endosomes. In spinal cord, synaptobrevin I and II were colocalized in many terminals. However, labeling for synaptobrevin I was more intense whereas labeling for synaptobrevin II was stronger in dorsal than in ventral horn terminals. Motor endplates contained only synaptobrevin I. In the sciatic nerve, synaptobrevin I was present predominantly in large, myelinated axons, whereas synoptobrevin II was virtually absent but abundant in small- and medium-sized axons. Lumbar sympathectomy, ventral rhizotomy, and double-labeling studies confirmed that synaptobrevin I is present predominantly in motor neurons whereas synaptobrevin II is present in adrenergic and sensory neurons. We conclude that synaptobrevin I and II are transported bidirectionally by fast axonal transport and are expressed heterogeneously in different neurons in the peripheral nervous system of the adult rat, suggesting that these isoforms have special functional roles in different sets of neurons.
In vivo, in vitro, and computational analysis of dendritic calcium currents in thalamic reticular neurons
Authors: Destexhe A , Contreras D , Steriade M , Sejnowski TJ , Huguenard JR
Published: April 2018

Thalamic reticular (RE) neurons are involved in the genesis of synchronized thalamocortical oscillations, which depend in part on their complex bursting properties. We have investigated the intrinsic properties of RE cells using computational models based on morphological and electrophysiological data. Simulations of a reconstructed RE cells were compared directly with recordings from the same cell to obtain precise values for the passive parameters. In a first series of experiments, the low-threshold calcium current (I(Ts)) was studied via voltage clamp in acutely dissociated RE cells that lack most of their dendrites. Simulations based on a cell with truncated dendrites and Hodgkin-Huxley kinetics reproduced these recordings with a relatively low density of I(Ts). In a second series of experiments, voltage-clamp recordings obtained in intact RE cells in slices showed a higher amplitude and slower kinetics of I(Ts). These properties could be reproduced from the reconstructed cell model assuming higher densities of I(Ts) in distal dendrites. In a third series of experiments, current-clamp recordings were obtained on RE cells in vivo. The marked differences with in vitro recordings could be reconciled by simulating synaptic bombardment in the dendrites of RE cells, but only if they contained high distal densities of I(Ts). In addition, simpler models with as few as three compartments could reproduce the same behavior assuming dendritic I(Ts). These models and experiments show how intrinsic bursting properties of RE cells, as recorded in vivo and in vitro, may be explained by dendritic calcium currents.
MN20, a D2 cyclin, is transiently expressed in selected neural populations during embryogenesis
Authors: Ross ME , Carter ML , Lee JH
Published: April 2018

Although the regulation of proliferation and differentiation during brain development has long been considered to be interrelated, the mechanisms that coordinate the control of cell division and histogenesis are poorly understood. The cell cycle is a dynamic process that is governed by the concerted action of numerous cell cycle regulatory proteins in response to signals both intrinsic and extrinsic to the cell. Thus, proteins that regulate the cell cycle are well suited to provide a link between processes that control neuroblast proliferation and differentiation. We reported previously the isolation from brain of a message form of D2 cyclin, one of several cyclin proteins known to promote the progression from G1 to S phase. This MN20/D2 cyclin mRNA is expressed in highly restricted neural populations at embryonic (E) day 15 and postnatal (P) day 6 in the mouse. To gain insight into the role(s) this cyclin may serve in brain formation, the spatial and temporal pattern of MN20/D2 cyclin expression was examined by in situ hybridization at 48 hr intervals from E10.5 to P8. MN20 mRNA was detected in developing cerebellum, dorsal mesencephalon, cerebral cortex, and epithalamus, but not hippocampus, striatum, or thalamus. Comparison with 5-bromodeoxyuridine labeling of cells in S phase indicated that MN20 expression in embryonic cerebellum and cerebral cortex was most pronounced in young neurons that recently had become postmitotic. Although expressed in other embryonic cerebellar neurons, MN20 was detected in granule precursors only postnatally, after their migration from the rhombic lip to the external germinal layer. This indicates that MN20/D2 cyclin is induced in cerebellar granule precursors as they become competent to differentiate. The spatial distribution of MN20 expression in the developing brain suggests that regional differences in cell cycle regulation depend in part on the selective use of cyclin proteins. Moreover, detection of MN20 mRNA in postmitotic neural cells indicates that cyclin D2 expression has effects beyond promoting cell cycle progression and may also have a role in the response of the neural precursor to terminal differentiation signals as the cells exits from proliferation.
Differential regulation of adrenergic receptor development by sympathetic innervation
Authors: Habecker BA , Malec NM , Landis SC
Published: April 2018

