bluuebunny/crossref_metadata_2025 huggingface dataset:
The actions of serotonergic agonists and antagonists on the activity of brisk ganglion cells in the rabbit retina
Authors: Brunken WJ , Daw NW
Published: March 2018

Extracellular electrophysiological recordings were obtained from rabbit retinal ganglion cells in either a superfused eyecup or an in vivo preparation. Selective antagonists or agonists of serotonin at the 5- HT2 or 5-HT1A receptors were applied, and the changes in light-evoked and spontaneous activity were studied. Both 5-HT1A agonists and 5-HT2 antagonists reduced the ON-components of the light-evoked responses of all classes of brisk ganglion cell; spontaneous activity was reduced in these cells as well. These results suggest that the indoleamine- accumulating amacrine cells of the rabbit retina serve to facilitate the output of the depolarizing rod bipolar cell and thereby increase the efficacy of transmission between this and other cells in the rabbit retina, and that this process is mediated by 5-HT2 receptors. On the basis of the similarity of the actions of the 2 classes of drug studied, we hypothesize further that 5-HT1A receptors mediate an inhibitory process that serves to terminate the indoleamine-induced facilitation. This process may be located either in the bipolar terminal or presynaptic to it in the terminal of the putative indoleaminergic cells.
Muscle activity pattern regulates postnatal development of acetylcholinesterase molecular forms in normal mice and mice with motor endplate disease
Authors: Yeakley JM , Janavs JL , Reiness CG
Published: March 2018

We have studied the relative contributions of muscle activity and nerve- supplied materials to the regulation of AChE molecular forms during postnatal development of muscles in normal mice and in mice with motor endplate disease (med mice). Onset of this hereditary disease causes a progressive failure of evoked release of ACh from the motor neuron, which prevents contraction in muscles such as biceps and soleus. In these innervated but inactive muscles, one can examine the consequences of inactivity on the distribution of AChE forms. In normal mouse biceps the distribution of AChE forms, as shown by sucrose-gradient analysis, change substantially after birth; the most dramatic alteration is an increase in G4 AChE from 15 to 45% of total AChE during the third postnatal week. AChE profiles in normal or med biceps are indistinguishable until 10–12 d after birth, but the changes in distribution of AChE forms does not occur in med biceps nor in normal biceps denervated 2 weeks after birth. In contrast, the distributions of AChE forms in a predominantly slow muscle, the soleus, are similar in med and normal mice both early (10 d) and late (20 d) in the course of the disease, and the distributions are affected little by denervation. The profiles of AChE forms seen in normal soleus at all times studied resembled those seen in newborn biceps or biceps inactivated by denervation or the med disease. We conclude that neither innervation, age-dependent changes intrinsic to muscle, nor muscle activity is sufficient to induce the changes we seen in AChE forms in biceps. These results support the hypothesis that neonatal, inactive, or tonically active muscles produce an intrinsic pattern of AChE molecular forms, and that a phasic pattern of activity induces a postnatal redistribution of the AChE molecular forms expressed by the muscle.
Dopaminergic innervation of A II amacrine cells in mammalian retina
Authors: Voigt T , Wassle H
Published: March 2018

Dopaminergic amacrine cells were stained in cat, rat, and rabbit retina using an antibody against tyrosine hydroxylase (TH). Following intraocular injection of DAPI (4,6,diamidino-2-phenylindole), subsequent retinal whole-mount preparations revealed that the dopaminergic fiber plexus formed rings around amacrine cell bodies. Intracellular injection of Lucifer yellow (LY) into A II amacrine cells confirmed that this rod-related, bistratified interneuron has its cell body within the dopaminergic rings. Using a photooxidation process, LY was transformed into an electron-dense reaction product, enabling ultrastructural examination of LY-injected A II amacrine cells. In retinae counterstained with an antibody against TH, it was possible to show synapses from TH-positive fibers onto these cells. The dopaminergic plexus was further investigated by injecting single dopaminergic cells with LY and thus revealing their branching pattern. The present results emphasize the role of dopamine in modulating the rod pathway in mammalian retina.
Physiological properties, time of development, and central projection are correlated in the wing mechanoreceptors of Drosophila
Authors: Dickinson MH , Palka J
Published: March 2018

The wing of Drosophila contains 8 sensory structures (campaniform sensilla), which lie in specific locations and possess identical surface morphology. The axons of the campaniform neurons follow either a medial or a lateral tract within the CNS. Previous studies (Palka et al., 1986) indicate that choice of central pathway correlates with the time of birth and differentiation of the neurons rather than with their topographic distribution on the wing. On the basis of the response properties revealed by mechanical and electrical stimulation, these sensory cells also fall into 2 physiological categories, rapidly and slowly adapting, that correlate exactly with central projection and birthdate. Thus, within this discrete population of sensory neurons there exists a precise 3-way correlation between physiology, central projection, and time of development.
Plasticity in the organization of adult cerebral cortical maps: a computer simulation based on neuronal group selection
Authors: Pearson JC , Finkel LH , Edelman GM
Published: March 2018

