bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of the medial prefrontal cortex (cingulate gyrus) in the regulation of hypothalamic-pituitary-adrenal responses to stress
Authors: Diorio D , Viau V , Meaney MJ
Published: April 2018

In the studies reported here we have examined the role of the medial prefrontal cortex (MpFC) in regulating hypothalamic-pituitary-adrenal (HPA) activity under basal and stressful conditions. In preliminary studies we characterized corticosteroid receptor binding in the rat MpFC. The results revealed high-affinity (Kd approximately 1 nM) binding with a moderate capacity (42.9 +/- 3 fmol/mg) for 3H- aldosterone (with a 50-fold excess of cold RU28362; mineralocorticoid receptor) and high-affinity (Kd approximately 0.5–1.0 nM) binding with higher capacity (183.2 +/- 22 fmol/mg) for 3H-RU 28362 (glucocorticoid receptor). Lesions of the MpFC (cingulate gyrus) significantly increased plasma levels of both adrenocorticotropin (ACTH) and corticosterone (CORT) in response to a 20 min restraint stress. The same lesions had no effect on hormone levels following a 2.5 min exposure to ether. Implants of crystalline CORT into the same region of the MpFC produced a significant decrease in plasma levels of both ACTH and CORT with restraint stress, but again, there was no effect with ether stress. Neither MpFC lesions nor CORT implants had any consistent effect on A.M. or P.M. levels of plasma ACTH or CORT. Manipulations of MpFC function were not associated with changes in the clearance rate for CORT or in corticosteroid receptor densities in the pituitary, hypothalamus, hippocampus, or amygdala. Taken together, these findings suggest that MpFC is a target site for the negative-feedback effects of glucocorticoids on stress-induced HPA activity, and that this effect is dependent upon the nature of the stress.
Adrenal medulla grafts in the hemiparkinsonian rat: profile of behavioral recovery predicts restoration of the symmetry between the two striata in measures of pre- and postsynaptic dopamine function
Authors: Curran EJ , Albin RL , Becker JB
Published: April 2018

Following unilateral striatal dopamine depletion, the hemiparkinsonian rat exhibits rotational behavior in response to amphetamine and apomorphine. The rotational behaviors induced by these drugs are thought to reflect an asymmetry in presynaptic striatal dopamine release and an asymmetry in postsynaptic striatal dopamine receptor function, respectively. Grafts of adrenal medulla cells in the lateral ventricle of hemiparkinsonian rats have been reported to reduce behavioral asymmetry. More than one profile of behavioral recovery, however, is observed. Some animals show a graft-induced decrease only in the response to apomorphine, but others show a decrease in the response to amphetamine, and still others show a decrease in the behavioral responses to amphetamine and apomorphine. In this report, amphetamine- and apomorphine-induced turning behaviors were determined in hemiparkinsonian rats prior to and following intraventricular grafts of adrenal medulla or control tissue. Both bilateral intrastriatal microdialysis in freely moving animals and quantitative dopamine receptor autoradiography procedures were conducted in each animal so as to determine the relations between pre- and postsynaptic dopaminergic measures as well as the association between these measures and the different profiles of behavioral recovery after adrenal medulla grafts. We report here that in animals with an adrenal medulla graft-induced decrease in the behavioral response to amphetamine, the balance between the two striata in extracellular striatal dopamine concentrations and D2 dopamine receptor binding was restored. Furthermore, enhanced extracellular striatal dopamine concentrations were highly correlated with the graft-induced symmetry in striatal D2 dopamine receptor binding. In contrast to animals with decreased amphetamine-induced turning, in animals with a graft-induced decrease exclusively in response to apomorphine, the presynaptic symmetry was not restored and there was a significantly smaller effect on D2 receptor binding. We conclude that those animals that show decreased amphetamine-induced turning after adrenal medulla grafts had the most effective grafts, and suggest that methods designed to optimize this behavioral profile are most likely to lead to enhanced clinical efficacy with this procedure.
Noradrenaline hyperpolarizes identified rat mesopontine cholinergic neurons in vitro
Authors: Williams JA , Reiner PB
Published: April 2018

Inhibition of brainstem cholinergic neurons by noradrenergic neurons of the locus ceruleus has long been suggested as a key mechanism of behavioral state control. In particular, the commonly held view is that noradrenaline (NA) plays a permissive role in rapid eye movement (REM) sleep generation by disinhibiting brainstem cholinergic neurons. While this notion has been supported by numerous investigations, the inhibition of cholinergic neurons by NA has never been directly demonstrated. The purpose of this study was to investigate the effects of NA upon identified cholinergic neurons in the rat mesopontine tegmentum. Using whole-cell patch-clamp recordings in slices, 175 cells were studied during bath application of 50 microM NA. Cholinergic neurons were positively identified by intracellular labeling with biocytin and subsequent staining with NADPH-diaphorase, a reliable marker for brainstem cholinergic neurons (Vincent et al., 1983). Successful intracellular labeling was obtained in 96 cells. Ninety-two percent (36 of 39) of cholinergic neurons hyperpolarized in response to NA, while noncholinergic cells (n = 57) exhibited mixed responses. Application of NA in a low-Ca2+, high-Mg2+ solution elicited the same hyperpolarizing effect as in normal solution, which indicated that the effect of NA on cholinergic neurons was direct. The noradrenergic hyperpolarization was mimicked by the alpha 2-adrenoceptor agonist UK- 14,304, and was blocked by the alpha 2-adrenoceptor antagonist idazoxan, which suggested an alpha 2-mediated response. Finally, voltage-clamp experiments revealed that NA activates the inwardly rectifying potassium current, IKG.(ABSTRACT TRUNCATED AT 250 WORDS)
Kinetic basis for the voltage-dependent inhibition of N-type calcium current by somatostatin and norepinephrine in chick sympathetic neurons
Authors: Golard A , Siegelbaum SA
Published: April 2018

