bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of quisqualate receptor desensitization in cultured postnatal rat hippocampal neurons
Authors: Thio LL , Clifford DB , Zorumski CF
Published: March 2018

The quisqualate class of glutamate receptors is thought to play an important role in excitatory synaptic transmission, synaptic plasticity, and neuronal death. Since desensitization is a prominent feature of the responses mediated by this class of receptors, we have characterized the rapidly desensitizing quisqualate response in cultured postnatal rat hippocampal neurons using the whole-cell patch- clamp technique. Quisqualate and its structural analogs elicit a peak current that rapidly decays to a steady-state level. In contrast, currents induced by kainate, NMDA, and their structural analogs exhibit either no decay or a much slower decay. The biophysical and pharmacological properties of the peak and steady-state quisqualate currents indicate that both are mediated by an ionotropic quisqualate receptor. Quisqualate currents desensitized monoexponentially by approximately 70% with a time constant near 80 msec. Both the rate and percentage of desensitization showed slight voltage dependence and were concentration dependent, reaching maximal values at saturation. Additionally, the overlap of the dose-response curves for activation of the steady-state current and desensitization of the peak current by a conditioning dose suggests that the two processes are related. Furthermore, desensitizing quisqualate currents were observed when Ca2+, Mg2+, Na+, K+, and Cl- were removed from the extracellular solution or their concentrations greatly reduced. These results suggest that the decline in the response is not caused by a simple open channel block mechanism. Despite the lack of desensitization by kainate, our observations are consistent with the hypothesis that quisqualate and kainate act at a single receptor-channel complex. Kainate and quisqualate appeared to interact competitively when applied simultaneously and noncompetitively when quisqualate was applied first. In addition, saturating doses of quisqualate and kainate gave steady- state currents of equal amplitude in neurons treated with the lectin WGA, an inhibitor of quisqualate receptor desensitization.
A mannose-specific recognition mediates the defasciculation of axons in the leech CNS
Authors: Zipser B , Cole RN
Published: March 2018

We are studying a mannose-specific recognition mediating the projection of axons in the synaptic neuropil of the embryonic leech CNS. A functional class of neurons, the sensory afferents, can be distinguished by a mannose-containing epitope that is asparagine-linked to a 130 kDa surface protein and is reactive with the monoclonal antibody Lan3-2. Sensory afferents project as a tightly fasciculated bundle through peripheral nerves but, upon arriving in the CNS, defasciculate into the synaptic neuropil. This defasciculation allows the previously bundled sensory afferents to form an arborization in the synaptic neuropil. Three lines of experimental evidence indicate that the defasciculation is mediated by the sensory afferent's mannose- containing Lan3-2 epitope. The defasciculation is inhibited (1) by blocking the Lan3-2 epitope with Lan3-2 Fab fragments, (2) by cleaving the asparagine-linked carbohydrate moieties from surface proteins with the glycosidase N-glycanase, and (3) by competing for a putative mannose-binding protein with the neoglycoprotein mannose-BSA [albumin, p-aminophenyl alpha-D-mannopyranoside (26 mol monosaccharide/mol albumin)]. In addition to inhibiting the defasciculation, the three perturbation reagents also elicited the refasciculation of axons that had defasciculated prior to their application. These three different experimental approaches provide strong evidence that carbohydrate recognition regulates the projections of sensory afferents in the leech synaptic neuropil. Carbohydrate interactions therefore can play a major role in regulating the neuronal architecture in the CNS.
Guidance of callosal axons by radial glia in the developing cerebral cortex
Authors: Norris CR , Kalil K
Published: March 2018

During development, columns of the mammalian cerebral cortex are formed by migration of neurons along fascicles of radial glia. Subsequently, axons of the corpus callosum connect reciprocal regions of each cerebral hemisphere. To determine whether the radial growth of callosal afferents through the developing cortex may be guided by particular cellular elements, we examined the ultrastructural relationship between callosal afferents and radial fibers in the early postnatal hamster sensorimotor cortex. Developing callosal axons and their growth cones were labeled with HRP injected into the cortex at 3 d postnatal when the growth cones have extended across the callosum and are just entering the contralateral cortex. An EM analysis of 30 HRP-labeled axons and their growth cones revealed that they extended upon fascicles of radial processes associated with migrating neurons. Reconstruction of seven of these growth cones, serially sectioned in their entirety, showed that growth cones were associated with the same radial fascicle as their axon. Growth cones also touched other cellular elements such as axons. However, the finding that callosal afferents, from the point at which they enter the cortex to their growth cones, were apposed to a continuous fascicle of radial fibers suggests that callosal axons are tracking along radial processes. We conclude that the majority of the radial processes within fascicles are likely to be glial, based on their relatively large diameters, electron-lucent cytoplasm with a regular array of microtubules, the presence of glycogen granules, occasional cytoplasmic protrusions lacking microtubules, and their consistent association with migrating neurons. We propose therefore that radial glia may serve a guidance function for growing callosal axons in their radial trajectory through the developing cerebral cortex.
Characterization of potential precursor populations in the mouse olfactory epithelium using immunocytochemistry and autoradiography
Authors: Schwartz Levey M , Chikaraishi DM , Kauer JS
Published: March 2018

