bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane properties of two types of basal cells in Necturus taste buds
Authors: Delay RJ , Mackay-Sim A , Roper SD
Published: April 2018

Necturus taste buds contain two types of basal cells: presumptive stem cells and Merkel-like basal cells. Both types of basal cells are small round cells located at the base of the taste bud, indistinguishable from each other with light microscopy. However, with electron microscopy, autoradiography, or immunocytochemistry, these two types of basal cells can be easily distinguished. We isolated basal cells from taste buds, characterized their voltage-dependent currents using gigaseal whole-cell recordings, and processed the cells for electron microscopy or immunocytochemistry. We were able to distinguish two cell types electrophysiologically and to correlate cell type with membrane properties. Isolated Merkel-like basal cells had several voltage- activated currents: transient, TTX-sensitive, inward Na+ current; sustained, saturating outward K+ current; and slowly inactivating inward Ca2+ current. These currents are similar to those observed in taste receptor cells. In contrast, presumptive stem cells from Necturus taste buds only had outward K+ currents.
The potency of mu-opioid hyperpolarization of hypothalamic arcuate neurons is rapidly attenuated by 17 beta-estradiol
Authors: Lagrange AH , Ronnekleiv OK , Kelly MJ
Published: April 2018

The mu-opioid agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) hyperpolarizes the majority of arcuate hypothalamic (ARC) neurons by opening an inwardly rectifying potassium conductance. The EC50 for the DAMGO- induced hyperpolarization was 60 +/- 3 nM in ARC neurons from ovariectomized guinea pigs. Superfusion of 17 beta-estradiol (E2; 100 nM) for 20 min in vitro resulted in a significant decrease in DAMGO potency (EC50 = 212 +/- 16 nM) in 40% of the neurons that were tested. This rapid effect of E2 on the mu-opioid response was not mimicked by the biologically inactive isomer 17 alpha-estradiol. Multiple concentrations of E2 were used to generate an E2 concentration-response curve, with an EC50 of 9 nM and a maximal increase in the DAMGO EX50 of 411% of controls. The membrane properties and firing rate of E2- sensitive and E2-insensitive neurons were not different. Streptavidin- FITC labeling did not reveal any significant morphological differences between the groups, but a higher number of E2-sensitive cells was found in the lateral ARC and cell-poor zone. Moreover, immunocytochemical staining of the recorded cells revealed that beta-endorphin neurons were among those sensitive to E2. Therefore, E2 could increase beta- endorphin release by decreasing the potency of beta-endorphinergic autoinhibition, thus increasing the tonic opioid inhibition of E2- insensitive cells. Furthermore, the diffuse projections of hypothalamic beta-endorphin neurons would allow E2 to alter processes throughout the brain, as well as having local effects in the hypothalamus.
Inactivation of Gi and G(o) proteins in nucleus accumbens reduces both cocaine and heroin reinforcement
Authors: Self DW , Terwilliger RZ , Nestler EJ , Stein L
Published: April 2018

The pertussis toxin (PTX)-sensitive G proteins Gi and G(o) may be implicated in drug reinforcement and addiction, since certain reward- related dopamine and opiate receptor subtypes are coupled to these G proteins, and since chronic exposure to cocaine or morphine alters levels of these G proteins in the nucleus accumbens (NAc). As a direct test of this hypothesis, Gi and G(o) proteins in the NAc were selectively inactivated by intra-accumbens injections of PTX in rats self-administering either cocaine or heroin. In control animals, bilateral injections of inactive PTX (0.1 microgram/1 microliter/side) in the NAc failed to alter baseline rates of cocaine and heroin self- administration. In contrast, the same dose of active PTX produced significant, long-lasting increases (up to 1 month) in the self- administration of both drugs, and shifted the dose-response curves to the right. These results suggest that PTX reduces or shortens the reinforcing efficacy of cocaine and heroin, leading to compensatory increases in drug self-administration. Similar NAc injections of PTX reduced the level of Gi alpha and G(o) alpha subunits as measured by both ADP-ribosylation and Western blot, without affecting levels of Gs alpha or G beta subunits. The effect of the toxin was mainly limited to the NAc, and no evidence of abnormal cell death or gliosis was observed. The onset of changes in self-administration rate coincided with the onset of changes in ADP-ribosylation, suggesting that, initially, the increased drug self-administration results directly from a reduction in functional Gi and G(o) proteins. After 28 d, self- administration baselines began to recover while levels of G protein ADP- ribosylation and immunoreactivity remained low, suggesting that adaptive mechanisms are involved at later time points. These results provide direct support for a common role of Gi and G(o) proteins in the NAc in the reinforcing and addictive properties of psychostimulant and opiate drugs.
AE3 anion exchanger isoforms in the vertebrate retina: developmental regulation and differential expression in neurons and glia
Authors: Kobayashi S , Morgans CW , Casey JR , Kopito RR
Published: April 2018