Rat sweat glands provide an interesting model system for a developmental study of adrenergic receptor expression because their sympathetic innervation undergoes a switch from a nonadrenergic to cholinergic and peptidergic phenotype. alpha 1B, alpha 2B, and beta 2 receptors are expressed in rat footpads; alpha 1 and beta 2 receptors are localized specifically to sweat glands, and alpha 2 receptors also are expressed in other tissues. alpha 1 and, to a lesser extent, beta 2 receptors decrease during development, whereas alpha 2 levels remain relatively constant. Decreased receptor expression is accompanied by the loss of alpha 1-stimulated inositol phosphate accumulation, but no change in beta-stimulated cAMP production. The number of alpha 1 and beta 2 receptors decreases after P21, when the sympathetic innervation no longer produces catecholamines. Neonatal sympathectomy causes a partial failure of alpha 1 downregulation, but has no effect on beta 2 or alpha 2 receptor levels. Therefore, at least two distinct mechanisms regulate development of adrenergic receptors in sweat glands. Innervation-independent processes control developmental expression of alpha 1, beta 2, and alpha 2 receptors, and an additional, innervation- dependent mechanism influences expression of alpha 1 receptors. Denervation at postnatal day 20, when the sympathetic innervation is cholinergic and peptidergic, results in retention of alpha 1 receptors, but cholinergic blockade begun at P20 does not. These results indicate that regulation of receptor expression in sweat glands is complex, and suggest that the innervation-dependent factors that decrease alpha 1 levels during development act through a nonadrenergic, noncholinergic mechanism.
The molecular composition of neuronal microfilaments is spatially and temporally regulated
Authors: Weinberger R , Schevzov G , Jeffrey P , Gordon K , Hill M , Gunning P
Published: April 2018

The actin-based microfilament system is thought to play a critical role in neuronal development. We have determined specific changes in the composition of microfilaments accompanying neuronal morphogenesis. By using specific antibodies against the isoforms for tropomyosin (Tm) (Tm- 5 and TmBr-1/-3) and actin (beta- and gamma-actin), we found that during early morphogenesis in vivo immature growing axons contain beta- and gamma-actin and Tm-5. In particular, Tm-5 is exclusively located in the immature axonal processes relative to the neuronal cell body. In contrast, beta-actin and Tm-5 are absent in mature, quiescent axons. This developmental loss from axons is associated with an approximately twofold downregulation of beta-actin and Tm-5 levels in the brain; gamma-actin levels do not change, and this molecule is widely distributed throughout neurons during development. The loss of beta- actin and Tm-5 from axons is accompanied by a progressive appearance of TmBr-1/-3. This apparent replacement of Tm-5 with TmBr-1/-3 occurs over a 2 d time period during rat embryonic hindbrain development and is conserved in evolution between birds and mammals. The loss of Tm-5 from axons involves a redistribution of this molecule to the cell soma and dendrites. These findings suggest that specialized microfilament domains are associated with the development and maintenance of neuronal polarity. We conclude that these Tm isoforms and beta-actin are subject to specific patterns of segregation associated with axonal development and neuronal differentiation. This provides a potential molecular basis for the temporal and spatial specificity of microfilament function during neuronal differentiation.
The interaction of imposed and inherent olfactory mucosal activity patterns and their composite representation in a mammalian species using voltage-sensitive dyes
Authors: Kent PF , Mozell MM , Murphy SJ , Hornung DE
Published: April 2018

From amphibian data, two mechanisms that could underlie the encoding of odorants by the mucosal activity patterns they engender are as follows (1) receptors with similar odorant selectivities could be aggregated spatially on the mucosa (inherent patterns); (2) in analogy to gas chromatography, as odorants are drawn along the surface of the mucosa the strongly sorbed ones could be deposited preferentially upstream, whereas the weakly sorbed ones could be distributed more evenly (imposed patterns). Do both of these possible coding mechanisms operate in mammals and, if so, how do they interact in giving composite patterns (imposed + inherent)? Fluorescence changes in di-4-ANEPPS applied to rat mucosas were monitored by a 10 x 10 pixel photodiode array. To observe the inherent patterns, three odorants of varying sorbabilities first were puffed uniformly onto the entire mucosa mounted in a Delrin chamber. To bring out the imposed patterns, the chamber was then sealed to replicate anatomically the rat's nasal cavity, and these same odorants were drawn at three flow rates along the mucosal flow path. The results demonstrated for the first time the existence of imposed patterns in a mammal. The strongly sorbed odorants, unlike the weakly sorbed one, showed marked imposed patterns. Within physiological limits, increasing the flow rate decreased the magnitude of the imposed patterns. One might consider strategies that the olfactory process could use either to negate or to take advantage of the chromatographic effect, because the lability of the composite patterns with changing stimulus conditions raises questions about their role in odorant encoding.
Hippocampal ensemble activity during spatial delayed-nonmatch-to-sample performance in rats
Authors: Deadwyler SA , Bunn T , Hampson RE
Published: April 2018

