bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracellular ATP modulates calcium uptake and transmitter release at the neuromuscular junction
Authors: Lindgren CA , Smith DO
Published: March 2018

Adenosine, AMP, ADP, and ATP were tested for their ability to modulate evoked quantal transmitter release at excitor-opener nerve terminals in the crayfish walking leg. Only ATP was found to have a significant effect, inhibiting release by 43%. To determine whether the effects of extracellular ATP on transmitter release were related to changes in free Ca2+ levels in the nerve terminal, net 45Ca uptake was measured in regions of the nerve near the terminal; although resting 45Ca uptake was increased by 5 mM exogenous ATP. Thus, extracellular ATP normally inhibits evoked transmitter release; this is associated with reduced stimulation-induced net Ca2+ uptake into the synaptic terminal. To explore the possibility that metabolic factors might be involved, nerve ATP levels were reduced by greater than or equal to 50% by omitting glucose from the bathing solution. Although quantal content and synaptic delay were unaffected by reduced intracellular ATP, under these conditions 5 mM exogenous ATP failed to reduce quantal release. Five millimolars of ATP did increase synaptic facilitation, however, which is consistent with a reduced ability to regulate intracellular free Ca2+. Therefore, under conditions of reduced intracellular ATP, the increased resting Ca2+ uptake produced by 5 mM ATP is not buffered completely. This leads to elevated free Ca2+ levels in the nerve terminal, increasing the amount of transmitter released following an action potential.
Tactile discrimination of shape: responses of slowly and rapidly adapting mechanoreceptive afferents to a step indented into the monkey fingerpad
Authors: Srinivasan MA , LaMotte RH
Published: March 2018

The representation of shape in the responses of monkey cutaneous mechanoreceptive afferents to steps of varying shape vertically indented into the fingerpad was studied. A series of flat plates was used, each with a step change in thickness in the middle so that one- half of the plate was thicker than the other. The cross-sectional shape of the step approximated that of a half-cycle sinusoid, 0.5 mm high, that was varied in half-cycle wavelength (step width) and hence in steepness and curvature. The steps fell into 2 categories, characterized as “steep” and “‘gradual.” Evoked action potentials were recorded from single, slowly adapting and from rapidly adapting Meissner corpuscle mechanoreceptive afferent fibers (SA and RA, respectively) innervating the fingerpad of the anesthetized monkey while each step was indented at a succession of lateral positions across the fiber's receptive field. The responses of each SA provided a spatial response profile (number of evoked impulses as a function of step position) that was directly related to the variation in curvature across the step. The rate of discharge was greatest under the sharpest (convex) portion of the step, least under the adjacent concave portion, and intermediate under the flat portions of the steps. The results indicated an exquisite sensitivity of the SA, even during the ramp phase of vertical indentation, to the changes in skin curvature. The spatial response profile remained relatively undistorted over time during the ensuing steady phase, while the contrast between the peak and the minimum response improved. RAs responded only during the ramp phase and with fewer responses, and gave rise to a poorly modulated spatial response profile, even though half of the RAs tested showed limited sensitivity to the amount or rate of change of skin curvature. It was hypothesized that RA responses are predominantly influenced by the vertical velocity of the most sensitive spot in the receptive field. When the same step stimuli were applied to the human fingerpad, the capacities of humans to discriminate differences in step shape were found to correlate with the discriminability of SAs, as opposed to the considerably poorer discriminability of RAs. It is concluded that information concerning the local curvature and hence the shape of objects indenting the skin is primarily coded by the SAs. In the 2 preceding papers (LaMotte and Srinivasan, 1987a, b), we investigated the responses of SAs and RAs to the same sinusoidal steps stroked back and forth across the fingerpad under constant compressional force.(ABSTRACT TRUNCATED AT 400 WORDS)
The Conus toxin geographutoxin IL distinguishes two functional sodium channel subtypes in rat muscle cells developing in vitro
Authors: Gonoi T , Ohizumi Y , Nakamura H , Kobayashi J , Catterall WA
Published: March 2018

Sodium currents in cultured rat muscle cells converted to myoballs by treatment with colchicine were recorded using a giga-ohm seal voltage- clamp procedure in the whole-cell configuration. Geographutoxin II (GTX II), a novel polypeptide toxin from the piscivorous marine snail Conus geographus, reduces sodium currents in rat myoballs without marked alteration of the time course or voltage dependence of activation of the remaining current. Titration of the inhibition of sodium currents by GTX II showed that, in individual myoballs, a fraction of the sodium current averaging 49 +/- 9% (SEM) was inhibited by saturating (25 microM) concentrations of GTX II. The concentration-effect curve fit a noncooperative, 1:1 binding isotherm with a single KD for GTX II of 19 nM characteristic of inhibition of the TTX-sensitive sodium channels of adult rat muscle. Titration of the sodium current remaining in the presence of 2.5 microM GTX II with TTX gave complete inhibition. The dose-response curve fit a noncooperative, 1:1 binding isotherm with a single KD for TTX of 1.3 microM characteristic of TTX-insensitive sodium channels of embryonic muscle. The action of GTX II was not frequency dependent. The all-or-none inhibition of these 2 sodium channel subtypes by GTX II suggests substantial structural differences in the region of neurotoxin receptor site 1 on TTX-sensitive and - insensitive sodium channels and provides definitive evidence that these 2 sodium channel subtypes function in parallel in muscle cells developing in the absence of innervation.
Recycling of plasmalemma in chick tectal growth cones
Authors: Cheng TP , Reese TS
Published: March 2018