Neurotransmitter inhibition of calcium currents (ICa) can be relieved by large depolarizing prepulses. This effect has been postulated to be due either to the voltage-dependent unbinding of an inhibitory molecule from the channel or to a slow voltage-dependent gating step intrinsic to the modulated channel. According to the first hypothesis, the rate of reinhibition (reblock) following a depolarizing prepulse should depend on the concentration of active inhibitory molecules and thus should increase with the extent of inhibition. To distinguish between these models we examined the actions of norepinephrine (NE) and somatostatin (SS) on high-threshold calcium currents in chick sympathetic ganglia, using whole-cell voltage-clamp methods. As previously described in other systems, both NE and SS inhibit omega- conotoxin-sensitive N-type Ca2+ current in a voltage-dependent manner. Pertussis toxin (PTX) pretreatment prevents the inhibition of the current, while replacing GTP in the patch pipette with GTP-gamma-S results in irreversible inhibition, consistent with the involvement of a PTX-sensitive G-protein. The inhibitory responses to NE and SS are not additive, suggesting that they act at a common locus. The inhibitory response to repeated applications of NE or SS desensitizes, with little evidence for cross desensitization. The inhibition of ICa is relieved by a 15 msec prepulse to +100 mV. Following repolarization to -80 mV, ICa slowly reblocks. During prolonged applications of NE or SS the extent of inhibition decreases due to desensitization and reblock kinetics are significantly slowed (time constant increases from 60 msec to > 100 msec for both NE and SS). These results are well fit by a quantitative model in which the kinetics of reblock reflect the binding of an inhibitory molecule to the channel.
Synaptotagmin: a membrane constituent of neuropeptide-containing large dense-core vesicles
Authors: Walch-Solimena C , Takei K , Marek KL , Midyett K , Sudhof TC , De Camilli P , Jahn R
Published: April 2018

Synaptotagmin is known to be a major membrane protein of synaptic vesicles (SVs) in neurons. We have now used an immunoisolation procedure to demonstrate that synaptotagmin is also present in the membranes of peptide containing large dense-core vesicles (LDCVs) of rat hypothalamus and bovine posterior pituitary. Synaptotagmin bead- immunoisolated organelles from these tissues primarily consisted of SVs but contained occasionally larger structures reminiscent of LDCVs that were absent from vesicle populations immunoisolated with a synaptophysin antibody. Furthermore, the vesicles immunoisolated with synaptotagmin beads contained significant amounts of neuropeptide Y (NPY). In contrast, vesicles immunoisolated with synaptophysin beads did not contain detectable levels of NPY. Sucrose density gradient fractionation of postnuclear supernatants obtained from the bovine posterior pituitary resulted in a bimodal distribution of synaptotagmin, corresponding to the positions of both SVs and neurosecretory granules. A similar distribution was found for cytochrome b561 and the 116 kDa subunit of the vacuolar proton pump. In contrast, the SV proteins synaptophysin, SV2, and p29 were restricted to the SV-containing fractions. Immunoisolation of small and large vesicles from the sucrose gradient confirmed the differential distribution of synaptotagmin and synaptophysin in the two types of secretory vesicles in nerve endings of the posterior pituitary. We conclude that synaptotagmin is a constituent of both SVs and peptide- containing secretory vesicles in the nervous system. Since both types of organelles undergo Ca(2+)-dependent exocytosis, these findings support a general role of synaptotagmin as an exocytotic Ca2+ receptor.
Evidence for in vivo interactions between neuropeptide Y-related peptides and sigma receptors in the mouse hippocampal formation
Authors: Bouchard P , Dumont Y , Fournier A , St.-Pierre S , Quirion R
Published: April 2018

Recently, it was proposed that neuropeptide Y (NPY) and peptide YY (PYY) could act as endogenous ligands for sigma binding sites, as both NPY and PYY competed with high affinity (nanomolar) for 3H-(+)SKF 10,047 binding sites in rat brain membrane homogenates (Roman et al., 1989). However, various laboratories failed to replicate these in vitro findings. In order to clarify this apparent discrepancy and investigate further possible interactions between NPY and sigma related sites, we evaluated the effects of NPY, PYY and homologs, as well as nonrelated peptides, on in vivo 3H-(+)SKF 10,047 binding parameters in the mouse hippocampal formation. As expected, haloperidol (2 mg/kg), a prototypical sigma receptor ligand, competed for 90% of in vivo hippocampal labeling observed following a peripheral intravenous injection of 3H-(+)SKF 10,047. Intracerebroventricular injections of 300–3000 pmol of either NPY, PYY, NPY2–36, or the Y1 agonist Leu31,Pro34-NPY inhibited significant proportions (17–35%) of haloperidol-sensitive in vivo 3H-(+)SKF 10,047 hippocampal labeling. However, a Y2 receptor agonist, NPY13–36, and nonrelated peptides such as neurotensin and vasoactive intestinal polypeptide, as well as adrenalin, failed to alter in vivo 3H-(+)SKF 10,047 hippocampal binding. It thus appears that NPY, PYY, and a selective Y1 agonist can interact in a concentration-dependent manner, with in vivo 3H-(+)SKF 10,047 labeling in the mouse hippocampal formation. This effect demonstrates selectivity as a Y2 agonist, unrelated peptides, and adrenalin failed to alter in vivo sigma labeling. This in vivo interaction may be relevant to some of the respective biological actions of NPY and sigma-related molecules.
Aspartate and glutamate mediate excitatory synaptic transmission in area CA1 of the hippocampus
Authors: Fleck MW , Henze DA , Barrionuevo G , Palmer AM
Published: April 2018

