bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and quantification of calcium-binding proteins in squid axoplasm
Authors: Krinks MH , Klee CB , Pant HC , Gainer H
Published: March 2018

The identities and quantities of calcium-binding proteins were determined in axoplasm isolated from the squid giant axon. 45Ca-binding assays on nitrocellulose filters containing axoplasm proteins separated by SDS-polyacrylamide electrophoresis revealed 4 major calcium-binding bands. These included the high-molecular-weight (Mr greater than 330 and 220 X 10(3] neurofilament proteins, an unidentified protein band that migrated around Mr 55,000, and a diverse group of proteins that migrated together around Mr 17,000. The low-molecular-weight (Mr 17,000) calcium-binding proteins could be resolved into calmodulin (ca. 120 mumol/kg axoplasm), 2 other Mr 17,000 calcium-binding proteins, and a small amount of calcineurin B. It is estimated that these calcium- binding proteins in squid axoplasm could theoretically bind about 1 mmol Ca2+/kg axoplasm. 125I-Calmodulin overlay and Western blot analyses disclosed a number of calmodulin-binding proteins in axoplasm. These included fodrin, calcineurin A, and Ca2+/CaM protein kinase II subunits.
A selective impairment of motion perception following lesions of the middle temporal visual area (MT)
Authors: Newsome WT , Pare EB
Published: March 2018

Physiological experiments indicate that the middle temporal visual area (MT) of primates plays a prominent role in the cortical analysis of visual motion. We investigated the role of MT in visual perception by examining the effect of chemical lesions of MT on psychophysical thresholds. We trained rhesus monkeys on psychophysical tasks that enabled us to assess their sensitivity to motion and to contrast. For motion psychophysics, we employed a dynamic random dot display that permitted us to vary the intensity of a motion signal in the midst of masking motion noise. We measured the threshold intensity for which the monkey could successfully complete a direction discrimination. In the contrast task, we measured the threshold contrast for which the monkeys could successfully discriminate the orientation of stationary gratings. Injections of ibotenic acid into MT caused striking elevations in motion thresholds, but had little or no effect on contrast thresholds. The results indicate that neural activity in MT contributes selectively to the perception of motion.
Habituation in Stentor: produced by mechanoreceptor channel modification
Authors: Wood DC
Published: March 2018

Habituation in Stentor was shown in the previous paper (Wood, 1988) to result from a progressive diminution in mechanoreceptor potential amplitude during the course of repetitive mechanical stimulation. The source of this diminution was analyzed in the present studies by eliciting mechanoreceptor currents from control and habituated cells under voltage clamp control. The I-V plots obtained have a voltage- dependent region between -60 and -20 mV, and a linear voltage- independent region between -20 and 20 mV, with a reversal potential near 20 mV. The slope of the linear region did not change as a result of habituation indicating that the maximum mechanoreceptor current, and hence the total number of mechanoreceptor channels, was the same in control and habituated cells. The mechanoreceptor current reversal potential was changed by only a few millivolts, from 21.6 to 17.2 mV, leaving the driving force term for the mechanoreceptor current at resting potential relatively unchanged. On the other hand, the voltage- dependent region of the I-V plot became significantly steeper after habituation (9.6 mV/e-fold change) compared to its value before habituation (12.3 mV/e-fold change). This shift in voltage dependence is sufficient to explain the diminution in receptor potential amplitude seen during normal recordings at resting potential. The importance of the voltage-dependent characteristic of the mechanoreceptor channels to the habituation process was corroborated by the observation that the amplitude of receptor potentials elicited from cells depolarized by current pulses habituated to only a very limited degree while receptor potentials elicited at resting potential underwent marked habituation.(ABSTRACT TRUNCATED AT 250 WORDS)
Quantitative autoradiographic measurement of local cerebral glucose utilization in freely moving rats during postnatal development
Authors: Nehlig A , de Vasconcelos AP , Boyet S
Published: March 2018

The quantitative 2–14C-deoxyglucose autoradiographic method of Sokoloff et al. (1977) was used to measure local cerebral glucose utilization in freely moving developing rats. The animals were studied at 10, 14, 17, 21, and 35 d and at the adult stage. Glucose utilization was very low and quite uniform in 10- and 14-d-old rats, ranging from 20 to 30 mumol/100 gm/min, except in a few posterior areas. Between these 2 stages, rates of glucose utilization significantly increased in 6 areas, among which 4 were belonging to the auditory system. Between 14 and 17 d, glucose utilization significantly changed in 9 structures out of the 68 studied, mainly auditory, visual, parietal, and thalamic areas. Between the stages of 17 and 21 d, glucose utilization was increased by 50 or 100% in all brain structures studied, except in the medial habenula and white matter areas. After weaning time, rates of glucose utilization still significantly changed in 50 areas, widely distributed through all studied systems. Between 35 d and the adult stage, the average rate of glucose utilization did not change and rates of energy metabolism significantly increased in 13 brain areas. In one structure, the medial habenula, glucose utilization was already high 10 d after birth and did not change over the whole studied period. These increases in the rates of glucose utilization are consistent with the behavioral, anatomical, and functional changes known to occur during this period of development in the rat.
Adenosine uptake sites in brain: regional distribution of putative subtypes in relationship to adenosine A1-receptors
Authors: Deckert J , Bisserbe JC , Klein E , Marangos PJ
Published: March 2018