Plasma membrane anion exchangers constitute a multigene family that contributes to the regulation of intracellular pH and chloride concentration in many cell types. We have characterized two polypeptide isoforms of the AE3 gene that are expressed in the rat retina. Using antipeptide antibodies specific for defined NH2-terminal and COOH- terminal epitopes, we have identified a 165 kDa polypeptide whose expression is restricted to the primary glial cell type of the retina, the Muller cell, and a 125 kDa polypeptide that is expressed in horizontal neurons. Expression of the Muller cell isoform exhibits a polarized distribution and is highest in basal endfoot processes. These AE3 isoforms exhibit a distinct developmental expression pattern in postnatal rat retina. The neuronal isoform is undetectable in neonatal retina until postnatal day 10–15, correlating strongly with the onset of retinal function.
Accelerated resensitization of the D1 dopamine receptor-mediated response in cultured cortical and striatal neurons from the rat: respective role of alpha 1-adrenergic and N-methyl-D-aspartate receptors
Authors: Trovero F , Marin P , Tassin JP , Premont J , Glowinski J
Published: April 2018

As previously shown in vivo, noradrenergic and glutamatergic neurons can regulate the denervation supersensitivity of D1 dopaminergic (DA) receptors in the rat prefrontal cortex and striatum respectively. Therefore, the effects of methoxamine (an alpha 1-adrenergic agonist) and glutamate on the resensitization of D1 DA receptors were investigated in cultured cortical and striatal neurons from the embryonic rat. In the presence of sulpiride and propranolol, DA stimulated the D1 DA receptor-mediated conversion of 3H-adenine into 3H- cAMP in both intact cortical and striatal cells and these responses were markedly desensitized in cells preexposed for 15 min to DA (50 microM). The complete recovery of the D1 DA response was more rapid in striatal (15 min) than in cortical (80 min) neurons. Methoxamine accelerated the resensitization of the D1 response in cortical but not in striatal neurons. The effect of the alpha 1-adrenergic agonist in cortical neurons was blocked by prazosin and chlorethylclonidine. In contrast, glutamate accelerated the resensitization of the D1 response in striatal but not in cortical neurons and the effect observed in striatal neurons was totally blocked by 2-amino-5-phosphonovaleric acid, an NMDA receptor antagonist. Protein kinase C was shown to be involved in the alpha 1-adrenergic-induced resensitization of the cortical D1 response but not in the glutamate-evoked resensitization of the striatal D1 response. Finally, for comparison, similar experiments were performed on beta-adrenergic receptors using isoproterenol (1 microM) as an agonist. Methoxamine did not modify the resensitization of the beta-adrenergic response in cortical neurons, but glutamate accelerated the resensitization of this response in striatal neurons.
Immunolocalization of NMDA receptors in the central nervous system of weakly electric fish: functional implications for the modulation of a neuronal oscillator
Authors: Spiro JE , Brose N , Heinemann SF , Heiligenberg W
Published: April 2018

