bluuebunny/crossref_metadata_2025 huggingface dataset:
Transfection with trk restores "slow" NGF binding, efficient NGF uptake, and multiple NGF responses to NGF-nonresponsive PC12 cell mutants
Authors: Loeb DM , Greene LA
Published: March 2018

NGF binds to and activates the protein tyrosine kinase gp 140prototrk. Expression of this receptor is required for at least some responses to NGF. Three outstanding issues are addressed in the present work. First, we determined whether expression of gp 140prototrk is required for all neuronal NGF responses. Second, we examined the role of gp 140prototrk in NGF binding and internalization. Third, we addressed the utility of NGF-nonresponsive PC12nnr5 cells for study of the NGF mechanism. In contrast to wild-type PC12 cells, PC12nnr5 cells do not express endogenous gp 140prototrk. We therefore asked whether they possess other defects that compromise NGF signaling pathways. To answer these questions, we transfected PC12nnr5 cells with a cDNA encoding full- length human gp 140prototrk and isolated cell lines permanently expressing the receptor. Introduction of trk rescued all of the many and varied NGF responses assessed, including enhanced protein tyrosine phosphorylation, induction of immediate-early and neural-specific genes, neurite outgrowth and regeneration, maintenance of survival in serum-free medium, and stimulation of AChE activity. In contrast to PC12nnr5 cells, the trk-transfected lines also bind and internalize NGF with wild-type PC12 cell characteristics. These findings indicate that gp 140prototrk is required for many, if not all, responses of neuronal cells to NGF and is necessary for proper NGF binding and internalization. Additionally, as no signaling defect other than the absence of trk expression was revealed in PC12nnr5 cells, this work supports the utility of this line for genetic dissection of the NGF mechanism of action.
Clustering and mobility of voltage-dependent sodium channels during myelination
Authors: Joe EH , Angelides KJ
Published: March 2018

In myelinated axons, voltage-dependent sodium channels are segregated at high density at nodes of Ranvier (Rosenbluth, 1976; Waxman and Quick, 1978; Black et al., 1990; Elmer et al., 1990), a distribution that is critical for the saltatory conduction of action potentials (Huxley and Stampfli, 1949). The factors that specifically control the organization and immobilization of sodium channels at nodes are unknown. Recently we have reported that segregation of sodium channels on axons is highly dependent on interactions with active Schwann cells and that continuing axon-glial interactions are necessary to maintain sodium channel distribution during differentiation of myelinated nerve (Joe and Angelides, 1992). The specific recruitment of sodium channels at these early stages of myelination and the conspicuous absence of other axon membrane components suggest that the factors governing sodium channel cluster formation show molecular specificity. However, it is not clear whether these clustered sodium channels originate from a redistribution of preexisting diffusely distributed sodium channels. To determine how Schwann cells might regulate sodium channel distribution during myelination we have examined the lateral mobility of fluorescently labeled sodium channels at defined stages of myelination by fluorescence photobleach recovery using tetramethylrhodamine (TmRhd)-labeled Tityus gamma, a sodium channel- specific fluorescent toxin. First, to test whether Schwann cells, in addition to modulating sodium channel distribution, affect the mobility of sodium channels, we cultured dorsal root ganglion neurons in the presence or absence of Schwann cells and monitored sodium channel mobility on cell bodies, axon hillocks, and axons. Even in the absence of Schwann cells, approximately 80% of the sodium channels were immobile on the time scale of the fluorescence photobleach recovery measurement (DL < or equal to 10(-12) cm2/sec), although the remaining fraction of channels are mobile with diffusion coefficients of 5–13 x 10(-11) cm2/sec. Most importantly, in contrast to the effects of Schwann cells on altering the distribution of sodium channels, we found that Schwann cells did not alter the rate of lateral mobility or the mobile fraction of axonal sodium channels. Therefore, although sodium channel distribution depends on Schwann cell contact, immobilization of sodium channels is independent of Schwann cell contact. These effects appear to be specific for sodium channels because 45% of the succinyl concanavalin-A receptors on the axon are mobile, a fraction that decreases to 25% in the presence of Schwann cells later in development. To determine how sodium channels might be immobilized other than by Schwann cell contact, TmRhd-Tityus gamma-labeled dorsal root neurons were treated with 0.5% Triton X-100.(ABSTRACT TRUNCATED AT 400 WORDS)
Molecular cloning of the murine JAK1 protein tyrosine kinase and its expression in the mouse central nervous system
Authors: Yang X , Chung D , Cepko CL
Published: March 2018

Degenerate oligonucleotide primers were employed in PCRs to clone protein tyrosine kinases that may play potential roles in the development of the mammalian CNS. Using one PCR clone to screen a mouse eye cDNA library, a full-length cDNA of a cytoplasmic tyrosine kinase, the homolog of human JAK1, was obtained. The murine JAK1 kinase belongs to a new family of cytoplasmic kinases that contain two tandem catalytic domains. Northern analyses indicated that murine JAK1 mRNA is expressed in a variety of tissues and cell lines. In the adult mouse eye, in situ hybridization and immunohistochemistry showed that JAK1 mRNA and protein were expressed in the retinal ganglion cell layer and the inner part of the inner nuclear layer, presumably in amacrine cells. JAK1 protein was also detected in horizontal cells and in the two synaptic layers of the adult retina. During retinal development, JAK1 protein was first detected in retinal ganglion cells and in their axons as early as embryonic day 14. Expression of JAK1 protein in amacrine cells and horizontal cells occurred only postnatally. This pattern of expression was also observed in the chick retina, suggesting an evolutionarily conserved function of JAK1 kinase in vertebrate retinal development and/or function. Immunohistochemical staining against JAK1 was detected in two areas of the adult mouse brain, the olfactory bulb and a group of cells in the hypothalamus. Together, these expression studies suggest a role for JAK1 kinase in the differentiation or function of a subset of CNS neurons.
A protein tyrosine phosphatase expressed within dopaminoceptive neurons of the basal ganglia and related structures
Authors: Lombroso PJ , Naegele JR , Sharma E , Lerner M
Published: March 2018

