bluuebunny/crossref_metadata_2025 huggingface dataset:
Forebrain origins and terminations of the medial forebrain bundle metabolically activated by rewarding stimulation or by reward-blocking doses of pimozide
Authors: Gallistel CR , Gomita Y , Yadin E , Campbell KA
Published: March 2018

Using [14C]-2-deoxyglucose autoradiography, we determined which forebrain and diencephalic areas showed metabolic alterations in response to unilateral electrical stimulation of the posterior medial forebrain bundle at parameters chosen to produce a just-submaximal rewarding effect. At these parameters, only a few areas were activated. There was no detectable activation anterior or dorsal to the genu of the corpus callosum. Just anterior to the anterior commissure, there was strong activation of the vertical limb of the diagonal band of Broca, with a focus in the nucleus of the diagonal band. Just posterior to the anterior commissure, there was strong activation of compartment “c” of the medial forebrain bundle (MFB), with weaker activation of the bed nucleus of the stria terminalis and the medial preoptic area. At midhypothalamic levels, the dorsolateral, dorsomedial, and ventral MFB all showed activation. There was bilateral suppression of activity in the lateral habenula. Activation appeared to end in the anterior ventral tegmental area of Tsai. Reward-blocking doses of the neuroleptic pimozide activated the caudate and the lateral habenula but did not alter any of the unilateral effects of stimulation. Using longer pulse durations and/or shifting the site of stimulation to the substantia nigra activated many of the systems not activated in the first experiment, including all of the major dopaminergic projection systems, proving the capacity of the technique to reveal activation of these systems. The results permit one to define a discrete projection system that merits electrophysiological investigation as a likely substrate for the rewarding effect of MFB stimulation. They also suggest that dopaminergic projection systems may not form part of the reward pathway itself.
Development and characteristics of airstepping in chronic spinal cats
Authors: Giuliani CA , Smith JL
Published: March 2018

Airstepping, walking-like movements of the hindlimbs, is a commonly observed behavior in chronic spinal animals when they are held vertically. The purpose of this study was to: describe the development of airstepping after spinalization and compare it to the onset of segmental reflexes, characterize the EMG pattern of muscle activity during spontaneous airstepping, and examine the effects of sensory perturbation on the characteristic pattern. Airstepping was analyzed during three conditions of tonic sensory perturbation which included: tail pinching, tape applied to one hindpaw, and immobilization of the ankle and knee in a plaster cast. Seven adult cats were spinalized at T- 12, and bipolar electrode wires were surgically implanted in selected hindlimb muscles at the hip, knee, and ankle. Testing began within 48 hr of transection. Segmental reflexes and paw-shake responses were present in the first week; however, the earliest observed airstepping occurred during tail pinching at 2 weeks after surgery, and the average onset of spontaneous airstepping (without exteroceptive stimuli) was at 33 days. The average cycle period of spontaneous airstepping (691 msec) was comparable to the shortest periods reported for fictive rhythms and to treadmill walking between 1 and 2 m/sec. Intralimb coordination was characterized by flexor and extensor synergies typical of locomotion, while interlimb coordination was characterized by alternating cycles similar to that reported for treadmill walking and fictive locomotion. Neither intralimb nor interlimb patterns of coordination were altered by conditions of sensory perturbation, although cycle period and EMG recruitment level were variable. Many characteristics of airstepping are similar to those of treadmill and fictive locomotion.
Reflex neurogenic inflammation. I. Contribution of the peripheral nervous system to spatially remote inflammatory responses that follow injury
Authors: Levine JD , Dardick SJ , Basbaum AI , Scipio E
Published: March 2018

Recent studies of the mechanism of neurogenic inflammation have focused on the contribution of neuropeptides released from peripheral terminals of primary afferent sensory neurons. In this study we addressed the contribution of humoral and neural factors to the hyperalgesia and swelling that are produced contralateral to an injured hindpaw, a phenomenon which we refer to as reflex neurogenic inflammation. The contralateral inflammatory response develops gradually, over a period of hours, and shows no tachyphylaxis with repeated application of the same stimulus. Denervation of either limb significantly attenuated the contralateral responses. Selective lesions of small-diameter, presumed nociceptive afferent fibers with capsaicin, or of sympathetic postganglionic efferents by immunosympathectomy, also reduced swelling and hyperalgesia of the uninjured paw. Interruption of venous circulation to the injured limb by vein ligation did not alter the response in the contralateral paw. Taken together, these data suggest that reflex neurogenic inflammation is neurally mediated, via connections across the spinal cord.
Octopamine enhances dark-adaptation in Limulus ventral photoreceptors
Authors: O'Day PM , Lisman JE
Published: March 2018

Limulus ventral photoreceptors receive synaptic input from fibers that emerge from the brain and that appear to use octopamine as a transmitter. Exogenous application of octopamine has been shown to increase levels of intracellular cAMP in ventral photoreceptors, but the resulting physiological effects have been unclear. In this report, we show that octopamine increases the rate of dark-adaptation following a bright light. Since a similar increase in the rate of dark-adaptation is produced by IBMX (1 mM) and forskolin (100 microM), drugs shown previously to raise the concentration of cAMP, the octopamine effect may be mediated by cAMP. Our results suggest that dark-adaptation, a fundamental process of photoreceptors, is under efferent neural control.
Expression of noradrenergic and cholinergic traits by sympathetic neurons cultured without serum
Authors: Wolinsky EJ , Landis SC , Patterson PH
Published: March 2018

