bluuebunny/crossref_metadata_2025 huggingface dataset:
Synaptic differentiation of a single motor neuron: conjoint definition of transmitter release, presynaptic calcium signals, and ultrastructure
Authors: Cooper RL , Marin L , Atwood HL
Published: April 2018

The opener muscle in the walking legs of the crayfish (Procambarus clarkii) is innervated by only one excitatory motor neuron, yet excitatory postsynaptic potentials (EPSPs) of proximal fibers are eightfold larger than those of central muscle fibers at low frequencies of activation, due in large measure to differences in presynaptic properties. We investigated quantal release properties, calcium signals, and ultrastructure of presynaptic terminals to elucidate factors that could account for the physiological differences. Focal macropatch electrodes were placed over individual visualized terminal varicosities to obtain records of quantal contributions to the excitatory junctional current (EJC). At low frequencies of activation, mean quantal content is greater for proximal than for central varicosities. This difference is due to a higher probability of release per synapse, and not to a larger number of active synapses. Recorded varicosities were labeled with fluorescent beads deposited by the electrode. These beads adhered to the muscle fibers, outlining the recorded site for subsequent serial thin sectioning and reconstruction from electron micrographs. Comparisons of structure and function were made for individual varicosities. The number of active zones per terminal surface area and the number of synapses with multiple active zones (complex synapses) were greater in high-output varicosities. Calcium indicators were loaded into proximal and central nerve terminals by axonal injection to compare the relative differences in calcium buildup during stimulation. Presynaptic calcium signals were larger for proximal varicosities than for central varicosities.
Nocodazole-dependent transport, and brefeldin A--sensitive processing and sorting, of newly synthesized membrane proteins in cultured neurons
Authors: Cid-Arregui A , Parton RG , Simons K , Dotti CG
Published: April 2018

The envelope glycoproteins of Semliki Forest virus (SFV), Vesicular Stomatitis virus (VSV), and Influenza Fowl Plague virus (FPV) are vectorially targeted in neurons to the plasma membrane of dendrites (SFV and VSV) and axons (FPV). To gain insight into the mechanisms responsible for such polarized delivery we have examined the effects on neurons of nocodazole and brefeldin A (BFA), which are known to cause microtubule depolymerization and disassembly of the Golgi apparatus, respectively. Nocodazole treatment blocked transport of all viral glycoproteins to both axons and dendrites. BFA treatment induced disruption of the Golgi complex, including the trans-Golgi network (TGN), and tubulation of endosomes. However, the delivery of the SFV and FPV glycoproteins to the cell surface was not affected significantly by BFA, although processing and sorting were altered, as revealed by surface biotinylation and immunofluorescence microscopy of fixed nonpermeabilized cells. These results demonstrate the involvement of microtubules in axonal and dendritic transport of integral membrane glycoproteins, and the existence of a BFA-sensitive component in the sorting but not in the transport machinery.
Extent of intracortical arborization of thalamocortical axons as a determinant of representational plasticity in monkey somatic sensory cortex
Authors: Rausell E , Jones EG
Published: April 2018

The extent of intracortical arborization of individual thalamocortical axons in area 3b of the somatic sensory cortex and the degree of overlap in the cortical projections of relay cells in the ventral posterior nucleus of the thalamus were examined in macaque monkeys. Paired intracortical deposits of Fast blue (FB) and Diamidino yellow (DY) separated by 100–1500 microns were made by inserting crystals of dye into the tracks of tungsten microelectrodes used to record receptive field data on area 3b cells. Each injection gave retrograde labeling of one or more clusters of cells extending in elongated anteroposterior arrays through the ventral posterior medial (VPM) or ventral posterior lateral (VPL) nucleus. Double-labeled cells were only found when the distance between the centers of the dye deposits was less than 600 microns. With interdeposit distances greater than 600 microns, most clusters of retrogradely labeled cells had a majority of cells labeled by FB or DY. However, even with interdeposit distances of 1–1.5 mm the labeled clusters also contained significant numbers of cells labeled with the other dye. These results and an accompanying regression analysis indicate that the extent of intracortical arborization of single thalamocortical axons in area 3b is no greater than 600 microns. However, adjoining cells in the same part of the thalamic body representation can project to cortical targets as discrepant as 1.5 mm. It is proposed that the fine grain of the cortical representation depends upon inputs from the majority population of each thalamic cell cluster.
The potassium channel subunit KV3.1b is localized to somatic and axonal membranes of specific populations of CNS neurons
Authors: Weiser M , Bueno E , Sekirnjak C , Martone ME , Baker H , Hillman D , Chen S , Thornhill W , Ellisman M , Rudy B
Published: April 2018

