bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of oxotremorine on local glucose utilization in the rat cerebral cortex
Authors: Dam M , Wamsley JK , Rapoport SI , London ED
Published: March 2018

The [14C]2-deoxy-D-glucose technique (Sokoloff, L., M. Reivich, C. Kennedy, M. Des Rosiers, C. Patlak, K. Pettigrew, O. Sakurada, and M. Shinohara (1977) J. Neurochem. 28: 897–916) was used to examine the effects of central muscarinic stimulation on local cerebral glucose utilization (LCGU) in the cerebral cortex of the unanesthetized rat. Systemic administration of the muscarinic agonist oxotremorine (OXO, 0.1 to 1.0 mg/kg, i.p.) increased LCGU in the neocortex, mesocortex, and paleocortex. In the neocortex, OXO was more potent in elevating LCGU of the auditory, frontal, and sensorimotor regions compared with the visual cortex. Within these neocortical regions, OXO effects were greatest in cortical layers IV and V. OXO effects were more dramatic in the neocortex than in the meso- or paleocortex, and no significant effect occurred in the perirhinal and pyriform cortices. OXO-induced LCGU increases were not influenced by methylatropine (1 mg/kg, s.c.) but were antagonized completely by scopolamine (2.5 mg/kg, i.p.). Scopolamine reduced LCGU in layer IV of the auditory cortex and in the retrosplenial cortex. The distribution and magnitude of the cortical LCGU response to OXO apparently were related to the distributions of cholinergic neurochemical markers, especially high affinity muscarinic binding sites.
Light microscopic localization of brain opiate receptors: a general autoradiographic method which preserves tissue quality
Authors: Herkenham M , Pert CB
Published: March 2018

A general technique is described for using slide-mounted unfixed tissue sections to characterize and visualize drug and neurotransmitter receptors in brain or other tissues. The preparation of material, from fresh frozen, unfixed brain to dried sections securely attached to slides, is described in detail. The tissue can be kept intact during incubation at varying temperatures in solutions containing radiolabeled ligand, ions, buffers, and allosteric effectors. Strategies are described for determining optimal stereospecific binding with highest signal-to-noise ratios and for determining that a “meaningful” receptor is being studied. Dry formaldehyde fixation by vapors from heated paraformaldehyde preserves the tissue quality and traps the ligand near its site on the receptor, permitting subsequent histological processing through alcohols, solvents, and aqueous media, including liquid nuclear track emulsion. Visualization of [3H]naloxone- or [3H]enkephalin- labeled opiate receptor distributions in rat and human brains is achieved by tritium-sensitive film or by classical “wet” emulsion autoradiography. The advantages of the film include its ease of use and the ability to quantify receptor density by densitometry which can be computer-assisted. The advantage of the emulsion is the greater resolution and the concomitant appearance of morphology in cell-stained sections. Examples of correlations of opiate receptor distributions which underlying cytoarchitecture illustrate the potential for receptor localization studies.
Hippocampal resections impair associative learning and recognition memory in the monkey
Authors: Mahut H , Zola-Morgan S , Moss M
Published: March 2018

Damage to the hippocampus has been implicated in the permanent loss of memory in patients with medial temporal lobe resections. In two previous studies, it was established that bilateral ablations of the hippocampus in the monkey impaired performance on an associative learning task and on an object discrimination retention task. The two objectives of the present study were to assess the long term effects of hippocampal resections in the monkey and to extend the analysis of the effects of these resections to recognition memory. Therefore, the performance of monkeys with either hippocampal ablations or fornix transections, sustained 5 years earlier, was compared (1) on a concurrent discrimination task--a previously unencountered associative learning task--and (2) on a nonmatching-to-sample recognition task with either delays interposed between the presentation of the sample object and the recognition trial or with lists of either 1-, 3-, 5-, or 10- object samples. Significant impairment on both tasks was found after hippocampal, but not after fornix, damage. Though monkeys in the hippocampal group were impaired on both delays and lists, the impairment was more severe on the lists, with abnormal sensitivity to pro- and retroactive interference as a possible source of difficulty. Thus, in parallel with clinical findings, ablations of the hippocampus in the nonhuman primate produce an enduring disruption of memory.
Autoradiographic localization of adenosine receptors in rat brain using [3H]cyclohexyladenosine
Authors: Goodman RR , Synder SH
Published: March 2018

Adenosine (A1) receptor binding sites have been localized in rat brain by an in vitro light microscopic autoradiographic method. The binding of [3H]N6-cyclohexyladenosine to slide-mounted rat brain tissue sections has the characteristics of A1 receptors. It is saturable with high affinity and has appropriate pharmacology and stereospecificity. The highest densities of adenosine receptors occur in the molecular layer of the cerebellum, the molecular and polymorphic layers of the hippocampus and dentate gyrus, the medial geniculate body, certain thalamic nuclei, and the lateral septum. High densities also are observed in certain layers of the cerebral cortex, the piriform cortex, the caudate-putamen, the nucleus accumbens, and the granule cell layer of the cerebellum. Most white matter areas, as well as certain gray matter areas, such as the hypothalamus, have negligible receptor concentrations. These localizations suggest possible central nervous system sites of action of adenosine.
Neuropeptide proctolin associated with an identified skeletal motoneuron
Authors: O'Shea M , Bishop CA
Published: March 2018