The ability to vary systematically the neuronal environment is one advantage afforded by the use of cell culture. Replacement of serum, a variable and undefined medium supplement, with known ingredients allows even greater control of culture conditions. We have studied biochemical and morphological properties related to neurotransmitter metabolism of rat sympathetic neurons cultured in a modified defined medium. Neuronal survival, ultrastructure, and expression of noradrenergic properties appear similar in serum-free and serum-supplemented cultures: small granular vesicles characteristic of norepinephrine storage were observed in both types of culture, and tyrosine hydroxylase activity, conversion of dopamine to norepinephrine, catecholamine production, and storage capacity are equivalent in serum-free and serum-containing cultures. Several of these properties were not exhibited at high levels in previous formulations of this defined medium. Acetylcholine production, however, was about 10-fold lower in serum-free compared to serum-supplemented cultures, consistent with the findings of lacovitti et al. (lacovitti, L., M. I. Johnson, T. H. Joh, and R. P. Bunge (1982) Neuroscience 7:2225–2239). Acetylcholine production can be induced under serum-free conditions by a previously characterized cholinergic inducing factor from heart cell conditioned medium. This responsiveness to serum-free heart cell conditioned medium indicates that serum-free cultures retain plasticity with respect to transmitter status, despite expression of noradrenergic characteristics, unlike cultured neurons of which the noradrenergic transmitter status is maintained by chronic depolarization. Thus, sympathetic neurons survive, express numerous differentiated properties, and display a novel transmitter status under serum-free conditions.
Rat serum contains a developmentally regulated cholinergic inducing activity
Authors: Wolinsky EJ , Patterson PH
Published: March 2018

Sympathetic neurons cultured in defined medium do not develop the ability to produce acetylcholine, as do neurons grown with serum supplementation (lacovitti, L., M. I. Johnson, T. H. Joh, and R. P. Bunge (1982) Neuroscience 7:2225–2239; Wolinsky, E. J., S. C. Landis, and P. H. Patterson (1985) J. Neurosci. 5: 1497–1508). The implication that rat serum contains cholinergic inducing activity is further explored here. Dependence of cholinergic induction on serum concentration is demonstrated, and the activity is shown to reside in a macromolecular fraction. Very little cholinergic inducing activity is present in serum obtained from animals younger than 9 postnatal days. This age dependence correlates with the time of transition from noradrenergic to cholinergic transmitter status by the sympathetic innervation of the rat sweat gland in vivo (Landis, S. C., and D. Keefe (1983) Dev. Biol. 98: 349–372).
Differential visualization of dopamine and norepinephrine uptake sites in rat brain using [3H]mazindol autoradiography
Authors: Javitch JA , Strittmatter SM , Snyder SH
Published: March 2018

Mazindol is a potent inhibitor of neuronal dopamine (DA) and norepinephrine (NE) uptake. DA and NE uptake sites in rat brain have been differentially visualized using [3H]mazindol autoradiography. At appropriate concentrations, desipramine (DMI) selectively inhibits [3H]mazindol binding to NE uptake sites without significantly affecting binding to DA uptake sites. The localization of DMI-insensitive specific [3H] mazindol binding, reflecting DA uptake sites, is densest in the caudate-putamen, the nucleus accumbens, the olfactory tubercle, the subthalamic nucleus, the ventral tegmental area, the substantia nigra (SN) pars compacta, and the anterior olfactory nuclei. In contrast, the localization of DMI-sensitive specific [3H]mazindol binding, representing NE uptake sites, is densest in the locus coeruleus, the nucleus of the solitary tract, the bed nucleus of the stria terminalis, the paraventricular and periventricular nuclei of the hypothalamus, and the anteroventral thalamus. The distribution of DMI- insensitive specific [3H]mazindol binding closely parallels that of dopaminergic terminal and somatodendritic regions, while the distribution of DMI-sensitive specific [3H]mazindol binding correlates well with the regional localization of noradrenergic terminals and cell bodies. Injection of 6-hydroxydopamine, ibotenic acid, or colchicine into the SN decreases [3H]mazindol binding to DA uptake sites in the ipsilateral caudate-putamen by 85%. In contrast, ibotenic acid lesions of the caudate-putamen do not reduce [3H]mazindol binding to either the ipsilateral or contralateral caudate-putamen. Thus, the DA uptake sites in the caudate-putamen are located on the presynaptic terminals of dopaminergic axons originating from the SN.
Myelination determines the caliber of dorsal root ganglion neurons in culture
Authors: Windebank AJ , Wood P , Bunge RP , Dyck PJ
Published: March 2018