Immunocytochemical and biochemical studies were conducted to characterize a brain-specific protein tyrosine phosphatase, designated STEP for striatal enriched phosphatase. STEP immunoreactivity was most intense in select regions of the CNS receiving a dopaminergic input, and was localized to cell bodies, dendrites, and axonal processes. Western blot analyses of rat brain homogenates revealed a triplet of polypeptides with relative mobilities (M(r)) of 46 kDa, 37 kDa, and 33 kDa enriched within the striatum. Phase separation of protein homogenates by Triton X-114 extraction indicated that this triplet was enriched in soluble but not membrane fractions. Affinity-purified STEP fusion protein exhibited phosphatase activity while a mutated form of the STEP fusion protein (Cys300Ser) showed no demonstrable phosphatase activity.
GM1 ganglioside improves dopaminergic markers of rat mesencephalic cultures treated with MPP+
Authors: Dalia A , Neff NH , Hadjiconstantinou M
Published: March 2018

Rat embryonic mesencephalic cultures were employed to evaluate the consequences of adding GM1 ganglioside to cultures lesioned with the selective neurotoxin 1-methyl-4-phenylpyridinium (MPP+). MPP+ reduced dopamine and DOPAC content, dopamine uptake, aromatic L-amino acid decarboxylase activity, and the number of tyrosine hydroxylase- immunopositive neurons. The immunopositive neurons that remained were aberrant. All of these parameters were partially restored by adding GM1 ganglioside to the cultures. The response to GM1 was not altered by prior treatment of the cultures with cytosine beta-D-arabinofuranoside to reduce the number of glial cells. Dopamine uptake activity restored by GM1 was lost if GM1 was removed from the culture.
Choroid tissue supports the survival of ciliary ganglion neurons in vitro
Authors: Wentzek LA , Bowers CW , Khairallah L , Pilar G
Published: March 2018

It is well established that during in vivo development the neurons of the avian ciliary ganglion are dependent for their survival on structures in the eye. Separate neuron populations innervate intraocular smooth and striated muscle targets. All ciliary neurons survive when cocultured with striated muscle. We demonstrate that when ciliary ganglion neurons are plated on explants of the choroid coat (a smooth muscle-containing target tissue) using a defined medium (N2), the neurons survive and grow vigorously into the tissue, forming contacts between axons and target cells identified as smooth muscle. Conditioned medium from choroid explants also rescues all the neurons, as does coculturing ciliary ganglion neurons with dissociated choroid cells. However, the presence of horse serum and chick embryo extract in the medium inhibits the choroid's ability to support ciliary neurons. The effects of these additives on the phenotypic expression of the smooth muscle may explain the inability of previous investigators to demonstrate target-derived support from smooth muscle preparations. Because the choroid contains cell types other than smooth muscle (e.g., fibroblasts and endothelial cells), we could not identify smooth muscle as the only cell type responsible for the release of the soluble trophic factor present in the target tissue. However, indirect evidence using avian primary fibroblast cultures, a fibroblast cell line, and an anatomically simple smooth muscle preparation, the avian amnion, suggests that smooth muscle cells are sufficient to account for the observed trophic activity, and that similar target-derived molecules support the survival of both types of ciliary ganglion cells.
The retinal fate of Xenopus cleavage stage progenitors is dependent upon blastomere position and competence: studies of normal and regulated clones
Authors: Huang S , Moody SA
Published: March 2018