Our objective was to find large and identifiable peptide-containing neurons and their postsynaptic targets. For this, a whole mount immunohistochemical method was used to locate cell bodies of neurons in the cockroach central nervous system immunoreactive to antibodies raised against the pentapeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH). The morphology and projections of the immunoreactive neurons were investigated by combining intracellular dye injection and electrophysiological and immunochemical procedures. The presence of proctolin in specific immunoreactive neurons was check by high pressure liquid chromatography combined with bioassay applied to extracts made from individually identified and isolated cell bodies. Using these approaches, we have identified a large proctolin-containing motoneuron which innervates muscles in a proximal segment of the cockroach leg. This study establishes a well characterized cellular preparation in which the physiological actions and functions of a peptide-containing neuron can be studied in vivo.
Septohippocampal cholinergic neurons are regulated trans-synaptically by endorphin and corticotropin neuropeptides
Authors: Botticelli LJ , Wurtman RJ
Published: March 2018

The content of acetylcholine (ACh) in nerve terminals or the dorsal hippocampus was examined after intraventricular, intraseptal, or intrahippocampal administration of a variety of endorphin/corticotropin neuropeptides. beta-Lipotropin, alpha-endorphin, gamma-endorphin, alpha- melanotropin, beta-melanotropin, adrenocorticotropin-1–39 (ACTH1–39), and ACTH4–10 (1, 3, 10, or 30 micrograms each) did not affect levels of ACh in the hippocampus 30 min after injection into the lateral ventricle. beta-Endorphin, administered intraventricularly (1, 3, 10, or 30 micrograms) or intraseptally (1 microgram), increased levels of ACh, while ACTH1–24, injected similarly, decreased levels of the neurotransmitter. ACh concentrations remained unchanged after direct application of beta-endorphin or ACTH1–24 (1, 3, 10, of 30 micrograms each) into Ammon's horn. Acute unilateral transection of the fimbria/superior fornix resulted in a time-related decrease in hippocampal ACh concentrations. Levels of ACh did not change 1 hr after transection; however, concentrations of hippocampal ACh decreased significantly 1 d or 1 week after deafferentation. ACh levels in the contralateral hippocampus remained unaffected at all times tested. Fimbrial transection blocked fully both endorphin- and corticotropin- induced changes in hippocampal ACh after the neuropeptides were injected into the lateral ventricle or the septal region. Naloxone, which, after subcutaneous (1 mg/kg) or intraventricular (100 micrograms) injection alone, failed to change levels of hippocampal ACh, antagonized the effects of intraventricular or intraseptal beta- endorphin or ACTH1–24 or hippocampal ACh levels. The results suggest a site of endorphin/corticotropin receptor interaction at the level of cholinergic cell bodies in the septal region for regulating the activity of septohippocampal cholinergic neurons.
The premotor cortex of the monkey
Authors: Weinrich M , Wise SP
Published: March 2018

The relationship of single unit activity to limb movements guided by visuospatial cues supports the view that the premotor cortex is a distinct cortical field within the somatic sensorimotor cortex. The premotor cortex is similar to the precentral motor cortex (MI) in that most of its units are clearly related to voluntary movements but differs from MI by its higher threshold for microstimulation-evoked movements, its cytoarchitecture, and the presence of a larger population of neurons with activity related to the occurrence of visuospatial signals rather than, or in addition to, the movement cued by those signals.
Defective cyclic adenosine 3':5'-monophosphate phosphodiesterase in the Drosophila memory mutant dunce
Authors: Kauvar LM
Published: March 2018

A detailed characterization of the cyclic nucleotide phosphodiesterase (PDEs) from normal Drosophila melanogaster was made, including purification of the two major enzymes to near homogeneity. A third more labile phosphodiesterase also was identified in crude homogenates. The total activity per fly of one of these three enzymes, PDE-II, is strongly influenced by the dunce locus. Two independently derived dunce mutants produce variations of PDE-II with modified intrinsic properties: a marked decrease of thermal stability in dunce and a 10- fold increase in the Michaelis kinetic constant in dunce. These defects, which persisted in purified preparations of PDE-II, were mapped genetically to dunce. The results support the identification of dunce as the structural locus for PDE-II. The tight connection between the dunce gene and the PDE-II enzyme indicates that defective cyclic adenosine 3′:5′-monophosphate metabolism is the primary lesion which leads to failure of dunce flies to learn in the olfactory associative conditioning paradigm of Quinn et al. (Quinn, W. G., W. A. Harris, and S. Benzer (1974) Proct. Natl. Acad. Sci. U. S. A. 71: 708–712).
Transneuronal and peripheral mechanisms for the induction of motor neuron sprouting
Authors: Rotshenker S
Published: March 2018