Growth cones from freeze-substituted intact chick optic tectum were analyzed in serial reconstructions of thin-section electron micrographs in order to determine which internal organelles might contribute membrane for plasmalemmal expansion. These growth cones contain numerous stacked and single lumenless membrane-limited disks; the stacks are arrays of single disks interconnected, and possibly organized, by intervening electron-dense cross-links. The single and stacked disks together account for 80% of the total intracellular membrane in the growth cones. Single disks frequently lie close to and occasionally contact the filopodial plasmalemma; regularly spaced electron-dense cross-links also occur at these juxtapositions between single disks and the plasmalemma. Some of the juxtaposed disk membranes contact the growth cone plasmalemma, and images of some of these contacts appear to indicate fusion of the disk membrane with the plasmalemma. When excised optic tecta are exposed to cationized ferritin for various times, ferritin micelles appear sequentially in coated pits, coated vesicles, smooth vesicles, vacuoles, and then in stacked and single disks, including some of those contacting the plasmalemma. Because the cytoplasmic disks filled only at the longest times after exposure to ferritin, the membrane continuities between the disks and the plasmalemma are thought to be indicative of exocytosis rather than endocytosis. We propose, therefore, that components of the plasma membrane are recycled through the stacks of lumenless disks in the chick tectal growth cones; the disks therefore represent a pool of internal membrane waiting to be added to the growth cone plasmalemma that could be used for filopodial extension or neuritic extension.
Spatial firing patterns of hippocampal complex-spike cells in a fixed environment
Authors: Muller RU , Kubie JL , Ranck JB
Published: March 2018

A TV/computer technique was used to simultaneously track a rat's position in a simple apparatus and record the firing of single hippocampal complex-spike neurons. The primary finding is that many of these neurons behave as “place cells,” as first described by O'Keefe and Dostrovsky (1971) and O'Keefe (1976). Each place cell fires rapidly only when the rat is in a delimited portion of the apparatus (the cell's “firing field”). In agreement with O'Keefe (1976) and many other authors, we have seen that the firing of place cells is highly correlated with the animal's position and is remarkably independent of other aspects of the animal's behavioral state. Several properties of firing fields were characterized. Firing fields are stable over long time intervals (days) if the environment is constant. They come in several shapes when the animal is in a cylindrical apparatus; moreover, the set of field shapes is different when the animal is in a rectangular apparatus. It also seems that a single cell may have more than one field in a given apparatus. By collecting a sample of 40 place cells in a fixed environment, it has been possible to describe certain features of the place cell population, including the spatial distribution of fields within the apparatus, the average size of fields, and the “intensity” of fields (as measured by maximum firing rate). We also tested the hypothesis that the firing rate of each place cell signals the animal's distance from a point (the field center) so that a weighted average of the firing of the individual cells encodes the animal's position within the apparatus. The animal's position, calculated according to this 'distance hypothesis,” is systematically different from the animal's true position; this implies that the hypothesis in its simplest form is wrong.
Heterogeneous distribution of neurotensin-like immunoreactive neurons and fibers in the midbrain periaqueductal gray of the rat
Authors: Shipley MT , McLean JH , Behbehani MM
Published: March 2018

The midbrain periaqueductal gray (PAG) has been shown to be a site where various manipulations induce pain suppression. Recent physiological evidence (Behbehani and Pert, 1984; Behbehani et al., 1987) suggests that neurotensin has pronounced physiological actions in PAG and effects pain suppression. We have performed immunohistochemical studies in order to determine the magnitude and distribution of neurotensin-like immunoreactive (NT-IR) cell bodies and fibers in PAG. NT-IR cell bodies were common throughout PAG, although there were more in the caudal than the rostral half. NT-IR neurons were much more numerous in the ventral than the dorsal half of PAG, and some appeared to be located within the dorsal raphe nucleus. The pattern of NT-IR fibers was analyzed with the aid of image enhancement/analysis and densitometry. The fibers were found to be heterogeneously distributed, being most heavily concentrated in the region adjacent to the cerebral aqueduct in the caudal two-thirds of PAG. The distribution of NT fibers closely matches sites where exogenously applied NT elicits long-lasting excitation of PAG neurons (Behbehani et al., 1987). Based on the known physiological and behavioral actions of NT in PAG, the present anatomical results suggest that NT acts on elements located predominantly in the medial and ventrolateral parts of PAG. Neurons activated by NT may project directly to the nucleus raphe magnus and adjacent ventral medulla (Behbehani and Pert, 1984) to activate neurons that project to the spinal cord and modulate nociceptive circuits.
Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion
Authors: Cohen RS , Pant HC , House S , Gainer H
Published: March 2018