In order to understand the relationship of supporting cells to the differentiation of neurons in culture, we have used morphometry to study myelination of dorsal root ganglion (DRG) neurons by central or peripheral supporting cells. Dissociated DRG cultures from 15-day rat embryos, free of Schwann cells and fibroblasts, were prepared, and supporting cells were added from spinal cord or DRG; myelination commenced after 2 weeks. Control cultures received no supporting cells. At 7, 14, and 24 days, a total of 22 cultures were processed for electron microscopy. Three fascicles from defined points were sampled from each culture. In cultures containing glial cells, smaller fibers (p less than 0.001) were myelinated (mean of median diameter, 1.13 +/- 0.13 (SD) micron) than in cultures containing Schwann cells (1.67 +/- 0.17 micron), although there was no difference (p greater than 0.1) in the degree of myelination expressed as number of myelin lamellae/fiber. A new finding concerned the relationship of axonal diameter to the presence or absence of myelinating cells. In control cultures without supporting cells or in areas where supporting cells were absent, the range of neurite diameter (0.05 to 1.25 micron) and the median diameter (mean of median, 0.24 +/- 0.03 micron) were similar at different times (7, 14, and 24 days), demonstrating a stable population of neurite diameters throughout the period. In myelinated fascicles, a different distribution of neurite diameters was present. Myelinated neurites had a greater median diameter (measured to inner border of myelin) and a different range of fiber diameters compared to bare neurites. For Schwann cells, this range was 0.7 to 3.4 micron, and the mean of median diameters was 1.67 +/- 0.17 micron; for glial cells, the range was 0.6 to 2.4 micron, and the mean of median diameters 1.13 +/- 0.13 micron. Differences between myelinated and bare fibers were all highly significant (p less than 0.001).
Response characteristics of mammalian cochlear hair cells
Authors: Dallos P
Published: March 2018

Intracellular recordings were made from the low frequency region (third turn) of the guinea pig cochlea. Response characteristics are compared to gross potentials obtained from the organ of Corti fluid space. Inner hair cells (IHCs) possess relatively low (median, -32 mV) initial membrane potentials, whereas that of outer hair cells (OHCs) is higher (median, -53.5 mV). In response to tone burst stimuli, both cell types produce a combination of AC and DC responses. The latter are depolarizing for IHCs but may be of either polarity for OHCs. In terms of their AC responses, IHCs are about 12 dB more sensitive than OHCs. At low sound levels these cells are more linear than high frequency hair cells (Russell, I. J., and P. M. Sellick (1978) J. Physiol. (Lond.) 284: 261–290), judging from the relation between AC and DC response components. At high sound levels pronounced response saturation is seen. The overall tuning properties of the two hair cell types are rather similar, even though IHCs exhibit low frequency velocity dependence, whereas OHCs are displacement sensitive and the cell membrane time constant is larger for IHCs. In order to fit IHC experimental data it is necessary to assume the presence of an underdamped complex pole above the best frequency. The electrical behavior of the OHC does not disqualify it as a conveyor of auditory information to the central nervous system, even though its primary function may be that of a mechanical effector (evidence summarized by Dallos, P. (1985) in Contemporary Sensory Neurobiology, Alan R. Liss, Inc., New York, pp. 207–230).
Appearance of the noradrenergic markers tyrosine hydroxylase and neuropeptide Y in cholinergic nerves of the iris following sympathectomy
Authors: Bjorklund H , Hokfelt T , Goldstein M , Terenius L , Olson L
Published: March 2018

Selective autonomic denervations of the iris have been used to study the possible redistribution of adrenergic markers within adult nerve fiber systems and to reveal the cellular origin of a nonsympathetic fiber plexus induced to express such markers. The presence and distribution of fibers showing neuropeptide Y (NPY)- and tyrosine hydroxylase (TH)-like immunoreactivity was studied in the rat iris using stretch-prepared whole mounts. Normal irides contained a dense regular network of NPY-positive varicose fibers. Such fibers were regularly seen innervating blood vessels. The choroid membrane had a high number of fluorescent fibers. A similar, although slightly denser TH-positive fiber system was visualized in the iris. One or 2 days after surgical removal of the superior cervical ganglion, almost all NPY- and TH-positive fibers had disappeared, suggesting that most, if not all, NPY-positive fibers in the iris originate in the superior cervical ganglion. In irides from long-term sympathectomized animals, a high number of TH- and NPY-immunoreactive fibers had reappeared, while such irides were devoid of catecholamine-containing fibers, as evidenced by Falck-Hillarp histochemistry. The appearance of TH- and NPY-positive fibers in sympathetically denervated irides was clearly time dependent. The distribution of fluorescent fibers in irides from intact and sympathectomized animals showed obvious dissimilarities such as a lower fluorescence intensity and fewer varicose fibers in denervated irides. Furthermore, in irides from sympathectomized rats, TH- and NPY-positive fibers were not associated with blood vessels. Unilateral removal of the parasympathetic ciliary ganglion, which supplies the iris with cholinergic fibers, 3 days prior to sacrifice in animals bilaterally sympathectomized 1 month earlier, led to a drastic reduction in numbers of TH- and NPY-positive iris fibers on the ciliarectomized/sympathectomized side as compared to the sympathectomized-alone side. The present experiments thus suggest that adult cholinergic neurons in vivo are capable of expressing adrenergic characteristics under experimental conditions.
The coordination of arm movements: an experimentally confirmed mathematical model
Authors: Flash T , Hogan N
Published: March 2018