The clonal origin of the stage 43–44 Xenopus retina from cleavage stage precursors was quantitatively assessed with lineage tracing techniques. The retina descends from a specific subset of those blastomeres that form forebrain. The most animal dorsal midline cell (D1.1.1) produced about half of the retina, the three other dorsal ipsilateral blastomeres each produce about an eighth of the retina, and the four contralateral dorsal blastomeres and an ipsilateral ventral-animal cell together produce the remaining eighth of the retina. There was no significant spatial segregation of the clones derived from different progenitors in either the anterior-posterior or dorsal-ventral axes of the retina and no boundaries between clones were observed. Instead, the clones intermixed to form multiple radial subclones that were equivalent to those demonstrated by marking optic vesicle progenitor cells (Holt et al., 1988; Wetts and Fraser, 1988). This mosaic pattern was initiated by the beginning of gastrulation, advanced in the neural plate, and virtually complete in the optic vesicle. At optic vesicle stages cell movement within subclones was restricted, resulting in the formation of lineally related columns of cells in the mature retina. To determine if the blastomere progenitors are determined to produce these retinal lineage patterns, the major retinal progenitor (D1.1.1) was deleted bilaterally. About 60% of the tadpoles developed normal- appearing eyes; of these the retinas in two-thirds were normal in size and the rest were smaller. The blastomeres surrounding the deleted D1.1.1 progenitors changed their contributions to retina in different ways to effect a complete or partial restoration. Ventral blastomeres, which normally contribute mainly to the tail, produced substantial amounts of the retina while dorsal blastomeres, which normally contribute mainly to the head, decreased their contribution to the retina. To determine whether these changes in retinal lineage were due to changes in blastomere position after the surgery, various other blastomeres were deleted prior to lineage mapping. Dorsal-animal blastomeres took over the retinal fate of their dorsal-vegetal neighbors after those neighbors were deleted, but did not change fate after the deletion of their ventral-animal neighbors. This result suggests that dorsal-animal blastomeres change positional values in only one direction (dorsal to vegetal) after neighbor cell deletion, and that retinal fate is dictated by blastomere position. To test this hypothesis directly, different ventral and vegetal blastomeres, which normally do not produce retina, were transplanted to the position of D1.1.1.(ABSTRACT TRUNCATED AT 400 WORDS)
Intracellular analysis of relations between the slow (< 1 Hz) neocortical oscillation and other sleep rhythms of the electroencephalogram
Authors: Steriade M , Nunez A , Amzica F
Published: March 2018

The newly described slow cortical rhythm (approximately 0.3 Hz), whose depolarizing-hyperpolarizing components are analyzed in the preceding article, is now investigated from the standpoint of its relations with delta (1–4 Hz) and spindle (7–14 Hz) rhythmicity. Regular-spiking and intrinsically bursting cortical neurons were mostly recorded from association suprasylvian areas 5 and 7; fewer neurons were also recorded from pericruciate motor and posterolateral visual areas. Although most cells were investigated under various anesthetics, a similar slow cortical rhythm was found in animals with brainstem transection at the low- or high-collicular levels. These cerveau isole (isolated forebrain) preparations display the major sleep rhythms of the EEG in the absence of general anesthetics. In 38% of recorded cortical neurons (n = 105), the slow rhythm was combined with delta oscillation. Both cellular rhythms were phase locked to the slow and delta oscillations in the surface- and depth-recorded EEG. In a group of this cell sample (n = 47), delta activity occurred as stereotyped, clock-like action potentials during the interdepolarization lulls of the slow rhythm. In another neuronal subsample (n = 58), delta events were grouped in sequences superimposed upon the depolarizing envelope of the slow rhythm, with such sequences recurring rhythmically at approximately 0.3–0.4 Hz. The associations between the two cellular and EEG rhythms (1–4 Hz and 0.3–0.4 Hz) were quantified by means of autocorrelograms, cross-correlograms, and spike-triggered averages. In 26% of recorded neurons (n = 72), the slow rhythm was combined with spindle oscillations. Regular-spiking cortical neurons fully reflected the whole frequency range of thalamically generated spindles (7–14 Hz). However, during similar patterns of EEG spindling, intrinsically bursting cells fired grouped action potentials (with intraburst frequencies of 100–200 Hz) at only 2–4 Hz. The dependence of the slow cortical oscillation upon the thalamus was studied by lesions and stimulation. The slow rhythm survived extensive ipsilateral thalamic destruction by means of electrolytic lesions or kainate-induced loss of perikarya in thalamic nuclei that were input sources to the recorded cortical neurons. To further prevent the possibility of a thalamic role in the genesis of the slow rhythm, through the contralateral thalamocortical systems and callosal projections, we also transected the corpus callosum in thalamically lesioned animals, and still recorded the slow rhythm in cortical neurons. These data indicate that the thalamus is not essentially implicated in the genesis of the slow rhythm.(ABSTRACT TRUNCATED AT 400 WORDS)
Specific induction of intracellular calcium oscillations by complement membrane attack on oligodendroglia
Authors: Wood A , Wing MG , Benham CD , Compston DA
Published: March 2018

Oligodendroglia (ODG) are unique among glial cell types in their capacity to activate complement in the absence of antibody, causing insertion of the potentially damaging membrane attack complex (MAC) into the plasma membrane. Using microfluorimetry of indo-1 fluorescence we have detected a complex oscillatory [Ca2+]i response in ODG following exposure to sublethal dilutions of serum-derived complement. Oscillations were transitory and preceded complete and stable return to resting [Ca2+]i levels, whereas nonoscillating ODG underwent rapid lysis. Depletion of the terminal complement component C9 from serum removed the oscillatory stimulus, which could be restored by reconstitution with purified C9. Exposure to the C9-homologous peptide melittin produced [Ca2+]i oscillations similar in pattern to those induced by whole serum. However, this type of response could not be reproduced by Ca2+ ionophores or mechanical wounding, suggesting that oscillations cannot be provoked by Ca2+ influx alone and depend on the presence of the MAC or a pore-forming lesion. Oscillations were not prevented in the continuous presence of caffeine, demonstrating independence from caffeine-releasable intracellular stores. Inhibition of the endoplasmic reticular Ca(2+)-ATPase with thapsigargin produced an abrupt elevation in [Ca2+]i but did not alter the latency between exposure to serum and the initial complement-induced transient. However, the slope of this initial transient was considerably reduced and oscillations suppressed, demonstrating dependence of the oscillatory mechanism on functional endoplasmic reticular Ca2+ stores. The coincidence of ODG recovery with oscillating [Ca2+]i suggests that the complex calcium signal that follows MAC attack may stimulate repair or protective mechanisms.
Inward-rectifying potassium channels in retinal glial (Muller) cells
Authors: Newman EA
Published: March 2018