Monoclonal antibodies to squid neurofilament (aNFP) and intermediate filament (aIFA) proteins were used as probes for the biochemical and immunocytochemical analyses of neurofilament structure and distribution in the squid giant axon and stellate ganglion. On Western blots the aNFP antibody stained exclusively the 220 kDa and high-molecular-weight (HMW) components of neurofilaments in the giant axon, whereas the aIFA antibody primarily labeled the 60 kDa protein in the giant axon and the 60 and 65 kDa proteins in the stellate ganglion. Dephosphorylation of axoplasmic proteins by alkaline phosphatase resulted in a decrease in the molecular weights of both the 220 kDa and HMW neurofilament proteins and a concomitant loss of reactivity with the aNFP antibody on Western blots. This indicated that the aNFP antibody is specific for a phosphorylated epitope in the neurofilament. Increased dephosphorylation of the 220 kDa protein led to an enhanced immunostaining of the resultant 190 kDa polypeptide by the aIFA antibody, suggesting that the phosphorylated epitope may mask the conserved epitope recognized by aIFA. Light and electron microscopic immunocytochemical studies show intense labeling by the aNFP antibody in the giant axon. In contrast, the aIFA antibody labeled the glial cells around the giant axon intensely, while labeling of the giant axon itself was considerably less than that with the aNFP antibody. Since the 60 kDa protein in axoplasm is intensely stained by the aIFA antibody on Western blots, the relatively low amounts of labeling seen on semithin and thin sections of the giant axon by this antibody may be due to the masking of the 60 kDa protein by in situ fixed axoplasmic proteins. However, the aIFA antibody intensely labeled glial cells within the stellate ganglion and “islands” of filaments and nuclear membranes within ganglion cells. No reactivity for either antibody was seen in synapses. The aNFP antibody specifically labeled “beadlike” portions and cross-bridges on the axonal neurofilaments, suggesting that these components consist of the 220 kDa and HMW proteins. In contrast, the aIFA antibody labeled relatively smooth filaments in ganglion and glial cells. These data suggest that the 65 kDa protein represents the squid glial filament protein and that the 60 kDa protein found in axoplasm represents the low-molecular-weight subunit in the axonal neurofilament. The latter appears to be formed and/or organized in “islands” of filaments within ganglion cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Effect of changes in neurofilament content on caliber of small axons: the beta,beta'-iminodipropionitrile model
Authors: Parhad IM , Clark AW , Griffin JW
Published: March 2018

The structural role of neurofilaments in the normal axon and the consequences of altered axonal transport of neurofilaments have been extensively studied in large axons. These studies suggest that neurofilament numbers and interneurofilament spacing are major determinants of axonal cross-sectional area. In contrast, in small axons and dendrites, microtubules and membranous organelles appear to be the most closely correlated with size and shape of the cell process. In this study we have examined the effect of impairment in neurofilament transport on small axons, typical of most CNS pathways. Neurofilament transport was impaired by administration of beta,beta'- iminodipropionitrile (IDPN), resulting in proximal accumulation and distal depletion of neurofilaments. The evolution of these changes was studied in the optic nerves of guinea pigs treated with IDPN, 1–35 weeks following intoxication. The effect of this redistribution of neurofilaments on cross-sectional area of small axons was evaluated using quantitative ultrastructural methods. Our results show that with the alteration in neurofilament transport seen with IDPN intoxication, there is a wide spectrum of neurofilament densities, ranging from a 5- fold increase above normal in the proximal axon, to a 5-fold decrease below normal in the distal axon. Although the optic nerve fibers enlarge with the increase in neurofilament content, they do not atrophy significantly with the continued loss of neurofilaments. We conclude that factors other than neurofilament content are capable of maintaining size and shape of these small axons. Candidate organelles include microtubules and membranous organelles and possibly other axonal elements.
Hypophysectomy increases vasoactive intestinal peptide-stimulated cyclic AMP generation in the hippocampus of the rat
Authors: Harrelson AL , McEwen BS
Published: March 2018

In investigating the feedback effects of circulating hormones on the brain, we showed previously that adrenalectomy (ADX) increases vasoactive intestinal peptide (VIP)-stimulated cAMP generation in slices from rat hippocampus, a brain structure with high levels of glucocorticoid receptors. This effect is reversed by replacement with glucocorticoids such as dexamethasone (DEX) and corticosterone (CORT). Here we report that, like ADX, hypophysectomy (HYPOX) also elevates VIP- stimulated cAMP generation, compared with sham-operated controls. Moreover, like glucocorticoid replacement, administration of ACTH to HYPOX rats causes a decrease in cAMP stimulation by VIP. Furthermore, ACTH had no effect when given to HYPOX + ADX rats, indicating that the effects of ACTH require the presence of adrenal steroid secretion. However, we find that ACTH may have a permissive role in this glucocorticoid effect because, in the absence of the pituitary, DEX treatment does not decrease VIP-stimulated cAMP levels in the hippocampus. In addition, hippocampal beta-adrenergic-stimulated cAMP accumulation was not suppressed by DEX treatment of HYPOX rats, which again is different from the effect of DEX treatment on ADX animals. These results are discussed in terms of possible synergism between pituitary hormones and steroid hormones in exerting feedback actions on brain function.
Fetal frontal cortex transplanted to injured motor/sensory cortex of adult rats. I. NADPH-diaphorase neurons
Authors: Gonzalez MF , Sharp FR
Published: March 2018