After injury to the nerve to one cutaneous pectoris muscle of the frog, the intact nerve to the contralateral muscle sprouts and forms additional synaptic connections with already innervated muscle fibers. It has been suggested (Rotshenker, S. (1979) J. Physiol. (Lond.) 292: 535–547; Rotshenker, S., and F. Reichert (1980) J. Comp. Neurol. 193: 413–422) that axotomy initiates a signal for sprouting in the injured neurons that is transferred transneuronally across the spinal cord to intact motor neurons. The present study was designed to test the hypothesis that axotomy initiates the signal for sprouting by interfering with some trophic signaling between the injured neurons and denervated muscle. Colchicine therefore was applied to the nerve to the left muscles to inhibit axonal transport on whose integrity trophic interactions depend. Consequently, supernumerary innervation developed in contralateral right intact muscles much the same as after axotom. Surprisingly, axons that were exposed to the drug also sprouted and formed synapses. Furthermore, the sprouting response of axons that were exposed to the drug also was produced in nerve fibers that were separated from their cell bodies. These results suggest two ways in which colchicine may produce sprouting and synapse formation and thereby suggest two mechanisms by which motor neurons may be induced to sprout: (a) transneuronally, by presenting growth stimuli to their cell bodies and central processes in the central nervous system and (b) by presenting growth stimuli to their peripheral extensions.
Discharge rate and excitability of cortically projecting intralaminar thalamic neurons during waking and sleep states
Authors: Glenn LL , Steriade M
Published: March 2018

Spontaneous firing and antidromically or synaptically evoked discharges of 89 single neurons in centralis lateralis-paracentralis (CL-Pc) intralaminar thalamic nuclei were examined during waking and sleep states in behaving cats with chronic pontine lesions. Twenty-four neurons were activated synaptically at short latencies from the midbrain reticular formation (MRF) after anterograde degeneration of passing fibers. Sixty-five neurons were identified antidromically as projecting to motor or parietal association cortical areas; of them, 23 also could be excited synaptically from the MRF. These neurons were regarded as possibly being involved in the transfer toward the neocortex of the tonic excitation from the MRF during EEG- desynchronized behavioral states. Rates of spontaneous discharge in CL- Pc neurons doubled from synchronized sleep (S) to either wakefulness (W) or desynchronized sleep (D). First order measures of discharge patterns indicated that interval modes in both W and D states (greater than 10 msec) are significantly different from those in S (2.5 msec). During S, the intervals found in the less than 5-msec class indicated the intraburst frequencies; a later minor mode (200 to 350 msec) reflected the interburst silent periods. All neurons tested for antidromic activation from cortical areas had enhanced responsiveness in both W and D states as compared to S sleep. In some cases, the enhanced antidromic excitability was observed in conjunction with a transformation from initial segment spikes during S to full spikes in EEG-desynchronized states. During both W and D states, compared to S sleep, the probability of monosynaptically elicited single discharges to MRF stimulation was increased, and the latency and duration of high frequency bursts evoked by MRF volleys were shortened. We conclude that the features of cortically projecting intralaminar neurons that relay MRF activity fit in well with their hypothesized role in the tonic activation processes that characterize both W and D states. Several lines of evidence suggest that sustained hyperpolarization prevails in intralaminar neurons during S sleep. This is the basic prerequisite for thalamic bursting. The effect of long lasting inhibitory potentials in thalamic neurons provides a mechanism for closing sensory channels during S sleep.
Estradiol receptor levels in rat hypothalamic and limbic nuclei
Authors: Rainbow TC , Parsons B , MacLusky NJ , McEwen BS
Published: March 2018

The amount of cytoplasmic receptor for the steroid hormone, estradiol (E2), was determined in 46 nuclei and subdivisions of rat brain. Individual nuclei were removed from 300-micrometers frozen sections according to the punch-out method of Palkovits (Palkovits, M. (1973) Brain Res. 59: 449–450), and the content of E2 receptor was measured with a sensitive radioligand binding method. Cytoplasmic receptors for E2 were distributed heterogeneously throughout the rat brain. The highest level of receptor (40 fmol/mg of protein) was found in the periventricular nucleus of the preoptic area, while low (1 fmol/mg) but detectable levels of receptors were found in such limbic regions as the nucleus of the diagonal band, the olfactory tubercle, and the cingulate cortex. Regions that were devoid of detectable receptor included the medial septum, the parietal cortex, and the ventral thalamus. Our results support the notion that E2 influences reproductive behavior and neuroendocrine function by binding to receptors in discrete areas of the brain and provide the first quantitative map of E2 receptors in individual rat brain nuclei.
Granule cell as a site of gene action in the weaver mouse cerebellum: evidence from heterozygous mutant chimeras
Authors: Goldowitz D , Mullen RJ
Published: March 2018