There are at least two basal cell populations in the olfactory epithelium that could give rise to olfactory neurons during development, in the normal adult, and after experimentally induced receptor cell death. These populations have been subdivided as horizontal (HBC) and globose (GBC) basal cells on the basis of morphological criteria and by staining with antibodies against cytokeratin. HBCs are positive for cytokeratin while GBCs are negative. We have studied which cell type is induced to divide during receptor cell regeneration stimulated by olfactory bulbectomy using a combination of immunocytochemistry and autoradiography. By examining which population increases its labeling index with 3H-thymidine (3H- TdR) at various times after bulbectomy, it is shown that there is an increase in 3H-TdR uptake in the cytokeratin-negative GBCs with no change in the cytokeratin-positive HBCs. This suggests that the GBCs are specifically induced to divide in response to cues that accompany receptor cell death, and it is thus concluded that these cells are among the precursors of new olfactory receptor neurons.
Calcium-activated chloride conductance in frog olfactory cilia
Authors: Kleene SJ , Gesteland RC
Published: March 2018

We have measured the effects of cytoplasmic Ca2+ on the conductance of single cilia excised from frog olfactory receptor neurons. When free cytoplasmic Ca2+ is buffered at 0.1 microM, ciliary conductance is low. As Ca2+ is increased, ciliary conductance increases. Maximal conductance averages sevenfold higher than that measured in the absence of Ca2+. We estimate that the K1/2 for Ca2+ activation is 5 microM; the dose-response curve indicates some positive cooperativity of Ca2+ binding. Activation by Ca2+ is rapid and fully reversible. Most of the Ca(2+)-activated current is carried by Cl- and persists in the absence of Na+ and K+. The Cl- channel inhibitor 3′,5-dichlorodiphenylamine-2- carboxylate (300 microM) reduces the Ca(2+)-activated current by 90%. Odorants induce a Ca2+ influx in some olfactory receptor neurons, but the consequences of this influx for neuronal function are not well understood. Our findings allow us to predict that a Ca2+ influx would increase the permeability of the olfactory cilia to Cl-. How this would affect the neuronal potential is uncertain, since the equilibrium potential for Cl- in olfactory receptor neurons is unknown.
Rapid and selective uptake, metabolism, and cellular distribution of docosahexaenoic acid among rod and cone photoreceptor cells in the frog retina
Authors: Rodriguez de Turco EB , Gordon WC , Bazan NG
Published: March 2018

The uptake, metabolism, and cellular distribution of 3H-docosahexaenoic acid (3H-22:6) in the frog retina during in vitro incubation were studied. An initial diffuse labeling throughout the retina was detected by autoradiography and was followed by an active steady increase in labeled photoreceptor cells. After 6 hr of incubation, 92% of the label was concentrated in photoreceptor cells. Among these cells, 435-rods (green rods) labeled heavily and showed two to three times higher uptake than the 502-rods (red rods). Cone uptake labeling was the lowest, showing negligible labeling throughout the cytoplasm. However, oil droplets of the 575-cones actively concentrated 22:6. The high uptake of 3H-22:6 by photoreceptor cells was followed by its rapid esterification into phospholipids. After 6 hr of labeling, only 5% of the radioactivity in the retina was free 22:6, whereas 88% was esterified into phospholipids. The remaining 22:6 was distributed equally in triacylglycerols (TAGs) and diacylglycerols. When 3H-22:6 (0.11 microM) of high specific activity was used, early incubation times showed phosphatidylinositol (PI) labeling to be of the same order of magnitude or greater than that of phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Although the amount of endogenous 22:6 esterified into PI accounted for less than 2% of the 22:6 in retinal phospholipids, 27% of 3H-22:6 labeling was recovered in this phospholipid. When 14C-22:6 at a final concentration of 70 microM was used, a different profile of lipid labeling was observed. Forty percent of the labeling remained in the free fatty acid pool, followed by TAG (24%), PC (14%), and PE (12%). PI showed the smallest increase in picomoles of 14C-22:6 incorporated, when compared with 3H-22:6. In conclusion, a selective and differential uptake of 3H-22:6 by photoreceptor cells is coupled to its active utilization for phospholipid biosynthesis, mainly that of PC, PE, and PI. The differential uptake of 3H-22:6 among photoreceptor cells may reflect involvement of this fatty acid in cell-specific functions.
Pathfinding and target selection by developing geniculocortical axons
Authors: Ghosh A , Shatz CJ
Published: March 2018