Fetal frontal cortex from 18-d-old embryonic rat brain was transplanted into cavities of juvenile host motor/sensory cortex. Two to seven months later, sections were reacted for NADPH-diaphorase (NADPH-d) enzyme histochemistry. NADPH-d-positive neurons survived in 11 of 13 grafts. All but one of the transplants had reduced numbers of these neurons, although in 3 transplants the reductions were moderate and not statistically significant. The distribution and morphology of NADPH-d neurons within most grafts was comparable to that of NADPH-d neurons in normal host cortex. At the margin of the 2 transplants with no NADPH-d neuronal perikarya, NADPH-d fibers crossed from host to transplant as far as a millimeter into the transplant, and on rare occasions, the host neurons that gave rise to these fibers were identified. This suggests that host-transplant interactions are possible. One transplant had an abnormally large number of NADPH-d-positive neurons and fibers, possibly due to selective survival of these neurons. The data reported here for NADPH-d in cortical transplants may also apply to neuropeptide Y (NPY), since nearly all neocortical NPY neurons also contain NADPH-d.
Fetal frontal cortex transplanted to injured motor/sensory cortex of adult rats. II. VIP-, somatostatin-, and NPY-immunoreactive neurons
Authors: Sharp FR , Gonzalez MF , Sagar SM
Published: March 2018

Fetal frontal cortex transplants that survived 2–9 months in cavities in adult rat motor/sensory cortex were processed for vasoactive intestinal polypeptide (VIP), somatostatin 14 (SS), and neuropeptide Y (NPY) immunocytochemistry, and NADPH-diaphorase (NADPH-d) histochemistry. All transplants had surviving VIP, SS, NPY, and NADPH-d neuronal perikarya and fibers with normal adult morphology. The number of peptidergic neurons within transplants, however, often appeared to be less than that in equivalent areas of host cortex. Most transplanted SS and VIP neuronal perikarya did not migrate to form the laminae characteristic of normal cortex. A few transplants had SS and VIP cells arranged in laminae in which the VIP processes were parallel to one another and perpendicular to one transplant surface, approximating normal host neocortex. VIP, NPY, and SS fibers crossed between host brains and transplants, suggesting that peptide host-transplant interactions are possible. All adult host cortical and most transplanted NPY neurons colocalized with NADPH-d. The failure of some transplanted NPY neurons to express NADPH-d suggests these transplanted cells may be functionally impaired, but that they can survive without the NADPH-d enzyme.
The role of striate cortex in the guidance of eye movements in the monkey
Authors: Segraves MA , Goldberg ME , Deng SY , Bruce CJ , Ungerleider LG , Mishkin M
Published: March 2018

We studied the effect of unilateral striate cortical ablations on smooth pursuit and saccadic eye movements in the monkey. The monkeys made quite accurate saccades to stationary stimuli in the field contralateral to the lesion, and they readily pursued foveal targets moving in all directions. However, when visual stimuli were stepped into the field contralateral to the lesion and then began to move, thus insuring that the moving stimulus was confined to the impaired visual hemifield, several oculomotor abnormalities emerged. Saccades to moving stimuli presented in the impaired field consistently undershot targets that moved away from the central fixation point after the step, and overshot targets that moved back towards the central fixation point. There was little or no smooth pursuit eye velocity generated in any direction to moving stimuli in the impaired field, and the monkeys could not generate smooth pursuit to stimuli maintained a few degrees from the fovea in the impaired field, although they were able to pursue such stimuli held in the normal field. Ablation of striate cortex also affected the latencies of saccades. When step-ramp stimuli were presented in the normal field, the monkeys delayed the initiation of saccades to targets moving towards the central fixation point, and hastened the initiation of saccades to targets moving away from the central fixation point. By contrast, changes in the direction of target movement did not affect the latencies of saccades into the impaired field. The deficits seemed permanent, lasting as long as the monkeys were tested--over 2 years in one case--but they were not total. Each monkey could use stimuli moving into the affected field to develop some eye velocity, although this residual ability had a much longer latency and lower gain than that provided by the intact visual system. These results show that striate cortex is intimately involved in the estimation of stimulus velocity critical to the genesis of smooth pursuit and saccadic eye movements.
Voltage-activated potassium channels in the plasma membrane of rod outer segments: a possible effect of enzymatic cell dissociation
Authors: Hestrin S , Korenbrot JI
Published: March 2018

