bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of neurotransmitter receptor desensitization: chlordiazepoxide stimulates fading of the GABA response
Authors: Mierlak D , Farb DH
Published: March 2018

Benzodiazepines are neuromodulatory drugs that potentiate GABA-mediated conductance increases. We report the findings of an investigation into the effect of a full benzodiazepine-positive modulator (agonist), chlordiazepoxide (CDPX), on desensitization of the GABA response in chick spinal cord neurons maintained in primary monolayer cell culture. GABA application initially increases cell conductance, which then desensitizes. CDPX increases the apparent rate constant and extent of desensitization for the GABA response. The observed values for rate constant and extent of desensitization in the presence of CDPX are significantly greater than the values predicted for an equivalent peak response to GABA alone. Flunitrazepam, another full positive modulator, also stimulates the rate constant for GABA-induced desensitization. Furthermore, a weak partial benzodiazepine-positive modulator, Ro 15- 1788 (an imidazodiazepine) (1) exhibits little or no potentiation of GABA-induced desensitization, and (2) antagonizes the ability of CDPX to stimulate GABA desensitization. The results demonstrate a novel form of neuromodulator action: the stimulation of receptor desensitization that cannot be explained by channel blockade. Thus, benzodiazepine modulators have the capacity to increase GABA receptor desensitization while increasing the peak response to GABA.
Expression of a surface-associated antigen on Y-cells in the cat lateral geniculate nucleus is regulated by visual experience
Authors: Sur M , Frost DO , Hockfield S
Published: March 2018

The monoclonal antibody Cat-301, generated against cat spinal cord (McKay and Hockfield, 1982), recognizes a surface-associated antigen that, in the cat lateral geniculate nucleus (LGN), is selectively expressed on Y-cells (Hockfield et al., 1983; Hendry et al., 1984; Sur et al., 1984). We now report that the antigen recognized by Cat-301 appears late in development, along a time course similar to that described for the maturation of the physiological properties of Y-cells in the LGN, and that its expression is sharply reduced by monocular lid suture or dark-rearing from birth, 2 visual deprivation procedures that lead to a reduction in the proportion of Y-cells recorded physiologically in the LGN (Sherman et al., 1972; Kratz et al., 1979; reviewed in Sherman and Spear, 1982). Monocular lid suture in the adult has no effect on Cat-301 antigen levels or, as previously reported (Sherman et al., 1972), on the proportion of physiologically recorded Y- cells. In addition, reversing the monocular deprivation in adulthood by opening the neonatally sutured eye and suturing closed the previously normal eye for 6 months restores neither normal levels of Cat-301 labeling nor, as previously reported (Geisert et al., 1982), the proportion of recordable Y-cells. The development of Cat-301 immunoreactivity thus parallels the development of LGN Y-cell physiology. The relative reduction in levels of immunoreactivity consequent to neonatal, but not adult, visual deprivation shows that Cat-301 antigen expression does not simply reflect the level of visually evoked electrical activity in the LGN, but rather reflects a process that depends on the nature of visual experience early in life.
A synapse-specific carbohydrate at the neuromuscular junction: association with both acetylcholinesterase and a glycolipid
Authors: Scott LJ , Bacou F , Sanes JR
Published: March 2018

With the aim of investigating the roles of carbohydrates in synapse formation, we have characterized a synapse-specific saccharide at the vertebrate neuromuscular junction. Two lectins of similar specificity (Dolichos biflorus agglutinin, DBA, and Vicia villosa-B4 agglutinin, VVA-B4) stain synaptic but not extrasynaptic regions of the rat muscle fiber surface and thus define a synapse-specific carbohydrate. Using these and other probes, we show that the carbohydrate moiety concentrated at the neuromuscular junction resembles N- acetylgalactosamine (GalNAc) linked in the beta-anomeric form to the termini of oligosaccharides. VVA-B4 also selectively stains neuromuscular junctions in human, mouse, rabbit, guinea pig, chick, frog, axolotl, snake, fish, and lamprey muscles, a phylogenetic conservatism that suggests a synapse-related role for GalNAc beta- terminal saccharides. AChE from muscle binds to DBA- and VVA-B4- agarose, and is thereby identified as a glycoconjugate bearing the synapse-specific carbohydrate. Assay of AChE isoforms reveals that asymmetric, collagen-tailed forms, known to be highly concentrated at the rat neuromuscular junction, bind DBA and VVA-B4, while globular forms, which are more widely distributed, do not. A second class of GalNAc-bearing glycoconjugates is demonstrable immunohistochemically with monoclonal antibodies to stage-specific embryonic antigen (SSEA)-3 (Shevinsky et al., 1982) and GM2 (Natoli et al., 1986), which recognize GalNAc-bearing glycolipids. These antibodies selectively stain neuromuscular junctions, where they recognize glycolipid-like molecules that bind VVA-B4 but are distinguishable from AChE. The association of a synapse-specific carbohydrate with at least 2 different synapse- specific molecules raises the possibility that the former plays a role in determining a property that the latter share, such as concentration at the synapse.
Migration and differentiation of neural crest and ventral neural tube cells in vitro: implications for in vitro and in vivo studies of the neural crest
Authors: Loring JF , Barker DL , Erickson CA
Published: March 2018