This paper presents studies of the coordination of voluntary human arm movements. A mathematical model is formulated which is shown to predict both the qualitative features and the quantitative details observed experimentally in planar, multijoint arm movements. Coordination is modeled mathematically by defining an objective function, a measure of performance for any possible movement. The unique trajectory which yields the best performance is determined using dynamic optimization theory. In the work presented here, the objective function is the square of the magnitude of jerk (rate of change of acceleration) of the hand integrated over the entire movement. This is equivalent to assuming that a major goal of motor coordination is the production of the smoothest possible movement of the hand. Experimental observations of human subjects performing voluntary unconstrained movements in a horizontal plane are presented. They confirm the following predictions of the mathematical model: unconstrained point-to-point motions are approximately straight with bell-shaped tangential velocity profiles; curved motions (through an intermediate point or around an obstacle) have portions of low curvature joined by portions of high curvature; at points of high curvature, the tangential velocity is reduced; the durations of the low-curvature portions are approximately equal. The theoretical analysis is based solely on the kinematics of movement independent of the dynamics of the musculoskeletal system and is successful only when formulated in terms of the motion of the hand in extracorporal space. The implications with respect to movement organization are discussed.
Quantitative comparison of the structural features of slow and fast neuromuscular junctions in Manduca
Authors: Rheuben MB
Published: March 2018

The multiterminal slow and fast neuromuscular junctions of the moth Manduca sexta were compared using scanning, thin-section, and freeze- fracture techniques to see what structural features might underlie their functional differences. Slow neuromuscular junctions, here formed on tonic muscle fibers, produce a facilitating e.j.p. the amplitude of which is 1/5 to 1/3 the size of a fast excitatory junction potential (EJP) and the duration of which is nearly four times longer. A slow junction consists of a single terminal branch that is shorter in length than either of the pair of branches that a fast junction forms close together on the muscle fiber. Within the junction, slow nerve terminals exhibit longer, more frequent constrictions and are very varicose compared with fast. Since fast larval junctions on tonic muscle fibers are also varicose (Schaner and Rheuben, 1985), this is unlikely to represent an intrinsic property of the nerve. However, calculations of the length constants of the varicose versus nonvaricose shapes indicate that the effect of passive cable properties on normal functioning may act to limit the length of the slow terminals more than that of fast. Even though the varicose shape can be predicted to prolong the time course of the EJP, calculations show that, at the measured length, this would not explain the very long EJP that is observed. Within the neuromuscular junctions, the synapses are characterized on the muscle membrane by a patch of densely packed particles on the external leaflet and on the nerve membrane by a single linear active zone. The total number of synapses per slow junction is about 1/3 that of fast junctions. There is a weak correlation between average area of the individual postsynaptic particle patches and cross-sectional area of the muscle fibers that transcends nerve and muscle fiber types. The average lengths of active zones from the two types do not differ significantly. However, the number of particles per active zone in slow junctions is about 55% of the number in fast active zones. Chemically fixed slow nerve terminals have a greater density of synaptic vesicles remaining than do fast. If a proportion of the active zone particles represent structures directly involved in the probability of transmitter release, such as Ca++ channels, then the latter two characteristics may jointly reflect differences in capability to release and mobilize transmitter that would partly explain the different EJP amplitude and facilitation properties.
Immunocytochemical mapping of 1B236, a brain-specific neuronal polypeptide deduced from the sequence of a cloned mRNA
Authors: Bloom FE , Battenberg EL , Milner RJ , Sutcliffe JG
Published: March 2018

The 318-amino acid, carboxy-terminal sequence of the putative brain- specific polypeptide 1B236 was deduced from the nucleotide sequence of its cloned brain-specific mRNA. Antisera raised against selected synthetic peptide fragments of this protein were used to map the cellular location of the presumptive gene product in the brains of normal or colchicine-pretreated adult rats. Antisera directed against any of three C-terminally located, but nonoverlapping, nonhomologous, synthetic peptide segments (P5, P6, or P7) produced virtually identical maps of intensely immunoreactive neuropil staining. The immunoreactivity was distributed heterogeneously and was most pronounced within olfactory, somatosensory, and limbic systems, and was more modest in certain motor and auditory structures. In colchicine- pretreated rats, large, multipolar perikarya were observed within the amygdala, caudate-putamen, cingulate, parietal, and piriform cortices, as well as in particular diencephalic and pontine nuclei. Smaller immunoreactive neurons with more limited dendritic extensions were observed in the olfactory bulb, the cerebellar cortex, and the dorsal horn and intermediolateral cell columns of the spinal cord. No immunoreactivity was observed in visceral structures innervated by the autonomic nervous system or in non-neural tissues. In addition to the virtually superimposable maps produced by antisera to all three synthetic fragments selected from the C-terminus of 1B236, some uniquely reactive sites were seen. Antisera to the most N-terminal of the three synthetic immunogens (P5) were reactive with neurons of the medial trapezoid nucleus and in nerve terminals surrounding the deep cerebellar nuclei. Antisera against the most C-terminal synthetic immunogen (P7) were reactive with neurons of the paraventricular and supraoptic hypothalamic nuclei. These data demonstrate that the 1B236 protein is located within selected neuronal elements within functionally related cellular circuits established more formally by other methods. Our data show that protein 1B236-immunoreactive cells share at least the expression of this protein and suggest that these cells may also be related epigenetically or evolutionarily. These data, together with other subcellular, ultrastructural, and electrophysiological properties of 1B236, suggest that this protein could be considered as a prohormone capable of yielding several final candidate transmitter products.
Immunocytochemical studies of the peptidergic content of fibers and terminals within the lateral spinal and lateral cervical nuclei
Authors: Giesler GJ , Elde RP
Published: March 2018