We examined whether L-aspartate (ASP) and L-glutamate (GLU) both function as endogenous neurotransmitters in area CA1 of the rat hippocampus. Radioligand displacement experiments using 3H-DL-alpha- amino-3-hydroxy-5-methylisoxazole-4-propionic acid (3H-AMPA) to label AMPA/kainate receptors and 3H-cis-4-phosphonomethyl-2-piperidine carboxylic acid (3H-CGS-19755) to label NMDA receptors confirmed that GLU (Ki approximately 500 nM) but not ASP (Ki > 1 mM) has high affinity for AMPA/kainate receptors whereas GLU (Ki approximately 250 nM) and ASP (Ki approximately 1.3 microM) both have high affinity for NMDA receptors. Elevating extracellular potassium concentration (50 mM, 1 min) evoked the calcium-dependent release of both ASP (approximately 50% increase) and GLU (approximately 200% increase) from hippocampal slices and from minislices of area CA1. Reducing extracellular glucose concentration (0.2 mM) reduced GLU release, enhanced ASP release, and reduced AMPA/kainate receptor-mediated responses more than NMDA receptor-mediated responses (to 7% and 34% of control, respectively). Fiber volleys, antidromic population spikes, membrane potential, input resistance, and ATP content all were not affected by glucose reduction. Unlike low glucose, the inhibitory neuromodulator adenosine (5 microM), which reduces ASP and GLU release to a similar extent, reduced AMPA/kainate and NMDA receptor-mediated population EPSPs similarly (to 11% and 12% of control, respectively). AMPA/kainate and NMDA receptor- mediated population EPSPs were also similarly reduced by 0.4 microM TTX (to 32% and 22% of control, respectively) and similarly enhanced by 10 microM 4-aminopyridine (to 206% and 248% of control, respectively). Finally, NMDA receptor-mediated EPSCs measured by whole-cell recording decayed faster in low glucose (73 msec vs 54 msec) but not in adenosine (73 msec vs 78 msec). Together, these results confirm that ASP and GLU are both involved in excitatory synaptic transmission at the Schaffer collateral-commissural terminals in area CA1 of the rat hippocampus.
Synaptic events that generate fast oscillations in piriform cortex
Authors: Ketchum KL , Haberly LB
Published: April 2018

Prominent, odor-evoked, fast (40–60 Hz) oscillations have been reported in the olfactory bulb and piriform (primary olfactory) cortex of both awake-behaving and anesthetized animals. The present study used current source-density analysis to examine the origin of the fast oscillations evoked by single weak shocks to afferent fibers. These shock-evoked oscillations closely resemble those evoked by odor. The results revealed that each cycle of the oscillatory field potential was generated by a stereotyped series of membrane currents similar to those previously characterized in the nonoscillatory response to strong afferent fiber shocks. Each cycle began with a strong inward current in layer la identified as an EPSC mediated by afferent fibers in distal apical dendrites of pyramidal cells. This afferent input was followed by a strong inward current in layer Ib identified as an EPSC mediated by intrinsic association fibers in middle apical dendritic segments. These excitatory events were followed by a smaller inward current at the depth of pyramidal cell somata (layers II and superficial III) that may be the depolarizing Cl(-)-mediated IPSC previously identified in the strong-shock response. Based on an analysis of the timing of the EPSCs it was concluded that the weak shock-evoked oscillation is generated in the olfactory bulb and that the resulting periodic activity in afferent fibers drives the oscillation in the piriform cortex.(ABSTRACT TRUNCATED AT 250 WORDS)
Rescue of axotomized immature rat facial motoneurons by R(-)-deprenyl: stereospecificity and independence from monoamine oxidase inhibition
Authors: Ansari KS , Yu PH , Kruck TP , Tatton WG
Published: April 2018