Experimental mouse chimeras were used to determine the site(s) of gene action in the weaver mutant cerebellum. Chimeras containing mixtures of heterozygous weaver (wv/+) and non-weaver (+/+) cells were produced by the standard embryo aggregation technique. The non-weaver component of the chimera was chosen so that Purkinje cells or granule cells could be distinguished histologically from weaver Purkinje or granule cells. Levels of beta-glucuronidase activity were used to mark Purkinje cells, with the weaver strain having a high beta-glucuronidase activity, while the non-weaver strain had low beta-glucuronidase activity. The increased centralized clumping of heterochromatin in ichthyosis (ic) mutant mice compared to non-ic mice was used to mark granule cell populations. In the weaver chimera, there was a decreased cerebellar size, decreased numbers of Purkinje and granule cells, and increased ectopic Purkinje and granule cells compared to non-weaver, control mice. With the glucuronidase cell marker, it was found that there was no correlation between ectopia and genotype; that is, genetically normal cells, as well as weaver cells, were found in ectopic positions. Thus, the weaver gene acts extrinsic to the Purkinje cells in creating the ectopia characteristic of heterozygous weaver mutants. Analysis of the ectopic granule cells, however, revealed that 100% of the ectopic granule cells were from the weaver component of the chimera. Thus, the weaver gene intrinsically affects granule cells in causing ectopia. Other hypothetical sites of gene action would produce a genetically mixed population of ectopic granule cells, which was not the case in this study. These findings are discussed in relation to other abnormalities in the heterozygous weaver mutant and in regard to the Bergmann glia and homozygous mutant. Finally, speculations on the nature of the granule cell deficit are discussed briefly.
Effects of temperature on identified central neurons that control jumping in the grasshopper
Authors: Abrams TW , Pearson KG
Published: March 2018

Grasshoppers, like many poikilotherms, are generally more active at warmer body temperatures. In particular, they jump more frequently when warm. To determine the neuronal basis of this increase in jumping activity, we investigated the effects of temperature on the properties of identified central neurons known to be involved in the control of the jump; these included the fast extensor tibiae (FETi) motoneuron and the C, G, and M interneurons. Heating did not result in a reduction in the current or voltage threshold for action potentials; in most cases, there was an increase in the current threshold with heating. At higher temperatures, the frequency-current relations of interneurons and motoneurons had steeper slopes. With strong current pulses, increasing the temperature resulted in an increase in the initial peak firing frequencies of central neurons and usually also in their steady state firing frequencies. A second temperature effect favoring increased CNS activity in warm grasshoppers was increased afferent input from the periphery. In a broad variety of sensory receptors, there was a dramatic increase in their sensitivity to sensory stimuli at both threshold and suprathreshold intensities. Various identified central neurons differed in the way in which some of their properties were influenced by temperature. The C and G interneurons showed a striking similarity in the unusual way in which their repetitive firing properties were influenced by heating. Since these neurons are sibling progeny of a single neuroblast, this shared physiological property is correlated with their developmental history.
Evidence that lectin-binding sites are present on the surface of isolated hypothalamic granules containing luteinizing hormone-releasing hormone
Authors: Barnea A , Cho G
Published: March 2018

In the present study, we wished to ascertain if lectin-binding sites are present on hypothalamic granules containing luteinizing hormone- releasing hormone (LHRH). LHRH granules (isolated from homogenates of hypothalami of adult male rats by means of hypo-osmotic shock and differential centrifugation) were subjected to affinity chromatography on columns of concanavalin A (Con A)-Sepharose. The amount of LHRH present in the unbound and bound fraction was measured by radioimmunoassay. Of the total LHRH present in the granule suspension chromatographed, 30 to 40% was bound to the columns, and this binding was not related to the binding of the bulk to the proteins. Synthetic LHRH by itself or synthetic LHRH added to the LHRH granules before chromatography did not bind to Con A-Sepharose, indicating that only LHRH contained within granules is retained on the columns. In addition, we found that the binding of LHRH granules to Con A-Sepharose has a requirement for Ca2+ and Mn2+: after equilibration of Con A-Sepharose with Ca2+ and Mn2+ (prior to the chromatography), all of the LHRH granules were bound to the columns, and this binding was prevented by EDTA. To examine the specificity of the binding of LHRH granules to Con A-Sepharose, a competing sugar (alpha-methyl-D-mannoside (alpha-MM)) was added to the LHRH granules, and the columns were equilibrated with alpha-MM prior to chromatography. Under this condition, the Ca2+- and Mn2+-dependent binding of LHRH granules to Con A-Sepharose was inhibited. In addition to Con A-Sepharose, we observed that LHRH granules bind to wheat germ agglutinin-Sepharose but not to Sepharose which does not contain a lectin. These findings are indicative that lectin-binding sites, carbohydrate in nature, are present on the surface of isolated LHRH granules. We propose that such binding sites may play a role in the release of LHRH from hypothalamic neurons.
Characterization and localization of adenosine 3':5'-monophosphate- binding proteins in the nervous system of Aplysia
Authors: Eppler CM , Palazzolo MJ , Schwartz JH
Published: March 2018

Earlier work in Aplysia californica has indicated that sensitization of the gill reflex, a simple form of learning, is produced by cAMP- dependent protein phosphorylation which regulates the flux of ions in sensory neurons of the abdominal ganglion. These changes in ion flux result in the enhanced release of neurotransmitter from synapses of the sensory neurons which, in turn, mediate the behavior. Because it can be presumed that protein phosphorylation regulates the functioning of ion channel proteins, we have characterized cAMP-binding proteins photoaffinity labeled with 8-N3-cAMP and have found that, unlike other tissues, the nervous system contains a great variety of binding species. Also unlike other tissues, several of the binding proteins in neurons are associated with membrane, and these components are concentrated in fractions enriched in nerve endings. Selectivity of phosphorylation, not only between substrates in cytosol and membrane but also between different regions of the cell, is thus possible because of the variety of cAMP-binding proteins in neurons. We think that these membrane-associated binding proteins are the most likely candidates for the regulatory subunits of the cAMP-dependent kinases that control the functioning of ion channel proteins at the synapse.
Regeneration and functional reconnection of an identified vertebrate central neuron
Authors: Lee MT
Published: March 2018