During vertebrate development, neural crest cells migrate from the dorsal neural tube and give rise to pigment cells and most peripheral ganglia. To study these complex processes it is helpful to make use of in vitro techniques, but the transient and morphologically ill-defined nature of neural crest cells makes it difficult to isolate a pure population of undifferentiated cells. We have used several established techniques to obtain neural crest-containing cultures from quail embryos and have compared their subsequent differentiation. We confirm earlier reports of neural crest cell differentiation in vitro into pigment cells and catecholamine-containing neurons. However, our results strongly suggest that the 5-HT-containing cells that develop in outgrowths from thoracic neural tube explants are not neural crest cells. Instead, these cells arise from ventral neural tube precursors that normally give rise to a population of serotonergic neurons in the spinal cord and, in vitro, migrate from the neural tube. Therefore, results based on previously accepted operational definitions of neural crest cells may not be valid and should be reexamined. Furthermore, the demonstration that cells from the ventral (non-neural crest) part of the neural tube migrate in vitro suggests that the same phenomenon may occur in vivo. We propose that the embryonic “neural trough,” as well as the neural crest, may contribute to the PNS of vertebrates.
Differentiation and maturation of embryonal carcinoma-derived neurons in cell culture
Authors: McBurney MW , Reuhl KR , Ally AI , Nasipuri S , Bell JC , Craig J
Published: March 2018

We have previously shown that retinoic acid-treated cultures of the P19 line of embryonal carcinoma cells differentiate into neurons, glia, and fibroblast-like cells (Jones-Villeneuve et al., 1982). We report here that the monoclonal antibody HNK-1 reacts with the neurons at a very early stage of their differentiation and is, therefore, an early marker of the neuronal lineage. Cells in differentiated P 19 cultures synthesized acetylcholine but not catecholamines, suggesting that at least some of the neurons are cholinergic. The neurons also carry high- affinity uptake sites for GABA but not for serotonin. In long-term cultures, neuronal processes differentiated into axons and dendrites, which formed synapses. This biological system should prove valuable for examining the development and maturation of cholinergic neurons, since their differentiation occurs in cell culture.
Analysis of discontinuity in visual contours in area 19 of the cat
Authors: Saito H , Tanaka K , Fukada Y , Oyamada H
Published: March 2018

Previous ablation studies have suggested that area 19 of the cat plays an important role in pattern discrimination. To clarify the functional roles unique to area 19, we studied the receptive-field properties of cells in area 19 and compared them with those of cells in area 17. Recordings were made of anesthetized and immobilized animals. The majority (72%) of the cells in area 17 responded maximally to an elongated bar at a particular orientation, while they responded only weakly or not at all to a small spot (elongation-requiring cells). In contrast, more than half (63%) of the cells in area 19 showed a good response to a nonoriented small stimulus moving in any direction (dot- responsive cells). Two-thirds of the dot-responsive cells in area 19 failed to respond when the moving slit was elongated to more than some length in any orientation. These dot-responsive cells of the “inhibited- by-length” type responded strongly to the end of a long bar, and many of them also responded strongly to a break point in the middle of a long bar. We suggest that these dot-responsive cells of the “inhibited- by-length” type detect discontinuities in contours. Though they are in the minority, elongation-requiring cells constitute a considerable population (37%) in area 19, and dot-responsive and elongation- requiring cells from columnar patches in the same area. We conclude that, in contrast to area 17, whose main role is the decomposition of patterns into oriented contours, area 19 analyzes both orientation and discontinuities, with a strong bias towards the latter.
Activity-dependent regulation of tachykinin-like immunoreactivity in neurons of monkey visual cortex
Authors: Hendry SH , Jones EG , Burstein N
Published: March 2018

A monoclonal antibody that recognizes the carboxyl terminus of substance P was used to localize tachykinin-like immunoreactivity in neurons of area 17 of the adult monkey cerebral cortex. Tachykinin immunostaining was examined in normal monkeys, in monkeys receiving monocular injections of the sodium channel blocker TTX for 10 or 15 d, and in monkeys from which the crystalline lens of one eye had been removed 3 or 6 months prior to death. The immunocytochemical staining in each monkey was compared with the histochemical staining for the mitochondrial enzyme cytochrome oxidase (CO). These forms of monocular deprivation produce the most profound changes in the staining of layers II-III and IVC. In layers II-III of normal monkeys, tachykinin- immunoreactive somata are uniformly distributed by immunostained puncta are densely packed in rows of patches that correspond to the rows of CO- stained patches. Following monocular TTX injections, both the patches of CO staining in the deprived-eye columns and the corresponding patches of intense tachykinin immunostaining shrink. Quantitative analyses indicate the numerical density of immunostained somata is reduced by 50% within the deprived-eye rows of patches and is also reduced within regions surrounding the patches in both sets of ocular dominance columns. Following the removal of the lens from one eye, the CO-stained patches and the immunostained patches in one set of rows shrink and the density of immunostained somata in these rows is reduced by 60%. In the alternating rows, the CO staining between patches increases so that many of the patches fuse to form long, continuous bands. Patches of immunostained puncta also enlarge and fuse; the density of immunostained somata in these rows of enlarged patches is approximately 30% greater than normal. In layer IVC of normal monkeys, the CO staining and the tachykinin immunostaining are relatively uniform. Following monocular TTX injections the CO staining and the tachykinin immunostaining are greatly reduced in columns dominated by the injected eye, corresponding to an 80% reduction in the numerical density of immunoreactive somata. By contrast, the CO staining in layer IVC of aphakic monkeys is changed only slightly from normal and the tachykinin immunostaining appears normal. The changes in the density of immunostained somata in both layers II-III and in IVC occur even through the total density of thionin-stained neurons remains normal.
Growth cone configuration and advance: a time-lapse study using video- enhanced differential interference contrast microscopy
Authors: Aletta JM , Greene LA
Published: March 2018