During development of the mammalian cerebral cortex, thalamic axons must grow into the telencephalon and select appropriate cortical targets. In order to begin to understand the cellular interactions that are important in cortical target selection by thalamic axons, we have examined the morphology of axons from the lateral geniculate nucleus (LGN) as they navigate their way to the primary visual cortex. The morphology of geniculocortical axons was revealed by placing the lipophilic tracer Dil into the LGN of paraformaldehyde-fixed brains from fetal and neonatal cats between embryonic day 26 (E26; gestation is 65 d) and postnatal day 7 (P7). This morphological approach has led to three major observations. (1) As LGN axons grow within the intermediate zone of the telencephalon toward future visual cortex (E30– 40), many give off distinct interstitial axon collaterals that penetrate the subplate of nonvisual cortical areas. These collaterals are transient and are not seen postnatally. (2) There is a prolonged period during which LGN axons are restricted to the visual subplate prior to their ingrowth into the cortical plate; the first LGN axons arrive within visual subplate by E36 but are not detected in layer 6 of visual cortex until about E50. (3) Within the visual subplate, LGN axons extend widespread terminal branches. This represents a marked change in their morphology from the simple growth cones present earlier as LGN axons navigate en route to visual cortex. The presence of interstitial collaterals suggests that there may be ongoing interactions between LGN axons and subplate neurons along the entire intracortical route traversed by the axons. From the extensive branching of LGN axons within the visual subplate during the waiting period, it appears that they are not simply “waiting.” Rather, LGN axons may participate in dynamic cellular interactions within the subplate long before they contact their ultimate target neurons in layer 4. These observations confirm the existence of a prolonged waiting period in the development of thalamocortical connections and provide important morphological evidence in support of the previous suggestion that interactions between thalamic axons and subplate neurons are necessary for cortical target selection.
Diffusional transport of macromolecules in developing nerve processes
Authors: Popov S , Poo MM
Published: March 2018

Passive transport of macromolecules in growing nerve processes was analyzed quantitatively by measuring the rate of diffusion of fluorescently labeled molecules injected into the soma of cultured Xenopus neurons. We found that the diffusion of globular proteins in the neurite's cytoplasm was about five times slower than that in aqueous solution, a rate considerably higher than those inferred from previous studies on cultured non-neuronal cells. The dependence of the diffusion coefficient, D, on the size of diffusing molecules was examined by measuring the diffusional spread of fluorescently labeled dextrans over a wide range of molecular weights. We found that the size dependence of D deviates considerably from that expected for diffusion in a viscous aqueous medium: larger dextrans encounter disproportionately higher viscous resistance. Treatment of the neuron with the microfilament-disrupting agent cytochalasin B, or pre-loading of the cells with dephospho-synapsin I, a molecule that induces bundling of actin filaments, significantly increased the diffusion rate for large dextrans without affecting that of small dextrans. Taken together, these results provide a quantitative basis for assessing diffusion as a potential transport mechanism along nerve processes, and suggest that the microfilament meshwork imposes a selective constraint on the diffusion of large macromolecular components within the neuronal cytoplasm.
4-aminopyridine-induced epileptiform activity and a GABA-mediated long- lasting depolarization in the rat hippocampus
Authors: Perreault P , Avoli M
Published: March 2018

Two types of spontaneous filed potentials were recorded in rat hippocampal slices after addition of 4-aminopyridine (4-AP; 50 microM). One consisted of brief, epileptiform discharges that occurred at 0.6 +/- 0.2 sec-1 in the CA3 and CA1 areas. The other type occurred less frequently (0.036 +/- 0.013 sec-1) and was recorded in CA1, CA3, and dentate areas. It corresponded in all regions to an intracellular long- lasting depolarization (LLD; duration, 300–1200 msec; peak amplitude, 2– 15 mV) that was abolished by bicuculline methiodide; therefore, it was mediated by GABAA receptors. Sectioning experiments and the occurrence of propagation failures indicated that LLDs could be initiated by any area of the slice. Furthermore, the propagation of LLDs did not follow any consistent or predictable pattern along known anatomical hippocampal pathways. Finally, neither the occurrence nor the propagation of LLDs was affected when excitatory synaptic transmission was blocked by NMDA and non-NMDA receptor antagonists. In the presence of antagonists of glutamatergic receptors, LLDs disappeared after the omission of Ca2+ or the addition of Cd2+ to the perfusing solution, suggesting that synaptic transmission was required for their generation. These data indicate that 4-AP discloses both interictal epileptiform discharges and LLDs in the rat hippocampus. The first type of activity is presumably related to certain properties of CA3 pyramidal neurons and the neuronal circuit, whereas LLDs originate from the spontaneous, periodic activity of GABAergic interneurons located in any area of the hippocampus, and can propagate to the other areas by the use of nonsynaptic mechanisms. We propose that 4-AP reveals a novel type of interaction among GABAergic interneurons that is based on the accumulation and the dispersion of K+.
Nuclear and cytoplasmic localization of basic fibroblast growth factor in astrocytes and CA2 hippocampal neurons
Authors: Woodward WR , Nishi R , Meshul CK , Williams TE , Coulombe M , Eckenstein FP
Published: March 2018