I have examined the axonal regeneration of a pair of identified central neurons, the Mauthner (M) neurons, in Xenopus laevis tadpoles. Lucifer Yellow injections reveal regenerative sprouts arising from the proximal stumps of the M axons within a few days after axotomy; some of these can cross the lesion within 1 week. Many specimens examined at later times (up to 21 weeks) have processes that extend more than 2 mm (equivalent to 5 to 10 spinal segments) beyond the lesion. M axons which have regenerated caudal to the lesion can re-establish functional synaptic contacts with their normal targets, spinal motor neurons. Functional reconnection has been demonstrated as early as 9 days after axotomy and as far as 10 segments caudal to the lesion. In most of the specimens tested, the regenerating M axons appear to exhibit the same degree of specificity for appropriate postsynaptic targets as normal, untransected M axons. M axons retain the ability to generate throughout the range of stages included in this study. The results provide evidence that a return of normal function in the transected vertebrate spinal cord can be mediated by the reconnection of a regenerating neuron with its normal targets.
Response of neurons in the cat's lateral geniculate nucleus to moving bars of different length
Authors: Cleland BG , Lee BB , Vidyasagar TR
Published: March 2018

It is well recognized that in the visual cortex of the cat, some of the cells (hypercomplex) are sharply tuned for the length of a bar moving backwards and forwards across their receptive fields. Other cells (simple) exhibit no such tuning but appear to respond proportionately over a range of bar lengths. The tuning seen in hypercomplex cells is already observable to a lesser degree in retinal ganglion cells. Our experiments were carried out to determine the extent of this tuning in the lateral geniculate nucleus, which relays information from the retina to the cortex. Results show that geniculate cells have tuning properties intermediate between those of ganglion cells and hypercomplex cells. By adding together a linear array of geniculate cells, it is possible to model the characteristics of a simple cell and to demonstrate that while an elongated bar gives a minimal response in hypercomplex cells, it should have little effect on the response of simple cells.
Chronic impairment of axonal transport eliminates taste responses and taste buds
Authors: Sloan HE , Hughes SE , Oakley B
Published: March 2018

A Silastic nerve cuff containing colchicine (1% w/v) was placed around the combined lingualchorda tympani nerve of the Mongolian gerbil (Meriones unguiculatus) to evaluate the role of axonal transport in the maintenance of taste buds. After 3 days the summated gustatory impulse discharges recorded from the chorda tympani nerve were reduced by 60%, while compound action potentials had not changed appreciably. The lingual-chorda tympani nerve underwent ultrastructural changes including a loss of microtubules, an increased prominence and disorientation of neurofilaments, and a significant shrinkage in the cross-sectional area of axoplasm. The shrinkage of axoplasm and the accumulation of mitochondria and cholinesterase at the nerve cuff provided evidence that the colchicine treatment acted to impair axonal transport. More substantial pathological changes were evident in nerve ultrastructure by 15 days when both the ipsilateral chorda tympani taste responses and fungiform taste buds were nearly absent. Control cuffs lacking colchicine had little effect on chorda tympani taste responses, taste buds, or nerve ultrastructure. Eight or 15 days of nerve exposure to lumicolchicine, an isomer of colchicine with low affinity for tubulin, had no significant effect on taste responses. [3H]Colchicine was used in the nerve cuff to demonstrate that colchicine must have acted directly upon the nerve trunk, rather than the taste buds, to cause the loss of taste responses and taste buds. [3H]Colchicine levels were equal in the two sides of the tongue, whereas both the functional and structural deterioration of the taste buds were restricted to the ipsilateral side. We conclude that the loss of taste responses and taste buds was caused by chronically impaired axonal transport in gustatory axons.
Regulation of synaptic position, size, and strength in anuran skeletal muscle
Authors: Nudell BM , Grinnell AD
Published: March 2018

An analysis of the physiology, morphology, and position of endplates on identified fibers in the Xenopus laevis pectoralis muscle has revealed the following. 1. The percentage of fibers with one endplate is lower in large muscles, and within the same muscle, singly innervated fibers are smaller than dually innervated fibers. 2. Single junctions tend to be stronger than junctions on dually innervated fibers. 3. Single junctions typically are located near the middle of their fibers, while the endplates on dually innervated fibers are located toward either end and usually are separated by at least 20% of the total fiber length. A significant proportion of dually innervated fibers appears to be innervated by the same axon at both junctions. 4. Junctions on the same dually innervated fiber tend to be more similar in length than do junctions on different fibers of the same input resistance. This observation is the same whether both junctions on a given fiber are formed by the same or different axons. There is no corresponding tendency for greater similarity in physiological strength of paired junctions, which frequently show large differences in endplate potential amplitude. 5. The total terminal length on dually innervated fibers of equivalent input resistance is inversely correlated with the mean release per unit length and total release of both junctions. There is no apparent correlation between the distance separating endplates and their strength or length. The data support a model of synaptic regulation in which nerve terminals are attracted, grow, and are maintained in proportion to the amount of a substance supplied by muscle fibers. Our findings suggest that such a substance is produced or distributed uniformly throughout each fiber in amounts proportional to the fiber size and inversely proportional to the total transmitter output of all junctions innervating the fiber. A form of competitive interaction between the terminals which helps to determine synaptic spacing may involve local depletion or inactivation of this substance.
Rapid lateral diffusion of extrajunctional acetylcholine receptors in the developing muscle membrane of Xenopus tadpole
Authors: Young SH , Poo MM
Published: March 2018