Recent experimental evidence from the somatosensory, auditory, and visual systems documents the existence of functional plasticity in topographic map organization in adult animals. This evidence suggests that an ongoing competitive organizing process controls the locations of map borders and the receptive field properties of neurons. A computer model based on the process of neuronal group selection has been constructed that accounts for reported results on map plasticity in somatosensory cortex. The simulations construct a network of locally connected excitatory and inhibitory cells that receives topographic projections from 2 receptor sheets corresponding to the glabrous and dorsal surfaces of the hand (a typical simulation involves approximately 1500 cells, 70,000 intrinsic and 100,000 extrinsic connections). Both intrinsic and extrinsic connections undergo activity- dependent modifications according to a synaptic rule based on heterosynaptic interactions. Repeated stimulation of the receptor sheet resulted in the formation of neuronal groups-local sets of strongly interconnected neurons in the network. Cells in most groups were found to have similar receptive fields: they were exclusively glabrous or dorsal despite equal numbers of anatomical connections from both surfaces. The sharpness of map borders was due to the sharpness of the underlying group structure; shifts in the locations of these borders resulted from competition between groups. Following perturbations of the input, the network underwent changes similar to those observed experimentally in monkey somatosensory cortex. Repeated local tapping on the receptor sheet resulted in a large increase in the magnification factor of the stimulated region. Transection of the connections from a glabrous region resulted in the organization of a new representation of corresponding dorsal region. The detailed simulations provide several insights into the mechanisms of such changes, as well as a series of predictions about cortical behavior for further experimental test.
Circadian clock in cell culture: I. Oscillation of melatonin release from dissociated chick pineal cells in flow-through microcarrier culture
Authors: Robertson LM , Takahashi JS
Published: March 2018

The avian pineal gland contains circadian oscillators that regulate the rhythmic release of melatonin. We have developed a dissociated chick pineal cell culture system in order to begin a cellular analysis of this vertebrate circadian oscillator. Dissociated pineal cells maintained in cyclic light conditions (LD 12:12) released melatonin rhythmically. The release of melatonin was elevated during the dark and low during the light. A circadian oscillation of melatonin release persisted for at least 5 cycles in constant darkness with a period close to 24 hr; however, there was a gradual damping of the amplitude. Analysis of the rhythm revealed that the observed damping was consistent with either desynchronization of multiple oscillators within the cultures or damping of individual oscillators. The circadian oscillation of melatonin release persisted for up to 4 cycles under conditions of constant light; however, the oscillation was strongly damped and the period of the oscillation was lengthened significantly. Thus, dissociated pineal cells express a persistent circadian oscillation of melatonin release in constant darkness, and properties of this oscillation are modulated by light treatment in vitro. This flow-through cell culture method for dissociated chick pineal cells should provide a useful model for the analysis of a vertebrate circadian system at the cellular level.
An identified histaminergic neuron can modulate the outputs of buccal- cerebral interneurons in Aplysia via presynaptic inhibition
Authors: Chiel HJ , Kupfermann I , Weiss KR
Published: March 2018

We have identified 2 buccal-cerebral interneurons (BCIs), B17 and B18, that appear to be involved in the coordination of feeding behavior in Aplysia. The BCIs have their cell bodies in the buccal ganglion, but send axons to the cerebral ganglion via the cerebral-buccal connectives. The BCIs appear to make monosynaptic connections with neurons in the cerebral ganglion that modulate extrinsic muscles involved in feeding behavior. B17 and B18 are activated antiphasically during a motor program induced by stimulating the esophageal nerve and appear to “read out” different phases of the buccal program to different cells in the cerebral ganglion. B17 and B18 are not necessary, and probably not sufficient, to generate the buccal program. These BCIs, and other cells like them in the buccal ganglion, may be capable of coordinating the activity of the intrinsic muscles of the buccal mass with the activity of its extrinsic muscles, and perhaps with those of the lips, mouth, and tentacles. Identified histaminergic neuron, C2, can modulate the outputs of the BCIs onto their synaptic followers in the cerebral ganglion. Firing of C2 inhibits spiking of the BCIs, probably via cerebral-buccal interneurons. C2 also decreases the size of the EPSP that B17 and B18 evoke in cerebral neuron C4. C2 appears to do so monosynaptically, and it decreases the conductance of C4, ruling out one possible postsynaptic mechanism of action. Variance analysis of the EPSPs evoked by B18 supports the hypothesis that C2 acts presynaptically to decrease the release of transmitter. Applications of histamine to the solution bathing the neuron mimic the effect of firing C2 and reduce the size of the EPSPs B18 induces in C4. The bath-applied histamine appears to act directly on B18, since it elicits a voltage-dependent increased conductance hyperpolarization recorded in the soma of B18, and the hyperpolarization persists in a solution in which synaptic transmission has been blocked. Histamine did not produce any marked changes of the duration of a TEA-broadened somatic action potential of B18. To the extent that the soma of B18 reflects the membrane properties of its synaptic terminal region, the data suggest that histamine may produce presynaptic inhibition by hyperpolarizing the synaptic terminal region.
Color constancy in the honeybee
Authors: Werner A , Menzel R , Wehrhahn C
Published: March 2018