Fibroblast growth factors (FGFs) are known to stimulate mitogenesis in a variety of non-neuronal cell types and to support the survival in vitro of many neuronal cell types. The physiological role of FGFs in the CNS is currently not known. The present study determined the distribution in the rat CNS of a prominent member of the FGF family, basic FGF (bFGF). Immunohistochemical analysis showed that bFGF immunoreactivity was found predominantly in astrocytes throughout all regions of the CNS. In contrast, only a few neuronal populations were found to contain bFGF immunoreactivity, most prominent among them, neurons in the CA2 area of the hippocampus. This predominant localization of bFGF to astrocytes was confirmed by two other observations: (1) highly enriched cultures of astrocytes contained bFGF immunoreactivity and bioactivity, whereas highly enriched cultures of cerebral cortical neurons contained no detectable bFGF, and (2) neonatal rat cerebral cortex, which contains only a few differentiated astrocytes, also contained no detectable bFGF immunoreactivity and only low amounts of bFGF bioactivity. Immunocytochemical analysis also suggested that bFGF immunoreactivity was present in the nucleus as well as the cytoplasm of astrocytes and CA2 neurons. This nuclear localization was confirmed by EM analysis of the intracellular distribution of the immunoperoxidase reaction product. In addition, preparations of both nuclear and soluble fractions of brain extracts contained bFGF immunoreactivity and bioactivity. These data suggest that bFGF might be involved in mediating astrocytic influences on the late postnatal maturation and plasticity in the CNS, and that the nuclear localization of bFGF within astrocytes may play an important role in the differentiation of these cells. In addition, bFGF may play a similar role in a few specific neuronal populations, such as CA2 hippocampal neurons.
Differential effects of neurotrophic factors on neurotransmitter development in the IMR-32 human neuroblastoma cell line
Authors: Rabinovsky ED , Le WD , McManaman JL
Published: March 2018

The human neuroblastoma cell line IMR-32 exhibits both cholinergic and adrenergic properties. We have used IMR-32 cells to study the effects of CDF (CAT development factor) and bFGF (basic fibroblast growth factor) on the development of neurotransmitter properties. CDF treatment increases CAT activity in a dose-dependent manner, independent of cell density. Time course studies show that there is a threefold increase in the specific CAT activity in IMR-32 cells treated with CDF for 6 d. CDF does not, however, affect the level of tyrosine hydroxylase (TH) activity, or the rate of cell proliferation. bFGF, on the other hand, induces TH activity and decreases CAT activity in a dose-dependent manner. bFGF's effect on TH is enhanced by increasing cell density, while its reduction of specific CAT activity is independent of cell density. Time course studies show a 30-fold increase in TH activity per cell and a threefold decrease in CAT activity per cell, after treatment with bFGF for 6 d. In contrast to the effects of CDF, bFGF enhances cell proliferation in IMR-32 cells. Double-labeled immunofluorescence studies showed that 95% of the cells stain for CAT and 65% stain for TH following treatment with CDF and bFGF, respectively. When these factors are combined, approximately 75% of the cells express both CAT and TH, demonstrating that IMR-32 cells are bipotential with regard to neurotransmitter-associated enzyme expression. We also show that insulin-like growth factor I and NGF selectively induce CAT activity and cell proliferation, respectively, whereas epidermal growth factor has no effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Postmitotic death is the fate of constitutively proliferating cells in the subependymal layer of the adult mouse brain
Authors: Morshead CM , van der Kooy D
Published: March 2018

The early development of the mammalian forebrain involves the massive proliferation of the ventricular zone cells lining the lateral ventricles. A remnant of this highly proliferative region persists into adult life, where it is known as the subependymal layer. We examined the proliferation kinetics and fates of the mitotically active cells in the subependyma of the adult mouse. The medial edge, the lateral edge, and the dorsolateral corner of the subependymal layer of the rostral portion of the lateral ventricle each contained mitotically active cells, but the dorsolateral region had the highest percentage of bromodeoxyuridine (BrdU)-labeled cells per unit area. Repeated injections of BrdU over 14 hr revealed a proliferation curve for the dorsolateral population with a growth fraction of 33%, indicating that 33% of the cells in this subependymal region make up the proliferating population. The total cell cycle time in this population was approximately 12.7 hr, with an S-phase of 4.2 hr. To examine the fate of these proliferating cells, we injected low concentrations of a replication-deficient, recombinant retrovirus directly into the lateral ventricles of adult mice for uptake by mitotically active subependymal cells. Regardless of the survival time postinjection (10 hr, 1 d, 2 d, or 8 d), the number of retrovirally labeled cells per clone remained the same (1 or 2 cells/clone). This suggests that one of the progeny from each cell division dies. Moreover, the clones remained confined to the subependyma and labeled cells were not seen in the surrounding brain tissue. Thus, while 33% of the dorsolateral subependymal cells continue to proliferate in adult life, the fate of the postmitotic progeny is death.
Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells
Authors: Yang XC , Labarca C , Nargeot J , Ho BY , Elroy-Stein O , Moss B , Davidson N , Lester HA
Published: March 2018

The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell- specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX- resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.
NGF/BDNF chimeric proteins: analysis of neurotrophin specificity by homolog-scanning mutagenesis
Authors: Suter U , Angst C , Tien CL , Drinkwater CC , Lindsay RM , Shooter EM
Published: March 2018