We have studied the lateral diffusion of acetylcholine (ACh) receptors in the extrajunctional region of developing myotomal muscle cell membrane of Xenopus tadpoles by a technique of local inactivation. The myotomal muscle cell surfaces of Xenopus tadpoles were exposed to external solution by gently removing the skin of the tail. The density of ACh receptors was monitored by membrane depolarizations in response to iontophoretically applied pulses of ACh. A pulse of alpha- bungarotoxin was pressure ejected onto the exposed fiber surface, resulting in a rapid local inactivation of the ACh receptors. With time, the functional ACh receptors diffused into the region of inactivation, producing a recovery of ACh response. That the observed recovery of ACh sensitivity is due to diffusion of ACh receptors from the unexposed undersurface of the fiber to the inactivated region was evidenced by the following: (1) no recovery was observed following prolonged toxin application; (2) pretreatment of the muscle cells with concanavalin A, which cross-links and immobilizes ACh receptors, prevented recovery; (3) mapping of ACh response along the muscle cell axis showed that the recovery cannot be accounted for by diffusion along the longitudinal axis of the fiber; and (4) the diffusion coefficients observed after scaling the recovery rate with fiber radius fell within a small range (1.5 to 4.0 X 10(-9) cm2/sec), consistent with diffusion of ACh receptors around the fiber circumference. This finding of rapid lateral diffusion within developing tadpole myotomal muscle membrane supports the notion that the localization of ACh receptors induced by innervation could be achieved by a “diffusion- trap” mechanism where the nerve contact region serves as a trap for rapidly diffusing receptors in the membrane.
Correlation of axonal regeneration and slow component B in two branches of a single axon
Authors: Wujek JR , Lasek RJ
Published: March 2018

We investigated the relationship between slow axonal transport and axonal regeneration in the rat dorsal root ganglion (DRG) cell. The DRG cell sends out a single axon which bifurcates within the ganglion; one axon proceeds centrally into the spinal cord and the other proceeds peripherally. The rate of axonal regeneration is approximately 2 times faster for the peripheral processes (4.6 +/- 0.9 mm/day) than for the central processes (2.1 +/- 0.5 mm/day). The peripheral and central processes regenerate through dissimilar environments (sciatic nerve and dorsal root, respectively); thus, environmental factors may account for the differences in regeneration rates. We tested this possibility by measuring the regeneration of motoneuron axons within the ventral root (histologically similar to the dorsal root). The motoneuron regeneration rate within the ventral root is similar to the motoneuron regeneration rate within the sciatic nerve, suggesting that factors within the DRG cell produce the differences in regeneration rate. Slow axonal transport is classified into two distinct components: slow component a (SCa), corresponding to the microtubule/neurofilament network of the axonal cytoskeleton, and slow component b (SCb), corresponding to the microfilament complex/axoplasmic matrix. The transport rate of SCa and SCb in the peripheral sensory axons is approximately 2 times faster than their counterparts in the central sensory axons. SCa moves at 1.0 to 3.0 mm/day in the peripheral processes and 0.5 to 1.0 mm/day in the central processes; SCb moves at 3.5 to 6.5 mm/day in the peripheral processes and 2.0 to 3.5 mm/day in the central processes. In each branch of the DRG cell, the rate of axonal regeneration is similar to the rate of SCb transport. These results support the hypothesis that SCb is a rate-limiting factor in axonal regeneration because of its role in providing the cytoskeletal elements which are directly involved in the motility of the growth cone and elongation of the axon.
Denervation-induced decrease in mitochondrial calcium transport in rat hippocampus
Authors: Baudry M , Gall C , Kessler M , Alapour H , Lynch G
Published: March 2018

Calcium accumulation by mitochondria and the activity and in vitro phosphorylation of pyruvate dehydrogenase were measured in control and partially denervated hippocampus. Calcium uptake was measured with a calcium-sensitive electrode and 45Ca2+ accumulation; both methods indicated that lesions of the entorhinal cortex produced a sizable reduction of calcium transport when mitochondria were fueled with pyruvate while much smaller changes were observed using succinate or ATP as energy sources. The decrease in calcium transport was evident by 24 hr after the lesion and was still present 6 months later. Synaptic and nonsynaptic mitochondria were similarly affected by the lesions. The activity and in vitro phosphorylation of pyruvate dehydrogenase were also significantly reduced following lesions of the entorhinal cortex, suggesting that denervation altered the endogenous state of phosphorylation of the mitochondrial enzyme. Commissural lesions but not septal lesions also resulted in a decrease in mitochondrial calcium transport when mitochondria were fueled with pyruvate. These findings suggest that denervation disturbs mitochondrial regulation of free calcium via an action on enzymes which regulate pyruvate dehydrogenase phosphorylation and activity. The potential relationship of this effect to degenerative changes associated with deafferentation and certain disease states is discussed.
Absence of [125I] alpha-bungarotoxin binding to motor nerve terminals of frog, lizard and mouse muscle
Authors: Jones SW , Salpeter MM
Published: March 2018