As part of a series of studies in which we are attempting to determine the roles of the lateral spinal (LSn) and lateral cervical (LCn) nuclei in somatic sensation, we have examined the fibers and terminals within these nuclei in the rat using the indirect immunofluorescence technique. Eleven antisera were used. Within the LSn, antisera against dynorphin 1–8 (DYN), substance P (SP), and Met-enkephalin (ENK) produced labeling of a large number of processes in all segmental levels examined. Processes labeled with these antisera frequently apposed the cell bodies and dendrites of LSn neurons. Antisera against somatostatin (SOM) and FMRF-NH2 (FMRF) labeled smaller numbers of processes within the LSn. Few, if any, processes in the LSn were labeled using antisera against serotonin, cholecystokinin octapeptide, oxytocin, neurotensin, corticotrophin-releasing factor, and vasoactive intestinal polypeptide. In contrast to the LSn, the LCn contained virtually no labeled processes irrespective of the antiserum employed. An area was found adjacent to the LCn in the medial portion of the dorsal lateral funiculus (DLf) of C2 that resembled the LSn in several of its anatomical characteristics: like the LSn, the medial portion of the C2 DLf contained small multipolar neurons; it was similar to the LSn in its medial-lateral extent; and following staining with each antiserum, the LSn and the medial DLf of C2 contained a similar number of labeled processes. The peptide-containing area in the medial DLf of C2 was found to be continuous with the LSn. We therefore propose that this region is a rostral extension of the LSn.
Effects of changes in motor unit size on transmitter release at the frog neuromuscular junction
Authors: Herrera AA , Grinnell AD
Published: March 2018

The dependence of transmitter release and synaptic effectiveness on the size of a neuron's peripheral field was studied using neuromuscular junctions in sartorius muscles of adult frogs (Rana pipiens). The size of the peripheral field (motor unit size) was reduced by crushing the sartorius nerve and surgically removing half of the muscle fibers. Synapses thus formed were compared with those formed when crushed nerves reinnervated intact whole muscles, as well as with synapses in normal unoperated muscles. Indirect observations suggested that all motor axons participated in reinnervation and that motor unit size was indeed smaller in half-muscles. Synaptic safety margins, as measured by the sensitivity of nerve stimulus-evoked twitching to low Ca2+, were substantially higher in muscles with reduced motor units. These higher safety margins were due to enhanced evoked transmitter release. In Ringer solution containing Mg2+ and lowered Ca2+, total evoked release and evoked release per unit nerve terminal length were approximately 2- fold higher in muscles with reduced motor units, when studied 7 to 18 weeks postoperatively. A similar difference was seen when unblocked release was measured in a normal physiological solution, after blocking excitation-contraction coupling and muscle fiber action potential generation with formamide. Miniature endplate potential frequency in half-muscles was 2 to 3 times higher than in controls when tested in normal physiological solution, but was not significantly different in low Ca2+, Mg2+-containing solution. By 34 weeks postoperatively, there was no longer a difference in evoked release, even though the difference in motor unit size persisted.
Glutamic acid decarboxylase immunoreactivity in layer IV of barrel cortex of rat and mouse
Authors: Lin CS , Lu SM , Schmechel DE
Published: March 2018

The morphology and distribution of neurons and terminals that are immunoreactive to glutamic acid decarboxylase (GAD) were investigated in barrel cortex of the rat and mouse. The morphology of the GAD- immunoreactive neurons located in layer IV of the barrel field resembles that of the large, smooth stellate neurons described previously in Golgi studies. Most of the somas of GAD-positive neurons are located along the sides of the barrels. They constitute about 13 to 15% of the total neuronal population in layer IV. The spatial distribution of GAD-positive terminals in layer IV is similar to the distribution of GAD-positive somas. Very few GAD-positive neurons and terminals are found in the septal regions. This unique distribution of GAD immunoreactivity in the barrel cortex may serve as a model to study cortical inhibitory mechanisms.
Localized survival of ciliary ganglionic neurons identifies neuronotrophic factor bands on nitrocellulose blots
Authors: Carnow TB , Manthorpe M , Davis GE , Varon S
Published: March 2018

A novel and sensitive method has been developed to identify ciliary neuronotrophic factors (CNTFs) from tissue extracts after blotting to nitrocellulose paper. The CNTF proteins are required for the in vitro survival of embryonic chick ciliary ganglionic neurons. Tissue extracts containing such CNTFs are electrophoresed using sodium dodecyl sulfate- polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Purified ciliary ganglionic neurons are seeded on the surface of the nitrocellulose blot, and the culture is incubated for 24 hr in medium lacking CNTF. CNTF can be localized on the blot because it retains its ability to support the survival of the neurons cultured on the nitrocellulose. A band of viable neurons, easily visualized by staining with a vital dye, is supported by the blotted CNTF polypeptide. The number of neurons surviving on the blotted CNTF is related to the amount of CNTF originally loaded on the electrophoretic gel. As little as 2 ng (16 trophic units) of CNTF protein contained in crude tissue extracts can be loaded on the sodium dodecyl sulfate gel and still be recognized by the cultured neurons. This method was used to identify CNTF polypeptides from extracts of adult rat nerve (24,000 and 19,000 daltons) and from tissue found near experimentally induced adult rat brain lesions (24,000 daltons). The electrophoretic mobilities of these peptides are distinct from the previously purified chick eye CNTF polypeptide (20,400 daltons).
Localization of choline acetyltransferase within identified motoneurons of the nematode Ascaris
Authors: Johnson CD , Stretton AO
Published: March 2018