Adenosine uptake sites have been characterized and localized in guinea pig and pointer dog brain by in vitro autoradiography, using as probes 3H-nitrobenzylthioinosine (3H-NBI) and the recently available 3H- dipyridamole (3H-DPR). In guinea pig brain and, to a lesser extent, in pointer dog brain, 3H-DPR was found to label more high-affinity binding sites than 3H-NBI and NBI inhibited 3H-DPR binding having pseudo-Hill coefficients smaller than 0.5. 3H-DPR and 3H-NBI labeled brain structures with different intensities in guinea pig brain, as was revealed by quantitative analysis. While the intensity of 3H-DPR binding varied about 4-fold in neuron-containing structures, 8-fold differences were observed for 3H-NBI binding with phylo- and ontogenetically older brain areas such as hypothalamus and various brain stem structures showing relatively higher densities. These findings raise the interesting possibility of adenosine uptake site heterogeneity (NBI-sensitive and insensitive) in guinea pig brain, complementing the well-established adenosine receptor heterogeneity (A1 and A2). As adenosine's neurodepressant effects are believed to be mainly mediated by adenosine A1-receptors, these were localized using 3H-cyclohexyl-adenosine (3H-CHA) as a ligand probe. In guinea pig brain, the highest receptor densities were seen in hippocampus and claustrum, while only relatively low levels were found in hypothalamus and various brain stem structures. As was previously described for rat brain, major discrepancies in the regional distribution of adenosine A1- receptors and adenosine uptake sites, as labeled by 3H-NBI, were seen in guinea pig brain. These discrepancies were only partly abolished (e.g., in cerebellum) by the use of 3H-DPR as an additional ligand probe for adenosine uptake sites. Adenosine uptake site heterogeneity, therefore, probably does not explain the previously described discrepancies in rodent brain between the distribution of adenosine A1- receptors and uptake sites. Because of the low affinity of 3H-DPR for adenosine uptake sites in rat and mouse brain, these species could not be investigated with this new radioligand probe. In pointer dog brain, as compared to guinea pig brain, a more similar distribution pattern of adenosine A1-receptors and adenosine uptake sites in the brain structures investigated (e.g., hippocampus) could be observed. The situation in guinea pig brain can, therefore, not be universalized to other species.(ABSTRACT TRUNCATED AT 400 WORDS)
Neuropeptide Y modulates neurotransmitter release and Ca2+ currents in rat sensory neurons
Authors: Walker MW , Ewald DA , Perney TM , Miller RJ
Published: March 2018

Using 125I-labeled neuropeptide Y (NPY) and peptide YY (PYY), we demonstrated the existence of specific receptors for these peptides on rat dorsal root ganglion (DRG) cells grown in primary culture. Scatchard analysis of membrane homogenates indicated that the peptides bound to 2 populations of sites, with approximate affinities of 0.08 and 6.5 nM. Only low levels of binding were detected on sympathetic neurons cultured from the same animals or on a variety of neuronal clonal cell lines. The binding of 125I-NPY and 125I-PYY to DRG cell membranes was considerably reduced by the nonhydrolyzable analog of GTP, Gpp(NH)p. The major effect of Gpp(NH)p was to reduce the number of lower-affinity NPY binding sites without altering the number of high- affinity binding sites. NPY potently inhibited Ca2+ currents recorded under voltage clamp in rat DRG cells. Both the transient and sustained portions of the Ca2+ current were inhibited. The inhibitory effects of NPY were completely blocked following treatment of the cells with pertussis toxin. Depolarization elicited a large influx of Ca2+ into DRG neurons as assessed using fura-2-based microspectrofluorimetry. This influx of Ca2+ could be partially inhibited by NPY. Furthermore, NPY effectively inhibited the depolarization-induced release of substance P from DRG cells in vitro. Thus, NPY may be an important regulator of sensory neuron function in vivo.
The effect of down regulation of protein kinase C on the inhibitory modulation of dorsal root ganglion neuron Ca2+ currents by neuropeptide Y
Authors: Ewald DA , Matthies HJ , Perney TM , Walker MW , Miller RJ
Published: March 2018

Dorsal root ganglion (DRG) neurons cultured from neonatal rats contained high concentrations of protein kinase C (PKC). Normally, the majority of the enzyme activity was found in the cytosol and considerably less was associated with the membrane fraction. Upon incubation with the phorbol ester phorbol dibutyrate (PDBu, 10(-6) M) for 20 min, PKC activity increased in the membrane-associated fraction and decreased in the cytoplasmic fraction. Longer incubations with phorbol ester also induced a decline in membrane-associated PKC activity. If incubations were continued for periods of over 10 hr, both membrane and cytosolic PKC activity declined essentially to zero. Down- regulation of PKC had no effect on the number or affinity of 125I- neuropeptide Y (NPY) binding sites on DRG cells or on the absolute magnitude of the DRG Ca2+ current. However, the ability of NPY to inhibit the DRG Ca2+ current was greatly reduced. When sustained Ca2+ currents were evoked from depolarized holding potentials (-40 mV), all concentrations of NPY (10(-10)-10(-7) M) were less effective. In contrast, higher concentrations of NPY still blocked the transient portion of the DRG Ca2+ current evoked from hyperpolarized holding potentials. These results support the suggestion that PKC is involved in the inhibitory modulation of DRG Ca2+ currents by neurotransmitters. The precise role of PKC may vary depending on the type of Ca2+ channel involved.
Electrical tuning in hair cells isolated from the chick cochlea
Authors: Fuchs PA , Nagai T , Evans MG
Published: March 2018