The role of monoamine oxidase B (MAO-B) in R(-)-deprenyl-mediated rescue of rat facial motoneurons axotomized at postnatal day 14 (P14) was investigated using the (+)- and (-)-enantiomers of deprenyl [S(+)- deprenyl and R(-)-deprenyl]. Previously, doses of R(-)-deprenyl sufficient to inhibit MAO-B were shown to increase the survival of motoneurons following an apparent loss of target-derived trophic support caused by axotomy in P14 rats. In the present experiments, motoneuronal survival was measured 21 d after unilateral facial nerve transection at P14. The animals were treated with saline or doses of R(- )- or S(+)-deprenyl ranging from 0.001 to 10 mg/kg every 2 days (/2d). Frontal serial 10 microns sections were taken through the length of the facial nuclei ipsilaterally and contralaterally to the facial nerve transections. Every third section was immunoreacted for an antibody against ChAT to identify the motoneuron somata, while every adjacent third section was Nissl stained to assess motoneuronal survival. A second series of P14 rats was treated with similar doses of the two deprenyl enantiomers or saline and the brainstems removed for measurement of MAO-A and MAO-B activity at 4 hr after the treatments. Averages of 24% of the facial motoneurons survived axotomy with either saline treatment or 0.001 mg/kg/2d doses of R(-)-deprenyl. Doses of R(- )-deprenyl of 0.005, 0.01, and 10.0 mg/kg/2d increased the surviving facial motoneuron to 38%, 51%, and 48%, respectively, indicating an ED50 of about 0.005 mg/kg/2d. Doses of S(+)-deprenyl as high as 10 mg/kg/2d did not increase motoneuronal survival, revealing a stereospecificity for the increased survival of at least 2000-fold. The ED50 for MAO-B inhibition in the P14 brainstem was approximately 0.1 mg/kg for the (-)-enantiomer and 2.0 mg/kg for the (+)-enantiomer, revealing a 20-fold higher sensitivity of the enzyme toward the (-)- enantiomer in the P14 rat brainstem. A dose of 10 mg/kg of S(+)- deprenyl inhibited about 65% of brainstem MAO-B activity without increasing motoneuronal survival, whereas 0.005 and 0.01 mg/kg of R(-)- deprenyl increased motoneuronal survival without significant inhibition of brainstem MAO-B activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Endogenous axoplasmic proteins and proteins containing nuclear localization signal sequences use the retrograde axonal transport/nuclear import pathway in Aplysia neurons
Authors: Schmied R , Huang CC , Zhang XP , Ambron DA , Ambron RT
Published: April 2018

When the nuclear localization signal peptide (sp) of the SV 40 large T antigen was coupled to human serum albumin (HSA), rhodaminated (r), and microinjected into axons of Aplysia neurons in vitro, the rHSA-sp was conveyed through the axon to the cell body and then into the nucleus (Ambron et al., 1992). But since rHSA-sp is an artificial construct, we needed to determine whether naturally occurring nuclear proteins use this pathway. We therefore injected calf thymus histone H-1 and Xenopus oocyte nucleoplasmin into axons. By 3 hr both were retrogradely transported and targeted into the nucleus, though histone H-1 less efficiently than rHSA-sp or nucleoplasmin. In contrast, neither rHSA, nor rHSA conjugated to a peptide with a random distribution of basic amino acids, was transported or imported. To see if proteins that use the pathway remain intact, we coupled sp to HRP. When injected into varicosities, the HRP-sp was transported/imported to the nucleus, where it was enzymatically active. A key issue was to determine whether endogenous proteins use this pathway. Consequently, axoplasm was extruded from Aplysia nerves and the proteins were fractionated by size. SDS-PAGE and Western blots showed that two fractions contained proteins that were recognized by an affinity-purified antibody to sp: fraction 3 included sp83, and fraction 4 contained sp75. In addition, these two proteins were found in nuclei isolated from neurons. To assess transport, the total proteins in the fractions were rhodaminated and injected into varicosities. Fraction 3, but not fraction 4, contained protein that was transported through the axon to the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)
Nonuniform expression of habituation in the activity of distinct classes of neurons in the Aplysia abdominal ganglion
Authors: Falk CX , Wu JY , Cohen LB , Tang AC
Published: April 2018

Global observations of neuronal response in the Aplysia abdominal ganglion were made during habituation of the gill withdrawal reflex using voltage-sensitive dye recording. This technique makes it possible to measure the spike activity of 30–50% of the 1000 neurons present in the ganglion. Our experiments address the issue of how habituation is expressed in the activity of the population of neurons responding to siphon stimulation. Several classes of neurons exhibited characteristically distinct responses to the stimuli and to habituation training. One class of neurons (group I) responded to the onset and offset of the sensory stimulus although they are probably not primary sensory neurons. They habituate only partially when the behavioral reflex has already habituated completely. Two other classes (groups II and III) both have sustained responses to the touch, but habituate differently. Members of group III habituate completely while those in group II habituate only partially. Another class of neurons are inhibited by the stimulus (group IV). They become less inhibited after habituation. The response of both group I and group IV are new classes of response that have not been previously reported.
GABA-induced chemokinesis and NGF-induced chemotaxis of embryonic spinal cord neurons
Authors: Behar TN , Schaffner AE , Colton CA , Somogyi R , Olah Z , Lehel C , Barker JL
Published: April 2018

During CNS development, neuroblasts proliferate within germinal zones of the neuroepithelium, and then migrate to their final positions. Although many neurons are thought to migrate along processes of radial glial fibers, increasing evidence suggests environmental factors also influence nerve cell movement. Extracellular matrix molecules are thought to be involved in guiding neuronal migration, and molecules such as NGF and GABA exert trophic effects on immature neurons. The nature of the signals that initiate and direct neuroblast migration, however, is unknown. In vitro, NGF and GABA promote neurite outgrowth from cultured cells, and NGF induces axonal chemotaxis (directed migration along a chemical gradient). At earlier developmental stages, these molecules could influence neuroblast movement. Therefore, we investigated whether these molecules induce embryonic neuronal migration. Using an in vitro microchemotaxis assay, we show that rat embryonic spinal cord neurons migrate toward picomolar NGF and femtomolar GABA beginning at embryonic day 13 (E13). Cells exhibit chemotactic responses to NGF while GABA stimulates chemokinesis (increased random movement). GABA effects are mimicked by muscimol and inhibited by bicuculline and picrotoxin, suggesting GABA motility signals are mediated by GABA receptor proteins. Expression of GABA receptors by embryonic cord cells has been previously reported (Mandler et al., 1990; Walton et al., 1993). We used polymerase chain reaction analysis to demonstrate the presence of NGF and trk mRNA in E13 and E14 cord cells, indicating the cells express message for both NGF and high- affinity NGF receptors. Immunohistochemistry of E13 spinal cord sections indicates that NGF and GABA colocalize in fibers close to the target destinations of migrating neurons, suggesting diffusible gradients of these molecules provide chemoattractant signals to migratory cells. Thus, in vitro, neuroblast migration is induced by specific signaling molecules that are present in the developing spinal cord, and may stimulate migration of embryonic neurons prior to synaptogenesis.
Tuning of MST neurons to spiral motions
Authors: Graziano MS , Andersen RA , Snowden RJ
Published: April 2018