We have analyzed the dynamics of growth cone configuration using video- enhanced contrast differential interference contrast microscopy. Regenerating neurites from NGF-treated PC12 cells and sympathetic neurons were observed in real time during their elongation and reviewed by time-lapse video recordings. This technique provided a high- resolution view of motile growth cone elements including filopodia, microspikes, lamellipodia and ruffles. On the basis of our observations, a multistage model for growth cone advance is proposed. Elongation commences with lamellipodial spreading. If the newly extended lamellipodium does not retract or lift off the substrate in the form of a ruffle, a second phase--consolidation--occurs, in which the lamellipodium thickens as it fills with cytoplasm and organelles. The consolidated area then undergoes further transformation into an area of neuritic shaft as new lamellipodia form at the leading and peripheral zones of the distal process. We never observed filopodia or microspikes contracting to propel the growth cone forward. We also noted that elongating tips generally had large varicosities within 20 micron of their leading edges. These may play a role in neurite outgrowth and in the formation of smaller, synaptic vesicle-containing varicosities. The dynamic behavior of the growth cone was under the control of NGF. Withdrawal of NGF resulted in the disappearance of motile structures and cessation of growth, while readdition of NGF triggered the rapid reappearance of these structures and the resumption of growth. The high-resolution video microscopy of living growth cones provides necessary baseline information, as well as a bioassay paradigm, for future studies on the molecular mechanisms of nerve growth.
Functional anatomy of macaque striate cortex. V. Spatial frequency
Authors: Tootell RB , Silverman MS , Hamilton SL , Switkes E , De Valois RL
Published: March 2018

When macaque monkeys view achromatic, sinusoidal gratings of a single spatial frequency, the pattern of 14C-2-deoxy-d-glucose (DG) uptake produced by the gratings is shown to depend on the spatial frequency chosen. When a relatively high (5–7 cycles/deg) spatial frequency is shown binocularly at systematically varied orientations, uptake in parafoveal striate cortex is highest between the cytochrome oxidase blobs (that is, in the interblobs) in layers 1, 2, and 3. In layers 4B, 5, and 6, where the cytochrome oxidase blobs are faint or absent, DG uptake is highest in a periodic pattern that lies in register with the interblobs of layers 2 + 3. When the grating is, instead, of relatively low (1–1.5 cycles/deg) spatial frequency, DG uptake is highest in the blobs, in the blob-aligned portions of layers 1–4B, and in the lower- layer blobs as well. These variations in DG topography are confirmed in stimulus comparisons within a single hemisphere. Presumably, this shift in functional topography within the extra-granular layer is the primate homolog of “spatial frequency columns” shown earlier in the cat (Tootell et al., 1981; Silverman, 1984). In the well-differentiated architecture of primate striate cortex, laminar differences produced by high- versus low-spatial-frequency gratings are visible as well. Gratings of very high spatial frequency produce much higher uptake in 4Cb (which receives input from the parvocellular LGN layers) than in 4Ca (which gets its input from the magnocellular LGN layers). Gratings of low spatial frequency produce the converse result. Presumably, cells in the magnocellular LGN layers and/or in the magnocellular-dominated layer 4Ca have lower average spatial frequency tuning (larger receptive fields) than their counterparts in the parvocellular LGN and/or in striate layer 4Cb. The DG patterns produced by various spatial frequencies also vary with eccentricity, in a manner consistent with known, eccentricity-dependent variations of receptive-field size and spatial frequency tuning. Thus, gratings of a “middle”-spatial- frequency range (4–5 cycles/deg) produce high uptake in the blobs near the foveal representation and high uptake in the interblobs at more peripheral eccentricities, including 5 degrees. This shift in DG topography also includes the transition zone near 3 degrees, where the level of stimulus-driven uptake is as high in the blob regions as it is in interblob regions. Variations in uptake between layers 4Ca and 4Cb, as a function of eccentricity, shift in parallel with the changes in the upper-layer topography.
Mechanisms of long-term potentiation: EPSP/spike dissociation, intradendritic recordings, and glutamate sensitivity
Authors: Taube JS , Schwartzkroin PA
Published: March 2018

Synaptic efficacy is modified following a brief train of high-frequency stimulation (HFS) to a cell's afferent fibers (long-term potentiation; LTP). An alteration in the postsynaptic response to endogenous neurotransmitter, as a result of an increase in the number of postsynaptic receptors, has been proposed (Baudry and Lynch, 1980). We tested this hypothesis in the CA1 hippocampus by intracellularly recording the postsynaptic response to localized application of glutamate before and after induction of LTP. When LTP was produced, there was no corresponding change in neuronal sensitivity to glutamate application. These findings are not consistent with the hypothesis that HFS of fibers in CA1 stratum radiatum induces an increase in the number of postsynaptic glutamate receptors in CA1 pyramidal cells. Previous reports concerning LTP have indicated a dissociation between the degree of potentiation in the population EPSP and population spike. Simultaneous recordings of the CA 1 population EPSP and population spike in hippocampal slices confirmed that the degree of potentiation of the population spike was not predicted by the degree of potentiation in the population EPSP. Intradendritic impalements were obtained to more accurately assess changes in the intracellular EPSP following HFS. When the population EPSP was potentiated, there was also a potentiated intradendritic EPSP. When the population spike was potentiated following HFS, however, the intradendritic EPSP was often unchanged; in the same cell, there was an increased probability of action potential discharge to stimulation which was originally (i.e., pre-HFS) subthreshold for spike initiation. These results indicate that the EPSP (intracellular or extracellular) may be potentiated following HFS, but this potentiation is not a prerequisite for, or a correlation of, potentiation in the population spike. Furthermore, these findings suggest that LTP is composed of 2 independent components--a synaptic component and an EPSP-to-spike coupling component.
Mechanisms of long-term potentiation: a current-source density analysis
Authors: Taube JS , Schwartzkroin PA
Published: March 2018