Tall (inner) hair cells were isolated from specific locations in the chick cochlea. The electrical membrane properties of these cells were recorded using the tight-seal whole-cell technique. Depolarizing current steps elicited damped voltage oscillations that ranged in frequency from 100 to 250 Hz among cells from the middle third of the cochlea (basal cells). The current-voltage relation obtained under voltage clamp was dominated by calcium-activated potassium current in the voltage range over which these oscillations occurred. Tall hair cells isolated from the apical tip of the cochlea (apical cells) exhibited action potentials and lower frequency voltage oscillations (5– 14 Hz) during depolarizing current steps. Outward currents in these cells were 20-fold slower than those found in the basal cells. These results suggest that electrical tuning of hair cells may play a role in determining the frequency selectivity of the chick cochlea.
Enrichment of a vasoactive neuropeptide (calcitonin gene related peptide) in the trigeminal sensory projection to the intracranial arteries
Authors: O'Connor TP , van der Kooy D
Published: March 2018

Trigeminal sensory innervation of cerebral vessels and the surrounding dura is responsible for most intracranial head pain. Small-diameter fibers containing substance P (Sub P) have been observed in the periadventitia around feline cerebral blood vessels, and it has been suggested that these fibers are the trigeminal substrate for vascular pain associated with cluster and migraine headaches. Calcitonin gene related peptide (CGRP) coexists with Sub P in some of these fibers and with some Sub P containing neurons in the trigeminal ganglion. In addition, a population of trigeminal neurons containing CGRP but not Sub P has been observed. We now report that the population of trigeminal ganglion cells projecting to the cerebral vasculature is enriched in CGRP-containing neurons, and especially in the population of neurons containing CGRP and not Sub P. Using retrograde tracing of fluorescent tracers combined with immunocytochemistry after explant culture, we found approximately 32% of trigeminal ganglion cells projecting to the cerebral vasculature contained CGRP. Approximately 18 and 17% of these cells contained Sub P and cholecystokinin (CCK), respectively. The 32% of ganglion cells projecting to the cerebral vasculature that contain CGRP stands in contrast to the 12% CGRP positive seen in the population of ganglion cells projecting out to another target (the forehead), and the 21 and 23% CGRP positive observed in the mandibular branch and entire ganglion, respectively. Sub P and CCK are not enriched in the trigeminal innervation of the vasculature compared with their presence in cells throughout the ganglia.(ABSTRACT TRUNCATED AT 250 WORDS)
Interleukin-1 injected into mammalian brain stimulates astrogliosis and neovascularization
Authors: Giulian D , Woodward J , Young DG , Krebs JF , Lachman LB
Published: March 2018

Interleukin-1 (IL-1), a protein produced by mononuclear phagocytes, helps to initiate the inflammatory response through its action upon a diverse population of cells. Recently this immunomodulator has been detected at sites of traumatized brain. As reported here, recombinant forms of IL-1 injected into the cerebral cortex of adult rats elicit not only astrogliosis but also new blood vessel growth. These responses are typical of brain injury and suggest that IL-1-secreting inflammatory cells may mediate wound healing in the CNS.
Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions
Authors: Walicke PA
Published: March 2018

Basic fibroblast growth factor (bFGF) supports the survival of neurons from many regions of the E18 fetal rat brain. Survival was significantly increased for neurons derived from the hippocampus, entorhinal cortex (EC), frontal cortex, parietal cortex (PC), occipital cortex, striatum, septum, and thalamus, but not from the subiculum (Sb). The proportion of neurons rescued by bFGF varied among brain regions, suggesting the existence of subpopulations of responsive neurons. Like hippocampal neurons, neurons from the EC and PC required about 1 pM bFGF (10–20 pg/ml) for half-maximal response; striatal neurons, in contrast, required about 3 pM bFGF. Neurite outgrowth after 24 hr exposure was significantly increased for neurons from the hippocampus, EC, and PC, while striatal neurons had only a marginal response. Although bFGF stimulated some astrocytic proliferation in the cultures, glial contamination was maintained at 2% or less. Acidic FGF (aFGF) supported smaller numbers of neurons from each region, although it significantly increased survival of neurons from hippocampus, EC, PC, striatum, and Sb. The concentration required for half-maximal survival was around 100–300 pM (2–5 ng/ml). It appears that bFGF and aFGF are potent trophic factors for many populations of CNS neurons and could potentially play a significant role in nervous system development.
Development of spatial and temporal selectivity in the suprasylvian visual cortex of the cat
Authors: Zumbroich T , Price DJ , Blakemore C
Published: March 2018