Potassium channels play major roles in the regulation of many aspects of neuronal excitability. These channels are particularly well suited for such multiplicity of roles since there is a large diversity of channel types. This diversity contributes to the ability of specific neurons (and possibly different regions of the same neuron) to respond uniquely to a given input. Neuronal integration depends on the local response of spatially segregated inputs to the cell and the communication of these integration centers with the axon. Therefore, the functional implications of a given set of K+ channels varies depending on their precise location on the neuronal surface. Site- specific antibodies were utilized to characterize the distribution of KV3.1b, a subunit of voltage-gated K+ channels in CNS neurons. KV3.1b subunits are expressed in specific neuronal populations of the rat brain, such as cerebellar granule cells, projecting neurons of deep cerebellar nuclei, the substantia nigra pars-reticulata, the globus pallidus, and the ventral thalamus (reticular thalamic nucleus, ventral lateral geniculate and zona incerta). The KV3.1b protein is also present in various neuronal populations involved in the processing of auditory signals, including the inferior colliculus, the nuclei of the lateral lemniscus, the superior olive, and some parts of the cochlear nuclei; as well as in several other neuronal groups in the brainstem (e.g., in the oculomotor nucleus, the pontine nuclei, the reticulotegmental nucleus of the pons, trigeminal and vestibular nuclei, and the reticular formation) and subsets of neurons in the neocortex, the hippocampus and the caudate-putamen shown by double staining to correspond to neurons containing parvalbumin. KV3.1b subunits are localized predominantly in somatic and axonal membranes (particularly in axonal terminal fields) but are much less prominent in dendritic arborizations. This distribution is different than that of other subunits of voltage gated K+ channels and is consistent with a role in the modulation of action potentials. KV3.1b proteins have a cellular and subcellular distribution different than the related KV3.2 subunits which express in Xenopus oocytes currents similar to those expressed by KV3.1b.
Androgen alters the dendritic arbors of SNB motoneurons by acting upon their target muscles
Authors: Rand MN , Breedlove SM
Published: April 2018

In adult male rats, motoneurons of the spinal nucleus of the bulbocavernosus (SNB) have been shown to retract and reextend their dendritic branches in response to systemic androgen deprivation and readministration. Furthermore, other studies have suggested that the dendritic complexity of neurons can be regulated by their targets. To assess whether androgens might act upon the target muscles to mediate changes in SNB dendrites, adult male rats were castrated and implanted with a small capsule filled with testosterone (T) next to the bulbocavernosus and levator ani muscle complex (BC/LA) on one side, while the muscles on the contralateral side were implanted with another capsule containing hydroxyflutamide (hFl), an anti-androgen. We have previously shown that after 30 d of this focused, lateralized androgen treatment the BC/LA complex is significantly larger on the T-treated side. We now report that the total dendritic lengths of SNB motoneurons innervating muscles given androgen blockade are reduced by 44% compared to SNB motoneurons innervating muscles given androgen stimulation. Dendrite lengths within three regions of the spinal cord were altered in a nonuniform manner: large changes occurred in the dorsal and contralateral dendritic fields while there was no difference in the ipsilateral dendritic field. These results suggest that BC/LA muscles, in response to androgen stimulation, produce a trophic substance which regulates the dendritic organization of SNB motoneurons in adulthood.
Quantification of excitatory amino acid uptake at intact glutamatergic synapses by immunocytochemistry of exogenous D-aspartate
Authors: Gundersen V , Shupliakov O , Brodin L , Ottersen OP , Storm-Mathisen J
Published: April 2018

To study the localization and efficiency of glutamate/aspartate membrane transport in the vicinity of intact glutamatergic synapses, the avascular lamprey spinal cord was incubated with D-aspartate, a metabolically inert transporter substrate. The exogenous D-aspartate was localized by immunocytochemistry after aldehyde fixation. Incubation at 50 or 500 microM D-aspartate for 1 hr caused a prominent D-aspartate labeling of glial processes at glutamatergic synapses, while presynaptic axons and postsynaptic dendrites remained unlabeled. The glial processes surrounding glutamatergic sensory axons with a predominantly tonical firing pattern contained significantly higher levels of D-aspartate than did processes surrounding glutamatergic reticulospinal axons, which fire rarely and in brief bursts. Preparations incubated for 10 hr with 500 microM D-aspartate showed D- aspartate immunolabeling in glia as well as in the two types of glutamatergic axon, but no evidence was obtained for uptake into synaptic vesicles. Nor was such evidence obtained after high-frequency electrical stimulation. The observations suggest that excitatory amino acids delivered diffusely to the extracellular space in the intact CNS are transported almost exclusively into glia. The avid uptake in glial processes, combined with their spatial arrangement around glutamatergic synapses, appears to limit the access of exogenous D-aspartate to the nerve terminal glutamate/aspartate transporter. In physiological conditions, the glial processes are likely to impede the exchange of glutamate between the synaptic cleft and the rest of the extracellular space. The transport was more efficient in glial processes located near tonically active synapses than in ones located near synapses releasing transmitter sporadically. D-Aspartate is not a substrate of vesicular glutamate transport sites at these intact synapses.
Mice doubly deficient in the genes for P0 and myelin basic protein show that both proteins contribute to the formation of the major dense line in peripheral nerve myelin
Authors: Martini R , Mohajeri MH , Kasper S , Giese KP , Schachner M
Published: April 2018