A multicolored display was illuminated by 3 bands of wavelengths corresponding to the maxima of the spectral sensitivities of the 3 types of photoreceptors found in the bee retina. The intensity of each band could be varied individually. The light fluxes emitted by the colored areas of the multicolored display were determined quantitatively. Free-flying honeybees were trained with sugar solution to choose one of the colored areas. The illumination was then changed in such a way that the light fluxes formerly emitted by the training area were now measured on another area. When the trained bees were tested under those conditions, they still chose the training area. The relative positions of the colored areas were changed in order to exclude learning of position. It is concluded that color vision in bees is, in a certain range, independent of the spectral content of the illumination. Model calculations show that the behavior observed in bees is consistent with the retinex theory (Land, 1977), i.e., an algorithm using long-range interactions is required to explain color constancy.
Dishabituation and sensitization emerge as separate processes during development in Aplysia
Authors: Rankin CH , Carew TJ
Published: March 2018

Until recently, dishabituation and sensitization have commonly been considered to reflect a unitary process: Sensitization refers to a general facilitation produced by strong or noxious stimuli that enhances subsequent responding; dishabituation has been thought to represent a special instance of sensitization in which the facilitation is simply superimposed on a habituated response level. The unitary process hypothesis was based on the observation that both decremented and nondecremented responses are facilitated by a common noxious or strong stimulus. However, this observation does not rule out the possibility that dishabituation and sensitization could reflect separate processes that are activated in parallel by a strong stimulus. Recent cellular experiments by Hochner et al. (1986) suggest that this, in fact, occurs in the sensory neurons of the gill withdrawal reflex in Aplysia. A developmental analysis of learning in the marine mollusc Aplysia permits a direct behavioral test of this hypothesis. If dishabituation and sensitization reflect a unitary process then they should emerge at the same time ontogenetically. On the other hand, if they reflect different processes, then they might emerge according to different ontogenetic timetables. In the present study we examined the temporal emergence of dishabituation and sensitization in the defensive siphon withdrawal reflex in 3 stages of juvenile Aplysia: stage 11, early stage 12, and late stage 12. Animals received one of 2 kinds of training: Dishabituation training, in which the effect of strong tail shock on habituated responses were observed, and Sensitization training, in which the effect of strong tail shock on nondecremented responses was observed. We found that, while dishabituation was present in all stages examined, sensitization did not emerge until several weeks later, in late stage 12. These results were confirmed and extended in a group of animals that were tested twice: first in stage 11, when they showed no sensitization, and again 13 weeks later, in late stage 12, when they then showed significant sensitization. Our analysis of nondecremented responses prior to the emergence of sensitization also revealed an unexpected inhibitory component of tail shock that produces reflex depression. Moreover, there was a clear progression in the net effects of tail shock during development: reflex depression was produced in stages 11 and early stage 12, followed by a transition to reflex facilitation (sensitization) in late stage 12. Finally, when sensitization emerged in late stage 12, the process of dishabituation showed a significant increase compared with previous developmental stages.
Development of sensitization in the escape locomotion system in Aplysia
Authors: Stopfer M , Carew TJ
Published: March 2018

The development of several forms of nonassociative learning (habituation, dishabituation, and sensitization) has previously been examined in the gill and siphon withdrawal reflex of Aplysia. In the present study we analyzed the development of one of these forms of learning, sensitization, in a different response system in Aplysia, escape locomotion. A broad range of juvenile stages was examined: stages 10, 11, early 12, late 12, and 13 (early adult). We found that sensitization was completely absent in early developmental stages, not appearing until late stage 12. This stage of development is particularly interesting because it is at this same point that (1) sensitization first appears in the gill and siphon withdrawal reflex (Rankin and Carew, 1987), and (2) the cellular analog of sensitization first emerges in the CNS (the abdominal ganglion) of juvenile Aplysia (Nolen and Carew, 1987). The fact that sensitization emerges synchronously in the escape locomotion system and the gill withdrawal system is striking because the 2 response systems differ markedly in their intrinsic developmental timetables, response topography, and underlying neural circuitry. Thus, the emergence of sensitization in both systems at the same late stage of juvenile development suggests the possibility that a single, unified process during development may be responsible for the simultaneous expression of sensitization.
Influences of peripheral nerve grafts on the survival and regrowth of axotomized retinal ganglion cells in adult rats
Authors: Villegas-Perez MP , Vidal-Sanz M , Bray GM , Aguayo AJ
Published: March 2018