The voltage- and K(+)-dependent properties of Muller cell currents and channels were characterized in freshly dissociated salamander Muller cells. In whole-cell voltage-clamp experiments, cells with endfeet intact and cells missing endfeet both displayed strong inward rectification. The rectification was similar in shape in both groups of cells but currents were 9.2 times larger in cells with endfeet. Ba2+ at 100 microM reduced the inward current to 6.8% of control amplitude. Decreasing external K+ concentration shifted the cell current-voltage (I-V) relation in a hyperpolarizing direction and reduced current magnitude. In multichannel, cell-attached patch-clamp experiments, patches from both endfoot and soma membrane displayed strong inward rectification. Currents were 38 times larger in endfoot patches. In single-channel, cell-attached patch-clamp experiments, inward- rectifying K+ channels were, in almost all cases, the only channels present in patches of endfoot, proximal process, and soma membrane. Channel conductance was 27.8 pS in 98 mM external K+. Reducing external K+ shifted the channel reversal potential in a hyperpolarizing direction and reduced channel conductance. Channel open probability varied as a function of voltage, being reduced at more negative potentials. Together, these observations demonstrate that the principal ion channel in all Muller cell regions is an inward-rectifying K+ channel. Channel density is far higher on the cell endfoot than in other cell regions. Whole-cell I-V plots of cells bathed in 12, 7, 4, and 2.5 mM K+ were fit by an equation including Boltzmann relation terms representing channel rectification and channel open probability. This equation was incorporated into a model of K+ dynamics in the retina to evaluate the significance of inward-rectifying channels to the spatial buffering/K+ siphoning mechanism of K+ regulation. Compared with ohmic channels, inward-rectifying channels increased the rate of K+ clearance from the retina by 23% for a 1 mM K+ increase and by 137% for a 9.5 mM K+ increase, demonstrating that Muller cell inward- rectifying channels enhance K+ regulation in the retina.
A spiking network model of short-term active memory
Authors: Zipser D , Kehoe B , Littlewort G , Fuster J
Published: March 2018

Studies of cortical neurons in monkeys performing short-term memory tasks have shown that information about a stimulus can be maintained by persistent neuron firing for periods of many seconds after removal of the stimulus. The mechanism by which this sustained activity is initiated and maintained is unknown. In this article we present a spiking neural network model of short-term memory and use it to investigate the hypothesis that recurrent, or “re-entrant,” networks with constant connection strengths are sufficient to store graded information temporarily. The synaptic weights that enable the network to mimic the input-output characteristics of an active memory module are computed using an optimization procedure for recurrent networks with non-spiking neurons. This network is then transformed into one with spiking neurons by interpreting the continuous output values of the nonspiking model neurons as spiking probabilities. The behavior of the model neurons in this spiking network is compared with that of 179 single units previously recorded in monkey inferotemporal (IT) cortex during the performance of a short-term memory task. The spiking patterns of almost every model neuron are found to resemble closely those of IT neurons. About 40% of the IT neuron firing patterns are also found to be of the same types as those of model neurons. A property of the spiking model is that the neurons cannot maintain precise graded activity levels indefinitely, but eventually relax to one of a few constant activities called fixed-point attractors. The noise introduced into the model by the randomness of spiking causes the network to jump between these attractors. This switching between attractor states generates spike trains with a characteristic statistical temporal structure. We found evidence for the same kind of structure in the spike trains from about half of the IT neurons in our test set. These results show that the behavior of many real cortical memory neurons is consistent with an active storage mechanism based on recurrent activity in networks with fixed synaptic strengths.
Tyrosine phosphorylation in early embryonic growth cones
Authors: Bixby JL , Jhabvala P
Published: March 2018

A large and growing body of evidence suggests that the regulation of tyrosine phosphorylation is important in the induction of axon growth. We have examined the subcellular distribution of enzymes regulating tyrosine phosphorylation in early embryonic brain, employing a preparation of isolated growth cone particles (GCPs). Because of the early developmental age and well-characterized nature of our tissue source, our GCP preparation offers some advantages over those described previously. As was found with other GCPs, our GCPs had relatively high levels of both the growth-associated protein GAP-43 and the intracellular tyrosine kinase pp60c-arc. In addition, we found that both total tyrosine kinase activity and total tyrosine phosphatase activity were concentrated two- to threefold in the GCPs relative to a neuronal membrane fraction. Two other nonreceptor tyrosine kinases, YES and FYN, were concentrated in the GCPs to a similar degree as that seen for SRC. In addition, we examined the developmental expression in brain of the three tyrosine kinases, using both a quantitative ELISA and Western blot analysis. Our results show that FYN, like SRC, reaches a peak of expression early in development, and declines thereafter. In contrast, expression of YES peaks later, and remains high in the adult brain. Immunofluorescence staining suggests that FYN is expressed both by neurons and by glia, and possibly by neuronal precursor cells. Our results implicate multiple tyrosine kinases as well as tyrosine phosphatases in growth cone function. In addition, the concentration of FYN in early embryonic growth cones combined with its early peak of expression suggests an important role for FYN in early neuronal development.
Activity-dependent release of endogenous adenosine modulates synaptic responses in the rat hippocampus
Authors: Mitchell JB , Lupica CR , Dunwiddie TV
Published: March 2018