In search for the molecular mechanisms underlying the formation of the major dense line in peripheral nerve myelin we investigated mice deficient in the myelin proteins P0 and MBP. In mice lacking both molecules axons were enwrapped by myelin-like processes devoid of the major dense line, while mice deficient in either protein showed, respectively, partial and normal compaction. Mice heterozygous for P0 but devoid of MBP showed myelin of reduced thickness around axons of normal caliber. Both molecules thus contribute to the formation of the major dense line and to the determination of myelin thickness. Furthermore, our observations modify the view that axon caliber is dependent on normal myelin.
Electrical activity and calcium influx regulate ion channel development in embryonic Xenopus skeletal muscle
Authors: Linsdell P , Moody WJ
Published: April 2018

The development of electrical excitability involves complex coordinated changes in ion channel activity. Part of this coordination appears to be due to the fact that the expression of some channels is dependent on electrical activity mediated by other channel types. For example, we have previously shown that normal potassium current development in embryonic skeletal muscle cells of the frog Xenopus laevis is dependent on sodium channel activity. To examine the interrelationships between the development of different ionic currents, we have made a detailed study of electrical development in cultured Xenopus myocytes using whole-cell patch-clamp recording. The initial expression of potassium, sodium, and calcium currents is followed by a brief period during which the densities of potassium currents decrease, while at the same time sodium and calcium current densities continue to increase, which may increase electrical excitability during this time. The normal developmental increase in both potassium and sodium currents is inhibited by the sodium channel blocker tetrodotoxin, suggesting that electrical activity normally stimulates the expression of both these currents. These effects of electrical activity appear to be mediated via activation of voltage-gated calcium channels. We suggest that the developmental acquisition of sodium and calcium channels by these cells, possibly coupled with a transient decrease in potassium current density, lead to an increase in electrical excitability and calcium entry, and that this calcium entry provides a critical developmental cue controlling the subsequent development of mature electrical properties.
Glia modulate the response of murine cortical neurons to excitotoxicity: glia exacerbate AMPA neurotoxicity
Authors: Dugan LL , Bruno VM , Amagasu SM , Giffard RG
Published: April 2018

We have developed “pure” neuronal cultures (< 1% astrocytes) from mouse neocortex to study the effect of glial cells on the response of neurons to injury. Cortical neurons were found to require glial-conditioned medium to survive. Immature neurons, 2–4 d in vitro, deprived of glial- conditioned medium, underwent apoptosis over 48 hr, as suggested by condensed nuclear morphology, DNA fragmentation, and protection by inhibition of macromolecular synthesis. Apoptosis induced by trophic factor deprivation has been described for other neuronal populations, such as superior cervical ganglion and dorsal root ganglion cells. Cortical neurons in pure culture provide another neuronal population for the study of apoptosis induced by trophic factor deprivation. We then studied the interaction of neurons and glia under excitotoxic conditions. Experiments on mature cultures showed that pure neuronal cultures were at least 10-fold more sensitive to acute glutamate exposure than were neuronal-glial (“mixed”) cocultures. The difference in sensitivity between pure neurons and mixed cultures was reduced when mixed cultures were treated with the glutamate uptake inhibitor, L- trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). In 24 hr exposure to N-methyl-D-aspartate (NMDA), or oxygen, glucose deprivation, pure neurons were more sensitive than mixed cultures; trans-PDC again increased the sensitivity of mixed cultures to nearly that of pure neuronal cultures. In contrast, mixed and pure neuronal cultures exposed to NMDA for 10 min, or to kainate for 24 hr, had similar injury dose-response curves, suggesting that glial glutamate uptake is a less important protective mechanism in these excitotoxic injuries. Surprisingly, pure neurons were less sensitive than mixed cultures to (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) toxicity at concentrations up to 100 microM. This does not reflect astrocyte toxicity, as AMPA at concentrations to 1 mM did not injure astrocyte cultures. Glial cultures showed increased levels of glutamate in the extracellular medium in response to exposure to AMPA, but not NMDA or kainate. However, pure neuronal and mixed cultures exposed to the same concentration of AMPA did not have elevated levels of glutamate in the media. We found that glia were generally neuroprotective under excitotoxic conditions, likely through their ability to clear extracellular glutamate. However, the presence of glia exacerbated AMPA neurotoxicity.
Lamina-specific expression of adhesion molecules in developing chick optic tectum
Authors: Yamagata M , Herman JP , Sanes JR
Published: April 2018