Choline acetyltransferase (CAT) activity has been measured in the nematode Ascaris lumbricoides. Strips of hypodermal tissue which contained branches of single identified motoneurons (Stretton, A.O.W., R.M. Fishpool, E. Southgate, J.E. Donmoyer, J.P. Walrond, and I.S. Kass (1978) Proc. Natl. Acad. Sci. U.S.A. 75: 3493–3497) as well as control strips containing no neural tissue were assayed. In strips of hypodermis which contained branches of excitatory motoneurons (classes DE1, DE2, and DE3), CAT activity was elevated 5 to 10 times compared to control levels. Branches of inhibitory motoneurons (classes D1 and V1) did not add to the level of CAT observed in the controls. These data provide additional support for the suggestion (e.g., del Castillo, J., W.C. deMello, and T. Morales (1963) Experientia 20: 141) that acetylcholine is an excitatory neurotransmitter at nematode neuromuscular junctions. The function of the low level of CAT observed throughout the hypodermis is unknown.
Dendritic transport. II. Somatofugal movement of neuronal lysosomes induced by colchicine: evidence for a novel transport system in dendrites
Authors: Gorenstein C , Ribak CE
Published: March 2018

The effect of colchicine injections on the ultrastructural localization of dipeptidyl peptidase II (Dpp II) was studied in the mitral cells of the rat olfactory bulb. In control animals, electron-dense reaction product representing Dpp II activity was observed in lysosomes, lipofuscin granules, short cisternae located close to the granular endoplasmic reticulum, and dense granules. Lysosomes and lipofuscin granules were the most intensely stained organelles. Dpp II-containing organelles were localized mainly to the cell body and were randomly distributed in the perikaryal cytoplasm. Twenty-four hours after a 100- micrograms intracerebroventricular colchicine injection, the distribution of Dpp II-containing organelles was drastically altered. Short cisternae and dense granules containing Dpp II reaction product were noticeably absent in these preparations. Lysosomes and lipofuscin granules were depleted from the perikaryal cytoplasm and were concentrated in dendrites. Lysosomes were observed to extend for considerable distances in dendrites where they acquired elongated and dumbbell shapes. The shapes of some of these labeled lysosomes gave the impression that they were actively being “pulled” into the dendrites. These results indicate that microtubules sequester lysosomes to the perikaryal cytoplasm and suggest the presence of a novel transport system responsible for the movement of lysosomes from the cell body to the dendrites.
Comparison of the distributions of ipsilaterally and contralaterally projecting corticocortical neurons in cat visual cortex using two fluorescent tracers
Authors: Segraves MA , Innocenti GM
Published: March 2018

Using the retrograde fluorescent tracers Fast Blue and Diamidino Yellow we have studied the callosal and ipsilateral corticocortical connections between the cat's area 17/18 border region and the posteromedial lateral suprasylvian visual area (PMLS), as well as the callosal connections of each of these regions with its contralateral homologue. The main goal was to determine whether single cortical neurons project with branching axons to more than one cortical target. In addition, the double-labeling technique enabled us to examine, within a single section of cortical tissue, the relative distributions of neurons with different cortical targets. Most corticocortical neurons labeled in the area 17/18 border region and in area PMLS projected to only one of the cortical injection sites tested. When two callosal neuron types were labeled in the same area, no double-labeled neurons were found. When ipsilateral corticocortical and callosal neurons were labeled in combination, a few double-labeled neurons were found in both cortical regions examined. The most common type of double- labeled neuron was located in area PMLS and projected bilaterally to the area 17/18 border region. Our findings regarding the laminar distributions of ipsi- and contralaterally projecting neurons are in agreement with previous studies. In addition, we have found that, for callosal neurons within the upper layers of areas 17 and 18, neurons projecting to the contralateral area 17/18 border are located in the lower half of layer II/III and in upper layer IV, whereas neurons projecting to contralateral area PMLS are restricted to the lower portion of layer II/III. In addition, for callosal neurons within the deep layers of area PMLS, neurons projecting to contralateral area PMLS are located throughout layers V and VI, whereas neurons projecting to the contralateral area 17/18 border are restricted to layer VI. There are numerous other possible targets for axon collaterals not examined in this paper. However, the scarcity of neurons with multiple projections demonstrated in this study reflects the high degree of specificity of cortical connectivity. This anatomical organization may be the basis for a precise channeling of differential information at the single neuron level.
Environmental influences in the development of neural crest derivatives: glucocorticoids, growth factors, and chromaffin cell plasticity
Authors: Doupe AJ , Landis SC , Patterson PH
Published: March 2018