Adenosine is a potent inhibitory modulator of synaptic transmission in the CNS, but its role in normal physiological function is unclear. In the present experiments, we have found electrophysiological evidence for activity-dependent release of adenosine from hippocampal slices evoked by physiologically relevant stimulation, and have demonstrated that this adenosine modifies synaptic activity in this brain region. When two independent excitatory pathways to the CA1 pyramidal neurons are used to evoke field EPSP responses, prior activation of one pathway will inhibit the EPSP evoked via the other input. This inhibition can be antagonized by the nonselective adenosine receptor antagonist theophylline, and by the selective A1 receptor antagonist 8- cyclopentyltheophylline, suggesting that the inhibitory response is due to the release of endogenous adenosine that activates presynaptic release-modulating A1 receptors. This inhibition can be observed following a single stimulus to the conditioning pathway, although it is more pronounced when a train of conditioning pulses is used, and is maximal following a train of 16–32 stimuli (at 100 Hz). When a train of four conditioning pulses is used, the inhibition appears with a latency of approximately 50 msec, peaks approximately 200–250 msec following the conditioning train, and recovers to baseline between 1 and 2 sec. Further evidence that this inhibition of excitatory transmission is mediated via adenosine is provided by the observation that superfusion with dipyridamole (an adenosine uptake inhibitor), and the adenosine deaminase inhibitor erythro-(2-hydroxy-3-nonyl)adenine, enhanced both the duration and amplitude of the inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
Estradiol attenuates alpha 2-adrenoceptor-mediated inhibition of hypothalamic norepinephrine release
Authors: Karkanias GB , Etgen AM
Published: March 2018

These studies tested the hypothesis that estradiol facilitates norepinephrine (NE) neurotransmission by modulating alpha 2- adrenoceptor-mediated inhibition of NE release. KCl-induced overflow of 3H from superfused slices preloaded with 3H-NE was Ca2+ dependent. Hypothalamic slices from estradiol-treated rats exposed to a single KCl pulse (S1) had modestly (20%) but significantly elevated NE release when compared to slices from ovariectomized (OVX) rats. Blockade of alpha 2-adrenoceptors by pretreatment with the imidazoline antagonists idazoxan (IDA) and RX821002 (RX) markedly facilitated NE release during S1 in hypothalamic slices from OVX rats; this facilitation was attenuated or absent in slices from estradiol-treated rats. In additional studies slices were stimulated twice, 24 min apart (S1 and S2), for 3 min with 20 mM KCl. In the absence of drug, the amount of 3H- NE released during S2 was always less than the amount released during S1 (i.e., S2:S1 approximately 0.6), regardless of whether slices were from OVX or estradiol-treated females. When 10 microM IDA was applied after S1 and 15 min prior to S2, the S2: S1 ratio increased to 1.8 +/- 0.1 in hypothalamic slices from OVX animals. In contrast, the S2:S1 ratio rose only to 1.1 +/- 0.2 in slices from estradiol-treated animals. RX applied before S2 markedly increased the S2:S1 ratio in both hypothalamic and preoptic area slices from OVX rats but failed to increase the S2:S1 ratio in slices from estradiol-treated rats. Interestingly, the modest effects of alkaloid alpha 2-antagonists such as yohimbine and rauwolscine on NE release in hypothalamic and preoptic area slices were not modified by estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)
Differential spatial and temporal gene expression in response to axotomy and deafferentation following transection of the medial forebrain bundle
Authors: Weiser M , Baker H , Wessel TC , Joh TH
Published: March 2018

Alterations in the levels of neurotransmitter biosynthetic enzymes are a concomitant of many neurodegenerative disorders. In order to elucidate potential mechanisms for longterm alterations in biosynthetic enzyme gene products in response to neuronal injury, an acute axotomy/deafferentation model was employed. A unilateral microknife transection of the medial forebrain bundle (MFB) axotomizes and/or deafferents phenotypically identified neuronal populations important in the function of the basal ganglia. Semi-quantitative in situ hybridization and immunohistochemical analysis demonstrated that the products of the immediate-early gene c-fos were induced postaxotomy in the noradrenergic neurons of the locus ceruleus (LC), but not in the dopaminergic neurons of the substantia nigra pars compacta (SNc). Analysis of the levels of mRNA, protein, and activity for tyrosine hydroxylase demonstrated that the LC neurons survive the injury while the SNc neurons degenerate. After MFB transection, Fos protein also was induced in the corpus striatum within 1 hr, first in large, putatively cholinergic neuronal populations followed at 3 hr by the small, putatively GABAergic neurons. The substantia nigra pars reticulata and the subthalamic nucleus neuronal populations, deafferented by the MFB transection, also exhibited Fos induction beginning at 3 hr. The data suggest that expression of Fos in a neuronal population is correlative with respect to cell survival following either axotomy or deafferentation. Whether Fos induction following injury is either a necessary mechanism of cell survival or merely a marker of increased neuronal activity requires further investigation.
Hippocampal neurons exhibit cyclothiazide-sensitive rapidly desensitizing responses to kainate
Authors: Patneau DK , Vyklicky L , Mayer ML
Published: March 2018