A current-source density (CSD) analysis was carried out in the CA 1 region of the hippocampal slice (1) to determine the pattern of current flow in pyramidal cells upon orthodromic stimulation and (2) to test the hypothesis that EPSP-to-spike potentiation is produced by an alteration in this distribution of current sinks and/or sources. The results indicated that 2 sinks occur near the cell body layer (in addition to the sink associated with the EPSP) in response to orthodromic stimulation of the apical dendrites. An early (i.e., short- latency) sink was present along the radiatum/pyramidale border and was evident throughout the time course of the evoked field potential. This sink peaked in magnitude just prior to the peak of the population spike and was associated with orthodromic stimulation; it was not seen with antidromic stimulation. A second, later, sink occurred in the proximal portion of the basal dendrites and had a characteristic time course similar to the population spike; this second sink was also present during antidromic stimulation. There was some suggestion that the earlier dendritic sink shifted apically with development of long-term potentiation (LTP). The existence and movement of such an active zone in these cells may help to explain the dissociation of EPSP and spike potentiation in LTP.
Neurite outgrowth in individual neurons of a neuronal population is differentially regulated by calcium and cyclic AMP
Authors: Mattson MP , Taylor-Hunter A , Kater SB
Published: March 2018

In identified Helisoma neurons, intracellular calcium can regulate neurite elongation and growth cone motility. Neurotransmitters such as 5-HT suppress both neurite elongation and the filopodial and lamellipodial movements of growth cones by causing increases in intracellular calcium (Haydon et al., 1984; Cohan et al., 1987; Mattson and Kater, 1987). Since an additional second messenger, cyclic AMP (cAMP), is known to mediate many physiological effects of neurotransmitters, we tested (1) the possible involvement of cAMP in the regulation of neurite outgrowth from Helisoma buccal neurons and (2) calcium-cAMP interrelationships in the regulation of outgrowth. The cAMP-elevating agents forskolin (5 x 10(-6)-10(-4) M) and dibutyryl cAMP (dbcAMP; 5 x 10(-3)-10(-2) M) suppressed neurite elongation and growth cone movements in identified neurons B19 (5-HT sensitive) and B5 (5-HT insensitive); the suppression was reversible. Exposure of these particular identified neurons to the calcium channel blocker La3+ (10(- 5) M) or a culture medium with reduced calcium prevented and reversed the suppressive effects of forskolin and dbcAMP. In order to determine if the results on neurons B5 and B19 were representative of all neurons or only a subset, we examined a larger population of neurons. Calcium ionophore A23187 suppressed outgrowth from all neurons in mass dissociate cultures of buccal neurons, while forskolin or dbcAMP plus IBMX suppressed outgrowth from only one-half of buccal neurons. Finally, we found that 2 subpopulations exist among the neurons whose outgrowth is suppressed by cAMP: One subpopulation requires calcium influx for cAMP to act, while the other does not. Thus, even within the relatively small population of neuronal types comprising the buccal ganglion of Helisoma, second messengers within different neurons can act and interact in different ways to regulate outgrowth.
Cytology and neuron-glial apposition of migrating cerebellar granule cells in vitro
Authors: Gregory WA , Edmondson JC , Hatten ME , Mason CA
Published: March 2018

In developing mammalian brain, many neurons migrate to their final position by moving in direct apposition to radially oriented glial cells. Glial-guided migration can be visualized in microcultures of mouse cerebellar cells by the combined use of cellular antigen markers and high resolution time-lapse video microscopy (Hatten et al., 1984; Edmondson and Hatten, 1987). Such studies have demonstrated the behavior of migrating cells and revealed a motile leading process on the migrating neuron that resembles an axonal growth cone and grows along extended glial fibers. To study the fine structural details of the migrating neuron and its neuron-glial apposition, we identified and monitored neurons in microcultures with video microscopy and examined the cytology and cellular contacts of the same cells with transmission electron microscopy. The cytology of the soma and leading process of migrating cells closely matches that described for granule cells in intact brain (Rakic, 1971). Newly observed structures include the presence of longitudinally oriented microtubules extending from a basal body in the soma into the leading process, and microfilament-rich filopodia arising from the soma and leading process. The most striking feature of actively migrating neurons is a specialized junction between the neuronal cell soma and apposing glial fibers. At this junction, here termed “interstitial density,” the extracellular space is dilated to 20 nm and filamentous material in the intracellular cleft either spans the cleft or runs parallel to the cell membranes. Some interstitial fibrils are contiguous with, or are transmembranous extensions of, submembranous cytoskeletal elements that attach to microtubules. Interstitial junctions were not found between neurons that did not translocate in the observation period before fixation. Instead, stationary cells formed desmosomes (puncta and macula adhaerentia) at appositions with glial processes.
B-50 phosphorylation and polyphosphoinositide metabolism in nerve growth cone membranes
Authors: Van Hooff CO , De Graan PN , Oestreicher AB , Gispen WH
Published: March 2018