Using patch-clamp techniques, we recorded single-channel currents from the plasma membrane of the outer segment of isolated light-adapted rods. The channels are potassium-selective and their conductance is about 87 pS. The channels are activated by depolarization and are not sensitive to cytoplasmic calcium, they are exclusively found in rods isolated with the proteolytic enzyme papain, and are not detected in rods isolated by mechanical means. Thus, these channels do not exist in an activatable form in the outer segment plasma membrane under physiological conditions. The channels might be derived from a normally inaccessible structure, such as the disk membrane, or, alternatively, they might be a modified form of a channel that is not active in the intact rod.
Fluorescence-activated cell sorting of embryonic mouse and rat motoneurons and their long-term survival in vitro
Authors: Schaffner AE , St. John PA , Barker JL
Published: March 2018

Motoneurons from embryonic mice and rats were labeled with retrogradely transported succinyl wheat germ agglutinin (WGA)-fluorescein isothiocyanate (FITC). After dissociation of the spinal cord, fluorescent motoneurons were isolated by flow cytometry. Sorted motoneurons were maintained for as long as 6 weeks in vitro on monolayers of astrocytes in muscle-conditioned medium. Immunocytochemical staining of the cultures for various neuronal antigens suggested that sorted motoneurons are receptive to GABAergic and glycinergic, as well as cholinergic, innervation. Many of the sorted cells were also labeled intracellularly with antibodies to choline acetyltransferase (ChAT) and GABA.
Fibronectin-like immunoreactivity in the developing cerebral cortex
Authors: Stewart GR , Pearlman AL
Published: March 2018

In the developing cerebral cortex of the mouse, binding of antibodies directed against the extracellular matrix glycoprotein fibronectin occurs with a distinct temporal and spatial pattern. On the 10th embryonic day (E10), when the wall of the telencephalic vesicle is made up of only the proliferating cells of the ventricular zone, antifibronectin (aFN) binding is restricted to the blood vessels and pia-arachnoid. Fibronectin-like immunoreactivity first appears in the neuropil as small points of immunofluorescence among the earliest postmitotic neurons that form the preplate (E11-12). A short time later (E12-13), aFN immunoreactivity becomes more diffuse but continues to be restricted to the preplate. As newly arriving neurons form the cortical plate within the preplate (E13-14), aFN binding is present in the marginal zone above the cortical plate and in the subplate below it. Both the marginal zone and the subplate contain early afferents and the cells that were previously part of the preplate. Binding of aFN is transient; by E18-19 it has diminished to the point where it is no longer detectable except in the blood vessels and pia-arachnoid. The transient appearance of fibronectin-like immunostaining in the zones that contain early cortical afferents suggests that fibronectin plays a role in forming the migratory pathway for the growth cones of these axons. In this role it may be acting in concert with other extracellular matrix components such as hyaluronectin, glycosaminoglycans, and laminin, which have been shown to have similar spatial distributions.(ABSTRACT TRUNCATED AT 250 WORDS)
Psychophysical evidence for separate channels for the perception of form, color, movement, and depth
Authors: Livingstone MS , Hubel DH
Published: March 2018

Physiological and anatomical findings in the primate visual system, as well as clinical evidence in humans, suggest that different components of visual information processing are segregated into largely independent parallel pathways. Such a segregation leads to certain predictions about human vision. In this paper we describe psychophysical experiments on the interactions of color, form, depth, and movement in human perception, and we attempt to correlate these aspects of visual perception with the different subdivisions of the visual system.
The interaction of ionic currents mediating single spike activity in retinal amacrine cells of the tiger salamander
Authors: Eliasof S , Barnes S , Werblin F
Published: March 2018

We investigated the ionic interactions responsible for the characteristic nonrepetitive spike activity of amacrine cells. First we measured 4 pharmacologically separable ionic components: a voltage- gated, transient inward sodium current, a voltage-gated, sustained inward calcium current, a calcium-gated, sustained outward potassium current, and a voltage-gated, transient outward potassium current. The measurements provided the time course and magnitudes of the underlying conductances as functions of voltage. Each current was simulated following conventional Hodgkin-Huxley theory. A composite of the simulated currents was analytically reassembled to generate an approximation of the voltage response to a current step. By artificially varying the magnitude and kinetics of the different conductances in the simulation, we determined the range of values that supported the nonrepetitive spike-like response. Amacrine cells tend to remain refractory following an initial spike because (1) the entire activation range for potassium is located at positive potentials with respect to sodium inactivation, so sodium inactivation is never fully extinguished, and (2) the fully activated sodium conductance is of insufficient magnitude to subsequently reach threshold, given this residual inactivation. Shifting the sodium inactivation range by 10 mV, or increasing sodium conductance by 5 times, leads to a more repetitive form of activity. Changes in the magnitude, time course, or activation range of the potassium conductance cannot alter these conditions.
Terminal arbors of axons projecting to the somatosensory cortex of the adult rat. I. The normal morphology of specific thalamocortical afferents
Authors: Jensen KF , Killackey HP
Published: March 2018