Despite their extensive sequence identities at the amino acid level (approximately 55%), NGF and brain-derived neurotrophic factor (BDNF) display distinct neuronal specificity toward neurons of both the PNS and CNS. To explore which region(s) within these neurotrophic factors might determine their differential actions on various subpopulations of peripheral neurons, a systematic series (homolog-scanning mutagenesis) of chimeric NGF/BDNF molecules was prepared using PCR overlap-extension techniques. After expression in COS-7 cells, the chimeric proteins were tested for their biological activities in neurite outgrowth and neuronal survival assays. This approach led to the functional expression of 12 NGF/BDNF chimeras. Surprisingly, despite replacing successive amino acid segments throughout the entire length of NGF with the corresponding parts of BDNF, all chimeras displayed full NGF-like activity in bioassays carried out with PC12 cells, embryonic chick dorsal root ganglion explants, sympathetic ganglion explants, and dissociated cultures of dorsal root ganglion neurons. Most of the chimeras additionally showed BDNF-like activity as defined by neurite outgrowth on chick nodose ganglion explants. However, none of the chimeras supported the survival of dissociated nodose ganglion neurons. Our results suggest that NGF and BDNF must share very similar higher- order protein structures, and we propose that the overall structure or conformation of NGF, in contrast to short amino acid –active-site” segments, may determine its exact neuronal specificity.
Basic FGF in adult rat brain: cellular distribution and response to entorhinal lesion and fimbria-fornix transection
Authors: Gomez-Pinilla F , Lee JW , Cotman CW
Published: March 2018

Basic fibroblast growth factor (bFGF) is a potent trophic factor for neurons and astrocytes and recently has been implicated in the pathology of Alzheimer's disease. In order to better understand the role of bFGF in normal brain function and during pathology, we have analyzed its anatomical distribution and its response to injury in the CNS. Double-staining immunohistochemistry showed that bFGF immunoreactivity was localized in astrocytes, in select neuronal populations, and occasionally in microglial cells throughout the normal rat brain. Neuronal populations that showed bFGF immunoreactivity included septohippocampal nucleus, cingulate cortex, subfield CA2 of the hippocampus, cerebellar Purkinje cells, cerebellar deep nuclei, facial nerve nucleus, and the motor and spinal subdivisions of the trigeminal nucleus and facial nerve nucleus. The pattern of bFGF immunoreactivity in the hippocampus was examined following entorhinal cortex lesion, or fimbria-fornix transection. After entorhinal cortex lesion, bFGF immunoreactivity increased in the outer molecular layer of the dentate gyrus ipsilateral to the lesion. The lesion effect on bFGF immunoreactivity was expressed as an increase in the number of bFGF astrocytes, as an increase in the intensity of bFGF immunoreactivity within astrocytes, and as an increase of bFGF immunoreactivity in the surrounding extracellular matrix, relative to the contralateral side. The time course and pattern of reorganization paralleled the sprouting of septal cholinergic terminals in response to the same type of lesion, suggesting that bFGF may play an important role in lesion-induced plasticity. After transection of the fimbria-fornix, chronic infusion of bFGF appeared to preserve NGF receptors on neurons within the medial septal complex and, as previously reported, prevent the death of medial septal neurons. Therefore, it appears that bFGF infusion, which has been shown to increase the synthesis of NGF by astrocytes (Yoshida and Gage, 1991), also helps enable neurons to respond to NGF. This suggests that after injury bFGF may participate in a cascade of neurotrophic events, directly and indirectly facilitating neuronal repair and/or promoting neuronal survival.
Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals
Authors: Betz WJ , Mao F , Bewick GS
Published: March 2018

Living motor nerve terminals from several species can be stained in an activity-dependent fashion by certain styryl dyes, such as RH414, RH795, and a new dye, FM1–43, which can be imaged independently of the others. The dyes evidently become trapped within recycled synaptic vesicles. In frog cutaneus pectoris muscle, bright fluorescent spots spaced regularly along the length of the nerve terminals appear after stimulation in the presence of the dye. The spots align well with postsynaptic ACh receptors and are persistent for many hours, unless further stimulation is given, in which case the spots disappear. Destaining, like staining, requires transmitter release and proceeds gradually over several minutes at high stimulus frequencies (e.g., 30 Hz), and fluorescent spots in the same terminal disappear at about the same rate. We suggest that each spot is a cluster of hundred of synaptic vesicles and that the mechanism of staining involves the ability of the dyes to partition reversibly into the outer leaflet of surface membranes, without being able to penetrate the entire membrane thickness. Then, during endocytosis following transmitter release, dye molecules become trapped in recycled synaptic vesicle membranes. The dyes therefore make it possible optically to study vesicle exocytosis and recycling in living nerve terminals in real time, and should be useful for marking terminals in a variety of preparations according to their level of activity.
Ionic mechanisms of anoxic injury in mammalian CNS white matter: role of Na+ channels and Na(+)-Ca2+ exchanger
Authors: Stys PK , Waxman SG , Ransom BR
Published: March 2018