We have studied the development of the spatial and temporal properties of neurons in the medial bank of the suprasylvian visual cortex (PMLS) in kittens aged between 9 d and 8 weeks. Quantitative measurements were made of the responses to drifting high-contrast gratings of optimum orientation and direction of motion, but varying in spatial and temporal frequency. The spatial resolution (“acuity”) of cells increased rapidly and was fully mature (over 2 cycles/deg for the best cells) at 3 weeks of age. The optimum spatial frequency also tended to improve and reached adult values (around 0.5 cycles/deg for the best cells) at about the end of the third week. In younger kittens, the spatial resolution of neurons was not obviously correlated with the eccentricity of their receptive fields, but in older animals acuity was clearly elevated for receptive fields in the central visual field. The proportion of “low-pass” cells (showing no obvious attenuation of response for gratings of low spatial frequency) decreased with age and simultaneously there was a slight increase in the mean spatial bandwidth of “bandpass” cells. Responses to drifting sinusoidal gratings were generally dominated by an unmodulated elevation of discharge at all ages. In tests with stationary, contrast-modulated gratings presented at different spatial positions, cells in the youngest kittens behaved nonlinearly and showed mainly an unmodulated increase in discharge, whereas in older kittens, as in adult cats, most neurons responded to contrast-modulated gratings with a small, phase- dependent response at the temporal frequency of modulation and a larger component at twice the fundamental frequency. None of the cells recorded at any age had a true “null position.” As in adult PMLS, the widths of receptive fields in kittens were, on average, about twice the size of the preferred spatial period (4 times the preferred bar width). At all ages, therefore, neurons in PMLS resembled striate complex cells with respect to the nonlinearity of their responses and the spatial structure of their receptive fields. The preferred temporal frequency and high-temporal-frequency cutoff also improved, on average, during the first 3 weeks of life, and the range of temporal frequencies over which cells responded continued to increase until at least 8 weeks. Although the low-spatial-frequency inhibition that creates spatial bandpass characteristics probably depends on cortical mechanisms, the postnatal development of both temporal and spatial resolution might well be limited by maturation at the level of the retina.
Evidence for the involvement of more than one mRNA species in controlling the inactivation process of rat and rabbit brain Na channels expressed in Xenopus oocytes
Authors: Krafte DS , Snutch TP , Leonard JP , Davidson N , Lester HA
Published: March 2018

The properties of rat and rabbit brain sodium (Na) channels expressed in Xenopus oocytes following either unfractionated or high-molecular- weight mRNA injections were compared to assess the relative contribution of different size messages to channel function. RNA was size-fractionated on a sucrose gradient and a high-molecular-weight fraction (7–10 kilobase) encoding the alpha-subunit gave rise to functional voltage-dependent Na channels in the oocyte membrane. Single- channel conductance, mean open time, and time to first opening were all similar to the values for channels following injection of unfractionated RNA. In contrast, inactivation properties were markedly different; Na currents from high-molecular-weight RNA inactivated with a several-fold smaller macroscopic inactivation rate and showed a steady-state voltage dependence that was shifted in the depolarizing direction by at least 10 mV relative to that for unfractionated RNA. Single-channel recording revealed that the kinetic difference arose from a greater probability for high-molecular-weight RNA induced channels to reopen during a depolarizing voltage step. Pooling all gradient fractions and injecting this RNA into oocytes led to the appearance of Na channels with inactivation properties indistinguishable from those following injection of unfractionated RNA. These results suggest that mRNA species not present in the high- molecular-weight fraction can influence the inactivation process of rat brain Na channels expressed in Xenopus oocytes. This mRNA may encode beta-subunits or other proteins that are involved in posttranslational processing of voltage-dependent Na channels.
Control by an identified modulatory neuron of the sequential expression of plateau properties of, and synaptic inputs to, a neuron in a central pattern generator
Authors: Nagy F , Dickinson PS , Moulins M
Published: March 2018

Recordings from the lateral gastric (LG) neuron, which forms part of the gastric mill central pattern generator in the red lobster, Palinurus vulgaris, indicate that regenerative membrane properties (plateau properties) and synaptic inputs interact sequentially rather than simultaneously to determine its discharge pattern. LG thus presents a composite discharge, consisting of 2 separate segments of firing and one silent period. The first firing segment depends on regenerative membrane properties; this is the endogenous component, or segment, of LG's discharge. The second firing segment is the result of extrinsic synaptic input, forming the synaptic component of LG's discharge. The relative importance of these 2 components can vary, and thus LG's discharge ranges from one in which LG fires only as a result of its endogenous component to one in which its endogenous component is entirely absent and only the synaptic component underlies action potentials. Activity in an identified modulatory neuron suppresses the endogenous segment and enhances the synaptic segment of LG's discharge. This long-lasting effect in turn changes phase relationships within the gastric mill network and provides mechanisms for producing flexibility in the gastric pattern generator and for ensuring that a specific motor output is generated by a flexible neural network.
Primate motor cortex and free arm movements to visual targets in three- dimensional space. I. Relations between single cell discharge and direction of movement
Authors: Schwartz AB , Kettner RE , Georgopoulos AP
Published: March 2018