A characteristic feature of the rat somatosensory neocortex is a discrete topographic representation of the facial whiskers. Afferent fibers projecting to this vibrissae representation were “bulk-labeled” by injecting horseradish peroxidase into the white matter. Terminal arbors with the morphological characteristics of Lorente de No's (1949) “specific” thalamocortical afferents were then reconstructed through serial sections. These terminal arbors, characterized by the discrete organization of their dense plexus in layer IV, have a laminar distribution of boutons that parallels the laminar pattern of terminal degeneration resulting from lesions of the ventral posterior nucleus of the thalamus. The regional distribution of different-sized arbors corresponds to the distribution of vibrissae-related clusters of different sizes. Larger arbors were found in the posteromedial region corresponding to the mystacial vibrissae representation, while smaller arbors were found in the anterolateral region corresponding to the representation of the anterior sinus hairs. Terminal arbors were also reconstructed from sections stained simultaneously to demonstrate the pattern of vibrissae-related clusters. The greatest concentration of boutons on these axons occurred within a single vibrissae-related cluster. Furthermore, when 2 fibers terminated within a single cluster, their terminal arbors appeared to be largely coextensive. The morphology, size, and distribution of these terminal arbors support the hypothesis that the layer IV plexus of a single specific thalamocortical afferent tends to fill a vibrissae-related cluster. Thus, the organization of specific thalamocortical afferents may be responsible for clustered organization within the somatotopic map of the rodent neocortex.
Phosphatase and carbocyanine dye binding define different types of phosphate groups in mammalian neurofilaments
Authors: Ksiezak-Reding H , Yen SH
Published: March 2018

The phosphorylation state of human and bovine spinal cord neurofilaments (NF) was studied by direct phosphate analysis and carbocyanine dye (“Stains-all”) binding to NF polypeptides resolved on SDS-polyacrylamide gels. Electrophoretically purified NF-H (200 kDa), NF-M (160 kDa), and NF-L (68 kDa) of human origin contained 24, 18, and 4 mol phosphate/mol protein, whereas bovine NF contained 53, 23, and 5 mol phosphate/mol protein, respectively. Incubation of NF preparations with E. coli alkaline phosphatase removed about 55% of the phosphate from NF-H, about 30% of the phosphate from both human and bovine NF-M, but did not change the phosphate content of NF-L. This treatment also inhibited or substantially reduced the binding of electroblotted NF-H and NF-M to 2 anti-NF monoclonal antibodies known to recognize phosphorylated sites on projection side arms. “Stains-all” was found to be a very sensitive probe for detection of phosphorylated cytoskeletal proteins. Without the phosphatase treatment, NF and other phosphoproteins, MAP1, MAP2, tubulin, and tau, all bound the carbocyanine dye on SDS gels, forming blue dye-protein complexes. Measured densitometrically at 615 nm, the staining intensity (relative units/mol protein) was 9, 9, and 3 for human and 10, 13, and 6 for bovine NF-H, NF-M, and NF-L, respectively. NF-H bound the dye less efficiently than was expected from its phosphate content. After phosphatase treatment, NF-H, with half of its phosphate residues remaining, no longer formed blue complex with “Stains-all,” the staining intensity of NF-M decreased by 20–40%, and the staining of NF- L was not changed.(ABSTRACT TRUNCATED AT 250 WORDS)
The properties and regulation of functional acetylcholine receptors on chick ciliary ganglion neurons
Authors: Margiotta JF , Berg DK , Dionne VE
Published: March 2018

The properties of acetylcholine receptor (AChR) channels on chick ciliary ganglion neurons in culture were examined using patch-clamp recording techniques. Acetylcholine (ACh) was applied by rapid microperfusion. Whole-cell current noise analysis revealed a single class of functional receptors on the neurons. Dose-response studies indicated a Kd of about 36 microM and a Hill coefficient of 1.5–1.7, predicting 2 ACh binding sites per receptor. Both fast and slow components of receptor desensitization were observed. Single-channel recordings from excised outside-out patches of soma membrane exposed to 2–5 microM ACh indicated a single-channel conductance of 40 pS, a reversal potential of -9 mV, a mean open duration of 1 msec, and an opening probability of 0.34. The kinetic behavior of the channels was provisionally described by a 3-closed, 1-open state model for receptor activation. In all of these properties, AChRs of ciliary ganglion neurons resemble those on skeletal muscle fibers. Growing the neurons in an elevated K+ concentration produced a 2–3-fold decrease in peak whole-cell currents induced by ACh under standard test conditions, without altering any of the single-channel properties described above. Neither changes in cholinesterase activity nor receptor distribution accounted for the decrease. Instead, calculations indicated that elevated K+ reduced the ACh response by decreasing the number of functional AChRs on the neurons. No K+-dependent decrease is observed, however, in the number of total receptors on the neurons detected either by a monoclonal antibody specific for the receptor or by an alpha-neurotoxin that binds to the receptor and blocks its function. Moreover, the number of receptors detected by the 2 probes is at least 10-fold greater than the calculated number of functional receptors. The findings suggest that only a small fraction of the AChRs on the neuronal surface is functional and that the cell can alter the ratio of functional and nonfunctional receptors in response to growth conditions.
Correlations between active zone ultrastructure and synaptic function studied with freeze-fracture of physiologically identified neuromuscular junctions
Authors: Propst JW , Ko CP
Published: March 2018

