bluuebunny/crossref_metadata_2025 huggingface dataset:
Retrograde labeling, enrichment, and characterization of retinal ganglion cells from the neonatal rat
Authors: Sarthy PV , Curtis BM , Catterall WA
Published: March 2018

We have developed a method for labeling retinal ganglion cells in neonatal rats by retrograde transport of the fluorescent dye, True Blue (TB), injected into the optic chiasm. Following proteolytic dissociation of labeled retinas into single cells, the labeled cells could be enriched 50- to 100-fold by centrifugation in a 5%/10% metrizamide gradient. When plated in Ham's F-10 medium in the presence of fetal calf serum and chick optic tectum-conditioned medium, the labeled cells could be maintained in vitro up to 48 hr. In these cultures, the ganglion cells (GCS) constituted 50 to 70% of the total cell population. When GC-rich fractions or GC cultures were stained with a monoclonal antibody to Thy-1 antigen, greater than 90% of the TB- labeled cells were reactive. In order to localize voltage-sensitive sodium channels, GC-rich cultures were reacted with 125I-scorpion toxin. Analysis of the autoradiograms showed that the density of silver grains was about 10-fold higher on TB-labeled cells than on nonfluorescent cells, or in controls which contained excess of unlabeled toxin. When GC cultures were incubated with micromolar concentrations of putative GC transmitters, aspartate and glutamate, the amino acids were accumulated by 15 to 20% of labeled cells. Several lectin receptors were also localized on TB-labeled cells in situ. Whereas the lectins wheat germ agglutinin, concanavalin A, peanut agglutinin, Dolichos biflorus agglutinin, and Limulus polyphemus agglutinin bound to TB-labeled cells, others such as Ricinus communis agglutinin I, Ulex, and Lotus lectins showed no binding. The lectin binding was specific since preincubation with the appropriate hapten sugar blocked lectin binding.
The connections of the middle temporal visual area (MT) and their relationship to a cortical hierarchy in the macaque monkey
Authors: Maunsell JH , van Essen DC
Published: March 2018

The cortical and subcortical connections of the middle temporal visual area (MT) of the macaque monkey were investigated using combined injections of [3H]proline and horseradish peroxidase within MT. Cortical connections were assigned to specific visual areas on the basis of their relationship to the pattern of interhemispheric connections, revealed by staining for degeneration following callosal transection. MT was shown to be reciprocally connected with many topographically organized cortical visual areas, including V1, V2, V3, and V4. These pathways link regions representing corresponding portions of the visual field in the different areas. In addition, MT has reciprocal connections with two previously unidentified cortical areas, which we have designated the medial superior temporal area (MST) and the ventral intraparietal area (VIP). The laminar distribution of terminals and cell bodies in cortical areas connected with MT follows a consistent pattern. In areas V1, V2, and V3, the projections to MT arise largely or exclusively from cells in supragranular layers, and the reciprocal connections from MT terminate mainly in supragranular and infragranular layers. In contrast, the projections to MST and VIP terminate mainly in layer IV, and the reciprocal pathways originate from cells in both superficial and deep layers. On the basis of this pattern, each connection can be designated as forward or feedback in nature, and a hierarchical arrangement of visual areas can be determined. In this hierarchy, MT is at a higher level than V1, V2, and V3, and at a lower level than MST and VIP. Subcortical projections were seen from MT to the claustrum, the putamen, the caudate nucleus, the inferior and lateral subdivisions of the pulvinar complex, the ventral lateral geniculate nucleus, the reticular nucleus of the thalamus, the superior colliculus, and the pontine nuclei.
Mapping and quantitation of proteins from discrete nuclei and other areas of the rat brain by two-dimensional gel electrophoresis
Authors: Heydorn WE , Creed GJ , Goldman D , Kanter D , Merril CR , Jacobowitz DM
Published: March 2018

A map of the location and relative concentration of a number of different proteins present in 25 distinct neuroanatomical regions of the male rat brain has been established utilizing two-dimensional polyacrylamide gel electrophoresis. The regions examined include cortical areas as well as nuclei from the hypothalamus, amygdala, thalamus, forebrain, and hindbrain. Tissue samples were obtained from each region of interest by microdissection. Proteins within these samples were first separated by charge using the technique of isoelectric focusing. In the second dimension, proteins were separated by mass on polyacrylamide slab gels containing sodium dodecyl sulfate. Proteins were visualized using a highly sensitive silver stain and quantitated by computerized scanning densitometry. The results demonstrate that all proteins examined varied somewhat in concentration among the different brain regions. The majority (53%) of polypeptides selected for quantitation were found to vary less than 4-fold in concentration between the neuroanatomical areas with the lowest and highest detected amounts. In contrast, approximately 10% of the proteins examined varied widely in the quantity measured in each brain region, with concentration values ranging more than 10-fold between the regions with the lowest and highest detected amounts. This atlas is a first attempt at systematically classifying the mass, charge, and relative concentration of proteins present in a variety of regions of the rat brain. The system presented here will serve as a basis for future studies in this area.
Motoneuron death and motor unit size during embryonic development of the rat
Authors: Harris AJ , McCaig CD
Published: March 2018