White matter of the mammalian CNS suffers irreversible injury when subjected to anoxia/ischemia. However, the mechanisms of anoxic injury in central myelinated tracts are not well understood. Although white matter injury depends on the presence of extracellular Ca2+, the mode of entry of Ca2+ into cells has not been fully characterized. We studied the mechanisms of anoxic injury using the in vitro rat optic nerve, a representative central white matter tract. Functional integrity of the nerves was monitored electrophysiologically by quantitatively measuring the area under the compound action potential, which recovered to 33.5 +/- 9.3% of control after a standard 60 min anoxic insult. Reducing Na+ influx through voltage-gated Na+ channels during anoxia by applying Na+ channel blockers (TTX, saxitoxin) substantially improved recovery; TTX was protective even at concentrations that had little effect on the control compound action potential. Conversely, increasing Na+ channel permeability during anoxia with veratridine resulted in greater injury. Manipulating the transmembrane Na+ gradient at various times before or during anoxia greatly affected the degree of resulting injury; applying zero-Na+ solution (choline or Li+ substituted) before anoxia significantly improved recovery; paradoxically, the same solution applied after the start of anoxia resulted in more injury than control. Thus, ionic conditions that favored reversal of the normal transmembrane Na+ gradient during anoxia promoted injury, suggesting that Ca2+ loading might occur via reverse operation of the Na+)-Ca2+ exchanger. Na(+)- Ca2+ exchanger blockers (bepridil, benzamil, dichlorobenzamil) significantly protected the optic nerve from anoxic injury. Together, these results suggest the following sequence of events leading to anoxic injury in the rat optic nerve: anoxia causes rapid depletion of ATP and membrane depolarization leading to Na+ influx through incompletely inactivated Na+ channels. The resulting rise in the intracellular [Na+], coupled with membrane depolarization, causes damaging levels of Ca2+ to be admitted into the intracellular compartment through reverse operation of the Na(+)-Ca2+ exchanger. These observations emphasize that differences in the pathophysiology of gray and white matter anoxic injury are likely to necessitate multiple strategies for optimal CNS protection.
Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding
Authors: Pompeiano M , Palacios JM , Mengod G
Published: March 2018

In order to localize the cells expressing 5-HT1A receptors in the rat brain, we used in situ hybridization histochemistry to visualize the distribution of the mRNA coding for 5-HT1A receptors. Oligonucleotides derived from different parts of the coding region of the rat 5-HT1A receptor gene were used as hybridization probes. 5-HT1A binding sites were visualized on consecutive sections by receptor autoradiography using 3H-8-hydroxy-2-(di-n-propylamino)tetralin as ligand. The highest levels of hybridization were observed in the dorsal raphe nucleus, septum, hippocampus, entorhinal cortex, and interpeduncular nucleus. Positive hybridization signals were also present in other areas, such as the olfactory bulb; cerebral cortex; some thalamic and hypothalamic nuclei; several nuclei of the brainstem, including all the remaining raphe nuclei, nucleus of the solitary tract, and nucleus of the spinal tract of the trigeminus; and the dorsal horn of the spinal cord. The distribution and abundance of 5-HT1A receptor mRNA in different rat brain areas generally correlate with those of the binding sites, suggesting that 5-HT1A receptors are predominantly somatodendritic receptors.
Opioids excite dopamine neurons by hyperpolarization of local interneurons
Authors: Johnson SW , North RA
Published: March 2018

Increased activity of dopamine-containing neurons in the ventral tegmental area is necessary for the reinforcing effects of opioids and other abused drugs. Intracellular recordings from these cells in slices of rat brain in vitro showed that opioids do not affect the principal (dopamine-containing) neurons but hyperpolarize secondary (GABA- containing) interneurons. Experiments with agonists and antagonists selective for opioid receptor subtypes indicated that the hyperpolarization of secondary cells involved the mu-receptor. Most principal cells showed spontaneous bicuculline-sensitive synaptic potentials when the extracellular potassium concentration was increased from 2.5 to 6.5 or 10.5 mM; these were prevented by TTX and assumed to result from action potentials arising in slightly depolarized local interneurons. The frequency of these synaptic potentials, but not their amplitudes, was reduced by opioids selective for mu-receptors. It is concluded that hyperpolarization of the interneurons by opioids reduces the spontaneous GABA-mediated synaptic input to the dopamine cells. In vivo, this would lead to excitation of the dopamine cells by disinhibition, which would be expected to contribute to the positive reinforcement seen with mu-receptor agonists such as morphine and heroin.
Increases in excitability of neurons of the motor cortex of cats after rapid acquisition of eye blink conditioning
Authors: Aou S , Woody CD , Birt D
Published: March 2018