The existence of nicotinic acetylcholine receptors (AChRs) on the motor nerve terminals of vertebrates has long been controversial. We have re- examined this issue by electron microscope autoradiography with [125I] alpha-bungarotoxin, following separation of nerve terminals from muscle fibers by collagenase and protease treatment. We found no label over nerve terminal membranes other than that due to background, and we calculate upper limits of less than 0.1% of the postsynaptic AChR density for nerve terminals in frogs, lizards, and mice. We conclude that there are essentially no presynaptic acetylcholine receptors that bind alpha-bungarotoxin at vertebrate neuromuscular junctions.
Dose-dependent effects of capsaicin on primary sensory neurons in the neonatal rat
Authors: Nagy JI , Iversen LL , Goedert M , Chapman D , Hunt SP
Published: March 2018

The numbers of myelinated and unmyelinated fibers were counted in dorsal roots of adult rats treated neonatally with capsaicin in doses ranging from 5 to 100 mg/kg. Substance P and somatostatin levels in the spinal cord, dorsal roots, and sensory ganglia also were determined in control and treated animals. Capsaicin administration lead to the loss of both small myelinated and unmyelinated fibers from dorsal roots. However, whereas a near total loss, up to 94%, of unmyelinated fibers was achieved after high doses of capsaicin, the reduction of myelinated fibers, even of the smallest caliber, did not exceed 40%. The degree of fiber loss showed a clear dose dependency, with little detectable damage to myelinated fibers at doses of less than 50 mg/kg and with an ED50 for damage to unmyelinated fibers of 5 to 10 mg/kg. In all of the structures examined, particularly the dorsal roots, a roughly parallel decrease of substance P and somatostatin was found with capsaicin dose. The depletions of spinal cord substance P (55%) and somatostatin (20%) produced by neonatal capsaicin treatment were similar to those produced by dorsal rhizotomy. Capsaicin does not appear to be specific for primary afferents containing either substance P or somatostatin.
Myasthenic patients' IgG causes redistribution of acetylcholine receptors: freeze-fracture studies
Authors: Pumplin DW , Drachman DB
Published: March 2018

Immunoglobulin from patients with myasthenia gravis (MG) accelerates the degradation of acetylcholine receptors (AChRs) of skeletal muscle by a mechanism dependent on receptor cross-linking. However, the intermediate steps between cross-linking and endocytosis and degradation are not known. We have used freeze-fracture electron microscopy to study the effects of myasthenic immunoglobulin on the distribution of AChRs in the surface membranes of cultured rat skeletal muscle. Large, angular intramembrane particles were identified as AChRs. These particles were present in three configurations: distributed individually; in “hot spots”; and in clusters of two to 60 particles. Large depressions (greater than 100 nm in diameter) were thought to be endocytotic pits; they occurred preferentially in hot spots, and approximately half of the depressions were closely associated with clusters of AChRs. In control myotubes, hot spots contained a high density of AChR particles that were evenly, rather than randomly, distributed. Exposure to myasthenic patients' IgG induced clustering of the particles within such hot spots. In non-hot spot regions, control myotubes had mainly individually distributed AChRs, while clusters were seen only rarely. The addition of myasthenic IgG caused a rapid increase, followed by a gradual decrease, in the incidence of individual clusters as well as an increase in cluster size. This redistribution of particles was dependent on cross-linking by the myasthenic anti-AChR antibodies. Monovalent Fab fragments of myasthenic IgG had no effect on the distribution of AChR particles. However, addition of a second “piggyback” antibody to cross-link Fab- AChR complexes caused a redistribution of AChR particles like that due to the original myasthenic IgG. These findings indicate that cross- linking of AChRs by divalent antibody causes clustering of AChR particles, which may play an important role in their accelerated endocytosis and degradation.
The peptidergic organization of the cat periaqueductal gray. I. The distribution of immunoreactive enkephalin-containing neurons and terminals
Authors: Moss MS , Glazer EJ , Basbaum AI
Published: March 2018