Chronic paralysis of rat embryos during the last 4 to 6 prenatal days causes a diminution in skeletal muscle fiber numbers but inhibits motoneuron death. Consequently, as paralyzed muscles develop, an increased number of motoneurons attempts to form synapses at a reduced number of synaptic sites. Paralyzed muscle fibers receive their synapses at a single endplate, as in control muscles, but these endplates are hyperinnervated, with about twice the normal number of inputs. Counts of axons, synaptic inputs, and muscle units showed that motoneurons normally contact a maximum number of muscle fibers shortly before birth, and this number remains stable for several days postnatal until it finally is reduced to the adult number. The average motor unit size in paralyzed embryos at the time of birth was the same as in controls. We suggest that it is not necessary to postulate the existence of competition between embryonic nerve terminals in order to explain regulation of the number of muscle fibers initially contacted by a motoneuron. Motoneuron death was not immediately affected by paralysis, but paralysis “rescued” all motoneurons whose death normally would have occurred 24 hr or more after the time when paralysis was initiated, regardless of when this was. This implies that the peak period for determination to die is during embryonic day 14, when myotube formation is just beginning and no recognizable endplate structures are present in muscles. When paralyzed, motoneurons normally destined to die are capable of forming a normal number of functional nerve-muscle contacts.
Amino acid uptake, content, and metabolism by neuronal and glial enriched cellular fractions from mouse cerebellum
Authors: Shank RP , Campbell GL
Published: March 2018

A series of biochemical determinations was performed on five cellular fractions obtained from the cerebellum of 8- to 14-day-old mice. Cerebellar tissue was dissociated by mild trypsinization and mechanical agitation. The dissociated cellular material was separated into five fractions using a series of continuous density gradients formed with Percoll. Three of the fractions were comprised primarily of cell bodies. One of these was dominated by cells having the size and morphological appearance of granule cells, and based on several criteria the other two were were enriched in astrocyte cell bodies. Morphological analysis indicated that the remaining two fractions were enriched, respectively, in nerve terminals and large nucleated cell bodies. The uptake of 12 amino acids and 4 other metabolites by these cellular fractions was examined, and Km and Vmax values were determined for 10 of the compounds studied. High affinity transport carriers (Km approximately 1 to 20 microM) for most of the compounds studied were evident in neuronal and astrocyte-enriched fractions; however, for glutamate and gamma-aminobutyric acid (GABA) additional carriers with higher substrate affinities (Km approximately 0.1 to 0.3 microM) were evident in the astrocyte-enriched fraction. The fraction enriched in granule cell bodies was distinguished by an exceptionally low uptake of GABA and citrate, and a comparatively low uptake of beta-alanine, taurine, alpha-ketoglutarate, and glutamate. An analysis of the content of nine amino acids in the five fractions revealed that only glutamate, aspartate, and GABA were concentrated in the fraction enriched in nerve terminals. GABA was concentrated also in the fraction enriched in large cell bodies and was present at a low concentration in the fraction enriched in granule cell bodies. The other amino acids measured were distributed nearly evenly among the five fractions. Several differences in metabolic activity among the five fractions were observed. Radiolabel from several precursors was incorporated into GABA preferentially in the fractions enriched in large cell bodies and nerve terminals. In contrast, the accumulation of label in glutamine occurred preferentially in the fractions enriched in astrocytes and granule cell bodies. Labeling of alanine from [14C]pyruvate and of proline from [14C]ornithine was most prominent in the fractions enriched in astrocytes and granule cell bodies.
Chemical and electrotonic connections between Aplysia neurons in primary culture
Authors: Bodmer R , Dagan D , Levitan IB
Published: March 2018

Dissociated Aplysia neurons will regenerate neurites and form functional connections in primary cell cultures. The specificity of intercellular connectivity in these cultures was investigated by coculturing neurosecretory bag cells with neurons dissociated from the buccal ganglion. It was found that bag-bag and buccal-buccal electrotonic synapses form with high frequency, consistent with previous findings in pure bag and buccal cultures. There is specificity in the formation of these connections, since no bag-buccal electrotonic synapses were observed. Chemical interactions, on the other hand, are present between bag and buccal neurons. In a buccal-bag pair, injection of sufficient depolarizing current into the buccal cell to elicit a train of action potentials leads to a slow hyperpolarizing response in the bag cell. The bag cell hyperpolarization is accompanied by an increase in the cell's input conductance. This connection appears to be unidirectional, produces a voltage shift in the bag cell which is opposite in sign to that in the buccal cell, and is blocked by the removal of Ca2+ from the extracellular medium, indicating that it is mediated by a chemical neurotransmitter. The selective formation of electrotonic synapses in these mixed bag-buccal cultures, together with the presence of chemically mediated interactions, make this system particularly useful for investigating the establishment of intercellular connectivity.
Steroid regulation of neuronal death in the moth nervous system
Authors: Truman JW , Schwartz LM
Published: March 2018