To investigate the role of extrinsic influences on the survival and growth of axotomized retinal ganglion cells (RGCs) in the mature mammalian CNS, both optic nerves (ONs) of adult rats were transected intraorbitally and, on one side, replaced by an autologous segment of peripheral nerve (PN) that had been left unconnected distally. The survival of RGCs and the regrowth of their cut axons into the PN grafts were assessed using morphometric techniques, neuroanatomic tracers, and immunologic cell markers to identify and count RGCs at times ranging from 15 d to 9 months. It was observed that (1) in the absence of a PN graft, more than 90% of the RGCs died by 1 month after axotomy; (2) between 1 and 3 months after axotomy, survival of RGCs in the PN- grafted retinas was enhanced 2–4-fold; (3) nearly 20% of the surviving RGCs regrew lengthy axons into the grafts; and (4) although the density of surviving RGCs in PN-grafted retinas decreased significantly between 1 and 3 months after axotomy, the densities of RGCs with axons extending into the graft remained relatively stable. These results confirm that in the adult rat retina, neuronal death is a major effect of axotomy near the cell soma. Although such lesions lead to the degeneration of many RGCs, we show that extrinsic influences introduced by the apposition of a PN segment at the time of severing the ON can rescue a substantial number of these neurons. Because the enhanced survival of many axotomized RGCs in the PN-grafted retinas appears to be limited to the first few weeks after injury, while those of RGCs that regenerate axons into the grafts do not show a parallel decline, it is possible that, in these experiments, neuronal viability depends on a spectrum of differently timed influences that may include the early diffusion of critical molecules arising from the graft and the subsequent establishment of more complex interactions with graft components.
Monoclonal antibodies reveal cell-type-specific antigens in the sexually dimorphic olfactory system of Manduca sexta. I. Generation of monoclonal antibodies and partial characterization of the antigens
Authors: Hishinuma A , Hockfield S , McKay R , Hildebrand JG
Published: March 2018

The olfactory system of the moth Manduca sexta is sexually dimorphic. Male moths possess a male-specific olfactory “subsystem,” comprising olfactory receptor cells (ORCs) and CNS neurons and synaptic areas associated with the detection of female sex pheromones, in addition to elements common to males and females. In order to explore the molecular differences between cells that subserve the sexual dimorphism and odor- specificity of components of the olfactory system, we generated monoclonal antibodies (Mabs) against tissue of the olfactory system of the moth. In 2 fusions, we screened 1105 hybridoma lines and obtained 272 lines that secreted antibodies against Manduca nervous tissue, as assayed immunocytochemically on sections of the primary olfactory center (the antennal lobe) in the brain of Manduca. We describe here 3 classes of Mabs exemplifying the several cell-type-specific antibodies obtained through the screening procedure. Seven hybridoma lines secrete antibodies that specifically recognize cell bodies, axons, and initial segments of dendrites of many or all ORCs of both males and females (classified as olfactory-specific antibodies, OSAs). Electron- microscopic studies of 2 of the Mabs in this class showed that they recognize antigens associated with the cell membrane and that the immunoreactive ORC axons are bundled together in fascicles in the antennal nerve. On immunoblots, one of the OSA Mabs recognizes 3 distinct protein bands of apparent Mrs 42,000, 59,000, and 66,000 Da. When tissue samples enriched in either receptor cell bodies, dendrites, and initial segments of axons or in distal segments of axons and their terminals and synapses were extracted separately, different patterns of bands were detected--42,000 and 59,000 Da bands from cell bodies and initial segments of axons and dendrites, and 42,000 and 66,000 Da bands from distal segments of axons and their terminals--suggesting that the 59,000 Da protein is modified to the 66,000 Da protein during axonal transport. The second Mab we describe here, the male olfactory-specific antibody (MOSA), selectively recognizes the sexually dimorphic ORCs that are present only in males. The antigen recognized by this antibody is found in cell bodies, dendrites, axons, and axon terminals. By electron-microscopic immunocytochemistry, the MOSA immunoreactivity is found in the cytoplasm and appears not to be associated with particular subcellular organelles. This antibody demonstrates that male-specific ORCs are molecularly distinct from other types of ORCs.
Monoclonal antibodies reveal cell-type-specific antigens in the sexually dimorphic olfactory system of Manduca sexta. II. Expression of antigens during postembryonic development
Authors: Hishinuma A , Hockfield S , McKay R , Hildebrand JG
Published: March 2018

Two classes of monoclonal antibodies specific to the olfactory system of Manduca sexta have been isolated: the olfactory-specific antibody (OSA), which specifically recognizes many or all olfactory receptor cells (ORCs) in both males and females, and the male olfactory-specific antibody (MOSA), which stains male-specific receptor cells (principally or exclusively sex-pheromone receptors present only in antennae of males; Hishinuma et al., 1988). In the investigation reported here, we examined the expression of the antigens during postembryonic development in order to correlate the presence of particular antigens with the status of differentiation of the ORCs or with their acquisition of particular functions. As assessed immunocytochemically, the OSA recognizes certain epithelial cells in the antennal imaginal disk of the fifth-instar larva. Later, during the first 70 hr of adult development, when differentiative cell divisions are occurring in the antennal epithelium to generate ORCs and the other cells that make up olfactory sensilla, no cells are stained. Immediately after this period of mitoses, the OSA immunoreactivity reappears exclusively in the ORCs, which begin to elaborate axons as an early event in their differentiation. On immunoblots, the OSA recognizes specific sets of molecules (distinguished on the basis of their apparent molecular weights): 53,000 and 59,000 Da antigens in the disk epithelial cells in the last-instar larva; 53,000, 59,000, and 66,000 Da antigens in the ORCs from 15 to 60% of metamorphic adult development; and 42,000, 59,000, and 66,000 Da antigens in the ORCs from 60 to 100% of adult development. The MOSA also recognizes a subset of the epithelial cells in the antennal disks in male and female larvae.
The effect of altered neuronal activity on the development of layers in the lateral geniculate nucleus
Authors: Casagrande VA , Condo GJ
Published: March 2018