The neuron-specific phosphoprotein B-50 (Mr 48 kDa, isoelectric point, IEP, 4.5), which is identical to GAP43, is a member of a family of growth-associated proteins. Protein B-50 is a major phosphoprotein in nerve growth cones isolated from fetal rat brain. In a growth cone particulate fraction (GCp), endogenous B-50 phosphorylation is Ca2+- dependent and is unaffected by cAMP. Addition of purified protein kinase C (PKC) to GCP enhances B-50 phosphorylation. In heat- inactivated GCp, B-50 is one of the major substrates of purified PKC. Endogenous B-50 phosphorylation in GCP is stimulated in a dose- dependent manner by 4 beta-phorbol diesters, known to activate PKC, but not by the inactive 4 alpha-phorbol derivatives. In synaptic plasma membranes (SPM) isolated from adult rat brain, the degree of B-50 phosphorylation has been implicated in the modulation of receptor- mediated polyphosphoinositide (PPI) hydrolysis. In addition to B-50 and its kinase, PKC, the GCp fraction was also shown to contain all other components of such a modulatory system: the phosphatidylinositol 4- phosphate (PIP)-kinase, as shown on Western blots with affinity- purified IgGs against PIP-kinase, and the polyphosphoinosides, PIP and phosphatidylinositol 4,5-bisphosphate (PIP2), since the addition of gamma-32P-ATP to the GCp fraction not only results in B-50 phosphorylation but also in the labeling of phosphatidic acid (PA), PIP, and PIP2. ACTH1–24, which inhibits B-50 phosphorylation in the GCp fraction in a dose-dependent manner (IC50 = 5 x 10(-6) M), stimulates PIP2 labeling dose-dependently in the same preparation.(ABSTRACT TRUNCATED AT 250 WORDS)
Visuotopic organization and extent of V3 and V4 of the macaque
Authors: Gattass R , Sousa AP , Gross CG
Published: March 2018

The representation of the visual field in areas V3 and V4 of the macaque was mapped with multiunit electrodes. Twelve Macaca fascicularis were studied in repeated recording sessions while immobilized and anesthetized. V3 is a narrow strip (4–5 mm wide) of myeloarchitectonically distinct cortex located immediately anterior to V2. It contains a systematic representation of the central 35–40 degrees of the contralateral visual field; the representation of the upper quadrant is located ventrally in the hemisphere and that of the lower quadrant, dorsally. There is a small gap between the dorsal (V3d) and ventral (V3v) portions of V3. The representation of the horizontal meridian is adjacent to that in V2 and forms the posterior border of both V3d and V3v. Most or all of the anterior border of V3d consists of the representation of the lower vertical meridian. The entire anterior border of V3v consists of the representation of the upper vertical meridian. V4 is a strip of myeloarchitectonically distinct cortex 5–8 mm wide, immediately anterior to V3. It contains a coarse, but systematic, representation of approximately the central 35–40 degrees of the contralateral visual field. The representation of the upper visual field is located ventrally in the hemisphere. Most of the representation of the lower visual field is located dorsally. The posterior border of V4 corresponds to the representation of the vertical meridian, and the representation of the horizontal meridian is located at or near its anterior border. In both V3 and V4, the representation of the central visual field is magnified relative to that of the periphery. In both areas, the size of receptive fields increases with increasing eccentricity; however, at a given eccentricity, the receptive fields of V4 are larger than those of V3.
Light and electron microscope localization of the microtubule- associated tau protein in rat brain
Authors: Migheli A , Butler M , Brown K , Shelanski ML
Published: March 2018

We have studied the distribution of microtubule-associated tau proteins in rat brain using monoclonal and affinity-purified polyclonal antibodies. Tau staining is prominent in axons in white matter areas of brain, as reported by Binder et al. (1985). In addition, we also find tau protein in neuron cell bodies, especially in the brain stem and basal ganglia and in the cell bodies of interfascicular oligodendroglia. Using electron microscopy, tau antibodies and colloidal gold-labeled second antibodies, gold particles are found associated with microtubules in axons and in the cytoplasm of cell bodies, while the nuclei, mitochondria, and myelin remain unlabeled. In double-staining experiments, tau staining co-localizes with that of tubulin. Our studies indicate that tau proteins are more widely distributed in brain than previously reported and cannot be used as an exclusive marker for axons.
Transient expression of laminin in the optic nerve of the developing rat
Authors: McLoon SC , McLoon LK , Palm SL , Furcht LT
Published: March 2018