In whole-cell recordings from mammalian CNS neurons, AMPA-preferring glutamate receptors exhibit strong desensitization in response to AMPA, glutamate, and quisqualate, but not to kainate or domoate. Such desensitization is reduced by lectins, by the nootropic drug aniracetam, and by diazoxide. None of these compounds strongly modulate responses to kainate and domoate, consistent with the apparent lack of desensitization to these agonists. We now report experiments on hippocampal neurons in which responses to kainate were strongly potentiated by cyclothiazide, a benzothiadiazine diuretic and antihypertensive drug structurally related to diazoxide. Cyclothiazide increased the maximum response to a saturating concentration of kainate by approximately 300% and produced a shift to the left in the kainate dose-response curve. Because cyclothiazide was considerably more effective than aniracetam in reducing desensitization evoked by glutamate, we tested the possibility that potentiation of responses to kainate was due to block of a previously undetected component of desensitization in the response to kainate itself. In outside-out patches responses to rapid perfusion of 3 mM kainate showed 34% desensitization, the onset of which developed with a time constant of 2.2 msec. Desensitization of responses to kainate was abolished by 100 microM cyclothiazide, as was the much stronger desensitization evoked by glutamate and AMPA. Cyclothiazide also slowed the rate of deactivation of responses to kainate recorded after return to agonist- free solution. Current-voltage plots for control responses to kainate exhibited outward rectification that was associated with a reduction in the amount of desensitization on depolarization. Both effects were absent in the presence of cyclothiazide, suggesting that rectification of responses to kainate was due to the voltage dependence of desensitization. The complete block of desensitization produced by cyclothiazide provides a powerful new tool for analysis of allosteric regulatory mechanisms at AMPA-preferring glutamate receptors.
Voltage-dependent ionic conductances of type I spiral ganglion cells from the guinea pig inner ear
Authors: Santos-Sacchi J
Published: March 2018

Type I spiral ganglion cells provide the afferent innervation to the inner hair cells of the mammalian organ of Corti and project centrally to the cochlear nucleus. While single-unit studies conducted over the past several decades have provided a wealth of information concerning the response characteristics of these neurons and, to some extent, their receptor targets, little is known about the neuron's intrinsic electrical properties. These properties undeniably will contribute to the firing patterns induced by acoustic stimuli. Type I spiral ganglion cell somata from the guinea pig inner ear were acutely isolated and the voltage-dependent conductances were analyzed with the whole-cell voltage clamp. Under conditions that mimic the normal intra- and extracellular ionic environments, type I spiral ganglion cells demonstrate fast inward TTX-sensitive Na currents (whose current density varied markedly among cells) and somewhat more slowly developing outward K currents. Resting potentials averaged -67.3 mV. Under current clamp, no spontaneous spike activity was noted, but short current injections produced graded action potentials with after hyperpolarizations lasting several milliseconds. The nondecaying outward K current activated at potentials near rest and was characterized by a pronounced rectification. The kinetics of the Na and K currents were rapid. Maximum peak inward Na currents occurred within 400 microseconds, between a voltage range of -10 and 0 mV, and inactivated within 4 msec. Recovery from inactivation was also rapid. At a holding potential of -80 mV, the time constant for recovery from an inactivating voltage step to -10 mV was 2.16 msec. Above -50 mV outward K currents reach half-maximal amplitude within 1.5 msec. In addition to these currents, a slow noninactivating TTX-sensitive inward current was observed that was blockable with Cd2+ or Gd3+. Problems encountered with blocking the tremendous outward K current hampered the characterization of this inward current. Similarities between the kinetics of ganglion cell currents and some of the rapid temporal characteristics of eighth nerve single-unit activity confirm the notion that intrinsic membrane properties help shape auditory neuron responses to sound.
Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid
Authors: Beal MF , Brouillet E , Jenkins BG , Ferrante RJ , Kowall NW , Miller JM , Storey E , Srivastava R , Rosen BR , Hyman BT
Published: March 2018

An impairment of energy metabolism may underlie slow excitotoxic neuronal death in neurodegenerative diseases. We therefore examined the effects of intrastriatal, subacute systemic, or chronic systemic administration of the mitochondrial toxin 3-nitropropionic acid (3-NP) in rats. Following intrastriatal injection 3-NP produced dose-dependent striatal lesions. Neurochemical and histologic evaluation showed that markers of both spiny projection neurons (GABA, substance P, calbindin) and aspiny interneurons (somatostatin, neuropeptide Y, NADPH- diaphorase) were equally affected. Subacute systemic administration of 3-NP produced age-dependent bilateral striatal lesions with a similar neurochemical profile. However, in contrast to the intrastriatal injections, striatal dopaminergic afferent projections were spared. Both freeze-clamp measurements and chemical shift magnetic resonance spectroscopy showed that 3-NP impairs energy metabolism in the striatum in vivo. Microdialysis showed no increase in extracellular glutamate concentrations after systemic administration of 3-NP. The lesions produced by intrastriatal injection or systemic administration of 3-NP were blocked by prior decortication. However, the NMDA antagonist MK- 801 did not block the effects of intrastriatal 3-NP, consistent with a non-NMDA excitotoxic mechanism. In contrast to subacute systemic administration of 3-NP, chronic (1 month) administration produced lesions confined to the striatum in which there was relative sparing of NADPH-diaphorase interneurons, consistent with an NMDA excitotoxic process. Chronic administration showed growth-related proliferative changes in dendrites of spiny neurons similar to changes in Huntington's disease (HD). These results are consistent with in vitro studies showing that mild metabolic compromise can selectively activate NMDA receptors while more severe compromise activates both NMDA and non- NMDA receptors. Chronic administration of 3-NP over 1 month produces selective striatal lesions that replicate many of the characteristic histologic and neurochemical features of HD.
Differential metabolic and electrical activity in the somatic sensory cortex of juvenile and adult rats
Authors: Riddle DR , Gutierrez G , Zheng D , White LE , Richards A , Purves D
Published: March 2018