The active zone is a unique presynaptic membrane specialization that is believed to be the site of neurotransmitter release. To examine directly the relationship between active zone ultrastructure and synaptic efficacy, frog neuromuscular junctions were studied with a new technique combining electrophysiology, light microscopy, and freeze- fracture of identified single muscle fibers. This technique allows correlations to be made between quantal content (measured in low Ca2+ and high Mg2+ Ringer solution), endplate size, and active zone structure at the same neuromuscular junctions. By measuring physiological and morphological variables at the same junctions, the validity of structure-function correlations is significantly improved. Synaptic quantal content in 91 physiologically identified muscle fibers varied considerably and was only poorly correlated with endplate size, as shown in previous studies. To measure the total length of endplate branches, either a modified cholinesterase stain or rhodamine-labeled peanut agglutinin stain was used. When the same identified muscle fibers were freeze-fractured, active zones were exposed in 17 junctions. In a replica that contained a large part of one nerve terminal, there was no detectable gradient in active zone structure along the length of 3 different nerve terminal branches identifiable with both light and electron microscopy. The results from these 17 identified junctions indicate that quantal content per unit terminal length is positively correlated with the amount of active zone per unit terminal length. The estimated total active zone length and total number of active zone particles per junction are also positively correlated with the quantal content in these identified junctions. This study suggests that active zone size and spacing are better indicators of transmitter release than is endplate size and that the active zone may play an important role in regulating synaptic efficacy at the neuromuscular junction.
Taxol interferes with the interaction of microtubule-associated proteins with microtubules in cultured neurons
Authors: Black MM
Published: March 2018

Treatment of neurons with taxol leads to the formation of microtubule bundles in which individual microtubules are much more closely spaced than in untreated neurons (Letourneau and Ressler, 1984). This suggests that taxol interferes with the mechanisms that regulate microtubule spacing in situ. I have determined whether treatment of neurons with taxol alters the composition of their microtubules, reasoning that such alterations may be related to the taxol-induced alterations in microtubule spacing. Cultures of sympathetic neurons were incubated with 35S-methionine and either taxol, podophyllotoxin, a potent microtubule-depolymerizing agent, or dimethyl sulfoxide (DMSO), the solvent for the drugs. The levels of labeled microtubule-associated proteins (MAPs) assembled into microtubules in the various cultures were then assayed biochemically. I focused on 4 MAPs: tau, chartins, MAP-2, and the MAP with a molecular mass of 210,000 Da (210 kDa). In untreated cultures, these MAPs are prominent components of microtubules. The levels of all MAPs, as well as tubulin, in microtubules were greatly reduced in cultures treated with podophyllotoxin, compared to controls. Taxol had varied effects on the interaction of MAPs with microtubules in situ. Microtubules formed in the presence of taxol contained normal or slightly elevated levels of tau and the 210 kDa MAP compared to microtubules in control cultures. In contrast, microtubules formed in the presence of taxol were almost completely devoid of chartin MAPs and MAP-2 compared to controls. These results show that taxol interferes with the interaction of some, but not all, MAPs with microtubules in situ. The altered MAP composition of microtubules in taxol-treated neurons may contribute to the abnormal spacing of microtubules seen in such neurons.
Organization of individual afferent axons in layer IV of striate cortex in a primate
Authors: Florence SL , Casagrande VA
Published: March 2018

Evidence from a number of anatomical and physiological studies shows that information is transmitted from the retina to visual cortex via physiologically and anatomically distinct populations of neurons in the lateral geniculate nucleus (LGN). In order to gain a better understanding of the functional roles of these parallel channels from the LGN to cortex in primates, individual afferent axons to layer IV of striate cortex of galagos were filled with HRP by bulk injection into the white matter underlying striate cortex. A total of 55 axons and their terminal arbors, from zones representing both the central and peripheral visual fields, were completely reconstructed through serial sections. Based upon the sublaminar distribution and the morphology of these axons, they have been categorized into 2 groups, designated type I and II axons. Evidence from both past work and the present study suggests that type I axons represent the projections from physiologically defined Y-like cells in the magnocellular layers of the LGN, while type II axons represent the projections from X-like cells in the parvocellular LGN layers. Our results show that type I (presumed Y- like) arbors occupy relatively more cortical space within their main terminal sublayer (IV alpha) than is the case for the type II (presumed X-like) arbors which ramify primarily in layer IV beta. In addition, type I arbors have larger parent axons, fewer boutons along a restricted length of axon, and a greater tendency to branch in layer VI than type II arbors. Finally, both axon types are larger in the area of cortex representing central vision than in the area representing peripheral vision. These morphological characteristics suggest that the physiological differences between magnocellular and parvocellular geniculate cells may be amplified in cortex by differences in the organization of their terminal arbors. Further, within each afferent population, the terminal organization of axons reflects their visuotopic relationships in striate cortex. Comparison of these findings with data from cats and monkeys supports the idea that the relationship between the size of the terminal arbors of LGN X-like or parvocellular cells and the size of the cortical spatial subunit varies with differences in visual acuity across species; for LGN Y-like (or magnocellular) cells this relationship remains constant.
Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin
Authors: Larocca JN , Ledeen RW , Dvorkin B , Makman MH
Published: March 2018