The emergence of the adult Manduca sexta moth is followed by the programmed degeneration of about 50% of the insect's abdominal interneurons and motoneurons. Neurons in implanted ganglia undergo degeneration in concert with neurons of the host, thereby indicating that a hormonal cue is important. The neuronal death follows the normal decline in the levels of the steroid hormones, ecdysteroids, that occurs at the end of metamorphosis. Manipulations that cause a precocious ecdysteroid decline result in an early death of the neurons. Also, prolongation of the ecdysteroid titer extend the life of the neurons. By properly timed steroid application it was possible to dissociate the neuronal degeneration from the behavioral events and muscle degeneration that also occur at emergence. Also the stereotyped sequence of death among identified motoneurons could be stopped at various points in the sequence by the carefully timed application of steroid. It was concluded that the signal for neuronal death was the withdrawal of ecdysteroids at the end of metamorphosis and that the death was probably a direct response of the individual neurons to the hormone withdrawal.
Sprouting and synapse formation produced by Carbocaine
Authors: Tal M , Rotshenker S
Published: March 2018

Injury to one cutaneous pectoris nerve of the frog induces sprouting of and synapse formation by the contralateral homologous intact nerve. It was previously suggested that axotomy initiates a signal for growth in the cell bodies of the injured motor neurons and that this signal is transferred transneuronally across the spinal cord to intact motor neurons (e.g., Rotshenker, S. (1982) J. Neurosci. 2: 1359–1368). The present study was designed to test the hypothesis that axotomy initiated the signal for growth by depriving neuronal somata of a trophic substance derived from the target muscle. The superficial layer of muscle fibers comprising one cutaneous pectoris muscle, the source of the hypothetical trophic substance, was removed by means of local application of the myotoxic local anesthetic Carbocaine. An increased supernumerary pattern of innervation developed in drug-treated muscles first and in contralateral intact muscles thereafter. These results raise the possibility that target muscle fibers play a role as regulators of a signal for growth in the cell bodies of their innervating motor neurons.
Brain extract induces synaptic characteristics in the basal lamina of cultured myotubes
Authors: Sanes JR , Feldman DH , Cheney JM , Lawrence JC
Published: March 2018

The basal lamina (BL) that occupies the synaptic cleft of the skeletal neuromuscular junction is antigenically distinct from extrasynaptic muscle fiber BL, rich in acetylcholinesterase (AChE), and bears projections that form junctional folds in the postsynaptic membrane. We report here that these synapse-specific features of BL are all present at low levels in embryonic rat myotubes cultured without nerve, and that their levels are markedly increased by addition of a soluble extract from adult rat brain. Light and electron microscopic methods show that: (1) antibodies which bind preferentially to synaptic BL in vivo stain small, discrete patches of the myotube's BL; (2) AChE accumulates in patches on the myotube surface; and (3) myotube BL and membrane form invaginations that resemble junctional folds. Patches of BL that bear synaptic antigens, AChE, or folds usually overlie clusters of acetylcholine receptors in the plasma membrane. Myotubes treated with a brain extract bear 5 to 20 times more junctional folds and patches rich in acetylcholine receptors, synaptic BL antigens, and AChE than control myotubes. Together with a previous demonstration that electrical and/or contractile activity can modulate the amount and composition of myotube BL (Sanes, J.R., and J.C. Lawrence, Jr. (1983) Dev. Biol. 97: 123–136), these results suggest that nerves could regulate differentiation of muscle fiber BL by a combination of activity-dependent and -independent mechanisms.
gamma-Aminobutyric acid directly depolarizes cultured oligodendrocytes
Authors: Gilbert P , Kettenmann H , Schachner M
Published: March 2018

gamma-Aminobutyric acid (GABA) depolarizes in a dose-dependent manner approximately one-third of all immunologically identified oligodendrocytes in cultures of mouse spinal cord. Measurements of [K+]o indicate that the response to GABA is not due to K+ released from active neurons. The depolarization is not accompanied by a change in cell input resistance. Replacement of sodium in the bathing solution abolishes the entire response, whereas ouabain only inhibits the repolarization phase. Current clamp experiments with two separate intracellular electrodes show that the depolarization increases at more positive potentials while the repolarization increases at more negative potentials. Bicuculline and picrotoxin but not nipecotic acid reduce the GABA effect. Pentobarbital and chlordiazepoxid also reduce the GABA- induced depolarization. Muscimol produces a depolarization similar to that of GABA. Heterogeneity in the oligodendrocyte population is indicated by the observation that some cells respond to both GABA and glutamate, while others respond only to one and some are not responsive to either.
Histamine as a neurotransmitter in the stomatogastric nervous system of the spiny lobster
Authors: Claiborne BJ , Selverston AI
Published: March 2018

Histamine is a putative neurotransmitter in mammals and molluscs, but its role in the nervous systems of other animals is not known. This study examines the possibility that histamine is a neurotransmitter in an arthropod. Results show that first, 14 neurons in the stomatogastric ganglion of the spiny lobster respond to histamine. The response is inhibitory, is mediated by an increased conductance to chloride, and desensitizes with repeated applications of histamine. These same 14 neurons receive one type of synaptic potential from two extrinsic neurons, the “through-fibers” of the inferior ventricular nerve. This synaptic potential is also inhibitory, is mediated by an increased conductance to chloride, and is blocked when histamine receptors are desensitized. Second, assays of endogenous histamine indicate that histamine is distributed nonuniformly throughout the stomatogastric nervous system and that its distribution correlates with the axonal pathways and terminal arborizations of the inferior ventricular nerve through-fibers. Lastly, histamine is present in relatively high concentrations in the cell bodies of the through-fibers, whereas it is not detectable in other neurons in the stomatogastric system. These results suggest that histamine may be a transmitter in the lobster.
Neuropeptide modulation of photosensitivity. I. Presence, distribution, and characterization of a substance P-like peptide in the lateral eye of Limulus
Authors: Mancillas JR , Brown MR
Published: March 2018