Using a monoclonal antibody raised against the R1 subunit of the rat NMDA receptor, we mapped the distribution of NMDA receptors in the brains of three genera of electric fish. On Western blots, the antibody recognized a glycoprotein of approximately 105 kDa throughout the CNS. On tissue sections, it strongly labeled a number of neuronal somata and dendrites in the medulla, with weaker immunoreactivity in the forebrain and across much of the rest of the nervous system. At the ultrastructural level, reaction product was localized, though not exclusively, to the postsynaptic region of synapses. To study the role of NMDA receptors in a specific neural circuit, we focused on the medullary pacemaker nucleus. Neurons in this nucleus, which fire action potentials regularly and trigger each electric organ discharge (EOD), receive glutamatergic input from identified premotor areas. Activity in these areas can cause the pacemaker nucleus to produce outputs with distinct temporal dynamics, which are observed in the behaving animal as modulations of the EOD. The projection cells of the pacemaker nucleus, the relay cells, were heavily labeled with the anti-NMDA R1 antibody in all genera studied. These results are consistent with the previous finding that a particular EOD modulation mediated by the connection from one premotor area of the brain to the relay cells is blocked by application to the pacemaker nucleus of NMDA receptor blockers. Our results complement ongoing efforts to study this nucleus and provide additional evidence for the role of NMDA receptors in diverse neural circuits.
Spontaneous neuronal calcium spikes and waves during early differentiation
Authors: Gu X , Olson EC , Spitzer NC
Published: April 2018

Calcium ions play critical roles in neuronal development, but the factors that govern spontaneous fluctuations in intracellular calcium are not well understood. Transient, repeated elevations of calcium in embryonic Xenopus spinal neurons have been recorded over periods of 1 hr in vitro and in vivo, confocally imaging fluo-3-loaded cells at 5 sec intervals. Calcium spikes and calcium waves are found both in neurons in culture and in the intact spinal cord. Spikes rise rapidly to approximately 400% of baseline fluorescence and have a characteristic double exponential decay, while waves rise slowly to approximately 200% of baseline fluorescence and decay slowly as well. Imaging of fura-2-loaded neurons indicates that intracellular calcium increases from 50 to 500 nM during spikes. Both spikes and waves are abolished by removal of extracellular calcium. Developmentally, the incidence and frequency of spikes decrease while the incidence and frequency of waves are constant. Spikes are generated by spontaneous calcium-dependent action potentials that can be triggered by low- threshold, T-type calcium current and are eliminated by agents that block voltage-dependent calcium channels. They can be elicited by depolarization, are generated in an all-or-none manner, and are rapidly and bidirectionally propagated. Spikes also utilize intracellular calcium stores, since blocking release from stores substantially reduces their amplitude. Waves are not elicited by depolarization nor by activation of glutamate receptors, and are propagated at a rate consistent with diffusion of calcium. Waves are blocked by Ni2+ at a higher concentration than required to block classical voltage-dependent calcium channels. Previous work now suggests that spikes are required for expression of the transmitter GABA and for potassium channel modulation. The present study indicates that waves in growth cones are likely to regulate neurite extension.
The functional organization of human extrastriate cortex: a PET-rCBF study of selective attention to faces and locations
Authors: Haxby JV , Horwitz B , Ungerleider LG , Maisog JM , Pietrini P , Grady CL
Published: April 2018

The functional dissociation of human extrastriate cortical processing streams for the perception of face identity and location was investigated in healthy men by measuring visual task-related changes in regional cerebral blood flow (rCBF) with positron emission tomography (PET) and H2(15)O. Separate scans were obtained while subjects performed face matching, location matching, or sensorimotor control tasks. The matching tasks used identical stimuli for some scans and stimuli of equivalent visual complexity for others. Face matching was associated with selective rCBF increases in the fusiform gyrus in occipital and occipitotemporal cortex bilaterally and in a right prefrontal area in the inferior frontal gyrus. Location matching was associated with selective rCBF increases in dorsal occipital, superior parietal, and intraparietal sulcus cortex bilaterally and in dorsal right premotor cortex. Decreases in rCBF, relative to the sensorimotor control task, were observed for both matching tasks in auditory, auditory association, somatosensory, and midcingulate cortex. These results suggest that, within a sensory modality, selective attention is associated with increased activity in those cortical areas that process the attended information but is not associated with decreased activity in areas that process unattended visual information. Selective attention to one sensory modality, on the other hand, is associated with decreased activity in cortical areas dedicated to processing input from other sensory modalities. Direct comparison of our results with those from other PET-rCBF studies of extrastriate cortex demonstrates agreement in the localization of cortical areas mediating face and location perception and dissociations between these areas and those mediating the perception of color and motion.
Hippocampal synaptogenesis in cell culture: developmental time course of synapse formation, calcium influx, and synaptic protein distribution
Authors: Basarsky TA , Parpura V , Haydon PG
Published: April 2018