The neural crest gives rise to three major adrenergic cell types: sympathetic principal neurons, adrenal chromaffin cells, and small intensely fluorescent (SIF) cells. All of these derivatives synthesize and store catecholamines, but they differ in numerous other characteristics. SIF cells appear intermediate in phenotype between the other two. We have examined the role of several environmental factors in the differentiation of sympathetic principal neurons and adrenal chromaffin cells. In previous studies of young rat adrenal chromaffin cells in dissociated cell culture, differentiated characteristics such as the presence of the enzyme phenylethanolamine N-methyltransferase (PNMT), epinephrine (E) synthesis, and large catecholamine storage vesicles were not well maintained. Here we describe long-term culture of chromaffin cells which, in the presence of micromolar glucocorticoid, maintained all of these characteristics. In addition, chromaffin cells of a variety of ages were found to be dependent on glucocorticoid for long-term survival in culture. In the absence of glucocorticoid, many adrenal chromaffin cells from neonatal rats could be rescued by nerve growth factor (NGF) administration. They extended neurites, as previously described by Unsicker and colleagues (Unsicker, K., B. Krisch, U. Otten, and H. Thoenen (1978) Proc. Natl. Acad. Sci. U.S.A. 75: 3498–3502). In contrast to previous studies, however, with long-term exposure to NGF these cells became indistinguishable from mature sympathetic neurons, as judged by the following morphological and biochemical criteria: increased cell size and loss of intense CA fluorescence in their cell bodies; acquisition of characteristic neuronal ultrastructure, including morphologically specialized synapses; loss of chromaffin granules, PNMT, and E synthesis; and acquisition of neuron markers, including tetanus toxin labeling and immunoreactivity to neurofilament protein. This conversion to neurons was markedly enhanced by addition of a non-neuronal cell conditioned medium (CM) containing a neurite-promoting factor, which acted by increasing the NGF responsiveness of the chromaffin cells. Even chromaffin cells from adult rats, which are known to grow few processes in response to NGF alone, became neuronal in the presence of this CM plus NGF. While converting to neurons, adrenal chromaffin cells transiently assumed an intermediate phenotype resembling type I SIF cells, which suggests particular developmental relationships between the different cell types of the sympathoadrenal lineage.
Small intensely fluorescent cells in culture: role of glucocorticoids and growth factors in their development and interconversions with other neural crest derivatives
Authors: Doupe AJ , Patterson PH , Landis SC
Published: March 2018

The neural crest gives rise to a number of adrenergic derivatives, including sympathetic neurons and adrenal chromaffin cells, which contain catecholamines (CAs) but differ in other morphological and functional characteristics. Small intensely fluorescent (SIF) cells, which exist primarily as a minority cell population in autonomic ganglia, are a third cell type in the sympathoadrenal branch of the neural crest lineage. In some respects these cells appear intermediate in phenotype between sympathetic neurons and adrenal chromaffin cells. We established pure dissociated cell cultures of SIF cells from rat superior cervical ganglia (SCG) and used these to study the role of environmental factors in SIF cell development and the relationship of these cells to the other cell types of the sympathoadrenal lineage. When cells from neonatal rat SCG were grown for 3 weeks in the presence of glucocorticoid and in the absence of nerve growth factor (NGF), pure cultures of SIF cells developed. The properties of the cells included (i) small cell size and the occasional presence of short neurites, (ii) intense CA histofluorescence and immunoreactivity for CA synthetic enzymes, (iii) synthesis and storage of CA from radioactive precursors, and (iv) characteristic ultrastructure. The concentration of the glucocorticoid and the presence or absence of non-neuronal cell factors influenced which types of SIF cells developed. In micromolar glucocorticoid most of the cells resembled adrenal chromaffin or type II SIF cells: they displayed immunohistochemically detectable phenylethanolamine-N-methyltransferase (PNMT), synthesized and stored epinephrine, and contained large granular vesicles (100 to 300 nm). When SCG cells were grown in 10(-8) M hormone, many fewer SIF cells developed and a higher percentage of these lacked PNMT immunoreactivity, had neurites, and contained vesicles of smaller mean diameter (70 to 130 nm), similar to those of type I SIF cells in vivo. In the presence of conditioned medium (medium conditioned by non- neuronal cells) as well as glucocorticoid, virtually all of the cells morphologically resembled type I SIF cells. In the absence of glucocorticoid, no SIF cells were ever observed after 3 weeks in culture. By following the development of CA histofluorescence and SIF cell ultrastructure in the cultures over time, we demonstrated that SIF cells were not present in large numbers in these cultures immediately after plating, but were induced from an apparently undifferentiated precursor by the hormonal environment, whereas most of the principal neurons died.
Glucocorticoid receptors and regulation of phenylethanolamine-N- methyltransferase activity in cultured chromaffin cells
Authors: Kelner KL , Pollard HB
Published: March 2018

Glucocorticoids are known to regulate the enzyme phenylethanolamine-N- methyltransferase (PNMT) in the adrenal medulla of the rat and are thereby thought to control the synthesis of epinephrine. We have examined the details of this relationship in a simplified system, chromaffin cell primary cultures derived from bovine adrenal medulla. Cultured chromaffin cells were found to have a cytosolic, high affinity, saturable glucocorticoid-binding protein with the steroid specificity of a classical glucocorticoid receptor and a Kd of approximately 1 nM. Treatment of cultured cells with dexamethasone or hydrocortisone at any time up to 21 days in culture increased PNMT activity in the soluble fraction of the cell. The concentration of hormone required to produce a half-maximal response was 10 nM dexamethasone when cells were cultured in the presence of 5% fetal calf serum, or 1 nM in a defined serum-free medium. These dose-response relationships are consistent with mediation of this effect by the glucocorticoid receptor. Unexpectedly, however, the glucocorticoid- induced increment in PNMT activity was not inhibited by cycloheximide at concentrations up to 50 microM, and an acceleration of protein synthesis by insulin treatment did not augment the glucocorticoid effect on PNMT. Treatment of the cells with dexamethasone (100 microM) prevented the decline in the epinephrine-to-norepinephrine ratio seen over time in culture, an effect consistent with increased PNMT activity. However, there was no effect of dexamethasone on the ability of the cells to secrete catecholamines in response to stimulation with high KCl or 30 microM nicotine.
On the mechanism of acetylcholine receptor accumulation at newly formed synapses on chick myotubes
Authors: Role LW , Matossian VR , O'Brien RJ , Fischbach GD
Published: March 2018