The lateral eye of Limulus is innervated by an efferent system of fibers containing a substance P-like peptide. They were detected and their distribution was studied using indirect immunocytochemical techniques and monoclonal and serum antibodies. These thin, efferent fibers travel up the optic nerve, cross the lateral plexus, and branch out profusely when reaching the ommatidial layer. Innervation is extended to more than one component of the ommatidia, including the pigment, retinular, and eccentric cells. Immunoreactive staining could be abolished by absorbing the antisera with as little as 1 microM synthetic substance P. The efferent character of the fibers was established by means of ligation experiments, a technique also used to determine their origin in the circumesophageal connectives. Radioimmunoassay with two different C-terminal serum antibodies confirmed the presence of substance P-like material in the eye in the amounts of 61.44 pg/micrograms of protein, or up to 18 ng/eye. Gel filtration chromatography of crude extracts of the lateral eye, followed by radioimmunoassay, revealed an elution pattern extremely similar but not identical to that of synthetic substance P. These results show that a system of efferent fibers containing a substance P- like peptide originates in cells in the circumesophageal ring and innervates the ommatidia of the lateral eye. Their distribution and origin suggest an involvement in the modulation of photosensitivity, as part of a larger, generalized, level-setting regulator that is driven by a circadian clock but can also be activated by other systems.
Efferent neurotransmission of circadian rhythms in Limulus lateral eye. I. Octopamine-induced increases in retinal sensitivity
Authors: Kass L , Barlow RB
Published: March 2018

Octopamine increases the sensitivity of the Limulus lateral eye in situ when injected beneath the cornea during the day. The effect of octopamine is dose-dependent with a threshold concentration of about 0.1 microM injected at 1 microliter/min for 15 min. Injection of 40 microM octopamine increases lateral eye sensitivity to approximately 70% of the nighttime level normally caused by the efferent output of a circadian clock. Injections of octopamine analogues and other candidate neurotransmitters indicate that the postsynaptic receptor mediating the increase of retinal sensitivity is relatively specific for the structure of octopamine. The postsynaptic receptor is tentatively classified as a type 2B octopamine receptor (Evans, P. D. (1981) J. Physiol. (Lond.) 318: 99–122). Clozapine suppresses the effects of both exogenous octopamine and the endogenous efferent neurotransmitter. Together with the results from Barlow et al. (Barlow, R. B., Jr., S. J. Bolanowski, Jr., and M. L. Brachman (1977) Science 197: 86–89) and Battelle et al. (Batelle, B. -A., J. A. Evans, and S. C. Chamberlain (1982) Science 216: 1250–1252) our study leads to the following conclusion: retinal efferents, driven by a circadian clock in Limulus brain, release octopamine that increases visual sensitivity.
Supplementary motor area of the monkey's cerebral cortex: short- and long-term deficits after unilateral ablation and the effects of subsequent callosal section
Authors: Brinkman C
Published: March 2018

The short-term and long-term behavioral effects of unilateral lesions of the supplementary motor area (SMA) were studied in five monkeys (Macaca fascicularis ssp). A monkey with a unilateral lesion of the premotor area (PM) served as a control. In all animals, general behavior was unaffected by the lesions. For a few weeks postoperatively, all monkeys showed a clumsiness of forelimb movements, bilaterally, which involved both the distal and proximal muscles. Two SMA-lesioned monkeys (but not the PM-lesioned one), studied for up to 1 year postoperatively, showed a characteristic deficit of bimanual coordination where the two hands tended to behave in a similar manner instead of sharing the task between them. This deficit was more pronounced after a lesion contralateral to the nonpreferred hand. The deficit was interpreted as indicating that the intact SMA now influenced the motor outflow of both the ipsilateral hemisphere and the contralateral one through the corpus callosum. Callosal section immediately abolished the bimanual deficit, although the clumsiness returned transiently. The results imply that SMA may give rise normally to discharges informing the contralateral hemisphere of intended and/or ongoing movements via the corpus callosum. This may prevent duplication of responses during unimanual tasks and help to coordinate the behavior of both hands during a bimanual task.
Monaural occlusion alters sound localization during a sensitive period in the barn owl
Authors: Knudsen EI , Esterly SD , Knudsen PF
Published: March 2018

Sound localization was disrupted in young barn owls by chronically plugging one ear. Owls that were younger than 8 weeks of age at the time of ear plugging recovered normal localization accuracy while plugged, whereas those that were older than 8 weeks at the time of ear plugging did not. The end of the sensitive period for the adjustment of sound localization accuracy coincides with the maturation of the head and ears, suggesting that the exposure of the auditory system to stable, adult-like acoustic cues could play a role in bringing the sensitive period to a close. The results demonstrate that, early in development, associations between auditory cues and locations in space can be altered by experience.
An evaluation of the hypothesis of shifting terminals in goldfish optic tectum
Authors: Easter SS , Stuermer CA
Published: March 2018