The main objective of this study was to examine the role of neural activity in the development of cell layers in the dorsal lateral geniculate nucleus (LGN). We studied this relationship in postnatal tree shrews either by completely blocking retinal ganglion cell activity with TTX or by selectively blocking activity to the developing ON-center LGN layers (1 and 2) with 2-amino-4-phosphonobutyric acid (APB), using unilateral and bilateral eye injections. All manipulations were carried out from birth (P0), when no LGN cell layers are evident, to or past the point when layers are recognizable (i.e., 1–2 weeks). Nissl-stained and cytochrome oxidase (CO)-reacted material was examined for all cases. Our results show that in the absence of activity produced by bilateral TTX injections, interlaminar spaces between cell layers do begin to develop. Retinal afferents, which are segregated at birth, remain segregated, and differential CO staining between matched sets of LGN layers is evident. The normal pace of LGN development, however, is slowed significantly: LGN cells are smaller and interlaminar spaces are narrower than are seen in age-matched controls. Unilateral TTX injections produce similar, but more dramatic and asymmetric, effects on LGN cells, perhaps because cells are at a competitive disadvantage relative to their normally innervated counterparts in cortex. Combining unilateral eye enucleation at P0 with subsequent TTX treatment of the other eye clearly demonstrates that axons from the remaining eye are capable of producing their normal complement of LGN layers. The development of the LGN ON-center layers, 1 and 2, and the interlaminar space between them are more affected by TTX treatment than are the other layers. By contrast, APB eye injections do not selectively affect the development of the ON-center layers, but do result in some slowing of overall LGN development. Taken together, these results suggest that activity of retinal afferents is not essential for initiating interlaminar space formation, but is important for the normal pace of maturation of LGN cell layers.
Parallel pathways can conduct visual CS information during classical conditioning of the NM response
Authors: Koutalidis O , Foster A , Weisz DJ
Published: March 2018

Single and combined lesions were made to the lateral geniculate nucleus (LGN), the superficial layers of the superior colliculus (SC), and the pretectal nuclei (Ptc) prior to conditioning of the nictitating membrane (NM) response in rabbit with a visual conditioned stimulus (CS). Due to technical considerations, lesions of the dorsal LGN were accompanied by lesions of the visual cortex, the only output of the dorsal LGN, in order to render the dorsal LGN nonfunctional. Single lesions to any one of the 3 target systems (LGN, SC, Ptc) did not alter the rate of conditioning. Furthermore, double lesions to any 2 of the systems did not prevent conditioning, although LGN + SC lesions significantly retarded acquisition. When all 3 systems were lesioned, however, animals never acquired to the visual CS, although they successfully conditioned to an auditory CS. The results indicate that in rabbit there are parallel visual pathways individually capable of supporting the acquisition of conditioned NM responses.
The roles of potassium currents in Drosophila flight muscles
Authors: Elkins T , Ganetzky B
Published: March 2018

The roles of different K+ currents in regulating the generation and waveform of action potentials in Drosophila dorsal longitudinal flight muscles (DLMs) were examined in current-clamp experiments. In response to depolarizing current, DLMs displayed an initial transient rectification of the electronic potential lasting for up to hundreds of milliseconds. This delay in excitation was followed by oscillations or graded spikes that finally gave way to sharply rising spikes. Previous voltage-clamp studies of DLMs have revealed an inward Ca2+ current and at least three K+ currents: IA and IK, which are voltage-dependent, and IC, which is Ca2+ dependent. IA and IC are early inactivating currents, while IK is a slow, noninactivating current. In mature adults, selective elimination of IA either with Shaker (Sh) mutations or with 4- aminopyridine (4-AP), had no effect on spike duration or on the delay in excitation. In contrast, when IC was specifically eliminated with the slowpoke (slo) mutation, there was no delay before excitation, the amplitude of the spikes was significantly increased, and the spike duration was increased by 10-fold. Similar results were obtained by reducing IC in normal muscle by intracellular injections of EGTA or by use of low Ca2+ saline. Furthermore, DLM spikes evoked in slo by stimulation of the motorneuron were also broadened, suggesting that IC functions in a similar manner during normal flight as in current- clamped muscles. Elimination of IK along with IA and IC in saline containing tetraethylammonium or Ba2+ resulted in further prolongation of the DLM spike. In Ba2+ saline, there was an additional increase in spike amplitude as well. We conclude that in mature adults, IC, rather than IA, plays the major role in repolarization of DLM spikes and in the delay before excitation.
Characterization of the electrically evoked release of substance P from dorsal root ganglion neurons: methods and dihydropyridine sensitivity
Authors: Holz GG , Dunlap K , Kream RM
Published: March 2018