Measurements were made of resting potentials, input resistance, and excitability to intracellularly applied, depolarizing current pulses in neurons of the pericruciate cortex of conscious cats before and after acquisition of a rapidly conditioned eye blink reflex (CR). Neuronal excitability increased after conditioning, and an increased input resistance was found to be correlated with the increased level of excitability. No associated changes were found in resting potentials as a consequence of conditioning. When cells were divided into groups according to the latency of spike activity elicited by a click conditioned stimulus (CS) in relation to four separate excitatory EMG components of the compound blink CR, excitability increases were found in cells with increased spike activity at alpha 1 (8–40 msec), alpha 2 (40–72 msec), beta (72–112 msec), and gamma (112–160 msec) latencies after delivery of the CS. Also, the proportion of cells with high excitability (less than 0.7 nA required for spike elicitation) was increased at each latency period after conditioning. Increases in later components of spike discharge could also be found in the cells with increases in earlier components of discharge and increased excitability. The findings suggested that excitability increases facilitated a responsiveness to the CS that supported production of long- as well as short-latency components of the blink CR. Many of the changes in neuronal properties found after rapid eye blink conditioning, such as the increases in excitability and resistance and in the proportion of CS-excitable cells, resembled changes found earlier after acquisition of a slowly developing Pavlovian blink CR, using the same click CS and tap unconditioned stimulus without addition of a hypothalamic stimulus. The possibility should be considered that the (10–100 times) more rapidly acquired form of eye blink conditioning does not represent a different form of conditioning, but instead a change in the rate of conditioning supported by the more rapid production of increases in neural excitability.
Computer simulations of EPSP-spike (E-S) potentiation in hippocampal CA1 pyramidal cells
Authors: Wathey JC , Lytton WW , Jester JM , Sejnowski TJ
Published: March 2018

Long-term potentiation of hippocampal excitatory synapses is often accompanied by an increase in the probability of spiking to an EPSP of fixed strength (E-S potentiation). We used computer simulations of a CA1 pyramidal neuron to test the plausibility of the hypothesis that E- S potentiation is caused by changes in dendritic excitability. These changes were simulated by adding “hot spots” of noninactivating voltage- sensitive Ca2+ conductance to various dendritic compartments. This typically caused spiking in response to previously subthreshold synaptic inputs. The magnitude of the simulated E-S potentiation depended on the passive electrical properties of the cell, the excitability of the soma, and the relative locations on the dendrites of the synaptic inputs and hot spots. The specificity of the simulated E-S potentiation was quantified by colocalizing the hot spots with a subset (40 of 80) of the synaptic contacts, denoted “tetanized,” and then comparing the effects of the hot spots on these and the remaining (untetanized) synaptic contacts. The simulated E-S potentiation tended to be specific to the tetanized input if the untetanized contacts were, on average, electrically closer to the soma than the tetanized contacts. Specificity was also high if the tetanized and untetanized contacts were segregated to different primary dendrites. The results also predict, however, that E-S potentiation by this mechanism will appear to be nonspecific (heterosynaptic) if the synapses of the untetanized input are sufficiently far from the soma relative to the tetanized synapses. Experimental confirmation of this prediction would support the hypothesis that changes in postsynaptic excitability can contribute to hippocampal E-S potentiation.
NMDA channel behavior depends on agonist affinity
Authors: Lester RA , Jahr CE
Published: March 2018

We have compared the kinetic properties of NMDA receptor channels activated by exogenous agonists with those activated synaptically. Short (4 msec) applications of L-glutamate to outside-out patches from hippocampal neurons evoked currents that decayed with a double exponential time course that was controlled by both the unbinding rate of agonist and receptor desensitization. Lower-affinity agonists evoked NMDA receptor-activated currents that had faster rates of decay and recovered from desensitization more quickly, consistent with the idea that agonists which dissociate faster allow the receptor to reach a desensitized state less often. Both synaptic and patch responses could be well fitted with a simple kinetic model comprised of two independent but identical binding sites, one open state, one closed state, and one desensitized state, all doubly liganded. Provided that the agonist has a slow unbinding rate relative to the rates into the open and desensitized states (e.g., L-glutamate), this model predicts a response with two decay phases and can thus account for the synaptic response. Since the unbinding rate is the critical determinant of the time course, different affinity transmitters would affect such properties as excitatory postsynaptic current (EPSC) duration. Of the known endogenous excitatory amino acids, only L-glutamate has an affinity for the NMDA receptor consistent with the time course of the EPSC recorded between hippocampal neurons in culture.
Visceral targets specify calcitonin gene-related peptide and substance P enrichment in trigeminal afferent projections
Authors: Horgan K , van der Kooy D
Published: March 2018