It has been proposed that the terminal arbors of retinal ganglion cells continually shift positions in the optic tectum as a natural consequence of growth in larval amphibians and teleost fish. Here, we develop a detailed anatomical model of this process, based on recent observations in goldfish. The model predicts some unusual structural features of optic axons in the tectum. It was tested in two ways by use of the tracer, horseradish peroxidase (HRP). In the first, optic axons in the tectum were visualized by anterogradely transported HRP applied in the nerve or retina. As predicted, the axons had three parts: (1) fascicular segment (in the stratum opticum), (2) extrafascicular segment (in the synaptic neuropil), and (3) terminal arbor (also in the neuropil). The extrafascicular segment extended up to 1 mm caudally from the fascicle through which it entered the tectum. At its terminus, the arbor was restricted to a single lamina in which it branched extensively over an elliptical area with major axes between 100 and 200 micron long. In the second test, retinal ganglion cell bodies were labeled by retrogradely transported HRP applied to the tectum in a manner intended to label all axons and terminals in the vicinity. The pattern and locations of labeled cells varied, depending on where the HRP was applied, in detailed accord with the model's predictions. The close agreement between the model and the observations supports the hypothesis of shifting terminals.
The distribution and morphology of opioid peptide immunoreactive neurons in the cerebral cortex of rats
Authors: McGinty JF , van der Kooy D , Bloom FE
Published: March 2018

Pharmacological and biochemical evidence has implied that a widespread opioid peptide system exists within the cerebral cortex to mediate a variety of opiate effects. However, immunocytochemical detection of opioid peptides in the cortex has been limited. Using antisera to enkephalin and bovine adrenal medullary peptide, both fragments of proenkephalin, and an antiserum to dynorphin A, a fragment of prodynorphin, we now describe the regional and laminar distribution of a widespread population of olfactory cortical and neocortical cell bodies and fibers with opioid immunoreactivity in rats. Neurons stained with each antiserum are distributed bimodally in layers II and III and V and VI of neocortex as well as in layers II and III of olfactory cortex. The widespread distribution and heterogeneous morphology of cortical cells containing proenkephalin and dynorphin-A immunoreactivity suggest that opioid peptide-containing neurons may influence the functioning of local, commissural, and projection neurons in rat cerebral cortex.
Corticotropin-releasing factor: co-expression within distinct subsets of oxytocin-, vasopressin-, and neurotensin-immunoreactive neurons in the hypothalamus of the male rat
Authors: Sawchenko PE , Swanson LW , Vale WW
Published: March 2018

Two immunohistochemical methods that allow the concurrent localization of neuroactive substances within individual neurons have been used to identify, count, and chart the distribution of corticotropin-releasing factor (CRF)-immunoreactive cells in the paraventricular nucleus of the hypothalamus (PVH) that may also contain an additional peptide. In colchicine-treated male rats a moderate number of oxytocin-stained cells, localized primarily in a discrete, anterior part of the magnocellular division of the nucleus, was found also to stain positively for CRF. Similarly, oxytocin and CRF immunoreactivity were jointly expressed in magnocellular neurons distributed diffusely in the supraoptic nucleus. Smaller numbers of vasopressin- and neurotensin- stained neurons centered in specific parts of the parvocellular division of the PVH were stained with antisera against CRF. Possible mechanisms whereby the function of subsets of magnocellular and parvocellular neurosecretory neurons can be modulated differentially are discussed.
Effects of chronic suppression of bioelectric activity on the development of sensory ganglion evoked responses in spinal cord explants
Authors: Baker RE , Corner MA , Habets AM
Published: March 2018

Dorsal root ganglion (DRG) afferent terminals were identified, using electrophysiological techniques, within fetal mouse spinal cord cross- sections cultured in vitro. Afferent distribution patterns were monitored in explants grown for 3 to 6 weeks either in a serum- supplemented or in a serum-free, chemically defined medium (CDM). Bioelectrically active control explants from both series were compared with explants which had been reversibly silenced by chronic exposure to tetrodotoxin (TTX). The control (serum-grown) cultures showed a significant dorsal cord innervation preference, whereas in the corresponding TTX series there was an equal dorsoventral distribution. In the CDM series the mean number of DRG evoked responses was lower at first in TTX-grown than in control cultures, but with age in vitro there was a rise in excitability to normal levels. Spontaneous neuronal activity was abnormally low in cultures (serum as well as CDM-grown) which had been exposed to TTX. It is concluded that bioelectric activity may be an important factor in the proper regulation of synaptic connectivity and functional responsiveness in the developing spinal cord.
A test of Hebb's postulate at identified synapses which mediate classical conditioning in Aplysia
Authors: Carew TJ , Hawkins RD , Abrams TW , Kandel ER
Published: March 2018

In 1949, D. O. Hebb proposed a novel mechanism for producing changes in the strength of synapses that could account for associative learning. According to Hebb , the strength of a synapse might increase when the use of that synapse contributes to the generation of action potentials in a postsynaptic neuron. Thus, an essential feature of this postulate is that action potentials must occur in both a postsynaptic cell and a presynaptic cell for associative synaptic changes to occur. We have directly tested Hebb 's postulate in Aplysia at identified synapses which are known to exhibit a temporally specific increase in efficacy during a cellular analogue of differential conditioning. We find that the mechanism postulated by Hebb is neither necessary nor sufficient to produce the associative change in synaptic strength that underlies conditioning in Aplysia. In contrast, impulse activity in the presynaptic cell must be paired with facilitatory input, supporting the hypothesis that the temporal specificity of classical conditioning in Aplysia can be accounted for by activity-dependent amplification of presynaptic facilitation.
The distribution of immunoreactive alpha-neo-endorphin in the central nervous system of the rat
Authors: Zamir N , Palkovits M , Brownstein MJ
Published: March 2018