We describe the relations between the neuronal activity in primate motor cortex and the direction of arm movement in three-dimensional (3- D) space. The electrical signs of discharge of 568 cells were recorded while monkeys made movements of equal amplitude from the same starting position to 8 visual targets in a reaction time task. The layout of the targets in 3-D space was such that the direction of the movement ranged over the whole 3-D directional continuum in approximately equal angular intervals. We found that the discharge rate of 475/568 (83.6%) cells varied in an orderly fashion with the direction of movement: discharge rate was highest with movements in a certain direction (the cell's “preferred direction”) and decreased progressively with movements in other directions, as a function of the cosine of the angle formed by the direction of the movement and the cell's preferred direction. The preferred directions of different cells were distributed throughout 3-D space. These findings generalize to 3-D space previous results obtained in 2-D space (Georgopoulos et al., 1982) and suggest that the motor cortex is a nodal point in the construction of patterns of output signals specifying the direction of arm movement in extrapersonal space.
Primate motor cortex and free arm movements to visual targets in three- dimensional space. II. Coding of the direction of movement by a neuronal population
Authors: Georgopoulos AP , Kettner RE , Schwartz AB
Published: March 2018

We describe a code by which a population of motor cortical neurons could determine uniquely the direction of reaching movements in three- dimensional space. The population consisted of 475 directionally tuned cells whose functional properties are described in the preceding paper (Schwartz et al., 1988). Each cell discharged at the highest rate with movements in its “preferred direction” and at progressively lower rates with movements in directions away from the preferred one. The neuronal population code assumes that for a particular movement direction each cell makes a vectorial contribution (“votes”) with direction in the cell's preferred direction and magnitude proportional to the change in the cell's discharge rate associated with the particular direction of movement. The vector sum of these contributions is the outcome of the population code (the “neuronal population vector”) and points in the direction of movement in space well before the movement begins.
Biochemical and physiological consequences of an age-related increase in acetylcholinesterase activity at the rat neuromuscular junction
Authors: Smith DO , Emmerling M
Published: March 2018

Acetylcholinesterase (AChE) specific activity was assayed using diaphragm muscles obtained from mature adult (10 months) and aged (25– 27 months) rats. Biochemical assays indicated significant age-related increases in the AChE specific activity of both noninnervated and innervated tissue. The different molecular forms of AChE were separated by velocity sedimentation and were further assayed. The age-related increase was manifest primarily in the 10S (G4) form in both noninnervated and innervated tissue and also the 16S (A12) form of the noninnervated samples. To ascertain more conclusively whether AChE activity in the end-plate junctional region of innervated tissue changed in the older rats, miniature end-plate currents (m.e.p.c.s) were recorded under voltage-clamp conditions before and after AChE inhibition. When AChE activity was inhibited by 10 microM echothiopate or 1 mM methanesulfonyl fluoride, m.e.p.c. amplitudes and decay time constants increased in both age groups. The magnitude of these increases was larger in the older animals. However, calculations of the relative change in m.e.p.c. amplitudes after AChE inhibition indicated that less ACh was hydrolyzed by AChE in the older animals. Inhibition of AChE did not affect mean channel open time, which was estimated from spectral analyses of ACh-induced membrane noise. These data indicate that the prolonged decay times in the older rats following AChE inhibition is quite likely due to an expanded field of postsynaptic ACh receptors and not exclusively to a change in junctional AChE.
Characterization of solubilized atrial natriuretic peptide receptors from rat olfactory bulb and A10 cultured smooth muscle cells
Authors: Gibson TR , Zyskind AD , Glembotski CC
Published: March 2018

Atrial natriuretic peptide (ANP) receptors from A10 cultured vascular smooth muscle cells (VSMC) and rat olfactory bulbs have been solubilized and then pharmacologically and biochemically compared. The dissociation constant for 125I-ANP(99–126) was 12.7 pM for the VSMC- derived receptor and 164 pM for the olfactory receptor. Competition binding between 125I-ANP(99–126) and several unlabeled ANP analogs with the soluble olfactory receptor, demonstrated a rank order potency of ANP(99–126) = ANP(103–126) much greater than ANP(103–123). However, the rank order potency of the soluble VSMC ANP receptor was ANP(99–126) = ANP(103–126) = ANP(103–123). Therefore, the olfactory ANP receptor appears to require the complete COOH-terminal sequence of ANP as compared with the VSMC ANP receptor. When the 2 soluble receptor preparations were applied to a GTP-agarose column, a portion of the olfactory ANP receptor was retained on the column and could be eluted with 5 mM GTP, while the VSMC ANP receptor did not adsorb to the column. Since the olfactory bulb ANP receptor has been shown to contain a binding component of 116 kDa, while the VSMC ANP receptor binding component is 66 kDa, these receptors appear to be similar to the 2 receptor classes described recently in which the 120 kDa receptor that binds GTP is postulated to be coupled to guanylate cyclase, while the 60 kDa receptor does not bind GTP, is not coupled to guanylate cyclase, and may possess a hormone clearance function. Taken together, these data indicate that cyclic GMP appears to be a second messenger for ANP in the brain.
Topographically selective reinnervation of adult mammalian skeletal muscles
Authors: Laskowski MB , Sanes JR
Published: March 2018