The optic nerve of the developing rat was examined for the presence of laminin, an adhesive glycoprotein, to assess whether it might serve as a substrate for retinal axon growth in vivo. The optic stalk and nerve of developing rats were screened immunohistochemically for the presence of laminin before, during, and after the period of retinal axon growth. On embryonic day 14 (E14), laminin immunoreactivity was present in the ventral portion of the optic stalk, the same region in which the first retinal axons grow. Between E16 and postnatal day 10 (P10), cells positive for laminin were distributed throughout the cross-sectional area of the nerve. There was a progressive appearance of glial fibrillary acidic protein (GFAP) immunoreactivity, a marker for astrocytes, from the optic nerve head towards the chiasm beginning on E20. At the advancing front of GFAP immunoreactivity, cells were positive for both laminin and GFAP. Behind the front, laminin immunoreactivity disappeared from the cells. By P12, the only laminin immunoreactivity that remained within the optic nerve surrounded the vasculature. This is a time after the last retinal axons grow through the optic nerve. Monolayer cell cultures were prepared from perinatal rat optic nerves and processed for immunohistochemistry to determine which astrocyte type was laminin-positive. Type 1 astrocytes, which primarily compose the immature nerve, are GFAP-positive, A2B5-negative, and laminin-positive. Type 2 astrocytes, a major component of the mature optic nerve, were GFAP-positive, A2B5-positive and laminin- negative. An extract of developing optic nerve was analyzed by immunoblot along with laminin purified from Engelbreth-Holm-Swarm (EHS) sarcoma. Purified laminin ran with SDS-PAGE under reducing conditions as 2 bands with Mrs of 200,000 and 4000,000. Both bands reacted with antibodies to laminin. A low-salt extraction of whole optic nerve from E18 rats resulted in 2 bands with the same Mr as seen with laminin from EHS sarcoma. When only the inside of the optic nerve (which lacked the basal lamina and meninges that surround the outside) was processed, there was a dark 200,000 D band, but the 400,000 D band was virtually absent. These results are consistent with the hypothesis that laminin, or a variant form of laminin, serves as a substrate for retinal axon growth in the developing rat optic nerve.
Characterization of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase
Authors: Chang YC , Gottlieb DI
Published: March 2018

Immunoaffinity columns are prepared from the monoclonal antibody (MAb) GAD-1. These columns are used to enrich glutamic acid decarboxylase (GAD) from the cytosolic fraction of rat brain homogenates and from Triton X-100 extracts of the brain membrane fraction. In each case enzyme activity is enriched over 400-fold. The immunopurified fractions were analyzed by SDS-PAGE. Fractions purified from the cytosol consisted of a quantitatively major band of 59 kDa, and one band of 63 kDa, as well as a group centered around 55 kDa. Fractions purified from membranes consisted primarily of the 59 and 63 kDa components; only traces of the lower-molecular-weight components were present. The entire set of proteins purified on GAD-1 immunoaffinity columns is strongly recognized by 2 widely used antisera to GAD, those described in Saito et al. (1974) and Oertel et al. (1981). The 59 kDa protein from the cytosolic fraction was purified to homogeneity by preparative SDS-PAGE; a partial amino acid sequence of this protein was obtained. The 59 kDa protein has a high degree of sequence homology with the deduced amino acid sequence of the protein that was coded for by a cDNA for feline GAD (Kaufman et al., 1986; Kobayashi et al., 1987). Thus, these proteins are either products of a single gene that diverged during the evolution of rat and cat from a common ancestor, or are members of a closely related set of genes found in both species. The MAb GAD-6 recognizes the 59 kDa band and the group of bands centered around 55 kDa on Western blots. Therefore, these proteins are immunochemically related. GAD-6 does not recognize the 63 kDa band. In Western blots of unfractionated homogenates of the whole brain, the only band recognized by GAD-6 is a 59 kDa band.(ABSTRACT TRUNCATED AT 250 WORDS)
Vulnerability of cultured cortical neurons to damage by excitotoxins: differential susceptibility of neurons containing NADPH-diaphorase
Authors: Koh JY , Choi DW
Published: March 2018

Quantitative concentration-toxicity relationships were determined for the injury of cultured murine cortical neurons by several excitatory amino acid (EAA) agonists. All tested agonists produced concentration- dependent neuronal injury at concentrations between 1 and 1000 microM. With 5 min exposure, glutamate, aspartate, N-methyl-D-aspartate (NMDA), L-homocysteate (HCA), and quisqualate all had similar potencies, destroying half of the neuronal population (LD50) at concentrations of 50–200 microM, and similar efficacies, with 88–92% neuronal loss produced by exposure to high agonist concentrations. Quinolinate and kainate were substantially weaker toxins, producing only 20–30% neuronal loss after 5 min exposure to 3 mM concentrations; with prolonged (24 hr) exposure, 85–95% neuronal loss could be attained. The comparative EAA vulnerability of a specific cortical neuronal subpopulation containing high concentrations of the enzyme, reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), was also examined. Glutamate had no differential toxicity on these cells, damaging them at all concentrations in proportion to the general population; however, other, more selective, agonists produced strikingly differential injuries. These NADPH-d-containing [NADPH- d(+)]neurons were selectively resistant to damage by low concentrations of the NMDA agonists quinolinate, HCA, aspartate, or NMDA itself. By contrast, NADPH-d(+)neurons were selectively destroyed by concentrations of quisqualate or kainate too low to produce much general neuronal injury. The differential susceptibility of these neurons was not absolute, as high concentrations of all tested agonists produced nonselective neuronal injury. In light of recent evidence that forebrain NADPH-d(+)neurons are selectively spared in Huntington's disease, the present study continues to support the hypothesis that neuronal loss in Huntington's disease might result from excessive NMDA- receptor stimulation by any selective NMDA agonist. Furthermore, the demonstration that the differential susceptibility of NADPH-d(+)neurons is agonist concentration-dependent, rather than absolute, could provide a basis for explaining some existing conflicting experimental data.
The ON-alpha ganglion cell of the cat retina and its presynaptic cell types
Authors: Freed MA , Sterling P
Published: March 2018