The mechanism by which dihydropyridines (DHPs) modulate the electrically evoked or KCI-induced release of substance P (SP) from embryonic chick dorsal root ganglion (DRG) neurons was investigated in the present study. The release of SP, as measured by radioimmunoassay (RIA), was characterized in terms of its dependence on extracellular calcium ion, its stimulus-response relationship, its sensitivity to the calcium-channel blocker omega conus toxin (omega-CgTx), and its modulation by the DHPs Bay K 8644 and nifedipine. Here it is reported that omega-CgTx (1 microM) blocked the electrically evoked release of SP. In contrast, the calcium-channel agonist Bay K 8644 (5 microM) facilitated the release of SP (by 45%), whereas the calcium-channel antagonist nifedipine (5 microM) was without effect. When the release of SP was triggered by depolarization of cultures with 60 mM KCI, the actions of the DHPs became much more pronounced. Under these conditions, Bay K 8644 facilitated (by 115%), whereas nifedipine inhibited (by 58%), peptide secretion. Voltage-clamp analysis of DRG cell calcium currents demonstrated that these actions of omega-CgTx, Bay K 8644, and nifedipine are explicable in terms of their effects on the slowly inactivating (L-type) calcium current. On the basis of these findings, it is suggested that the SP release mechanism exhibits DHP sensitivity due to the involvement of L-type calcium channels in the neurosecretory process. This model predicts that the voltage and time- dependent antagonist actions of nifedipine are sufficient to explain its failure to inhibit the electrically evoked release of SP.
A developmentally regulated antigen associated with neural cell and process migration
Authors: Mendez-Otero R , Schlosshauer B , Barnstable CJ , Constantine-Paton M
Published: March 2018

The distribution of an epitope recognized by the monoclonal antibody JONES has been studied immunohistochemically in the developing nervous system of the rat. In the present report, we survey selected regions of the fetal, postnatal, and adult rat nervous system to test the hypothesis that JONES binding is invariably associated with neural cell migration and axon growth in the developing rat. A series of selected developmental stages extending from embryonic day (E) 9 to adult were used in this investigation. The distribution of JONES binding was examined using indirect immunofluorescence, as well as the immunogold procedure. Particular attention was paid to regions where the positions and timing of cell and axon migrations have been well described for the rat. JONES immunoreactivity first appears at E11–12, when it is localized to the lamina terminalis, the telencephalic-diencephalic junction, the midbrain, and the rhombic lip regions of the cytologically undifferentiated neural tube. In all the regions studied, during embryonic and early postnatal life, the labeling is very intense in the ventricular zone and shows a radial array in the adjacent intermediate and marginal zones. The expression of JONES epitope correlates particularly with times of cell migration in the retina, superior colliculus, cerebellum, and telencephalon and in regions undergoing neurite extension, such as the developing optic tract, the white matter of the cerebellum, the dorsal roots, the trigeminal system, and olfactory nerve. JONES binding becomes progressively restricted in the postnatal period. In the adult brain, immunoreactivity is present only in the retina and cerebellum. In the retina, JONES labeling is present in the outer plexiform layer and optic fiber layer. The labeling in the optic fiber layer extends to the optic nerve head and stops abruptly outside the orbit. In the cerebellum, JONES shows a radially oriented pattern throughout the molecular layer and delineates the cell bodies in the Purkinje cell layer. The only non-neural regions that show JONES immunoreactivity are the adrenal medulla and the kidney glomeruli. We conclude that the antigens recognized by the JONES monoclonal antibody are associated with the migration of subsets of cells and axons within the developing rat nervous system and, consequently, may play a role in conveying selectivity to these processes.
Developmental regulation of ganglioside antigens recognized by the JONES antibody
Authors: Schlosshauer B , Blum AS , Mendez-Otero R , Barnstable CJ , Constantine-Paton M
Published: March 2018

The JONES monoclonal antibody has been immunocytochemically associated with regions of the developing rat brain where cell and axon migrations are occurring (Mendez-Otero et al., 1986, 1988). In the present study the antigens recognized by the JONES antibody were analyzed in a variety of brain regions and at developmental ages selected to correspond to the preceding immunocytochemical observations. In accordance with earlier results from retina, JONES binding could not be detected in SDS gels from developing brain. Binding of the antibody was, however, prominent in chloroform/methanol extracts of the same tissues, and it was completely removed from tissue sections by brief chloroform/methanol form/methanol treatment. Enzymatic analyses of chloroform/methanol extracts indicated that the JONES epitope was sensitive to neuraminidase but insensitive to proteases. Overlay assays on developed high-performance thin-layer chromatographic plates (HPTLC) indicate that in all regions the JONES epitope resides on 2 or 3 ganglioside bands, depending on the age examined. These bands migrate between ganglioside standards GD 1a and GM2 on HPTLC plates and have been designated GJ1, GJ2, and GJ3, with the higher number designating the more rapidly migrating species. Occasionally, additional bands migrating in the range of polysialogangliosides were observed. The pattern of expression of GJ species was studied in forebrain, retina, and cerebellar tissue taken from embryonic day 18 (E18), postnatal day 0 (P0), P7, P14, and adult animals. Both region-specific differences in the relative prominence of each band and stage-specific differences in the total amount of the JONES gangliosides were detected. The stage- specific differences in the amount of JONES antigens are well correlated with the developmental periods of maximal cell migration in each region. While the JONES gangliosides are most prominent in forebrain before birth, in they are most prominent during the first 2 postnatal weeks. In cerebellum, JONES antigen expression is more pronounced during the 2 periods of cell migration in this tissue. In retina, the more rapidly migrating GJ3 band was the most prominent band at all stages examined, and this same band is retained in the adult. In cerebellum and forebrain GJ3 is also the most pronounced band during development. However, in contrast to the retina, the more slowly migrating GJ1 band is retained in adult forebrain and cerebellum. A variety of non-brain tissues have also been examined for the presence of the JONES antigens.
Experimental localization of muscarinic receptor subtypes to cingulate cortical afferents and neurons
Authors: Vogt BA , Burns DL
Published: March 2018