The optic tectum is the major synaptic target of retinal axons in birds. In the chick, retinal ganglion cell axons enter the optic tectum through a superficial lamina (the stratum opticum), extended branches into deeper laminae, and arborize in specific “retinorecipient” laminae, where they form synapses. Studies using an organotypic culture system have provided evidence that the tectum bears a series of distinct, lamina-specific, cell surface-associated cues that direct these axonal behaviors (Yamagata and Sanes, 1995). Here, we have used a panel of antibodies to 30 membrane and matrix components to ask whether known adhesive molecules are distributed in lamina-specific patterns. Among many spatiotemporal pattern of expression documented, three were particularly noteworthy: (1) The cell adhesion molecules NgCAM/L1 and TAG-1/axonin-1 were concentrated in the stratum opticum. (2) SC1/JC7/DM- GRASP/BEN, N-cadherin, neuropilin, polysialylated N-CAM, and glycoconjugates recognized by the lectin VVA-B4 were concentrated in retinorecipient laminae. (3) Neurofascin, tenascin-C/cytotactin, and a matrix molecule defined by the “Sigma” antibody were present at highest levels in areas that border the retinorecipient laminae. Some members of each group (NgCAM/L1, TAG-1/axonin, SC1/JC7, polysialic acid, VVA-B4- receptors, and neurofascin) appeared on schedule and in lamina- restricted patterns in tecta from embryos that had been enucleated before retinal axons left the eye. Thus, molecules in these three categories could provide signals to retinal axons that promote extension through the stratum opticum, induce arborization or synaptogenesis in retinorecipient laminae, and prevent sprouting into adjoining laminae. Interestingly, N-cadherin accumulated in retinorecipient laminae only following the onset of synapse formation, and failed to accumulate in enucleated tecta. Immunoelectron microscopy of normal tecta demonstrated the presence of N-cadherin in the synaptic cleft, suggesting a role for this molecule in synaptic maintenance.
Evidence for glycine modulation of excitatory synaptic inputs to retinal ganglion cells
Authors: Lukasiewicz PD , Roeder RC
Published: April 2018

The actions of glycine on the NMDA receptor-mediated synaptic responses of ganglion cells were studied in the tiger salamander retinal slice. Ganglion cell excitatory postsynaptic currents (EPSCs) were elicited either by exciting bipolar cells with potassium puffs or by light stimulation, and were measured using whole-cell patch-clamp techniques. Increasing bath glycine concentrations to 10 microM had little effect on the amplitude of the puff-evoked EPSCs, indicating either that synaptic glycine concentrations were saturating or that the added glycine was buffered by uptake mechanisms. However, 5,7- dichlorokynurenic acid (5,7-DCK), an antagonist for the glycine site on the NMDA receptor, reduced the ganglion cell responses to NMDA puffs, and reduced the potassium puff- and light-elicited EPSCs. The IC50 values for 5,7-DCK became larger with increasing glycine concentrations, but not with increasing NMDA concentrations, indicating that 5,7-DCK acted at the glycine site. The IC50 values for 5,7-DCK were increased with stronger potassium puffs or light stimuli, suggesting that synaptic glycine levels increased with the strength of the stimuli. EPSCs measured in ON-OFF ganglion cells at light ON and OFF were reduced by 5,7-DCK. For dim light stimuli, the IC50 values were lower for the OFF EPSCs compared to the ON EPSCs, indicating that glycine concentrations were different at the times of OFF and ON activity. Estimates of synaptic glycine concentrations suggest that for dim light stimuli, concentrations at the OFF synapses were not saturated, but concentrations at the ON synapses were saturated.
Gas chromatographic-mass fragmentographic quantitation of 3 alpha- hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and its precursors in blood and brain of adrenalectomized and castrated rats
Authors: Cheney DL , Uzunov D , Costa E , Guidotti A
Published: April 2018

Coupling high performance liquid chromatography with gas chromatography- mass fragmentography has made it possible to simultaneously measure subpicomolar concentrations of allopregnanolone and its precursors, pregnenolone, progesterone and 5 alpha-dihydroprogesterone (5 alpha- DHP) in various brain areas. Allopregnanolone was measured in the brain of adrenalectomized/castrated (ADX/CX) rats in nanomolar concentrations long after peripheral sources of allopregnanolone were removed. A partial decrease (approximately 30%) in the content of allopregnanolone was found in the brains of ADX/CX rats compared to sham-operated rats. Moreover, the content of allopregnanolone in brains of sham-operated as well as ADX/CX rats was nonuniformly distributed (olfactory bulb > striatum > cortex > hippocampus) and was one to two orders of magnitude higher than in plasma or liver. Infusion of pregnenolone sulfate in ADX/CX rats elicited a fourfold increase in 5 alpha-DHP and progesterone content and a seven- to eightfold increase in the content of allopregnanolone in brain but not in liver or plasma. Furthermore, the content of allopregnanolone in brain increased to the same extent in both sham-operated and ADX/CX rats following pregnenolone sulfate infusion. The 5 alpha-reductase inhibitor, (17 beta)17[[bis(1- methylethyl)amino]carbonyl] androstane-3,5-diene-3-carboxylic acid (SKF 105111), reduced the brain content of allopregnanolone and blocked the increased formation of allopregnanolone in brain following pregnenolone sulfate infusion. The results clearly demonstrate that the synthesis of allopregnanolone from 5 alpha-DHP and progesterone occurs in the brain and that a significant amount of allopregnanolone is synthesized locally in brain from its precursors. These experiments suggest that the brain, like adrenals and gonads, is a steroidogenic organ which produces allopregnanolone as one of its own most important physiologically relevant steroids.
Regulation of a potassium conductance in rat midbrain dopamine neurons by intracellular adenosine triphosphate (ATP) and the sulfonylureas tolbutamide and glibenclamide
Authors: Stanford IM , Lacey MG
Published: April 2018