We have examined relative levels of metabolic and electrical activity across layer IV in the primary somatic sensory cortex (S1) of the rat in relation to regions of differential postnatal cortical growth. Each of several indices used--mitochondrial enzyme histochemistry, microvessel density, Na+/K+ pump activity, action potential frequency, and deoxyglucose uptake--indicate regional variations of metabolic and electrical activity in this part of the brain in both juvenile (1-week- old) and adult (10–12-week-old) animals. At both ages, areas of the somatic sensory map related to special sensors such as whiskers and digital pads showed evidence of the most intense activity. Thus, mitochondrial enzyme staining, blood vessel density, and Na+/K+ ATPase activity were all greatest in the barrels and barrel-like structures within S1, and least in the adjacent interbarrel cortex and the cortex surrounding S1. Multiunit recordings in and around the posteromedial barrel subfield of anesthetized animals also showed that the average ratio of evoked to spontaneous activity was greater in barrels than in the surrounding, metabolically less active cortex. Furthermore, autoradiograms of labeled deoxyglucose accumulation in awake behaving animals indicated systematic differences in neural activity across S1 barrels and barrel-like structures showed more deoxyglucose accumulation than interbarrel, nonbarrel, or peri-S1 cortex. These regional differences in neural activity correspond to regional differences in neocortical growth (Riddle et al., 1992). The correlation of greater electrical activity, increased metabolism, and enhanced cortical growth during postnatal maturation suggests that neural activity foments the elaboration of circuitry in the developing brain.
Transport of BC1 RNA in hypothalamo-neurohypophyseal axons
Authors: Tiedge H , Zhou A , Thorn NA , Brosius J
Published: March 2018

Ample evidence indicates that in nerve cells, several individual proteins are locally synthesized in postsynaptic domains in dendrites. By contrast, axonal terminals, at least in mammals, are generally thought to lack protein synthetic capacity. However, axonal nerve endings of the hypothalamo-neurohypophyseal tract have recently been shown to contain mRNAs encoding vasopressin, oxytocin, dynorphin, and neurofilament. In this report, we identify BC1 RNA, a small RNA polymerase III transcript that is specifically expressed in neurons, in hypothalamo-neurohypophyseal axons. BC1 RNA has previously been shown to be located in somatic and dendritic domains of various types of neurons in the rat nervous system. Here we present evidence to show that BC 1 RNA, like several neuropeptide mRNAs, is axonally transported from magnocellular hypothalamic neurons to neurosecretory nerve endings in the posterior pituitary. BC1 RNA, which has been reported to be a component of a ribonucleoprotein particle, is thus colocalized with dendritic mRNAs in dendritic domains and with axonal mRNAs in axonal domains, respectively. Such colocalization is indicative of functional interactions of BC1 RNA with those mRNAs that are targeted to extrasomatic domains of nerve cells.
Intraneostriatal administration of glutamate antagonists increases behavioral activation and decreases neostriatal ascorbate via nondopaminergic mechanisms
Authors: Pierce RC , Rebec GV
Published: March 2018

Behavioral findings suggest that the effects of neostriatal glutamate and ascorbate are opposed to those of neostriatal dopamine. Recent evidence also indicates that glutamate and ascorbate are linked via a carrier-mediated heteroexchange process, suggesting that ascorbate may act through the glutamate system to influence behavior. In order to assess glutamate-ascorbate interactions and their influence on the behavioral output of the basal ganglia, glutamate and homocysteic acid (a glutamate reuptake blocker) as well as NMDA antagonists were infused into the neostriatum of freely moving rats while extracellular neostriatal ascorbate was monitored via electrochemically modified carbon-fiber electrodes. Neostriatal 3,4-dihydroxyphenylacetic acid (DOPAC), a major dopamine metabolite, also was recorded in order to assess the dependency of any drug effect on the nigrostriatal dopamine system. Intraneostriatal infusions of L-glutamate (1 micrograms/microliters), but not L-homocysteic acid (30 micrograms/microliters), elevated extracellular neostriatal ascorbate levels. Neither of these drugs had any effect on neostriatal DOPAC or overt behavioral activity. Intraneostriatal infusion of the noncompetitive NMDA antagonist dizocilpine (MK-801; 3 micrograms/microliters) or the competitive NMDA antagonist 3-(2- carboxypiperazin-4-yl)-propyl-1-phosphonene (CPPene; 5 micrograms/microliters) decreased neostriatal ascorbate but had no effect on neostriatal DOPAC. Both dizocilpine and CPPene activated behavior in intact and sham-lesioned animals as well as in animals with near-total depletions of neostriatal dopamine following a 6- hydroxydopamine lesion. When administered systemically, however, dizocilpine (1.0 mg/kg) significantly increased neostriatal DOPAC. This effect appears to be regulated via midbrain NMDA receptors, in that this effect was completely abolished by electrolytic lesions of the substantia nigra pars reticulata.(ABSTRACT TRUNCATED AT 250 WORDS)
Purification and properties of m1-toxin, a specific antagonist of m1 muscarinic receptors
Authors: Max SI , Liang JS , Potter LT
Published: March 2018