Using a specific radioimmunoassay, we have found that immunoreactive (ir) alpha-neo-endorphin has a widespread and unique distribution in the rat brain and spinal cord. The highest concentration in brain is in the substantia nigra (1692.1 fmol/mg of protein). Very high concentrations of ir-alpha- neo-endorphin (greater than 500 fmol/mg of protein) are also found in the lateral preoptic nucleus, dentate gyrus, parabrachial nuclei, nucleus accumbens, globus pallidus, median eminence, and anterior hypothalamic nucleus. Relatively high concentrations of ir-alpha-neo-endorphin (250 to 500 fmol/mg of protein) are present in the bed nucleus of the stria terminalis, paraventricular nucleus, nucleus of the solitary tract, dorsomedial nucleus, central amygdaloid nucleus, periaqueductal gray matter, suprachiasmatic nucleus, periventricular nucleus, hippocampus, prepositus hypoglossal nucleus, arcuate nucleus, ventromedial nucleus, medial preoptic nucleus, zona incerta, dorsal premamillary nucleus, medial forebrain bundle (hypothalamic and preoptic), nucleus of the diagonal band, locus ceruleus, lateral septal nucleus, and nucleus ambiguus . Moderate levels (100 to 250 fmol/mg of protein) are found in the posterior hypothalamic nucleus, ventral premamillary nucleus, dorsal raphe nucleus, motor hypoglossal nucleus, caudate-putamen, periventricular thalamic nucleus, subcommissural organ, sensory trigeminal nucleus, perifornical nucleus, area postrema, supraoptic nucleus, cuneate nucleus, medial amygdaloid nucleus, and organum vasculosum laminae terminalis. Low concentrations of ir-alpha-neo- endorphin (less than 100 fmol/mg of protein) are found in many cortical structures, claustrum, thalamic nuclei, habenular nuclei, lateral geniculate body, red nucleus, superior and inferior colliculi, paramedian reticular nucleus, pontine nuclei, superior olive, vestibular nuclei, motor facial nucleus, gigantocellular reticular nucleus, and subfornical organ.(ABSTRACT TRUNCATED AT 250 WORDS)
Distribution of immunoreactive beta-neo-endorphin in discrete areas of the rat brain and pituitary gland: comparison with alpha-neo-endorphin
Authors: Zamir N , Palkovits M , Brownstein MJ
Published: March 2018

The distribution of immunoreactive (ir)-beta-neo-endorphin in 101 microdissected rat brain and spinal cord regions as well as in the neurointermediate lobe of pituitary gland was determined using a highly specific radioimmunoassay. The highest concentration of beta-neo- endorphin in brain was found in the median eminence (341.4 fmol/mg of protein). High concentrations of ir-beta-neo-endorphin (greater than 250 fmol/mg of protein) were found in 11 nuclei, including dorsomedial nucleus, substantia nigra, parabrachial nuclei, periaqueductal gray matter, anterior hypothalamic nucleus, and lateral preoptic areas. Moderate concentrations of the peptide (between 100 and 250 fmol/mg of protein) were found in 66 brain nuclei such as the amygdaloid and septal nuclei, most of the diencephalic structures (not including the hypothalamus), and the majority of the medulla oblongata nuclei and others. Low concentrations of ir-beta-neo-endorphin (less than 100 fmol/mg of protein) were found in 21 nuclei, e.g., cortical structures (frontal., cingulate, piriform, parietal, entorhinal, occipital), olfactory tubercle, and cerebellum (nuclei and cortex). The olfactory bulb has the lowest beta-neo-endorphin concentration (21.3 fmol/mg of protein). Spinal cord segments exhibit low peptide concentrations. The neurointermediate lobe of the pituitary gland is extremely rich in ir- beta-neo-endorphin.
Proctolin in the lobster: the distribution, release, and chemical characterization of a likely neurohormone
Authors: Schwarz TL , Lee GM , Siwicki KK , Standaert DG , Kravitz EA
Published: March 2018

A radioimmunoassay, immunohistochemical techniques, and high pressure liquid chromatography (HPLC) methods have been developed for the study of the pentapeptide proctolin in the lobster Homarus americanus. Proctolin-like immunoreactivity is present in nearly every portion of the lobster nervous system; immunoreactivity is found in the brain, in each of the ganglia and connectives of the ventral nerve cord, and in many of the nerve roots that emerge from the cord. The greatest amounts are found in the pericardial organs, which are well known neurosecretory structures, and these structures have been selected for more detailed study. The immunoreactive material in the pericardial organs appears to be authentic proctolin. This material co-migrates with synthetic proctolin in two HPLC systems. Furthermore, a peptide that is purified from pericardial organs by HPLC is indistinguishable from synthetic proctolin in high resolution fast atom bombardment mass spectrometry. Cytochemistry reveals that the surface of the pericardial organs is densely covered with immunoreactive varicosities. No cell bodies that stain for proctolin are found in the pericardial organs, and the cells that give rise to the varicosities have not yet been located. The nerve endings in pericardial organs are capable of releasing proctolin-like material when depolarized in the presence of Ca++. These findings suggest that proctolin is a neurohormone in the lobster.
Light microscopic autoradiographic localization of mu and delta opioid binding sites in the mouse central nervous system
Authors: Moskowitz AS , Goodman RR
Published: March 2018