In 2 rat muscles, serratus anterior and the diaphragm, the rostrocaudal axis of the motor pool is mapped onto the rostrocaudal axis of the muscle's surface (Laskowski and Sanes, 1987a). One possible basis for this orderly topography is that motor axons and intramuscular structures bear labels that favor connectivity among positionally matched partners. To test for the existence of such labels, we asked whether axons would selectively reinnervate appropriate portions of the muscles following nerve transection. We found that, on average, rostral and caudal halves of each muscle were preferentially reinnervated by axons from the rostral and caudal halves of its motor pool, respectively. In the serratus anterior, reinnervation was more selective following denervation in neonates than following denervation in adults, although in neither case was the normal pattern of innervation reestablished completely. These results show that motor axons can selectively reinnervate adult rat muscles, and support the idea that positional cues play a role in organizing neuromuscular topography.
Cultured smooth muscle targets lack survival activity for ciliary ganglion neurons
Authors: Creedon DJ , Tuttle JB
Published: March 2018

Cultured neurons require specific trophic agents in order to survive. This dependence is thought to resemble the neuron-target interdependence that develops in vivo during synaptogenesis and neuronal cell death. The notion that neurons in general derive trophic support from their synaptic targets is based primarily on studies of peripheral neurons and motor neurons. To assess the general applicability of this nerve-target relationship, we tested the ability of vascular smooth muscle (VSM) to support dissociated neurons from the chick ciliary ganglion. The ciliary ganglion contains 2 distinct neuronal populations, one of which innervates striated muscle, the other VSM. Striated muscle cocultures are known to support all of the neurons in the ganglion for extended periods. Dissociated neurons were therefore cocultured in microwells containing VSM derived from the rat or chick aorta and from the choroid coat of the chick eye. Surviving neurons were counted after 1, 2, 5, and 7 d. Striated muscle is able to support full neuronal survival in the same assay. However, in no case was VSM capable of contributing to neuronal survival in vitro. The neurons in the VSM cocultures were able to form neurites and make contacts with their putative targets, as confirmed by scanning electron and light microscopy. The presence of viable and differentiated smooth muscle cells was demonstrated in the cultures by transmission electron microscopy and analysis of smooth muscle alpha-actin. The failure of VSM and even the choroid target tissue to support the survival of their innervating neurons suggests that novel mechanisms may operate to provide trophic support for neurons innervating VSM targets.
Ultrastructural characterization of gerbil olivocochlear neurons based on differential uptake of 3H-D-aspartic acid and a wheatgerm agglutinin- horseradish peroxidase conjugate from the cochlea
Authors: Helfert RH , Schwartz IR , Ryan AF
Published: March 2018

Two populations of olivocochlear (OC) neurons have been identified in the gerbil brain stem on the basis of differential labeling patterns of 3H-D-aspartic acid (D-ASP) and wheatgerm agglutinin-horseradish peroxidase conjugate (WGA/HRP) from the cochlear perilymph. While both populations are capable of uptake and retrograde uptake of WGA/HRP, one population accumulates and retrogradely transports D-ASP (D-ASP OC neurons) and the other does not (non-D-ASP OC neurons). D-ASP OC neurons are found in or near the lateral superior olive, are small in size, and receive very few synaptic contacts. The vast majority of these synapses contain small, mildly pleomorphic vesicles with scattered dense core vesicles. Synapses with distinctly larger pleomorphic vesicles have also been observed. These neurons possess all of the features common to neurons of the lateral olivocochlear system. Non-D-ASP OC neurons are found primarily in the ventral nucleus of the trapezoid body, as well as in the area between the medial superior olive and the medial nucleus of the trapezoid body. These neurons are larger and receive greater numbers and types of synaptic contacts than those found on D-ASP OC neurons. The 2 most common synapses found on non-D-ASP OC neurons are axosomatic ones containing small, mildly pleomorphic vesicles and scattered dense core vesicles similar to those seen on the D-ASP OC neurons, and axodendritic synapses containing large, round vesicles. Much less frequently observed are synapses containing small, round vesicles or ones containing predominantly flat vesicles. The ultrastructural features of the non-D-ASP OC neurons correspond to those described for neurons of the medial olivocochlear system.
Rat astroglial somatomedin/insulin-like growth factor binding proteins: characterization and evidence of biologic function
Authors: Han VK , Lauder JM , D'Ercole AJ
Published: March 2018

Specific binding proteins (BPs) to somatomedin/insulin-like growth factors (Sm/IGFs) have been identified in conditioned media from a variety of cells in culture. By affinity cross-linking using disuccinimidyl suberate, we have covalently cross-linked radiolabeled somatomedin-C/insulin-like growth factor I (Sm-C/IGF I), insulin-like growth factor II (IGF II) and insulin to BPs in conditioned medium (CM) from cultured astroglial cells derived from cerebral cortices of neonatal rats. Two species of radiolabeled Sm/IGF BP complexes of 40,000 Da (40K) and 45K were identified. Competition with unlabeled Sm- C/IGF I and IGF II demonstrated that the BPs in each complex have similar affinities for Sm-C/IGF I and IGF II. The BP in the 45K complex was about 5-fold more sensitive to competition with unlabeled Sm/IGFs than the BP in the 40K complex, suggesting that it either has a higher affinity for Sm/IGFs or is less abundant. Evidence that the BPs in each complex are distinct includes the following findings: (1) insulin competed with Sm/IGF for binding to the 45K complex, but not the 40K complex, and (2) the BP in the 40K complex, but not the 45K complex, was recognized by antibodies raised against a BP purified from CM of buffalo rat liver (BRL) 3A cells. Growth hormone did not affect the apparent secretion of either BP. The binding activity of both BPs was retained after mild heat treatment, changes to extremes of pH (2–10), and prolonged storage at -20 degrees C, but was destroyed after heating to higher temperatures (80 degrees C and greater), reduction, and proteolytic treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
The concentration of cytosolic free calcium in vertebrate rod outer segments measured with fura-2
Authors: Ratto GM , Payne R , Owen WG , Tsien RY
Published: March 2018