Cells in the dorsal division of the medial superior temporal area (MSTd) have large receptive fields and respond to expansion/contraction, rotation, and translation motions. These same motions are generated as we move through the environment, leading investigators to suggest that area MSTd analyzes the optical flow. One influential idea suggests that navigation is achieved by decomposing the optical flow into the separate and discrete channels mentioned above, that is, expansion/contraction, rotation, and translation. We directly tested whether MSTd neurons perform such a decomposition by examining whether there are cells that are preferentially tuned to intermediate spiral motions, which combine both expansion/contraction and rotation components. The finding that many cells in MSTd are preferentially selective for spiral motions indicates that this simple three-channel decomposition hypothesis for MSTd does not appear to be correct. Instead, there is a continuum of patterns to which MSTd cells are selective. In addition, we find that MSTd cells maintain their selectivity when stimuli are moved to different locations in their large receptive fields. This position invariance indicates that MSTd cells selective for expansion cannot give precise information about the retinal location of the focus of expansion. Thus, individual MSTd neurons cannot code, in a precise fashion, the direction of heading by using the location of the focus of expansion. The only way this navigational information could be accurately derived from MSTd is through the use of a coarse, population encoding. Positional invariance and selectivity for a wide array of stimuli suggest that MSTd neurons encode patterns of motion per se, regardless of whether these motions are generated by moving objects or by motion induced by observer locomotion.
Memory through metamorphosis in normal and mutant Drosophila
Authors: Tully T , Cambiazo V , Kruse L
Published: April 2018

To establish that a stable, long-lasting form of memory exists in Drosophila, we trained third-instar larvae by electroshocking them in the presence of a specific odor using a Pavlovian conditioning procedure. We show that conditioned odor avoidance produced in larvae still was present in adults 8 d later. Such memory through metamorphosis was specific to the temporal pairing of odor and shock; presentations of odors alone or shock alone did not produce a change. Thus, the memory involved associative processes. We also show that similar training of the single-gene memory mutants dunce and amnesiac did not yield any detectable learning in larvae or memory retention in adults, suggesting that these mutations interfere with long-term memory (LTM) formation even if LTM is induced independently of earlier memory retention processes.
Death of developing septal cholinergic neurons following NGF withdrawal in vitro: protection by protein synthesis inhibition
Authors: Svendsen CN , Kew JN , Staley K , Sofroniew MV
Published: April 2018

Fetal septal neurons were grown in vitro under glass coverslips. This sandwich culture method significantly increased general neuronal survival, reduced glial proliferation, and permitted the removal of serum from the growth medium after 5 d in vitro. Thereafter, a simple, and completely defined, medium was used, and the effects of NGF, NGF withdrawal, and protein synthesis inhibition were examined on septal cholinergic neurons. NGF added to septal cultures at the time of plating resulted in a threefold increase in the number of cholinergic neurons seen at 14 d in vitro but had no effect on the survival of non- cholinergic cells. Cholinergic neurons identified by staining for AChE, ChAT, and p75NGFR could be maintained in serum-free, NGF-supplemented medium for over 40 d. When NGF was removed and NGF antibodies added to 14-d-old cultures, less than 30% of cholinergic neurons survived a further 4 d, but when NGF was similarly withdrawn from 35-d-old cultures, over 75% of cholinergic neurons survived. Reapplication of NGF after 3 but not after 12 or more hours of NGF withdrawal from 14-d- old cultures prevented the death of most cholinergic neurons. When NGF was withdrawn from 14-d-old cultures in the presence of the protein synthesis inhibitor cycloheximide, over 75% of the cholinergic neurons survived. These findings suggest that septal cholinergic neurons are dependent on NGF for survival only during a critical period of development and that growth factor-regulated developmental cell death may occur in CNS neurons by activation of programmed cell death requiring protein synthesis.
Analysis of the feeding motor pattern in the pond snail, Lymnaea stagnalis: photoinactivation of axonally stained pattern-generating interneurons
Authors: Kemenes G , Elliott CJ
Published: April 2018