The presence of adenosine triphosphate-regulated potassium channels (K- ATPs) in midbrain dopamine neurons is currently in dispute. This was investigated using whole-cell patch-clamp recordings from dopamine neurons in slices of midbrain from 9–12-d-old rats. Intracellular dialysis with Mg2+ ATP-free solutions resulted in a membrane hyperpolarization (14 +/- 6 mV), or outward current (102 +/- 27 pA) in voltage clamp, which developed over 14 +/- 1.6 min. These hyperpolarizations and outward currents were reversed by the K-ATP- blocking sulfonylureas tolbutamide (100 microM) and glibenclamide (3 microM). This sulfonylurea-sensitive outward current was associated with an increase in a nonrectifying (between -50 and -130 mV) conductance of approximately 2 nS, with a reversal potential of -100 mV (in 2.5 mM extracellular potassium), consistent with a potassium conductance increase. When the dialyzate contained Mg2+ATP (2 mM), no slowly developing hyperpolarization or outward current occurred, and tolbutamide (200 microM) and glibenclamide (10 microM) did not affect membrane potential or current. Additionally, the “potassium channel activators” (KCAs) lemakalim (200 microM) and pinacidil (50 microM) were also without effect on the membrane potential or holding current in these cells. The hyperpolarizations and outward currents caused by baclofen and quinpirole, agonists at GABAB and D2 receptors, respectively, were neither blocked by sulfonylureas nor occluded by the current resulting from depletion of intracellular ATP. Thus, these K- ATPs appear independent of the potassium channels coupled to GABAB and D2 receptors in these cells. This ATP-regulated potassium conductance may constitute a protective mechanism during anoxia or hypoglycemia, by restricting membrane depolarization of dopamine neurons when intracellular ATP levels fall.
Negative feedback neuroendocrine control of the inflammatory response in rats
Authors: Green PG , Miao FJ , Janig W , Levine JD
Published: April 2018

We describe that an ongoing inflammatory response at one site (produced by complete Freund's adjuvant injection in the hindpaw) produces a negative feedback inhibition on plasma extravasation, produced by perfusion of the inflammatory mediator, bradykinin (160 nM), at a second site (the knee joint). This negative feedback process is abolished in rats that have been neonatally treated with capsaicin to deplete most of their unmyelinated primary afferent fibers, which suggests that this negative feedback process is mediated by activation of primary afferent fibers. Electrical stimulation of the hindpaw at intensities that excite C-fibers also inhibited bradykinin-induced plasma extravasation. Stimulation at intensities that only excite A- fibers had no effect on bradykinin-induced plasma extravasation. Platelet activating factor-induced plasma extravasation, which is not dependent on the innervation of the joint, was not inhibited by stimulation of C-fibers from the hindpaw. Acute surgical interruption of the lumbar sympathetic outflow to the hind limb (the paravertebral ganglia between L2 and L4) did not attenuate the depression of bradykinin-induced plasma extravasation produced by C-fiber stimulation. This indicates that the depression is not mediated by activity in the sympathetic outflow. Transection of the spinal cord, hypophysectomy, inhibition of corticosterone synthesis, and adrenalectomy (but not adrenal medullectomy) all prevented the inhibition of BK-induced plasma extravasation by electrical simulation, indicating that the negative feedback inhibition on plasma extravasation is dependent on an intact neuraxis and an intact hypothalamic-pituitary-adrenocortical axis. In summary, our data demonstrate a negative feedback inhibition of an inflammatory process, which is elicited by stimulation of C-fiber afferents.
Analysis of synaptic distribution within single retinal axonal arbors after chronic NMDA treatment
Authors: Yen L , Sibley JT , Constantine-Paton M
Published: April 2018

Activation of the NMDA subtype of glutamate receptor has been implicated in structural synaptic plasticity in many developing sensory systems. In the frog retinotectal system, chronic exposure of the optic tectum to NMDA, which decreases the effectiveness of NMDA receptors (Debski et al., 1991), results in the pruning of the branches of retinal terminal arbors (Cline and Constantine-Paton, 1990). However, it is difficult from these studies to relate the involvement of NMDA receptors to changes in synapse distribution. In this study, we have developed an EM sampling procedure to quantitatively compare the amount and the distribution of synaptic contact within single retinal arbors. We report that within each retinal arbor, synaptic contact gradually increases from the main branches to the end branches of the arbor. Chronic NMDA treatment, however, significantly reduces the total amount of synaptic contact within each arbor. This reduced synaptic contact appears to be due to the pruning of the end branches, and the synapses these branches bear. The results are consistent with the hypothesis that NMDA receptor is an integral part of the mechanism that stabilizes coactive synapses, and that maintenance of an axonal branch requires a minimum density of synapses that are correlated with converging neighbors.
Developmental determinants at the mammalian optic chiasm
Authors: Guillery RW , Mason CA , Taylor JS
Published: April 2018