High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands 3H-N- methylscopolamine (3H-NMS), 3H-quinuclidinyl benzilate (3H-QNB), and 3H- pirenzepine. 3H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, 3H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by 3H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for 3H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine- nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20–25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure. In conclusion, both M1 and M2 muscarinic receptor subtypes and an adenylate cyclase system linked to at least some of these receptors are present as intrinsic components of myelin. The possibility that some of these muscarinic receptors may be involved in regulation of phosphinositide metabolism and the protein kinase activities of myelin is considered.
Role of phosphoinositide metabolites in the prolongation of afterhyperpolarizations by alpha 1-adrenoceptors in rat dorsal raphe neurons
Authors: Freedman JE , Aghajanian GK
Published: March 2018

Afterhyperpolarizations and outward tail currents of rat dorsal raphe neurons were measured by intracellular recording and single-electrode voltage clamping in the brain slice preparation. The alpha 1-agonist phenylephrine, and (in the presence of propranolol) norepinephrine, elicited an increase in the duration, but not of the initial amplitude, of afterhyperpolarizations and associated outward tail currents which followed depolarizing pulses. These effects were antagonized by prazosin, indicating that they were mediated by alpha 1-adrenoceptors. The outward tail currents were sensitive to apamin, a blocker of certain Ca2+-activated K+ currents. A prolongation of afterhyperpolarizations would offset the major excitatory alpha 1 effects, which were associated with suppression of resting K+ currents and of the A-current. Since polyphosphoinositide metabolites have been reported to be second messengers for Ca2+-dependent receptor actions, we compared their effects with those of alpha 1-receptor stimulation on these cells. Intracellular ejection of the putative second messenger myo-inositol-1,4,5-trisphosphate from the recording electrode transiently mimicked the actions of alpha 1-agonists on the afterhyperpolarization. Superfusion with 1 mM LiCl, simulating therapeutic levels of lithium, had no effect on the rate of recovery from inositol trisphosphate ejection. Superfusion with water-soluble phorbol esters (which mimic actions of another phosphoinositide metabolite, 1,2-diacylglycerol) suppressed rather than mimicked the activation of raphe cell firing by phenylephrine; this occurred with a rank-order potency consistent with activation of protein kinase C and was associated with suppression of a slow inward current and of the outward tail current. Our results suggest that phosphoinositide turnover is more likely to mediate modulatory or negative-feedback effects of alpha 1-adrenoceptors than to mediate the major excitatory effects.
The neostriatal mosaic: II. Patch- and matrix-directed mesostriatal dopaminergic and non-dopaminergic systems
Authors: Gerfen CR , Herkenham M , Thibault J
Published: March 2018

Mesostriatal projections, which arise from dopaminergic and non- dopaminergic neurons in the ventral tegmental area, substantia nigra, and retrorubral area, are compartmentally organized in the striatum. Anterograde axonal tract tracing with Phaseolus vulgaris- leucoagglutinin (PHA-L), combined with immunohistochemical localization of tyrosine hydroxylase (TH) and autoradiographic localization of mu- opiate receptor binding sites, shows that midbrain projections to the striatum are distributed to either the mu-opiate receptor-rich “patch” or the receptor-poor “matrix” striatal compartments. Three morphologically distinct mesostriatal afferent fiber types are labeled. The first type, type A, forms a plexus of relatively thin (0.1–0.4 micron), smooth fibers with small varicosities (0.3–0.6 micron). A second type, type B, is similar to the first in forming a plexus of fibers, but is slightly thicker (0.2–0.6 micron), with more frequent varicosities (0.4–1.0 micron) that give this fiber type a crinkled appearance. The third type, type C, constitutes a minority of striatal afferents and is characterized by its large caliber (0.4–0.7 micron) with large bulbous varicosities (1.2–2.0 micron). Projections of the ventral tegmental area (A10 cell group) are primarily dopaminergic type A fibers directed to the matrix of the ventromedial striatum, including the nucleus accumbens. The retrorubral area (A8 cell group) also provides predominantly dopaminergic type A fibers to the striatal matrix, but these are distributed dorsally. The substantia nigra contains a mixed population of neurons that project to the striatum. Some, located in the dorsal tier of the pars compacta (dorsal A9 cell group), provide dopaminergic type A fibers to the striatal matrix. Others, in the ventral tier of the pars compacta (ventral A9 cell group) and in the ventral tier of the pars reticulata (displaced A9 cells), provide dopaminergic type B fibers to the striatal patches. An additional set of substantia nigra neurons that are non-dopaminergic is the source of type C fibers to the striatal matrix. Thus, distinct dorsal and ventral sets of midbrain dopaminergic neurons project, respectively, to striatal matrix and patches, and there is a non- dopaminergic mesostriatal projection to the matrix.