We have photoinactivated identified feeding interneurons known as N1 and N2 neurons. These are pattern-generating neurons that are active in the protraction of the radula and rasping phases, respectively, of the feeding cycle of the pond snail. The N1 or N2 feeding interneurons in the buccal ganglia were filled with the fluorescent dye 5(6)- carboxyfluorescein (5-CF) from the cut end of the nerve that contains their axon. Filling the cerebrobuccal connective (N = 151) stained just one N1 cell in the contralateral buccal ganglion. Filling the postbuccal nerve stained neurons symmetrically in both buccal ganglia (N = 75): only one labeled cell in each ganglion is an N2 interneuron. The feeding rhythm was evoked by depolarizing a modulatory neuron, the SO, located in the buccal ganglia. The axonally filled N1 interneuron was irradiated at its axon in the buccal commissure with blue laser light (intensity of 0.5 MW.m-2). Irradiation of just one N1 completely blocked the feeding rhythm (seven preparations). In seven further preparations, N1 ablation slowed the SO-driven feeding rhythm and weakened the N1 input to the feeding neurons. Irradiation of the cell bodies of both the filled left and right N2 interneurons killed the cells but did not produce any consistent change in the feeding rate (15 preparations). The feeding interneurons and motoneurons still showed the characteristic N2 phase synaptic inputs, so more, as yet unidentified, N2 neurons must be located in other parts of the buccal ganglia. We conclude that the participation of the identified N1 interneurons is essential for the normal feeding pattern while other, still to be identified N2 neurons must be present and must contribute to the feeding rhythm. We suggest that the extra redundancy of the N2 network may be related to the greater necessity of sensory feedback control during rasping than during protraction of the radula.
Activity-dependent retinotopic refinement in a low-density retinotectal projection in the goldfish: evidence favoring synaptic cooperation over competition
Authors: Olson MD , Meyer RL
Published: April 2018

During optic nerve regeneration in goldfish, the label from a small retinal spot injection of WGA-HRP has been previously reported to be initially widely dispersed in the tectum and subsequently to condense into a small patch in the retinotopically appropriate location of tectum. This refinement involves two separate processes: one is activity independent and generates gross retinotopy; the other is activity dependent and mediates the formation of fine retinotopy. Since the number of synapses remains constant during this refinement, one or both of these processes may involve some form of competition for a limited number of synaptic sites. To clarify the role of synaptic competition, we created a low-density retinotectal projection in goldfish by deflecting about 20% of the optic fibers from one tectum into the opposite tectum, which was denervated of all other optic fibers. Under this condition, it was previously shown that less than half the normal density of synapses is formed. If competition for synaptic sites is a requirement of refinement, refinement should be prevented or significantly hindered. To monitor refinement during regeneration, 2 nl spot injections of WGA-HRP were made into the retina at various times after deflection. To distinguish between activity- dependent and activity-independent refinement, retinal impulse activity was blocked in some fish with repeated injections of TTX into the eye for the duration of the experiment. It was found that considerable activity-independent refinement occurred under continuous TTX blockade although the fibers remained more dispersed than in previous TTX studies when normal numbers of fibers were present. Surprisingly, in fish with normal impulse activity, the degree of activity-dependent refinement was almost normal. Labeled fibers condensed into a small area roughly comparable in size to that observed when the full complement of fibers was regenerating into tectum. These results suggest that competition for limited synaptic sites is not essential for activity-dependent refinement, which may, instead, be mediated by a cooperative process that actively promotes convergence. The findings further suggest that if synaptic competition plays a role in this system, it is in regulating activity-independent mechanisms that determine the large-scale distribution of fibers within tectum.
Segmental patterning of rat and chicken sympathetic preganglionic neurons: correlation between soma position and axon projection pathway
Authors: Forehand CJ , Ezerman EB , Rubin E , Glover JC
Published: April 2018

The segmental organization of midthoracic rat and chicken sympathetic preganglionic neurons was examined by retrograde labeling in vivo and in vitro. The results demonstrate that individual sympathetic preganglionic neurons project only rostrally or caudally within the sympathetic chain, even though the spinal segment in which they reside provides innervation to both rostral and caudal ganglia. In addition, there is both a segmental and an intrasegmental pattern in the thoracic sympathetic column, in which the position of preganglionic neurons is related to the direction they project in the sympathetic chain. Thoracic sympathetic preganglionic neurons are organized into discrete segmental units, each of which exhibits an internal rostrocaudal polarity with respect to the direction of axon projection in the sympathetic chain. The rostrocaudal bias of this internal polarity is graded from segment to segment along the longitudinal axis. Since there is minimal overlap between thoracic segments, the transition from one segment to another entails a sharp change in the pathway choice of the preganglionic neurons. The organization of the preganglionic projections thus includes (1) segmental periodicity, (2) intrasegmental gradients of neuronal identity, and (3) an axial gradient of segment identity. The significance of these findings is twofold. First, they suggest a functional organization that may be related to the specificity of sympathetic reflex action. Second, they reveal a cellular organization that suggests an underlying segmental pattern in the developing spinal cord.
Neuronal characterization, compartmental distribution, and activity- dependent regulation of glutamate immunoreactivity in adult monkey striate cortex
Authors: Carder RK , Hendry SH
Published: April 2018