Much work has been done on opioid systems in the rat CNS. Although the mouse is widely used in pharmacological studies of opioid action, little has been done to characterize opioid systems in this species. In the present study the distribution of mu and delta opioid binding sites in the mouse CNS was examined using a quantitative in vitro autoradiography procedure. Tritiated dihydromorphine was used to visualize mu sites and [3H-d-Ala2-d-Leu5]enkephalin with a low concentration of morphine was used to visualize delta sites. Mu and delta site localizations in the mouse are very similar to those previously described in the rat ( Goodman , R.R., S.H. Snyder, M.J. Kuhar , and W.S. Young, 3d (1980) Proc. Natl. Acad. Sci. U.S.A. 77:6239– 6243), with certain exceptions and additions. Mu and delta sites were observed in sensory processing areas, limbic system, extrapyramidal motor system, and cranial parasympathetic system. Differential distributions of mu and delta sites were noted in many areas. Mu sites were prominent in laminae I, IV, and VI of the neocortex, in patches in the striatum, and in the ventral pallidum, nucleus accumbens, medial and midline thalamic nuclei, medial habenular nucleus, interpeduncular nucleus, and laminae I and II of the spinal cord. In contrast, delta sites were prominent in all laminae of the neocortex, olfactory tubercle, diffusely throughout the striatum, and in the basal, lateral, and cortical nuclei of the amygdala. The determination of the differential distributions of opioid binding sites should prove useful in suggesting anatomical substrates for the actions of opiates and opioids.
Purification and immunological characterization of a calcium pump from bovine brain synaptosomal vesicles
Authors: Chan SY , Hess EJ , Rahamimoff H , Goldin SM
Published: March 2018

In previous work ( Papazian , D., H. Rahamimoff , and S. M. Goldin (1979) Proc. Natl. Acad. Sci. U. S. A. 76: 3708–3712), an ATP-dependent calcium transport activity derived from rat brain synaptosomes was reconstituted into artificial lipid vesicles and substantially purified by transport-specific fractionation. When this procedure was applied to bovine brain synaptosomes, the approximately 70-fold purified, reconstituted Ca2+ uptake system contained two major polypeptides of Mr = 230,000 (“C230”) and 94,000 (“C94”) as observed on sodium dodecyl sulfate (SDS) gels. Evidence is presented here that these polypeptides are immunologically related to one another and that the synaptosomal Ca2+ pump is immunologically distinct from Ca2+ pumps in non-neuronal cells. Antisera and monoclonal antibodies to the purified, reconstituted protein did not significantly cross-react with the Ca2+ pumps or any other components of bovine sarcoplasmic reticulum or erythrocytes. However, these antibodies did cross-react with a component of bovine brain axolemma-enriched membranes. A monoclonal antibody was produced that immunoprecipitated the Ca2+ transport activity, both in native, synaptosomal vesicles and in liposomes containing the reconstituted transport system. This antibody bound C230 more prominently than C94 on Western blots of SDS gels. An antiserum raised against C94 alone, obtained by elution from SDS gels, was also found to bind most prominently to C230 on Western blots. These results suggest that this synaptosomal Ca2+ pump is specific to nerve tissue and that C94 and C230 are structurally homologous components of this transport activity.
Biochemical interruption of membrane phospholipid renewal in retinal photoreceptor cells
Authors: Pu GA , Masland RH
Published: March 2018

The rabbit retina's synthesis of new phosphatidylcholine from extracellular choline was interrupted by an intravitreal injection of the choline analogue hemicholinium-3. This disrupted the process by which new membrane is added to the rod photoreceptor outer segments and eventually caused outer segment degeneration. During the first 2 days after hemicholinium-3 was injected, rows of vesicles replaced the newly formed membrane discs at the outer segment's base. The region of vesicles then expanded, and the distal outer segment detached and was quickly phagocytosed by the pigment epithelium. Two weeks after hemicholinium-3 injection, all of the retina's outer segments had been lost and the inner segments were reduced in length. The threshold concentration of hemicholinium-3 was approximately 20 microM intraocularly. At this dose, the cell bodies, intracellular organelles, and synapses of the rod cells survived. A small group of amacrine cells, possibly those that synthesize acetylcholine, became pyknotic; but the other retinal neurons remained normal to both light and electron microscopy even upon exposure to intraocular concentrations as high as 1 mM. Biochemical experiments indicated that at 20 microM hemicholinium-3, the perturbation of choline metabolism is partial and transient. That it has major selective consequences for the outer segments probably reflects the large amount of new phospholipid required for renewal of their membranes. The selectivity of the lesion was also evidenced by electrophysiological activity recorded from hemicholinium-3-treated retinas. Hemicholinium-3 was injected in vivo, and at various times retinas were isolated and incubated in vitro. The normal components of the electroretinogram were observed, but its amplitude rapidly declined; 12 days after injection, no response to light could be detected. Spontaneous firing of single ganglion cells was observed at all times following hemicholinium-3 injection. As the outer segments degenerated and the threshold of the electroretinogram rose, the thresholds of the ganglion cells also rose, but normal ganglion cell receptive fields could sometimes be plotted. Fourteen days after injection, when all of the outer segments were gone, ganglion cell responses to bright light could still be recorded; their thresholds were about 3.5 log units above normal. This finding is consistent with a previous report of light-evoked responses in mice after outer segment degeneration.(ABSTRACT TRUNCATED AT 400 WORDS)
Combination of immunocytochemistry and radioligand receptor assay to identify beta-adrenergic receptor subtypes on astroglia in vitro
Authors: Trimmer PA , Evans T , Smith MM , Harden TK , McCarthy KD
Published: March 2018