Despite the significant contribution of the periaqueductal gray (PAG) to an endogenous pain suppression system, little is known about its neurochemical organization. Previous pharmacological and physiological studies have indicated regional variations in the effectiveness with which the midbrain PAG can generate potent analgesia in response to either opiate microinjection or electrical stimulation. There is, however, no anatomical correlate of this regional variation. As a first step toward elucidating the neural circuitry underlying the PAG's contribution to endogenous pain suppression systems, we have mapped the distribution of leucine enkephalin (ENK)-like immunoreactivity in the cat PAG. Throughout the rostral-caudal extent of the PAG, ENK- containing neurons are clustered in discrete populations. ENK terminal field staining is somewhat more diffuse; however, there are several regions where terminal staining is consistently more intense. The distribution of ENK perikarya and terminals undergoes a ventral to dorsal shift from caudal to rostral PAG. Conceivably, the clustered distribution of ENK cells and terminals contributes to the differential effectiveness of various PAG regions in generating analgesia. The ventral-dorsal shift of ENK immunoreactivity may (1) correspond to a somatotopic organization within the PAG or (2) mirror the topographic relationship of the PAG's interactions with other components of the endogenous analgesia system. In addition, the changing pattern of ENK immunoreactivity may also reflect the involvement of the PAG and of endogenous opiates in systems other than those of pain control.
Electron microscopic radioautographic localization of iodinated nerve growth factor bound to and internalized by PC12 cells
Authors: Bernd P , Greene LA
Published: March 2018

Nerve growth factor (NGF) has many effects on sympathetic and sensory neurons, but the mechanisms by which NGF exerts its actions are unknown. We have determined the localization of bound and internalized 125I-NGF by light and electron microscopic radioautography on a cell line derived from a rat pheochromocytoma (PC12). In response to NGF, PC12 cells cease mitosis and develop neuron-like processes. We have localized 125I-NGF (5 ng/ml) in cells without previous exposure to NGF (naive) after a continuous incubation with 125I-NGF for 2 min, 1 hr, 6 hr, 1 day and 1 week, as well as in cells that were grown with NGF (50 ng/ml) for 1 week (primed) and then exposed to 125I-NGF for 15 min, 1 hr, 6 hr, and 1 day. Examination of whole mount radioautographs revealed that all cells were labeled and that the distribution of grains was homogeneous. Primed cells were labeled on neurites and growth cones, as well as on cell bodies, and also had a greater density of labeling than naive cells. These patterns were identical for all time points studied, with the exception that “hot spots” of radioactivity appeared along neurites following a long incubation with 126I-NGF (longer than 6 hr). Electron microscopic radioautography revealed that these “hot spots” correspond to accumulation of grains in aggregates of lysosomes. The binding of 125I-NGF was specific, since control cultures (excess nonradioactive NGF) exhibited only background levels of grains. Quantification of electron microscopic radioautographs revealed that 70 to 95% of the grains were internalized after 15 min. The only cellular components labeled above random distribution levels were the plasma membrane, lysosomes, and the nuclear membrane. Each of these structures exhibited “down-regulation” of specific binding in naive cells after 6 hr of incubation. The cytosol, polyribosomes, dense cored granules, heterochromatin, and euchromatin showed labeling at or below the levels expected from random distribution of grains within the cell. The distribution of grains about the nuclear membrane also suggested an association with 125I-NGF. These data indicate that a large proportion of NGF “bound” to target cells is in fact internalized, and are consistent with some, but not other models for NGF's mechanism of action.
Fine structural distribution of acetylcholine receptors at developing mouse neuromuscular junctions
Authors: Matthews-Bellinger JA , Salpeter MM
Published: March 2018

The distribution of acetylcholine receptors (AChRs) in neuromuscular junctions of embryonic and newborn mice (strain 129/ReJ) was examined on the fine structural level using [125I]alpha-bungarotoxin labeling and quantitative electron microscope autoradiography. Changes in junctional receptor site density were related to changes in the structure of the postjunctional membrane, in particular the differentiation of morphologically distinct regions of thickened membrane and the formation of junctional folds. The following sequence of development is described: (1) At the earliest age examined, embryonic day 16 of gestation (i.e., at approximately the time when junctional receptor accumulations are first detected), subneural receptor aggregates have poorly defined boundaries and seem to extend beyond the region of direct axonal or Schwann cell contact. At that time the subneural AChR site density is somewhat variable but averages approximately 2000 to 3000 sites/micron 2, and the subneural muscle membrane shows discontinuous membrane specialization (membrane thickening plus a cytoplasmic amorphous layer). There seems to be no preferential labeling of contact regions rich in such specialization compared with those where membrane thickening was less obvious or absent. (2) By birth, junctional AChRs are strongly correlated with morphologically specialized membrane, at a constant density of approximately 9000/micron 2 (comparable to that in adult animals of this strain of mouse). During the first postnatal week, a large fraction of the primary cleft is covered by Schwann cell rather than axon terminal. Receptor-rich dense membrane and incipient junctional folds are found under axon terminals and, to a lesser extent, under Schwann cell extensions, but they are not seen beyond the edge of the “junctional complex.” Coated vesicles are a prominent feature of the subjunctional sarcoplasm in neonatal junctions, and their number declines sharply during the first week. (3) Junctional folds differentiate mainly after the first postnatal week. Development of folds is associated with a 1.5- to 1.8-fold increase in length of specialized thickened membrane and a parallel increase in number of receptor sites per endplate. Since this is also a period of rapid overall endplate growth (Steinbach, J.H. (1981) Dev. Biol. 84: 267–276) the total number of endplate receptors increases greater than 30-fold during the maturation period following the first week while the concentration of receptors/micron 2 of thickened membrane remains constant.