Monospecific antibodies to glutamate were used to characterize the organization of excitatory neurons and the plasticity of glutamate expression in the macaque striate cortex. Somata and processes immunoreactive for glutamate were densely and unevenly distributed in layers II-III, IVA, IVC. In tangential sections through layers II and III, patches of intense glutamate immunostaining were observed and were found to coincide with regions of the cytochrome oxidase (CO)-rich puffs. By contrast, clusters of intense immunostaining were surrounded by the lightly immunostained but intensely CO-stained lattice in layer IVA. Similarly, in layer IVC, focal aggregates of intense glutamate immunoreactivity were interspersed among regions of light immunostaining but intense CO staining. Glutamate immunoreactivity was also intense in layer VI but was much lighter in layers I, IVB, and V. Throughout the striate cortex, neurons resembling pyramidal cells and spiny stellate cells and processes that included dendrites and axons were immunostained. None of the glutamate-positive neurons was GABA immunoreactive. Following monocular deprivation of adult monkeys by intravitreal injections of TTX into one eye, glutamate immunoreactivity in layers IVC was distributed in alternating intensely and lightly stained stripes. The stripes of reduced immunostaining, which contained an abnormally low concentration of glutamate neurons and pale neuropil, corresponded to columns dominated by the TTX-injected eye. Similar stripes of alternating intense and light immunoreactivity were seen in layers II-III, where they corresponded to rows of puffs at the centers of intact-eye and deprived-eye columns, respectively. These findings demonstrate that glutamate-immunoreactive neurons and terminals in monkey striate cortex are densely concentrated in layers receiving direct geniculocortical innervation. In addition, the glutamate neurons and terminals form discrete units, which in layers II and III coincide precisely with regions receiving geniculocortical terminations but in layers IVA are segregated from these terminations. The findings also indicate that glutamate immunoreactivity is regulated by visually driven activity, and suggest that monocular deprivation in adulthood leads to a reduction in the major excitatory neurotransmitter in visual cortex as well as previously indicated reductions in GABA, the major inhibitory neurotransmitter.
Nitric oxide-dependent efflux of cGMP in rat cerebellar cortex: an in vivo microdialysis study
Authors: Luo D , Leung E , Vincent SR
Published: April 2018

The stimulation of excitatory amino acid receptors in the cerebellar cortex results in the Ca2+/calmodulin-dependent activation of nitric oxide synthase. This leads to an increase in tissue levels of cGMP following the interaction of nitric oxide with soluble guanylyl cyclase. The cerebellar cortex has the highest levels of nitric oxide synthase and cGMP in the brain; however, the levels of guanylyl cyclase and cGMP-phosphodiesterase are remarkably low. Thus, the mechanisms regulating cGMP levels in cerebellar cells are unclear. One report has noted that cGMP can be released from cerebellar slices. We have therefore used intracerebellar microdialysis in awake, freely moving rats to test the hypothesis that activation of nitric oxide synthase in the cerebellar cortex results in the release of cGMP. Climbing fibers, which release excitatory amino acids in the cerebellum, were activated with systemic harmaline. This resulted in an immediate increase in extracellular cGMP, which was blocked by TTX or the removal of extracellular Ca2+, and attenuated by prior lesion of the climbing fibers. Blockade of N-type calcium channels with omega-conotoxin also antagonized the harmaline-induced increase. In contrast, blockade of L- type calcium channels, or inhibition of anion transport with probenecid or bromosulfophthalein, potentiated the increase in cGMP seen in response to harmaline. Inhibitors of nitric oxide synthase or guanylyl cyclase prevented the harmaline-induced increase in extracellular cGMP, while phosphodiesterase inhibitors potentiated the increase. Local application of the NMDA antagonist 2-amino-5-phosphonopentanoic acid or the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione attenuated the effect of harmaline.(ABSTRACT TRUNCATED AT 250 WORDS)
Target and neurotransmitter specificity of fetal central nervous system transplants: importance for functional reinnervation
Authors: Hudson JL , Bickford P , Johansson M , Hoffer BJ , Stromberg I
Published: April 2018

The ability of grafted fetal ventral mesencephalic dopaminergic (DAergic) neuroblasts to reinnervate the unilaterally DA denervated rat striatum and improve motoric asymmetry has been well documented in several laboratories. The importance of host target specificity, and catecholamine (CA) neurotransmitter species, in the ability of grafts to ameliorate rotational responses to apomorphine and to affect electrophysiological characteristics of striatal neurons has not been systematically studied. We unilaterally lesioned Sprague-Dawley rats with 6-hydroxydopamine (6-OHDA) and verified the lesions using apomorphine (0.05 mg/kg, s.c.)-induced rotational behavior. Some of the animals subsequently received, intrastriatally, either DA neuroblasts from ventral mesencephalon that normally innervate the striatum, or from arcuate nucleus that do not. Additionally, two other groups were included that received either a CAergic graft from the noradrenergic nucleus locus coeruleus or a graft of cerebral cortex, which normally projects to the striatum but does not contain CAergic neurons. Only the fetal ventral mesencephalic grafts were able to reduce apomorphine- induced rotations and normalize striatal cell firing rates; striatal cell firing rates with ventral mesencephalic grafts were 1.43 Hz +/- 0.22, with arcuate nucleus grafts were 6.03 +/- 0.73, with locus coeruleus grafts were 4.71 +/- 0.74, and with cerebral cortex grafts were 4.36 +/- 0.45. Moreover, only the ventral mesencephalic grafts produced a dense tyrosine hydroxylase (TH)-immunoreactive nerve terminal network in the striatum; in contrast, the arcuate nucleus grafts did not reinnervate the striatum. In locus coeruleus grafted striata, few very long TH-positive axons were seen. We thus conclude that target specificity and neurotransmitter type are critically important in the ability of a graft to functionally reinnervate the 6- OHDA denervated striatum.
Increased transmitter release at excitatory synapses produced by direct activation of adenylate cyclase in rat hippocampal slices
Authors: Chavez-Noriega LE , Stevens CF
Published: April 2018