The mammalian optic chiasm is widely and properly regarded as a region where axons from the temporal retina take an uncrossed course and separate from axons arising in the nasal retina that take a crossed course. However, this is but a rough approximation of the adult situation, and developmental studies must take account of several distinctive stages and axon rearrangements that characterize the region of the chiasm. At the early and late stages of development of nonprimate species the axons do not segregate in accordance with a strict naso-temporal rule at all, and their behavior at the chiasm is not relevant to the formation of the naso-temporal division. As the axons pass from the eye to the chiasm they tend to lose their retinotopic order, to gain a chronotopic order, and then, in the region of the chiasm, to regain some aspects of the retinotopic order before reaching their terminal sites. Molecular or cellular cues that allow the several distinct organizational steps to occur must be expected in the retina, on the axons themselves, and also along the pathway of the axons, prechiasmatically and at the chiasm. Some of these cues will be associated with local nerve cells, some with specialized glial elements and some with the retinofugal axons themselves. Several candidate molecules have been identified in the retina and along the path of the axons, but to date no clearly defined role in the specific events of the pathway determination have been identified. The sequence of developmental processes that characterizes the formation of the optic chiasm provides an interesting and useful challenge to experimentalists, because the advancing axons can now be observed in vitro and in the living brain. The pattern of growth changes as development proceeds, it shows distinctive properties in different species and in their genetic mutants, and it can be readily modified by simple experimental procedures. These all provide opportunities for investigating the function of proposed molecular cues that act in the development of the chiasm.
Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia
Authors: Stone J , Itin A , Alon T , Pe'er J , Gnessin H , Chan-Ling T , Keshet E
Published: April 2018

We have studied the role of the hypoxia-inducible angiogenic growth factor vascular endothelial growth factor (VEGF) in the induction and control of vessel growth in the developing retina of rats and cats, using in situ hybridization techniques. VEGF is expressed successively in two layers of neural retina, the innermost (axon) layer and the inner nuclear layer (INL). In the axon layer, VEGF is expressed transiently by astrocytes as they spread across the layer, closely preceding the formation of superficial vessels. In the INL, VEGF is expressed transiently by somas at the middle of the layer (presumably Muller cells), closely preceding the formation of the deep layer of retinal vessels. We propose that hypoxia caused by the onset of neuronal activity is detected by strategically located populations of neuroglia, first astrocytes, then Muller cells. In response they secrete VEGF, inducing formation of the superficial and deep layers of retinal vessels, respectively. As the vessels become patent, they relieve the hypoxic stimulus, so vessel formation is matched to oxygen demand. This hypothesis was tested experimentally in three ways. Expression of the high affinity flk-1 receptor for VEGF was demonstrated in newly formed retinal vessels, confirming that the secreted VEGF acts on the vessels, in a paracrine fashion. Direct hypoxic regulation of VEGF expression by macroglia was demonstrated in primary cultures of astrocytes and in cells of a glioma line. Hypoxic regulation of VEGF expression in the intact developing retina was demonstrated by showing that oxygen-enriched atmospheres that inhibit vessel formation also suppress endogenous VEGF production.
Muscle atonia is triggered by cholinergic stimulation of the basal forebrain: implication for the pathophysiology of canine narcolepsy
Authors: Nishino S , Tafti M , Reid MS , Shelton J , Siegel JM , Dement WC , Mignot E
Published: April 2018

Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness and rapid eye movement (REM) sleep-related symptoms, such as cataplexy. The exact pathophysiology underlying the disease is unknown but may involve central cholinergic systems. It is known that the brainstem cholinergic system is activated during REM sleep. Furthermore, REM sleep and REM sleep atonia similar to cataplexy can be triggered in normal and narcoleptic dogs by stimulating cholinergic receptors within the pontine brainstem. The pontine cholinergic system is, therefore, likely to play a role in triggering cataplexy and other REM-related abnormalities seen in narcolepsy. The other cholinergic system that could be involved in the pathophysiology of narcolepsy is located in the basal forebrain (BF). This system sends projections to the entire cerebral cortex. Since acetylcholine release in the cortex is increased both during REM and wake, the basocortical cholinergic system is believed to be involved in cortical desynchrony. In the current study, we analyzed the effect of cholinergic compounds injected into the forebrain structures of narcoleptic and control dogs. We found that carbachol (a cholinergic agonist) injected into the BF triggers cataplexy in narcoleptic dogs while it increases wakefulness in control dogs. Much higher doses of carbachol bilaterally injected in the BF were, however, shown to trigger muscle atonia even in control dogs. These results suggest that a cholinoceptive site in the BF is critically implicated in triggering muscle atonia and cataplexy. Together with similar results previously obtained in the pontine brainstem, it appears that a widespread hypersensitivity to cholinergic stimulation may be central to the pathophysiology of canine narcolepsy.
Comparison of ionic currents expressed in immature and mature muscle cells of an ascidian larva
Authors: Davis AK , Greaves AA , Dallman JE , Moody WJ
Published: April 2018