We have examined the specificity and the mechanism of acetylcholine receptor (AChR) accumulation at embryonic chick nerve-muscle contacts that form in culture. Spinal cord motoneurons were identified in vitro after labeling them in vivo with Lucifer Yellow-wheat germ agglutinin conjugates. All of their processes induced receptor clusters on contacted myotubes; after 24 to 48 hr of co-culture, the incidence of neurite-associated receptor patches (NARPs) was approximately 1.2/100 microns of contact. In contrast, NARPs were rarely associated with spinal cord interneurons (approximately 0.1/100 microns of contact). Neurons dissociated from ciliary ganglia induce NARPS to the same extent as motoneurons. The relative contribution to NARPs of AChRs present in the membrane prior to plating ciliary ganglion neurons and of “new” AChRs inserted 8, 11, or 17 hr after addition of neurons was assessed with two fluorescent receptor probes. Rhodamine-conjugated alpha-bungarotoxin was used to label either old or new receptors; a monoclonal, anti-receptor antibody visualized with fluorescein-second antibody was used to label all (new and old) receptors. Analysis of digitized fluorescence images showed that NARPs contained both new and old receptors but that within the first 24 hr of co-culture the majority (60 to 80%) were new. We estimate that cholinergic neurites increase the rate of receptor insertion 4- to 5-fold during the first 8 hr of NARP formation. The contribution of new receptors to NARPs declines with time. After 3 days of co-culture, receptors inserted over an 8-hr interval comprised only 20% of the total NARP complement.
Synaptic transmission between dorsal root ganglion and dorsal horn neurons in culture: antagonism of monosynaptic excitatory postsynaptic potentials and glutamate excitation by kynurenate
Authors: Jahr CE , Jessell TM
Published: March 2018

Intracellular recording techniques have been used to provide information on the identity of excitatory sensory transmitters released at synapses formed between dorsal root ganglion (DRG) and dorsal horn neurons maintained in cell culture. Explants of embryonic rat DRG were added to dissociated cultures of embryonic dorsal horn neurons and synaptic potentials were recorded intracellularly from dorsal horn neurons after DRG explant stimulation. More than 80% of dorsal horn neurons within 1 mm of DRG explants received at least one fast, DRG- evoked, monosynaptic input. In the presence of high divalent cation concentrations, the acidic amino acid receptor agonists, L-glutamate, kainate, and quisqualate excited all dorsal horn neurons which received a monosynaptic DRG neuron input, whereas aspartate and N-methyl-D- aspartate (NMDA) had little or no action. Several compounds reported to antagonize the actions of acidic amino acids were tested for their ability to block DRG-evoked synaptic potentials and glutamate-evoked responses in dorsal horn neurons. 2-Amino-5-phosphonovalerate, a selective NMDA receptor antagonist, was relatively ineffective at antagonizing DRG-evoked synaptic potentials and glutamate-evoked responses. In contrast, kynurenate was found to be a potent antagonist of amino acid-evoked responses and of synaptic transmission at all DRG- dorsal horn synapses examined. The blockade of synaptic transmission by kynurenate appeared to result from a postsynaptic action on dorsal horn neurons. These findings indicate that glutamate, or a glutamate-like compound, but not aspartate, is the excitatory transmitter that mediates fast excitatory postsynaptic potentials at the DRG-dorsal horn synapses examined in this study.
FMRF-amide-like substances in the leech. II. Bioactivity on the heartbeat system
Authors: Kuhlman JR , Li C , Calabrese RL
Published: March 2018

In the preceding paper (Kuhlman, J. R., C. Li, and R. L. Calabrese (1985) J. Neurosci. 5: 2301–2309) FMRF-amide-like immunoreactivity was localized to a specific set of neurons in the leech. Three types of these neurons are involved in controlling the animal's heartbeat: HE motor neurons and HA modulatory neurons which directly innervate the hearts, and the swim-initiating interneurons (cells 204) which can accelerate the heartbeat central pattern generator. Application of synthetic FMRF-amide had effects on the hearts and the heartbeat central pattern generator that mimicked the actions of the HA and cell 204 neurons. Bath application of FMRF-amide (10(-7) to 10(-6) M) to the hearts activated their myogenic rhythm and increased their beat tension, thus mimicking the effects of activity in HA cells. Bath application of lower concentrations of FMRF-amide (10(-9) to 10(-8) M) to the isolated central nervous system dramatically accelerated the central motor program for heartbeat, thus mimicking the effects of activity in cell 204. These observations suggest that an FMRF-amide- like substance may be used as a chemical signal by HA and cell 204 neurons. The role of the FMRF-amide-like substance contained in HE motor neurons remains unclear, but it may be released along with the HE cell's neuromuscular transmitter, acetylcholine.