Anatomical circuits converging onto the ON-alpha (Y) ganglion cell were studied by computer-assisted reconstruction of substantial portions of 2 alpha cells from electron micrographs of serial sections. The alpha cells in the area centralis were labeled by a Golgi-like retrograde filling with horseradish peroxidase, and certain presynaptic amacrine processes were labeled by uptake of 3H-glycine. About 4400 synapses contacted the alpha cell. Eighty-six percent were from amacrine cells; the rest were from bipolar cells. About one-quarter of the amacrine synapses were specifically labeled by 3H-glycine and probably belong to the A4 amacrine. The bipolar inputs were provided by several types: cone bipolar CBb1 (85%), cone bipolar CBb5 (2%), the rod bipolar (5%), and some unidentified cone bipolars (11%). Contacts from each type occurred in specific strata, with the consequence that they tended to form spots or annulli over the alpha dendritic field. The CBb1 bipolars formed a moderately dense array (8000/mm2), with a nearest-neighbor distance of 8.6 +/- 1.3 microns. Most members of the array (84%) contacted the alpha cell, providing 1-7 synapses (average, 2.7 +/- 1.6). The placement of contacts from an individual CBb1 followed certain rules: they were restricted to a parent branch of the alpha arbor or to 2 daughter branches, but almost never crossed a branch of the alpha arbor. The synaptic territory of an individual CBb1 was not shared with other b1s (or cone bipolars of any sort), although it was shared with amacrine contacts. Rod bipolar cells also formed a very dense array (54,500/mm2) in the alpha dendritic field, but only a few of these (3%) contacted the alpha cell. The concentric receptive field of the CBb1, combined with the spatial organization of its array, is used to predict the CBb1 contribution to the alpha cell receptive field; this contribution resembles the spatial and temporal organization of the alpha receptive field itself.
Nerve growth factors (NGF, BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurons
Authors: Lindsay RM
Published: March 2018

Largely on the basis of studies with nerve growth factor (NGF), it is now widely accepted that development of the peripheral nervous system of vertebrates is dependent in part on the interaction of immature sensory and autonomic neurons with specific survival factors that are derived from peripheral target fields. I have found, in marked contrast to an absolute requirement for NGF during development, that adult rat dorsal root ganglion sensory neurons are not dependent on NGF or other survival factors for long-term (3-4 weeks) maintenance in vitro. When dissociated and enriched, at least 70-80% of adult DRG neurons survived and extended long processes either in the absence of exogenously added NGF or upon the removal of any possible source of endogenous NGF or other neurotrophic activity (i.e., nonneuronal cells, in chemically defined culture medium, in the presence of an excess of anti-NGF antibodies, or when cultured as single neurons in microwells). Although not required for survival or expression of a range of complex morphologies, both NGF and brain-derived neurotrophic factor (BDNF) were found to stimulate the regeneration of axons from adult DRG neurons.
Selective effects of hyperprolactinemia on in vitro dopamine release from median eminence synaptosomes
Authors: Gregerson KA , Selmanoff M
Published: March 2018

Prolactin is thought to exert an autoregulatory, negative feedback effect on its own secretion via stimulation of the tuberoinfundibular dopaminergic (TIDA) neurons. To investigate possible mechanisms involved in this feedback, the effects of experimentally induced hyperprolactinemia on the release of 3H-dopamine (3H-DA) were studied in nerve terminals (synaptosomes) isolated from rat median eminence (ME), the TIDA neuronal projection field. Synaptosomes were prepared from adult male rats treated with ovine prolactin (oPRL) or the vehicle for 48 hr. Synaptosomes were incubated in 0.1 microM 3H-DA at 30 degrees C until steady-state conditions were achieved, and then release of the preaccumulated transmitter was measured over 1–20 sec time intervals under basal and depolarizing conditions. Release of 3H-DA elicited by depolarization of the terminals was significantly greater in ME synaptosomes prepared from oPRL-treated animals as compared with preparations from controls. This effect of the hyperprolactinemia appeared to be specific to the TIDA neurons since oPRL treatment did not result in increased evoked release of 3H-DA from terminals prepared from the mesolimbic, tuberohypophyseal, or nigrostriatal dopaminergic neurons. Basal efflux in all preparations was not changed from controls. The increased evoked release in oPRL-treated ME occurred when depolarization was induced either with high external [KCl] or veratridine. The enhanced 3H-DA efflux was evident during depolarization over a wide range of external calcium concentrations (0.01–3.0 mM), in the presence of 20 nM Ni2+ to block Ca2+ influx through voltage-gated channels, or when all external Ca2+ had been chelated, indicating that this effect of oPRL involves DA released through a mechanism independent of external calcium.
The development of sensorimotor synaptic connections in the lumbosacral cord of the chick embryo
Authors: Lee MT , Koebbe MJ , O'Donovan MJ
Published: March 2018