Rat trigeminal ganglion projections to a visceral target (intracranial blood vessels) are enriched in calcitonin gene-related peptide (CGRP) and substance P (Sub P) compared to trigeminal ganglion projections to a cutaneous target (the forehead skin). We asked if transplants of a novel visceral target (fetal stomach antrum tissue) into the path of the neonatal rat trigeminal frontal nerve projection to forehead skin would induce neuronal CGRP and Sub P enrichment. By postnatal day (P) 25, the percentage of nerves containing CGRP increased from 14–15% in the control trigeminal projection to forehead skin to 20–31% (in different experiments) in the trigeminal projection to transplanted stomach antrum. The percentage of Sub P-containing neurons increased from 10% in the control forehead skin projection to 22% in the trigeminal projection to stomach transplants over the same time period. The number of neurons in the trigeminal frontal nerve projection to stomach antrum transplants was not significantly different from the number of frontal neurons projecting to control forehead skin. We suggest that respecification of trigeminal neurons to the CGRP and Sub P phenotype, not selective survival of CGRP- and Sub P-positive afferents, is the mechanism by which stomach antrum induces enrichment of CGRP and Sub P. A subpopulation of rat trigeminal neurons with cutaneous forehead skin projections also sends a transient axon collateral projection to a visceral target (the cerebral arteries) during early postnatal development. Postnatal maintenance of an axonal projection to a cutaneous target (forehead skin) may be incompatible with a neuron also maintaining a visceral collateral to the cerebral arteries.(ABSTRACT TRUNCATED AT 250 WORDS)
Asymmetric modulation of cytosolic cAMP activity induces growth cone turning
Authors: Lohof AM , Quillan M , Dan Y , Poo MM
Published: March 2018

The possible role of cyclic nucleotides as second messengers mediating growth cone turning was studied by producing an asymmetric distribution of cyclic nucleotides across the growth cone. A repetitive pulse application method was developed to produce microscopic chemical gradients near the growth cone of embryonic Xenopus neurons in cell culture. When picoliters of a solution containing 20 mM dibutyryl cAMP (dB-cAMP), a membrane-permeable analog of cAMP, were repetitively ejected from a micropipette near the growth cone, neurite growth was consistently directed toward the pipette. Theoretical analysis of the diffusion gradient showed that the neurite is capable of detecting a 10% difference in dB-cAMP concentration across the growth cone. Similar responses were also observed using gradients of the phosphodiesterase inhibitor isobutylmethylxanthine, or of forskolin, which activates adenylate cyclase. Dibutyryl cGMP, however, produced no significant turning. These results suggest that a cytoplasmic gradient of cAMP across the growth cone is sufficient to initiate its turning response, and that cAMP in the growth cone could serve as a second messenger in mediating the action of extracellular guidance cues.
Differential localization of G-proteins Gi and Go in the accessory olfactory bulb of the rat
Authors: Shinohara H , Asano T , Kato K
Published: March 2018

To clarify the functional differences among G-proteins, we investigated the localization of Gi and Go in the olfactory bulb of rats by both immunohistochemical and immunochemical techniques, using purified antibodies specific to the alpha-subunits of Gi1 (Gi1 alpha), Gi2 (Gi2 alpha), and Go (Go alpha), respectively. We found that Gi2 alpha is localized exclusively in the accessory olfactory bulb, but it is present at only low levels in the main olfactory bulb. The unique pattern of immunoreactivity specific for Gi2 alpha and Go alpha within the glomeruli of the accessory olfactory bulb and the results of immunoassays indicate that the accessory olfactory bulb is divided into two parts: the anterior region is rich in Gi2, while the posterior region is rich in Go. These findings suggest that the accessory olfactory bulb has two different functions. In addition, we found that the concentration of Gi2 alpha in the accessory olfactory bulb increases during puberty and reaches the adult level at 12 weeks after birth, while that in the main olfactory bulb remains constant. By contrast, the concentrations of Go alpha in the accessory olfactory bulb and the main olfactory bulb increase with similar kinetics. These findings suggest that Gi2 is a key protein in signal transduction in the accessory olfactory bulb, and increases in its level seem to be related to sexual maturation.
The morphological basis for binocular and ON/OFF convergence in tree shrew striate cortex
Authors: Muly EC , Fitzpatrick D
Published: March 2018

We used retrograde and anterograde transport methods and single-cell reconstructions to examine the projection from layer IV to supragranular layers in the tree shrew's striate cortex. We found that neurons in the ON and OFF subdivisions of layer IV (IVa and IVb, respectively) have overlapping terminal fields throughout layers II and III. Despite their overlap, these projections are organized in a highly stratified, mirror-symmetric fashion that respects the vertical position of neurons within each sublayer. Neurons in the middle of layer IV (lower IVa and upper IVb) project to layers IIIa/b, II, and I; neurons located at the edges of layer IV (upper IVa and lower IVb) project to the lower half of layer IIIc; and neurons in the middle of IVa and the middle of IVb project to upper IIIc. The stratified nature of the projections from layer IV to layer III is reminiscent of the pattern of ipsilateral and contralateral eye inputs to layer IV. Inputs from the ipsilateral eye are limited to the edges of layer IV (upper IVa and lower IVb), while those from the contralateral eye terminate throughout the depth of IVa and IVb. Thus, cells near the edges of layer IV should receive strong input from both eyes, while those in the middle of layer IV should receive mostly contralateral input. Taken together, these results suggest that the projections from layer IV to layer III bring together the information conveyed by the ON and OFF pathways, but do so in a way that matches the ocular dominance characteristics for each pathway.