The field EPSP recorded in the CA1 region of rat hippocampal slices is potentiated by bath application of the direct adenylate cyclase activator forskolin (Chavez-Noriega and Stevens, 1992a). We have now used the whole-cell patch-clamp technique to analyze the effect of forskolin on evoked synaptic currents and on spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) recorded in rat hippocampal slices in order to determine the relative contributions of pre- and postsynaptic mechanisms to this increased synaptic strength. Application of 50 microM forskolin in the presence of 3-isobutyl-1- methylxanthine (IBMX; a phosphodiesterase inhibitor) enhanced the evoked EPSC (eEPSC) peak amplitude to 230 +/- 43% of control (n = 13). No significant change in sEPSC or in mEPSC amplitude was detected after forskolin addition (106 +/- 7%, n = 9), indicating that postsynaptic receptor sensitivity at synaptic junctions is not greatly affected. In contrast, a large increase in sEPSC and mEPSC frequency was noted in all cells (299 +/- 81%). Following forskolin application, the amplitude distribution of evoked synaptic currents shifted to larger values, but more significantly, a sharp decrease in failure rate was produced in all cells tested. Also, a significant correlation was found between the potentiation produced by forskolin in IBMX on the eEPSC and the ratio of the squared coefficient of variation (CV = SD/mean). Finally, a quantal analysis of four cells was consistent with the hypothesis that transmitter release was increased by forskolin/IBMX with, if anything, a concomitant decrease in quantal size. Together, these observations indicate that presynaptic mechanisms significantly contribute to the enhancement produced by this diterpene.(ABSTRACT TRUNCATED AT 250 WORDS)
Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons
Authors: Werth JL , Thayer SA
Published: April 2018

We sought to determine whether low-affinity, high-capacity mitochondrial Ca2+ uptake contributes to buffering physiological Ca2+ loads in sensory neurons. Intracellular free calcium concentration ([Ca2+]i) and intracellular free hydrogen ion concentration ([H+]i) were measured in single rat dorsal root ganglion (DRG) neurons grown in primary culture using indo-1 and carboxy-SNARF-based dual emission microfluorimetry. Field potential stimulation evoked action potential- mediated increases in [Ca2+]. Brief trains of action potentials elicited [Ca2+]i transients that recovered to basal levels by a single exponential process. Trains of > 25 action potentials elicited larger increases in [Ca2+]i, recovery from which consisted of three distinct phases. During a rapid initial phase [Ca2+]i decreased to a plateau level (450–550 nM). The plateau was followed by a slow return to basal [Ca2+]i [Ca2+]i transients elicited by 40–50 action potentials in the presence of the mitochondrial uncoupler carbonyl cyanide chlorophenyl hydrazone (CCCP), or the electron transport inhibitor antimycin A1, lacked the plateau, and the recovery to basal [Ca2+]i consisted of a single slow phase. Depolarization with 50 mM K+ produced a multiphasic [Ca2+]i transient and increased [H+]i from 74 +/- 3 to 107 +/- 8 nM. The rise in [H+]i was dependent upon extracellular Ca2+ and was inhibited by mitochondrial poisons. With mitochondrial Ca2+ buffering pharmacologically blocked, the recovery to basal [Ca2+]i was unaffected by removal of extracellular Na+. We conclude that large Ca2+ loads are initially buffered by fast mitochondrial sequestration that effectively uncouples electron transport from ATP synthesis, leading to an increase in [H+]i. Small Ca2+ loads are buffered by a nonmitochondrial, Na(+)- independent process.
A quantitative measure for short-term cortical plasticity in human vision
Authors: Kapadia MK , Gilbert CD , Westheimer G
Published: April 2018

The human visual system is normally very good at determining the relative positions of objects in space, but under certain conditions contextual influences can cause significant errors in this process. We studied spatial localization around an artificial scotoma, a small mask that occludes part of the visual field while a dynamic pattern is shown over a surrounding region, and found that the ability to determine the position of short line segments was strongly biased toward the interior of the scotoma. We attribute this “shift” or misassignment of position to receptive field (RF) expansions within the artificial scotoma as seen in recent physiological studies. Furthermore, our findings show that this shift begins within 1 sec of stimulus presentation, suggesting that RFs are constantly altered by their local context and that these dynamics are a part of normal vision.
Estrogen differentially regulates estrogen and nerve growth factor receptor mRNAs in adult sensory neurons
Authors: Sohrabji F , Miranda RC , Toran-Allerand CD
Published: April 2018

We have previously shown that neurons in the basal forebrain colocalize the neurotrophin receptor p75NGFR and estrogen receptors. The present study was designed to examine (1) if neural neurotrophin targets respond to estrogen as a general phenotypic feature and (2) if NGF receptor mRNAs are regulated by estrogen, using a prototypical target of NGF, the dorsal root ganglion (DRG) (sensory) neuron. We demonstrate, for the first time, the presence of estrogen receptor mRNA and protein (binding sites) in adult female rat DRG. Moreover, estrogen receptor mRNA expression, while present in DRG neurons from both the ovariectomized (OVX; estrogen deficient) and intact female rat, was downregulated, as in the adult CNS, during proestrus (high estrogen levels) and in OVX animals replaced with proestrus levels of estrogen, as compared to OVX controls. In contrast, although the mRNAs for the NGF receptors p75NGFR and trkA were also expressed in DRG neurons from OVX and intact animals, expression of both NGF receptor mRNAs was upregulated in sensory neurons during proestrus, as compared to the OVX condition. Estrogen replacement, on the other hand, resulted in a transient downregulation of p75NGFR mRNA and a time-dependent upregulation of trkA mRNA. Estrogen regulation of NGF receptor mRNA in adult peripheral neural targets of the neurotrophins supports the hypothesis that estrogen may regulate neuronal sensitivity to neurotrophins such as NGF and may be an important mediator of neurotrophin actions in normal neural function and following neural trauma.