The formation of chemical synapses between hippocampal neurons in primary cell culture was studied using electrophysiology, calcium imaging, and immunocytochemical approaches. Inhibitory and excitatory synapses formed within 12 d in cell culture (DIC) that were sensitive to the N-type calcium channel blocker omega-conotoxin GVIA (omega- CgTx). At 4 DIC, immature connections were present in which spontaneous, but rarely evoked, synaptic currents were detected. At both 4 and 12 DIC, the synaptic proteins rab3a, synapsin I, and synaptotagmin were present in hippocampal neurons, but the subcellular distribution changed from one in which immunoreactivity was initially distributed within soma and neurites to a punctate varicose appearance. Correlated with the transformation from immature to mature synaptic states was the onset of omega-CgTx-sensitive calcium influx. Taken together, these data suggest that the expression of functional omega- CgTx-sensitive calcium influx is temporally coincident with synapse formation, and that during the maturation of the synapse there is a redistribution of synaptic proteins.
Correlates of hippocampal complex-spike cell activity in rats performing a nonspatial radial maze task
Authors: Young BJ , Fox GD , Eichenbaum H
Published: April 2018

The observation of hippocampal place cells forms a major line of evidence supporting the view that the hippocampus is dedicated to spatial processing. However, most studies demonstrating the spatial properties of hippocampal unit activity have employed tasks that emphasize spatial cues but minimize nonspatial cues. In the present experiment we recorded the activity of hippocampal complex-spike cells from rats performing a nonspatial radial maze task. Performance in this task was guided by local visual-tactile cues on the maze arms, while distal spatial cues were minimized and made irrelevant. The influence of three variables on unit activity was examined:type of cue on an arm, spatial location of an arm, and the relative position of the animal on an arm. Of the units recorded, almost one-fifth were classified as “cue cells” in that their activity was associated with cue type but not spatial location. Conversely, a similar proportion of the units were classified as “place cells” in that their activity was associated with location, but not cue type. In an additional similar proportion of units, firing was influenced only by relative position and not by local cues or spatial locations. For the majority of units, however, firing was related to combinations of these three variables, indicating that most hippocampal neurons encoded conjunctions or relations between spatial and local cue information. This pattern of results indicates that when local rather than distal spatial cues are emphasized, hippocampal neural activity is strongly influenced by salient nonspatial cues and shows no overwhelming predominance of place coding. These findings are at odds with the hypothesis that the hippocampus is selectively involved in spatial processing and, conversely, support the broader view that the hippocampus encodes both spatial and nonspatial relations among important experimental variables.
Genomic organization of the FMRFamide gene in Lymnaea: multiple exons encoding novel neuropeptides
Authors: Kellett E , Saunders SE , Li KW , Staddon JW , Benjamin PR , Burke JF
Published: April 2018

Based on the sequencing of genomic and cDNA clones, we were able to determine that the FMRFamide gene consists of five exons covering at least 20 kb and predict the presence of further novel peptides. The exons are alternatively spliced: exon I (hydrophobic leader sequence) to exon II (tetrapeptides) and exon I to exons III (heptapeptides), IV, and V. A cDNA clone encoding the heptapeptides is described and has also been shown to encode further novel peptides SKPYMRFamide, HDYMRFamide, and SSFPRYamide. Analysis of the right internal parietal nerve using mass spectrometry showed that the novel peptide SKPYMRFamide was cleaved from the precursor. This peptide excites neurons, suggesting a physiological function in the CNS.
Dose-dependent reduction of tissue loss and functional impairment after spinal cord trauma with the AMPA/kainate antagonist NBQX
Authors: Wrathall JR , Choiniere D , Teng YD
Published: April 2018