Multiple-cell recording from specially designed arrays of microwire electrodes allowed analysis of anatomically defined ensemble activity from 10 different locations within the hippocampus of rats (n = 7) performing a two-lever operant version of a spatial delayed-nonmatch-to- sample task (DNMS). Application of population analysis procedures to ensembles of single-neuron activity within the CA1 and CA3 fields revealed firing patterns related to task-relevant events within a DNMS trial. The patterns were extracted via a canonical discriminant analysis in the form of “roots” that represented sources of variance in firing within the ensemble, such as phase of the task (Sample or Nonmatch), spatial position of the lever press response (left or right), and correct versus error trials. Comparison of the ensemble firing on correct versus error trials revealed important insight into ensemble information encoding, such as “miscoding” of the response position and lack of distinct encoding of the response in the Sample phase, which became increasingly vulnerable to error as a function of the duration of delay interval. The extracted discriminant scores were reflective of multiple representations within ensembles and suggested that “conjunctions” of task-relevant features could be represented effectively by small numbers of hippocampal neurons. The findings support the long-held supposition that hippocampal neurons play a critical role in the encoding and retrieval of information in recognition memory tasks.
Neuronal circuitry for comparison of timing in the electrosensory lateral line lobe of the African wave-type electric fish Gymnarchus niloticus
Authors: Kawasaki M , Guo YX
Published: April 2018

An African wave-type electric fish, Gymnarchus, compares timing on the order of microseconds of sensory feedback from from its high-frequency (approximately 400 Hz) electric organ discharges (EODs) received at different parts of its body surfaces. This capability is essential for and demonstrated by the jamming avoidance response (JAR). The organization of the timing comparison mechanisms was identified in the electrosensory lateral line lobe (ELL) in the hindbrain by field potential, extra- and intracellular recordings, and intracellular labeling with biotinylated agents. Timing of phase of the EOD feedback is carried by action potentials of S-type primary afferent fibers that project to the inner cellular layer (ICL) of the medial zone of the ELL and to the giant neurons in the ELL. The giant neurons bilaterally project to the ICL, where neurons sensitive to phase differences between different parts of the body occur. Although sensitive to dynamic phase changes of several microseconds, these differential-phase- sensitive neurons showed adaptation to steady-state changes of phase difference over a wide range (greater than +/- 100 microseconds) and continued to respond to small modulations after the mean difference was shifted. Gymnarchus and an independently evolved South American electric fish, Eigenmannia, exhibit nearly identical JARs and share a rather complex but identical set of computational algorithms for JAR. This study showed that one of the computational steps, the timing comparison between body surfaces, occurs in the hindbrain in Gymnarchus, in contrast to the midbrain in Eigenmannia. Thus, similar systems with a similar overall function may have evolved differently in different genera by assigning a subfunction to different substructures within the brain.
The selective neuronal NO synthase inhibitor 7-nitro-indazole blocks both long-term potentiation and depotentiation of field EPSPs in rat hippocampal CA1 in vivo
Authors: Doyle C , Holscher C , Rowan MJ , Anwyl R
Published: April 2018

The membrane-permeant gas NO is a putative intercellular messenger that has been proposed on the basis of previous in vitro studies to be involved in synaptic plasticity, especially the induction of long-term potentiation (LTP) of excitatory synaptic transmission in the hippocampus and cortex. In the present study, the role of NO in synaptic plasticity has been investigated in vivo. In particular, the action of the novel and selective neuronal NO synthase (nNOS) inhibitor 7-nitro-indazole (7-NI) has been investigated on the induction of LTP and depotentiation (DP) of field EPSPs in CA1 of the hippocampus in vivo. Unlike previously studied nonselective NOS inhibitors, 7-NI does not increase arterial blood pressure. In vehicle-injected rats, high- frequency stimulation consisting of a series of trains at 200 Hz induced LTP. However, LTP induction was strongly inhibited in 7-NI (30 mg/kg, i.p.)-treated animals. The inhibitory effect of 7-NI on the induction of LTP was prevented by pretreatment with L-arginine, the substrate amino acid used by NOS. In control animals, low-frequency stimulation consisting of 900 stimuli at 10 Hz induced DP of previously established LTP, whereas in 7-HI-treated animals only a short-term depression was induced. This effect of 7-NI also was prevented by D- arginine. The LTP and DP induced in control animals in this study were NMDA receptor-dependent, the NMDA receptor antagonist 3-(R,S)-2- carboxypiperazin-4-yl-propyl-1- phosphonic acid inhibiting the induction of both forms of synaptic plasticity. The present experiments are the first to demonstrate that an NOS inhibitor blocks the induction of the synaptic component of LTP and DP in vivo and, therefore, these results strengthen evidence that the production of NO is necessary for the induction of LTP and DP.
DNA mismatch repair and DNA methylation in adult brain neurons
Authors: Brooks PJ , Marietta C , Goldman D
Published: April 2018