There is an increasing need to assess the distribution of receptors for neuroactive substances on specific neural cell types. This study describes the establishment of methodology that combines the quantification of beta-adrenergic receptor subtypes by radioligand binding assays with immunocytochemical analysis of the contribution of astroglia (identified by the presence of glial fibrillary acidic protein) and fibroblasts (identified by the presence of fibronectin) to cultures prepared from neonatal rat cerebral cortex. The effects of subtle changes in culture methodology on the cellular composition of cerebral cortical cultures and the distribution of beta-adrenergic receptor subtypes were examined. The data indicate that (1) a decrease in the density of the initial plating suspension, (2) an increase in the age of the animals, or (3) supplementation of the cortical cell suspension with meningeal fibroblasts all result in an increase in fibronectin staining and a decrease in glial fibrillary acidic protein antibody staining. This change in the cellular composition of the cortical cultures correlated with an increase in the number of beta 2- adrenergic receptors and a corresponding decrease in the number of beta 1-adrenergic receptors. These observations point out the care which must be exercised when preparing primary astroglial cultures of sufficient purity for large-scale biochemical and pharmacological studies.
The effect of norepinephrine on visual cortical neurons in kittens and adult cats
Authors: Videen TO , Daw NW , Rader RK
Published: March 2018

The responses of visual cortical neurons during iontophoresis of norepinephrine (NE) were compared in kittens and adult cats. One type of neuronal “plasticity” (the ability of visual cortical neurons to have their response properties markedly altered during exposure to a restricted visual environment) normally occurs only in young kittens and has been hypothesized to depend on the presence of NE. We investigated this hypothesis in terms of a model of synaptic plasticity which suggested that NE would selectively enhance the evoked responses of neurons and that this enhancement might exist to a greater extent in kittens than in adult cats. The responses of 72% (43 of 60) of all neurons sampled decreased during NE iontophoresis, while the responses of only a few neurons (3 of 60) increased. Reduced activity occurred in roughly equal percentages of simple and complex cells. We also looked for differences in NE modulation of the “signal-to-noise” ratio of neurons. Most neurons had little spontaneous activity, but, of those that did, the signal-to-noise ratio increased in some instances and decreased in others. No difference in any of the effects of NE was observed between kittens and adult cats. The role of NE in cortical plasticity is discussed.
The ventral pallidal projection to the mediodorsal thalamus: a study with fluorescent retrograde tracers and immunohistofluorescence
Authors: Young WS , Alheid GF , Heimer L
Published: March 2018

We have examined rat basal forebrain projections to the mediodorsal thalamic nucleus (MD) by making injections of retrogradely transported fluorescent tracers into the MD. Additionally, in some animals, we also stained sections for glutamate decarboxylase (GAD) by the indirect fluorescent antibody technique. Our results demonstrate that the following basal forebrain areas project to the MD: lateral orbital cortex, agranular insular cortex superficial to claustrum, primary olfactory cortex, diagonal band nuclei, ventral pallidum, and amygdala. A large number of labeled cells are present in the olfactory tubercle, and these cells are almost without exception located in dense GAD- positive ventral pallidal areas rather than in striatal regions of the tubercle. This ventral pallidal projection to the MD strengthens the concept of a ventral striatal-pallidal system in parallel to the classic striatal-pallidal system which projects to the ventral thalamus. These results are also discussed in relationship to the olfactory system.
The suprachiasmatic nuclei of the fetal rat: characterization of a functional circadian clock using 14C-labeled deoxyglucose
Authors: Reppert SM , Schwartz WJ
Published: March 2018

The circadian clock located in the suprachiasmatic nuclei (SCN) was characterized in the fetal rat by using 14C-labeled deoxyglucose to monitor glucose utilization (metabolic activity) of the nuclei. A clear day-night oscillation of metabolic activity was detectable in the fetal SCN from the 19th through the 21st days of gestation; the nuclei were metabolically active during the subjective day and metabolically inactive during the subjective night. During the subjective day on gestational day 21, the fetal SCN were found to manifest high metabolic activity for most of the subjective day. We were able to acutely dissociate SCN metabolic activity in the mother rat from that in the fetus by exposing the pregnant animals to light during the normal dark period of diurnal lighting on gestational day 20. The results show the utility of the deoxyglucose method for directly investigating prenatally the function of the biological clock located in the SCN.