Initial studies on the role of glutamate receptors in traumatic spinal cord injury (SCI) implicated the NMDA subclass of ionotropic glutamate receptors in contributing to functional deficits. Recently we obtained evidence suggesting that non-NMDA ionotropic receptors may participate in producing a portion of the behavioral impairment after SCI. To test this hypothesis we have conducted a dose-response experiment, focally injecting 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX; 1.5, 5, or 15 nmol), a highly selective antagonist of alpha-amino-3- hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors, or vehicle alone, into the injury site beginning at 15 min after a standardized contusive SCI. Behavioral tests of hindlimb reflex and coordinated sensorimotor function were performed 1 d after injury and weekly thereafter. At 4 weeks, spinal cord tissue was examined using quantitative histopathological and immunocytochemical techniques. We found a dose-dependent reduction in tissue loss at the thoracic injury site, with greater residual amounts of both gray matter and myelinated white matter. The maximum dose (15 nmol) significantly reduced the average length of the lesions and doubled the area of residual white matter at the epicenter. Serotonin immunoreactivity caudal to the lesion, used as a marker for descending motor control axons, was also increased in a dose-related manner and nearly tripled with the highest dose of NBQX as compared to controls. Most importantly, the reduced tissue loss in NBQX-treated groups was correlated with reduced functional deficits. There was a dose-dependent enhancement of speed and degree of recovery of both reflex and coordinated hindlimb motor activity, and reduction in the time required for establishing a reflex bladder. The long-term functional deficits at 4 weeks after SCI were reduced in a dose-related manner. Further, regression analyses demonstrated a significant correlation between the increase in amount of residual tissue and improvement in hindlimb function. Our results suggest that in this type of incomplete contusive SCI, a large and functionally important proportion of the tissue loss appears to be due to secondary injury mediated by local AMPA/kainate receptors.
Extent and mechanism of sealing in transected giant axons of squid and earthworms
Authors: Krause TL , Fishman HM , Ballinger ML , Bittner GD
Published: April 2018

Transected axons are often assumed to seal at their cut ends by the formation of continuous membrane barriers that allow for the restoration of function in the axonal stumps. We have used several electrophysiological measures (membrane potential, input resistance, injury current density) and several morphological measures (phase- contrast, video-enhanced differential interference contrast, light, and electron microscopies) of living and fixed material to assess the extent and mechanism of sealing within hours after transecting giant axons of squid (Loligo pealei and Sepioteuthis lessoniana) and earthworms (Lumbricus terrestris). Our electrophysiological data suggest that the proximal and distal ends of transected squid giant axons do not completely seal within 2.5 hr in physiological saline. In contrast, the same set of measures suggest that proximal and distal ends of transected earthworm giant axons seal within 1 hr in physiological saline. Our morphological data show that the cut ends of both squid and earthworm axons constrict, but that a 20–70-microns- diameter opening always remains at the cut end that is filled with vesicles. Axonal transection induces the formation of vesicles that are observed in the axoplasm within minutes in standard salines and that rapidly migrate to the cut ends. These injury-induced vesicles are loosely packed near the cut ends of squid giant axons, which do not functionally seal within 2.5 hr of transection. In contrast, vesicles formed a tightly packed plug at the cut ends of earthworm medial giant axons, which do functionally seal within 1 hr of transection in physiological saline. Since we detect no single continuous membrane that spans the cut end, sealing does not appear to occur by the fusion of constricted axolemmal membrane or the formation of a membranous partition at the cut end. Rather, our data are consistent with the hypothesis that a tightly packed vesicular plug is responsible for sealing of earthworm giant axons.
Song-induced ZENK gene expression in auditory pathways of songbird brain and its relation to the song control system
Authors: Mello CV , Clayton DF
Published: April 2018