The use of fluorescent indicators such as fura-2 (Grynkiewicz et al., 1985) to measure the cytosolic free calcium activity in retinal rods is complicated by the rods' sensitivity both to the fluorescence and to the light that excites it. By stimulating fluorescence from large numbers of rods in whole, loaded retinas and averaging repeated measurements, however, we have been able to monitor changes in free [Ca2+]i during exposure to nonsaturating lights under physiological conditions. Retinas, isolated from the bullfrog Rana catesbeiana, were loaded with fura-2 by incubation and mounted, receptor-side up, in a perfusion chamber placed on the stage of a specially designed apparatus. A step of light delivered from above, whose wavelength alternated between 340 and 380 nm every 110 msec, excited fluorescence from 24 mm2 of retina and evoked a light response (the aspartate- isolated pIII component of the electroretinogram--ERG). By comparing the fluorescence intensities excited by the 2 wavelengths (corrected for background and dark-noise), the free [Ca2+]i of the rod outer segment was determined. In darkness, the [Ca2+]i of the outer segment was found to be approximately 220 nM. A bright light caused it to fall exponentially to approximately 140 nM, with a time constant of approximately 1.6 sec. The value of [Ca2+]i at the onset of illumination was independent of stimulus intensity over a 2 log-unit range, and in all cases the fall was monotonic. After terminating the illumination, [Ca2+]i rose again to its time-zero value.(ABSTRACT TRUNCATED AT 250 WORDS)
Nicotinic receptors of frog ganglia resemble pharmacologically those of skeletal muscle
Authors: Lipscombe D , Rang HP
Published: March 2018

The actions of ACh antagonists were studied on synaptic currents of autonomic ganglia of the frog. Fast excitatory synaptic currents (ESCs) were recorded from cardiac and paravertebral neurons with the use of the 2-microelectrode voltage-clamp method. The actions of 4 ACh antagonists, tubocurarine, hexamethonium, trimetaphan, and decamethonium were studied. Tubocurarine was effective at reducing the peak amplitude of ESCs (50% inhibition at 3 microM). In contrast, tubocurarine (1–30 microM) reduced the time constant of ESC decay by only 9% compared with controls. Both of these effects of tubocurarine were independent of membrane potential. Hexamethonium was a weak inhibitor of ESCs; at 600 microM peak amplitude was reduced only to about 60% of controls and decay time constants were unaffected at concentrations between 10 and 600 microM. These effects of tubocurarine and hexamethonium are consistent with these drugs being receptor antagonists with no evidence of ion channel block. Trimetaphan (3–100 microM) and decamethonium (100 microM) reduced the peak amplitude of ESCs. In the presence of 100 microM trimetaphan or 10 microM decamethonium, ESC decays were biexponential. The 2 exponential components induced by the presence of these drugs were faster and slower, respectively, than the single-exponential component of control ESC decays. The effects of these 2 drugs were more pronounced at hyperpolarized potentials and are consistent with a channel-blocking action. The actions of the 4 ACh antagonists on frog autonomic ganglia are similar to their effects at the neuromuscular junction but dissimilar to their effects on the rat submandibular ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)
Neuronal proteoglycans: biosynthesis and functional interaction with neurons in vitro
Authors: Dow KE , Mirski SE , Roder JC , Riopelle RJ
Published: March 2018

Glycosaminoglycans (GAGs) with electrophoretic mobilities on cellulose acetate similar to heparin (H), heparan sulfate (HS), and chondroitin sulfate (CS) were detected in cell extracts and in conditioned medium of high-density, neuron-enriched cultures labeled with 35SO4. Heparitinase digestion revealed that heparan sulfate proteoglycans (HSPGs) were heterogeneous in charge density and were responsible for neurite outgrowth activity for sensory neurons in conditioned medium. In the presence of beta-D-xyloside, an inhibitor of proteoglycan assembly, there was an increase of released GAGs with the mobility of CS and heavily sulfated HS but a decrease in neurite outgrowth activity on a laminin substrate at times greater than 14 hr. In the presence of beta-D-xyloside or the monoclonal antibody HNK-1 (Leu 7), which recognizes a neuronal cell surface epitope, there was a time-dependent inhibition of process formation that was half-maximal at 7–8 hr and independent of laminin concentration or cell adhesion to the laminin substrate. The kinetics and magnitude of the inhibitory effects of beta- D-xyloside and HNK-1 (Leu 7) were similar, and the influence of HNK-1 (Leu 7) could no longer be observed in the presence of beta-D-xyloside. Pretreatment of the laminin substrate with conditioned medium from high- density neuron cultures resulted in an increased rate of neurite formation compared with untreated laminin. Where the laminin substrate had been pretreated with conditioned medium, maximal inhibition by HNK- 1 (Leu 7) was apparent from the earliest times. However, if the conditioned medium had been digested with heparitinase, neither enhanced neurite outgrowth nor the inhibitory influence of HNK-1 (Leu 7) were observed. The present biosynthetic and functional studies suggest that neurons are one source of HSPGs. These data complement and extend earlier studies suggesting a role for HSPGs in neurite formation. The experiments also provide evidence for modulation of laminin by HSPGs which interact with laminin and promote neurite outgrowth that is mediated by a cell surface receptor at or in juxtaposition to the HNK-1 (Leu 7) epitope.
Embryonic assembly of a complex muscle is directed by a single identified cell in the medicinal leech
Authors: Jellies J , Kristan WB
Published: March 2018