The venom of the Eastern green mamba from Africa, Dendroaspis angusticeps, was found to block the binding of 3H-quinuclidinyl benzilate to pure m1 and m4 muscarinic ACh receptors expressed in Chinese hamster ovary cells. The principal toxin in the venom with anti- m1 muscarinic activity was purified by gel filtration and reversed- phase HPLC. This toxin has 64 amino acids, a molecular mass of 7361 Da, and an isoelectric point of 7.04. Its cysteine residues are homologous with those in curare-mimetic alpha-neurotoxins, and with those in fasciculin, which inhibits AChE. At low concentrations the toxin blocked m1 receptors fully and pseudoirreversibly while having no antagonist activity on m2-m5 receptors; the toxin is therefore named “m1-toxin.” At higher concentrations m1-toxin interacted reversibly with m4 receptors, and half of the toxin dissociated in 20 min at 25 degrees C. The affinity of m1-toxin is therefore much higher for m1 than for m4 receptors. By comparison with m1-toxin, pirenzepine has sixfold higher affinity for m1 than for m4 receptors. Autoradiographs of muscarinic receptors in the rat brain demonstrated that m1-toxin blocked the binding of 2 nM 3H-pirenzepine only in regions known to bind m1-specific antibodies. Thus, m1-toxin is a much more selective ligand than pirenzepine for functional and binding studies of m1 muscarinic receptors.
Response of caudate neurons to stimulation of intrinsic and peripheral afferents in normal, symptomatic, and recovered MPTP-treated cats
Authors: Rothblat DS , Schneider JS
Published: March 2018

Cat caudate nucleus (CD) neurons were tested for changes in spontaneous activity, response to peripheral sensory stimuli (tactile, auditory, and visual), and electrical stimulation of monosynaptic afferents (pericruciate cortex and nucleus centralis lateralis) in normal cats and in the same cats after induction of and spontaneous recovery from parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). After normal baseline data were collected, cats were given MPTP (7.5 mg/kg, 5–7 d) to induce a parkinsonian syndrome consisting of rigidity, akinesia, and decreased orienting to sensory stimuli. During this symptomatic period, the mean spontaneous activity of CD units increased (6.20 spikes/sec vs 2.25 spikes/sec in normal cats). In these same animals, the percentage of units responding to peripheral sensory stimulation was significantly decreased (compared to normal) while the percentage of units responding to electrical stimulation of monosynaptic afferents increased. By 6 weeks after MPTP administration, cats had recovered gross motor and sensorimotor function and CD unit recordings were reinitiated. In functionally recovered animals, all electrophysiological measures returned to levels resembling those seen in normal animals. These data suggest that the processing of peripheral sensory information is an important part of basal ganglia function and that the sensory responsiveness of the CD may reflect the overall motor condition of the animal. The changes observed in the responsiveness of CD neurons to direct electrical stimulation of monosynaptic afferents may indicate that the defect in the processing of polysynaptic sensory information observed in the striatum in parkinsonian animals may be occurring, at least in part, extrastriatally.
Adenosine triphosphate depletion reverses sodium-dependent, neuronal uptake of glutamate in rat hippocampal slices
Authors: Madl JE , Burgesser K
Published: March 2018

Extracellular accumulations of excitatory amino acids (EAAs) may mediate ischemic neuronal damage. Metabolic insults can decrease Na+ and K+ plasma membrane gradients, thereby reducing the driving force for uptake of EAAs into cells by Na(+)-dependent EAA cotransporters. EAA accumulations could result from decreased uptake and increased release due to reversal of these cotransporters. ATP depletion, uptake, and release of EAAs were measured by HPLC in slices treated with metabolic inhibitors. Inhibition and reversal of cotransporters were determined by uptake or release of D,L-threo-beta-hydroxyaspartate (OH- Asp), an EAA analog with high affinity for cotransporters. Moderate ATP depletion (7 > ATP nmol/mg protein > 3) reduced uptake by cotransporters without increasing release of EAAs. When ATP was severely depleted (ATP < 2 nmol/mg protein), increased release of EAAs and preloaded OH-Asp occurred, consistent with reversal of cotransporters. Release of glutamine and asparagine was not increased, confirming that release was not primarily due to nonselective increased membrane permeability. ATP depletion and ouabain acted synergistically to produce EAA release, strongly suggesting release was largely mediated by inhibition of Na/K-ATPases. Severe ATP depletion decreased glutamate-like immunoreactivity primarily in axonal terminal-like structures, suggesting release occurred primarily from terminals. Moderate ATP depletion may increase extracellular EAAs by decreasing uptake. Severe ATP depletion may further increase EAAs by reversing uptake, thereby releasing cytosolic neuronal pools of EAAs.