DNA repair is essential for maintaining the integrity of the nucleotide sequence of cellular DNA over time. Although much information has accumulated recently on the mechanisms of DNA repair in cultured cells, little is known about the DNA repair capabilities of cells in the adult brain. In the present study, we have investigated the capacity of nuclear extracts from adult rodent brain neurons to carry out DNA mismatch repair. We focused on the repair of G.T and G.U mismatches, which are caused by deamination of 5-methyl cytosine to thymine, or cytosine to uracil, respectively, because these are the only types of mismatches that can arise in nondividing cells. We found that nuclear extracts from adult brain neurons can correct G.T and G.U mismatches, restoring them to G:C base pairs. Several other types of DNA mismatches could not be processed. These data provide the first direct demonstration that neurons in the adult mammalian brain have the capability to carry out DNA mismatch repair. We also we report that adult brain contains high levels of DNA methyltransferase (MTase) activity. We propose that one function of DNA MTase in the adult brain is to remethylate newly incorporated cytosine residues from G.T mismatch repair after deamination of 5-methyl cytosine, thereby maintaining the original pattern of DNA methylation. The high levels of brain DNA MTase suggest further that this enzyme has additional functions in the brain.
Axons regulate the distribution of Schwann cell microtubules
Authors: Kidd G , Andrews SB , Trapp BD
Published: April 2018

It is well established that axons regulate Schwann cell phenotype. The purpose of the present study was to determine whether axons influence the arrangement of Schwann cell microtubules (MTs). Using double- labeling immunocytochemistry and confocal microscopy, we show that MTs in undifferentiated Schwann cells are nucleated from and attached to a single MT organizing center (MTOC) that is associated with the centrosome. Physical contact with appropriate axons initiates a myelin- forming phenotype that disperses MT minus ends and induces multiple MT- nucleating sites in Schwann cell perinuclear cytoplasm. The axonal signal that initiates myelin breakdown during Wallerian degeneration induces multiple MTOCs and MT bundles in Schwann cell perinuclear cytoplasm and in cytoplasm between degenerating myelin ovoids. These results establish that axons influence Schwann cell MT distribution by regulating the location and number of MT-nucleation sites.
Distribution of high-conductance Ca(2+)-activated K+ channels in rat brain: targeting to axons and nerve terminals
Authors: Knaus HG , Schwarzer C , Koch RO , Eberhart A , Kaczorowski GJ , Glossmann H , Wunder F , Pongs O , Garcia ML , Sperk G
Published: April 2018

Tissue expression and distribution of the high-conductance Ca(2+)- activated K+ channel Slo was investigated in rat brain by immunocytochemistry, in situ hybridization, and radioligand binding using the novel high-affinity (Kd 22 pM) ligand [3H]iberiotoxin-D19C ([3H]IbTX-D19C), which is an analog of the selective maxi-K peptidyl blocker IbTX. A sequence-directed antibody directed against Slo revealed the expression of a 125 kDa polypeptide in rat brain by Western blotting and precipitated the specifically bound [3H]IbTX-D19C in solubilized brain membranes. Slo immunoreactivity was highly concentrated in terminal areas of prominent fiber tracts: the substantia nigra pars reticulata, globus pallidus, olfactory system, interpeduncular nucleus, hippocampal formation including mossy fibers and perforant path terminals, medial forebrain bundle and pyramidal tract, as well as cerebellar Purkinje cells. In situ hybridization indicated high levels of Slo mRNA in the neocortex, olfactory system, habenula, striatum, granule and pyramidal cell layer of the hippocampus, and Purkinje cells. The distribution of Slo protein was confirmed in microdissected brain areas by Western blotting and radioligand-binding studies. The latter studies also established the pharmacological profile of neuronal Slo channels. The expression pattern of Slo is consistent with its targeting into a presynaptic compartment, which implies an important role in neural transmission.