The present study examines the morphological development of a highly organized muscle layer in the leech Hirudo medicinalis, in an effort to characterize those factors that are important in directing its assembly. The tubular body wall of the leech contains 3 major muscle layers that are anatomically distinct: an inner layer of longitudinal muscle, an outer layer of circular muscle, and a grid of oblique muscle sandwiched between them. The oblique muscle layer appears later in development than the other 2 and is preceded by several days by the development of a single, complex cell (here called the comb, or C-cell) whose shape strongly resembles the organization of the oblique muscle grid. There is a bilateral, mirror-image pair of C-cells in each segment. The C-cell has a central, longitudinally oriented soma and projects about 35 fine, parallel processes both medially and laterally at approximately 45 degrees to the long axis. Using a combination of intracellular and antibody labels, it was found that individual muscle cells align themselves with these processes to form correctly oriented fascicles during development. Photoablation of the C-cell at early stages resulted in the complete absence of all oblique muscle fascicles that would have corresponded to that cell; therefore, this discrete muscle-associated cell is considered to be an identified “muscle organizer.” Such cellular organizers may direct muscular and neuromuscular assembly in many species.
Structure of the intraspinal projections of single, identified muscle spindle afferents from neck muscles of the cat
Authors: Keirstead SA , Rose PK
Published: March 2018

The morphology and frequency of collaterals originating from single afferents supplying primary endings of muscle spindles in dorsal neck muscles have been examined using intra-axonal injections of HRP. Within the segment in which the afferent entered the spinal cord, one collateral was found for every 3.3 mm of stained axon. In contrast, afferents--one of more segments rostral to the segment in which they entered the spinal cord--had fewer collaterals: One collateral was found for every 6.3 mm of stained axon. The branching structure and terminal distribution of the collaterals were generally similar regardless of the muscle from which the afferent originated and the segment in which the collateral was found. Boutons were found in 2 zones: One of these was located in the intermediate zone, within and around the central cervical nucleus, and the other was found in laminae VIII and IX, including the motoneuron nuclei. The ventral termination zone of collaterals in the same segment as their parent axon entered the spinal cord was larger and had more boutons than the same projection of collaterals whose parent axon entered the spinal cord 1 or 2 segments caudal to the segment in which the collateral was found. These results indicate that afferents supplying primary endings of neck muscle spindles are more likely to contact neurons in the same segment in which the afferent enters the spinal cord than in more rostral segments. However, even within the same segment in which the afferent enters the spinal cord, the projection of neck muscle afferents to the ventral horn is less dense than the corresponding projection of hindlimb muscle spindle afferents in the lumbosacral spinal cord.
The location of cues promoting selective reinnervation of axolotl muscles
Authors: Wigston DJ , Donahue SP
Published: March 2018

The selective reinnervation of muscles suggests that muscles have intrinsic recognition cues that promote selective synaptogenesis. For example, the anterior and posterior heads of the axolotl iliotibialis (ILT) muscle are preferentially reinnervated by their original motoneurons even after surgically exchanging them. The nature and location of cues that promote such selectivity are unknown, although previous work suggests that the muscle fibers themselves might harbor the relevant molecules. To address this question, we removed anterior and posterior ILT muscles, destroyed their myofibers by surgically damaging them and treating them with bupivacaine, and reimplanted them in either a normal or a reversed anterior/posterior orientation. After the regenerated myofibers became innervated, we stimulated different spinal nerves and recorded the synaptic potentials evoked in muscle fibers. Our results showed that if the muscles were removed, damaged, and reimplanted in their original positions, the segmental origin of inputs to the regenerated myofibers was similar to that seen in normal muscles and in muscles reimplanted with their myofibers intact. However, muscles that were removed and damaged but regenerated in new positions were innervated differently from normal muscles and from muscles whose myofibers survived transplantation. Thus, the site at which a muscle regenerates has an influence on the source of the muscle's reinnervation. Nevertheless, the innervation of muscles that regenerated after transplantation to a foreign site was not strictly appropriate for the new position, but was biased towards the muscle's original innervation pattern. Therefore, some, but not all, of the cues that reflect the original identity of the transplanted muscles survive the replacement of its myofibers.