We have compared the voltage-gated ion channels present in larval ascidian muscle at two developmental stages: muscle precursor cells just after the terminal cell division and mature contractile muscle, 7- 11 hr later. All precursor cells express a high-threshold transient Ca current and a slowly activating delayed K current, and about half the cells express a low-threshold transient Ca current. An inwardly rectifying K current, which had been present from fertilization until just before the terminal cell division, is absent. Mature muscle retains two of the tailbud currents: the low-threshold transient Ca current and the slow delayed K current, although at larger densities, and also expresses a high-threshold Ca current that is similar in most respects to the precursor cell current but that lacks inactivation. In addition, mature muscle expresses two rapidly activating outward K currents, one voltage and one Ca dependent, that generate a composite outward K current that is eight times larger and activates eight times faster than the tailbud K current. Mature muscle also reexpresses the inward rectifier. We propose that the transient absence of the inward rectifier and the slow activation of the delayed K current early in development create a window of developmental time when spontaneous electrical activity is likely.
Cellular localization of synaptotagmin I, II, and III mRNAs in the central nervous system and pituitary and adrenal glands of the rat
Authors: Marqueze B , Boudier JA , Mizuta M , Inagaki N , Seino S , Seagar M
Published: April 2018

Three isoforms of synaptotagmin, a synaptic vesicle protein involved in neurotransmitter release, have been characterized in the rat, although functional differences between these isoforms have not been reported. In situ hybridization was used to define the localization of synaptotagmin I, II, and III transcripts in the rat CNS and pituitary and adrenal glands. Each of the three synaptotagmin genes has a unique expression pattern. The synaptotagmin III gene is expressed in most neurons, but transcripts are much less abundant than the products of the synaptotagmin I and II genes. A majority of neurons in the forebrain expressed both synaptotagmin I and III mRNAs while synaptotagmin II gene expression was confined to subsets of neurons in layers IV-VI of the cerebral cortex, in the dentate granule cell region, the hilus, and the CA1-CA3 areas of the hippocampus. In the cerebellum, all three transcripts were visualized in the granule cell layer. Furthermore, synaptotagmin I probes revealed striking differences between distinct populations of neurons, as in addition to moderate labeling of granule cells, much more prominent hybridization signals were detected on scattered cell bodies likely to be Golgi interneurons. In the most caudal part of the brain, synaptotagmin II transcripts were abundant and were coexpressed with synaptotagmin III mRNAs. This pattern was found in putative motoneurons of the spinal cord, suggesting that the two isoforms might be involved in exocytosis at the neuromuscular junction. Only synaptotagmin I mRNAs were detected in the anterior and intermediate pituitary and in adrenal medullary cells. These data reveal an unexpectedly subtle segregation of the expression of synaptotagmin genes and the existence of multiple combinations of synaptotagmin isoforms which may provide diversity in the regulation of neurosecretion.
Neuronal activity in the primate hippocampal formation during a conditional association task based on the subject's location
Authors: Eifuku S , Nishijo H , Kita T , Ono T
Published: April 2018

The hippocampal formation (HF) functions in two domains of memory: spatial and nonspatial associative memory. The HF includes the hippocampus proper, the dentate gyrus, and the subicular complex. Studies of spatial correlates of HF neuronal activity have revealed that a subject's location in space can impose critical constraints on patterns of neuronal activity in the HF. This report compares monkey HF neuronal responses in two kinds of stimulus-response association tasks (go/no-go tasks with symmetrical reinforcement). In a place-dependent, conditional, stimulus-response association (PCA) task, the subject's location was the condition upon which stimulus (object)-behavioral response association depended. In a place-in-dependent, simple, stimulus-response association (ISA) task, the object-behavioral response contingency was independent of the subject's location. Of 329 neurons recorded, the activity of 88 increased or decreased significantly in response to the presentation of an object during the PCA task. Responses of 17 neurons depended differentially on specific combinations of object, place, and behavior in the PCA task (specific- combination neurons). These specific-combination responses do not simply reflect object-behavioral response association in the PCA task, since neuronal responses in the same object-behavioral response association were not restored in the ISA task in which there was no dependence on the subject's location. This suggests the influence of location on HF neuronal responses in object-behavioral response association when the subject's location is imposed as a condition. Responses of 12 neurons differentiated the kind of object in the PCA task (object-differential neurons). In the ISA task, most object- differential responses diminished or disappeared. Since the HF object- differential responses elicited by the conditions imposed by the PCA task. Characteristics of the two neuron types suggest that HF neurons encode both stimulus percept and attributes such as the place where the stimulus is presented and the conditional relation imposed in the task.
Expression, secretion, and age-related downregulation of pigment epithelium-derived factor, a serpin with neurotrophic activity
Authors: Tombran-Tink J , Shivaram SM , Chader GJ , Johnson LV , Bok D
Published: April 2018