We have examined the development of synaptic connections between afferents and motoneurons in the lumbosacral spinal cord of the chick embryo between stages 28 and 39. The central projection of afferents was visualized following injection of dorsal root ganglia with HRP. Afferent fibers first entered the dorsal gray matter between stages 29 and 31. They grew in a ventrolateral direction, reaching motoneuron dendrites by stage 32. Quantitative analysis of axon numbers suggested that individual axons did not begin to branch extensively until they approached the lateral motor column at stage 36. Connectivity between afferents and motoneurons was assessed by stimulating dorsal roots or nerves supplying the femorotibialis muscle and recording the resulting motoneuron synaptic potentials intracellularly or from the cut ventral roots. At stages 37–39, low-intensity stimulation produced a short- latency positive potential that was followed at higher stimulus currents by slower positive potentials. All of these potentials were abolished in solutions that block chemical synaptic transmission (zero Ca2+/2 mM Mn2+). The early potential, which includes the monosynaptic EPSP produced by muscle afferents, persisted in the presence of the N- methyl-D-aspartate antagonist, 2-amino-5-phosphonovaleric acid (APV), but was largely eliminated by the more general excitatory amino acid antagonist, kynurenic acid. Therefore, in the chick, as in other species, a glutamate-like transmitter appears to be released at the synapses between muscle afferents and motoneurons. The APV-resistant potential was reduced in amplitude during bath application of the glycine and GABA antagonists, strychnine and picrotoxin, suggesting that it was composed of depolarizing inhibitory as well as excitatory components at these stages. The monosynaptic EPSP could be recorded in ventral roots as early as stages 32–33, when muscle afferents first grew into the vicinity of motoneuron dendrites. The EPSP in these young embryos was unaffected by picrotoxin and strychnine, but responded to APV and kynurenate in a manner similar to that at later stages. Between stages 28 and 32, only long-latency, slowly rising potentials could be evoked in the ventral roots by afferent activation. These potentials were abolished by superfusion with zero Ca2+/2 mM Mn2+, APV, or kynurenic acid, and could be revealed before stage 31 only by removing Mg2+ from the bath.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization and localization of a peripheral neural 5- hydroxytryptamine receptor subtype (5-HT1P) with a selective agonist, 3H-5-hydroxyindalpine
Authors: Branchek TA , Mawe GM , Gershon MD
Published: March 2018

Peripheral neural 5-hydroxytryptamine (5-HT) receptors are different from both classes 5-HT1 and 5-HT2, which have been described from studies of 5-HT receptors in the brain. Recently, it has been shown that, as in the CNS, there is more than a single type of neural receptor for 5-HT in the enteric nervous system. One of these, called 5- HT1P, has a high affinity for 3H-5-HT, initiates a long-lasting depolarization of enteric neurons associated with an increase in membrane resistance, and is the physiological receptor through which enteric serotoninergic neurons mediate slow EPSPs. The other receptor, called 5-HT3 (5-HT2P), does not bind 3H-5-HT with high affinity, and initiates a brief depolarization of enteric neurons with decreased input resistance, but a physiological action of 5-HT mediated by these receptors has not yet been identified. Hydroxylated indalpines have been found to be agonists at 5-HT1P receptors. We have now examined 5- HT1P receptors using 5-hydroxyindalpine (5-OHIP) as a probe. The action of 5-OHIP on enteric neurons was determined electrophysiologically and compared with that of 5-HT; the binding of 3H-5-OHIP to isolated enteric membranes was studied by rapid filtration, and to frozen sections of tissue by radioautography. 3H-5-OHIP binding was compared with that of 3H-5-HT. 5-OHIP, like 5-HT, induced a triphasic response in most enteric neurons: an initial short-lived depolarization, during which input resistance fell, followed by recovery, and then a long- lasting depolarization, during which the input resistance increased. 5- OHIP bound saturably, reversibly, and with high affinity to enteric membranes (Kd = 7.6 +/- 0.7 nM; Bmax = 76 +/- 14 fmol/mg protein). Binding of 3H-5-OHIP was not inhibited by agents that bind to alpha- or beta-adrenoceptors, nicotinic or muscarinic receptors, histamine H1 or H2 receptors, or 5-HT1(A,B,C, or D), 5-HT2, or 5-HT3 receptors, but was displaced by substances, such as hydroxylated indoles and a dipeptide of 5-hydroxytryptophan (5-HTP-DP), that antagonize the binding of 3H-5- HT to enteric membranes or tissue sections. It is concluded that 5-OHIP is an agonist at peripheral neural 5-HT1P receptors and can be used to label these receptors selectively outside the brain. Radioautographs demonstrated enteric 5-HT1P receptors in the lamina propria of the intestinal mucosa and in the submucosal and myenteric plexuses. Extraenteric 5-HT1P receptors were also found in the skin and heart. It is suggested that 5-HT1P receptors may be found on subtypes of primary afferent nerve fibers.
A neural map of interaural intensity differences in the brain stem of the barn owl
Authors: Manley GA , Koppl C , Konishi M
Published: March 2018

The nucleus ventralis lemnisci lateralis pars posterior (VLVp) is the first binaural station in the intensity-processing pathway of the barn owl. Contralateral stimulation excites and ipsilateral stimulation inhibits VLVp cells. The strength of the inhibition declines systematically from dorsal to ventral within the nucleus. Cells selective for different intensity disparities occur in an orderly sequence from dorsal to ventral within each isofrequency lamina. Cells at intermediate depths in the nucleus are selective for a particular narrow range of interaural intensity differences independently of the absolute sound-pressure level. A simple model of the interaction between inhibition and excitation can explain most of the response properties of VLVp neurons. The map of selectivity for intensity disparity is mainly based on the gradient of inhibition.