A technique was developed to evaluate the potency of ligand binding at M2 ACh receptors and to experimentally localize the M1 and M2 subtypes to specific neuronal processes. Normal and experimental material was prepared with tritiated ligand binding to cryostat-sectioned area 29c of posterior cingulate cortex in rat, coverslip autoradiography, and single-grain-counting techniques. Three fundamental issues were addressed. 1. A morphological criterion termed an index of heterogeneity was developed by which the specificity of M2 binding by different ligands could be assessed. The index was calculated by first determining the laminar distribution of pirenzepine (PZ) binding sites and then summing absolute laminar variations from this distribution for each ligand. According to this measure the most efficient protocol for assaying M2 sites was tritiated oxotremorine-M (OXO) coincubated in unlabeled PZ (5 x 10(-8) M). The classical muscarinic antagonist propylbenzilylcholine mustard (Pr-BCM), however, when coincubated in PZ, was almost as efficient as PZ-blocked OXO binding. 2. Terminal axons of neurons in the anterior thalamic nuclei (ATN) have M2 receptors based on the following observations. First, specific binding of the M1 ligand PZ was unaffected by ATN lesions. Second, tritiated OXO and PrBCM binding blocked with unlabeled PZ, conditions favoring M2 receptor binding, showed significant reductions in binding in layers la, lb, and IV following ATN ablations. Third, IC50 values as determined by competition of PZ for PrBCM binding sites were shifted to lower concentrations in superficial layers by ATN lesions but not in deep layers where the thalamus does not terminate. Finally, in contrast to PZ-blocked OXO and PrBCM binding, binding of PZ-blocked 3H- quinuclidinyl benzilate (QNB) was reduced to homogeneity following ATN lesions. 3. Cortical pyramidal neurons have dendritic receptors that are primarily of the M1 subtype but may also include M2 sites. Thus, full depth ibotenic acid lesions reduced PZ binding by almost 70%. Neurotoxin lesions of neurons in layers II-IV or Vb-VI were followed by degeneration of the apical dendrites of pyramids in layer I and 78 and 15% reductions, respectively, in PZ binding. Also, full-depth neurotoxin lesions combined with ATN ablations completely abolished heterogeneities in PrBCM and PZ-blocked OXO binding. These data demonstrate that experimental techniques can be used in conjunction with normal material to make morphological assessments of the efficiency of binding of putative M2 ligands.
Schwann cell surfaces but not extracellular matrix organized by Schwann cells support neurite outgrowth from embryonic rat retina
Authors: Kleitman N , Wood P , Johnson MI , Bunge RP
Published: March 2018

Despite evidence that glial cell surfaces and components of the extracellular matrix (ECM) support neurite outgrowth in many culture systems, the relative contributions of these factors have rarely been compared directly. Specifically, it remains to be determined which components of peripheral nerve support growth of central nerve fibers. We have directly compared neurite outgrowth from embryonic day 15 rat retinal explants placed onto beds of (1) Schwann cells without ECM, (2) Schwann cells expressing ECM (including a basal lamina), (3) cell-free ECM prepared from neuron-Schwann cell cultures, (4) nonglial cells (fibroblasts), and (5) 2 isolated ECM components, laminin and type I collagen. From the first day in culture, retinal explants extended neurites when placed on Schwann cells without ECM. Outgrowth on Schwann cells expressing ECM was also extensive, but not obviously different form that on Schwann cells alone. Ultrastructural study revealed that 95% of retinal neurites in ECM-containing cultures contacted other neurites and Schwann cell surfaces exclusively. On cell-free ECM prepared from neuron-Schwann cell cultures, neurite extension was poor to nonexistent. No neurite outgrowth occurred on fibroblasts. Retinal explants also failed to extend neurites onto purified laminin and ammoniated type I collagen substrata; however, growth was rapid and extensive on air-dried type I collagen. In cultures containing islands of air-dried type I collagen on a laminin-coated coverslip, retinal explants attached and extended neurites on collagen, but these neurites did not extend off the island onto the laminin substratum. We conclude from these experiments that neurite extension from embryonic rat retina is supported by a factor found on the surface of Schwann cells and that neither organized nor isolated ECM components provide this neurite promotion. These findings are discussed in relation to possible species differences in growth requirements for retinal ganglion cell neurites and to the specificity of response of different CNS neurites to ECM substrata.
Immunocytochemical localization of the guanine nucleotide-binding protein Go in primary cultures of neuronal and glial cells
Authors: Brabet P , Dumuis A , Sebben M , Pantaloni C , Bockaert J , Homburger V
Published: March 2018