Retinal pigment epithelial (RPE) cells form a functional complex with photoreceptor neurons of the retina, interacting through the interphotoreceptor matrix (IPM). We now provide evidence that the gene for pigment epithelium-derived factor (PEDF), a protein possessing neurotrophic and neuronal-survival activities, is highly expressed by both fetal and young adult RPE cells. PEDF mRNA is present in RPE cells of the human eye at 17 weeks of gestation, demonstrating its potential for action in vivo during early retinal development. The PEDF protein is secreted in vivo where it constitutes a part of the fetal and adult IPM surrounding photoreceptor outer segments. A polyclonal PEDF antibody recognizes at least four isoforms of secreted human and bovine PEDF by two dimensional gel analysis, and detects a similar 50 kDa protein in the IPM of several other vertebrate species. Within soluble extracts of RPE cells, however, where little, if any, of the 50 kDa species can be detected, an immunoreactive 36 kDa protein is observed by Western blot analysis. By immunofluorescence, PEDF is localized intracellularly in association with the nucleus, presumptive secretory granules, and cytoskeletal elements of cultured RPE cells with PEDF and actin antibodies colocalizing to the same cytoskeletal structures. During initial stages of attachment, PEDF and actin also concentrate at the tips of pseudopods extended by the cultured RPE cells. However, with successive passages, synthesis, and secretion of the PEDF protein as well as transcription of its mRNA decrease and are lost by about 10 passages. In parallel, cultured RPE cells lose their proliferative potential and change from an epithelial-like morphology in early passages to a more fibroblast-like appearance by about the 10th passage. PEDF is thus apparently present intracellularly and extracellularly in both fetal and early adult periods where it could be involved in cellular differentiation and survival and with its loss, in the onset of senescence.
Persistence of early-generated neurons in the rodent subplate: assessment of cell death in neocortex during the early postnatal period
Authors: Valverde F , Lopez-Mascaraque L , Santacana M , De Carlos JA
Published: April 2018

In the rat, the deepest neocortical layer forms a conspicuous cell band known as layer Vlb. Cells in layer Vlb are among the first to differentiate, and it has been regarded as an homolog to the subplate of primates and carnivores. Cell death has been considered a universal feature of subplate cells. In order to assess the validity of this assertion, we examined the sequence of generation and the extent of cell death in layer Vlb. This was achieved using injections of 3H- thymidine and two methods for the direct visualization of apoptotic figures. Single injections of 3H-thymidine were performed between E12 and E15 (E0 is the day of insemination), and brains were examined at different postnatal ages between P1 and P63. The number of heavily labeled cells were counted in layer Vlb in six standard, equally spaced coronal sections in each brain. Single injections at E12 labels about 3% of the entire population of layer Vlb cells, 17% at E13, 30% at E14, and < 1% at E15. Our results indicate that the absolute number of heavily labeled cells in layer Vlb remains constant. The analysis of variance (one-way ANOVA) showed that the difference among the group means was not significant from P1 to P63 after injections at either E12, E13, or E14. In order to confirm these results, we evaluated the distribution of pyknotic (apoptotic) cell bodies in the neocortex. Apoptotic cells were visualized in Nissl preparations and by histochemical staining using an in situ apoptosis detection kit. The analysis was performed in rats from E18 to P15. Both methods gave comparable results. We found that the amount of cell death in layer Vlb is neither particularly prominent nor significantly different from that which occurs in the remaining neocortical layers, apart from layer II and in the white matter of the corpus callosum. We conclude that neuronal death does not play any significant role in the rodent subplate.
Oxytocin receptor mRNA expression in the ventromedial hypothalamus during the estrous cycle
Authors: Bale TL , Dorsa DM , Johnston CA
Published: April 2018

Changes in OR mRNA expression in the ventromedial hypothalamus (VMH) in relation to the estrous cycle were measured by in situ hybridization with a rat oxytocin receptor (OR) probe. Binding studies have localized ORs to various brain regions, and have detected a high density of receptors in the VMH, a nucleus containing large numbers of estrogen responsive neurons. Previous studies in this lab have reported a significant increase in OR mRNA expression in the VMH in ovariectomized rats treated with estrogen. The present study was designed to determine whether changes in steroid hormone levels across the estrous cycle result in induction of OR mRNA expression. Autoradiographic studies revealed differences in OR mRNA expression in the rostral and caudal as well as medial and lateral aspects of the VMH. OR mRNA levels were highest in the caudal portion of the vIVMH on the afternoon (16:00 hr) of proestrus. The rostral region exhibited a high level of expression in the ventrolateral region of the VMH on the morning (9:00 hr) of proestrus and in the dorsomedial region of the VMH on the afternoon of proestrus. Little or no OR mRNA expression was evident in the rostral or caudal VMH on the morning or evening of diestrus. These results support previous findings which showed a regulation of OR binding by gonadal steroids and suggest that this may be due to altered expression of the OR gene. These effects suggest a possible role of ORs in the oxytocin stimulated release of luteinizing hormone.
Expression and activity-dependent changes of a novel limbic-serine protease gene in the hippocampus
Authors: Chen ZL , Yoshida S , Kato K , Momota Y , Suzuki J , Tanaka T , Ito J , Nishino H , Aimoto S , Kiyama H
Published: April 2018

A novel murine cDNA which encodes a protein designated neuropsin was cloned. Northern and in situ hybridization analyses demonstrated that neuropsin mRNA is expressed specifically in the limbic system of mouse brain and is localized at highest concentration in pyramidal neurons of the hippocampal CA1–3 subfields. Direct hippocampal stimulation and kindling induced by amygdaloid stimulation caused a significant bilateral change in neuropsin mRNA level in the hippocampal pyramidal neurons. The activity-dependent changes and the specific localization indicate that neuropsin is involved in hippocampal plasticity.