We have localized the guanine nucleotide-binding protein, Go, in primary cultures of pure neuronal and glial cells prepared from different mouse brain areas. Immunoblotting experiments with selective affinity-purified polyclonal rabbit antibodies to the 39 kDa alpha subunit of Go (Go alpha) indicated that Go is distributed in both neurons and glial cells. Go alpha accounts for 0.3% of total membrane proteins in striatal neurons. High specific Go immunoreactivity was also detected in cortical neurons and cerebellar granule cells. Similarly, striatal glial cells contain large amounts of Go (0.2% of total membrane proteins), as do glial cells from cerebral cortex and colliculi. Surprisingly, Go was barely detectable in cerebellar glial cells. 32P-ADP-ribosylation of the same neuronal and glial cell membranes with pertussis toxin indicated the presence of at least 3 substrates related to Go alpha, Gi alpha (41 kDa), and a 40 kDa protein. This 40 kDa protein is the major pertussis toxin substrate in glial cells, while Go alpha is predominant in neuronal membranes. Confirming immunoblotting, no labeled band was detected at 39 kDa in cerebellar glial cells with pertussis toxin. Indirect immunofluorescence staining of cerebellar granule cells and striatal neurons with purified Go alpha antibodies was pronounced at the plasma membrane level, particularly at cell-cell contact areas, and in neurite arborization. More discrete staining was also apparent in the cytoplasm, whereas nuclei remained unstained. In striatal glial cells, specific immunolabeling was more diffused over the whole cell, and dense around the nucleus. The localization of Go suggests that this protein must perform important functions in both the neuronal and glial cells that are discussed.
Interleukin-1 is an astroglial growth factor in the developing brain
Authors: Giulian D , Young DG , Woodward J , Brown DC , Lachman LB
Published: March 2018

The immunomodulator interleukin-1 (IL-1) is found to be an astroglial growth factor during development of the mammalian brain. In vitro studies indicate that ameboid microglia, a class of brain mononuclear phagocytes, are the likely source of IL-1. Examination of different brain regions during development shows IL-1 production only after the appearance of ameboid microglia. These observations suggest that brain mononuclear phagocytes secrete growth factors that regulate normal growth and development of the CNS.
Giant Aplysia neuron R2 has distal dendrites: evidence for protein sorting and a second spike initiation site
Authors: Ambron RT , Rayport SG , Babiarz J
Published: March 2018

Because of its anatomy, the neuron R2 of Aplysia has been used to study how proteins are distributed to their appropriate destinations within the cell. The R2 cell body resides in the abdominal ganglion, while its axons terminate on glands in the skin. Using intracellular injection of HRP and intraxonal recordings, we found that R2 has a dendritic (receptive) arborization in the pleural ganglion. The structure of these dendrites was examined after injecting the soma with 3H-L-fucose, thereby labeling glycoproteins that are transported to all regions of the cell. Light- and electron-microscope autoradiography show that the openings to the dendrites are not on the periphery, but are suspended inside the axon by glial cell infoldings. All of the organelles seen in the axon are found in the dendrites, including 2 types of vesicles. Neither the axon nor the dendrites contain ribosomes. Thus, R2 has 3 functionally distinct regions--cell body, dendrites, presynaptic terminals--that are separated from each other by at least 4 cm. This implies that pre- and postsynaptic proteins made in the cell body are transported along the axon to the pleural ganglion, where they are sorted. To investigate this idea, we exposed the abdominal ganglion to 35S-methionine to label R2–s proteins. Analyses by SDS-PAGE of the rapidly transported labeled proteins from R2 consistently showed a 78 kDa band that accumulated in the pleural ganglion and did not move into the peripheral nerves. This then is a putative dendritic constituent.
Hormonal regulation of the shape of identified motoneurons in the moth Manduca sexta
Authors: Truman JW , Reiss SE
Published: March 2018

Motoneurons MN-1 and MN-3 in the moth Manduca sexta go through 3 distinct phases during postembryonic life. During larval life their neuritic fields show allometric growth in concert with larval growth. Larval neurites are then lost during the larval-pupal transition, which is followed by the outgrowth of adult-specific neurites during adult development. In MN-1, the adult-specific arbor typically results from the outgrowth of neurites ipsilateral to the cell body. However, in a small percentage of cells, ipsilateral branches are not present and contralateral branches extend across the midline to fill the vacant space. This altered form of MN-1 is thought to result from the early failure of ipsilateral neurite extension. The steroid hormones, the ecdysteroids, are responsible for the outgrowth of adult neurites. The onset of the latter is correlated with the ecdysteroid increase that promotes adult differentiation and does not occur under conditions such as diapause, in which the normal steroid rise is absent. Artificial replacement of ecdysteroids, however, induces the adult-specific growth. This action of ecdysteroids to cause a change in neuronal form requires the absence of juvenile hormone (JH). Application of JH mimics prior to the onset of the program of adult outgrowth blocks this outgrowth. MN-1 and MN-3 show different times of JH sensitivity, which appear to be correlated with different times of neurite outgrowth. It is concluded that the role of JH is to maintain the status quo of central neurons and prevent changes in form in response to ecdysteroids. In the absence of JH, the ecdysteroids can then exert morphogenetic changes, but the nature of these actions, neurite outgrowth or regression, is likely a function of the developmental history of the cell.