The ZENK gene encodes a zinc-finger-containing transcriptional regulator and can be rapidly activated in songbird brain by presentation of birdsong (Mello et al., 1992). Here we map the areas of the songbird forebrain that show this genomic response to birdsong, using in situ hybridization. After 30 min of song presentation ZENK mRNA levels reach a peak in the caudomedial telencephalon, in areas adjacent to or closely related with primary auditory structures. These areas include subfields of field L (L1 and L3), the caudomedial neostriatum (NCM), the caudomedial hyperstriatum ventrale (CMHV) anterior to field L, the caudal paleostriatum, and two field L targets, HVC shelf and RA cup. In contrast, ZENK induction is absent in some areas that show a response to song by other measures and where ZENK induction might have been expected. These include the direct thalamo- recipient field L subfield L2, and the nuclei of the circuit involved in the acquisition and production of learned song. These results demonstrate that ZENK induction following song presentation occurs only in a subset of areas physiologically activated by song, and draw attention to areas previously unsuspected as related to processing of complex auditory stimuli. Based on what is known about ZENK function in mammalian systems (Christy et al., 1989; Cole et al., 1989; Wisden et al., 1990), we speculate that areas revealed by ZENK induction might correspond to sites where critical neuronal modifications occur in response to birdsong presentation, possibly leading to the formation of song-related memories.
Cellular and molecular characterization of Ca2+ currents in acutely isolated, adult rat neostriatal neurons
Authors: Bargas J , Howe A , Eberwine J , Cao Y , Surmeier DJ
Published: April 2018

Ca2+ currents in acutely isolated, adult rat neostriatal neurons were studied with whole-cell voltage-clamp techniques. In the vast majority of neurons (approximately 90%, n > 250), currents were exclusively of the high-voltage-activated (HVA) type. HVA currents activated near -40 mV and reached their maximum amplitude near 0 mV. Quasi-steady-state inactivation curves in many neurons were well fitted only with a sum of Boltzmann functions, suggesting that the HVA currents were heterogeneous. Although the block of whole-cell current by Cd2+ was well fitted with a single isotherm having an IC50 of near 1 microM, experiments with organic channel antagonists suggested that at least four types of HVA channels were expressed by most cells. On average, the L-channel antagonist nifedipine (5–10 microM) blocked 31 +/- 10% of the whole-cell current (n = 20), the N-channel antagonist omega- conotoxin GVIA (omega-CgTx) (2–5 microM) blocked 27 +/- 11% (n = 20), and the P-channel antagonist omega-agatoxin IVA (100–500 nM) blocked 21 +/- 10% (n = 18). In many neurons, the block by omega-CgTx was partially or completely reversible. In cells tested with a combination of these antagonists, 34 +/- 17% of the peak Ca2+ current remained unblocked (n = 13). Single-cell expression profiling of medium-sized neurons revealed the presence of rbA and rbB Ca2+ channel alpha 1 subunit mRNAs but low or undetectable levels of rbC mRNA (n = 12). These findings suggest that although adult neostriatal projection neurons do not express significant levels of LVA Ca2+ current, they do express a pharmacologically and structurally heterogeneous population of HVA currents.
Sun compass-based spatial learning impaired in homing pigeons with hippocampal lesions
Authors: Bingman VP , Jones TJ
Published: April 2018

The hippocampal formation is known to be critical for spatial cognition, for example, regulating the learning of environmental maps. But how is a spatial map learned, and what is the role of the hippocampal formation in the learning process? The sun compass is perhaps the most ubiquitous, naturally occurring spatial orientation mechanism found in the animal kingdom. The sun compass may also serve as a directional reference that supports spatial learning. We report that homing pigeons with hippocampal lesions were unable to use the sun compass to learn the directional location of food in an outdoor, experimental arena. Homing pigeons with lesions of the caudal neostriatum readily learned the same task, and showed appropriately shifted directional responses following a clock-shift manipulation demonstrating that they were indeed using the sun compass to learn the task. Finally, both hippocampal and control lesioned birds quickly learned a procedurally similar task where a color cue identified the location of food in the same experimental arena. The results indicate that hippocampal lesions impair sun compass use in the context of learning. As such, the results support the hypothesis that the importance of the hippocampal formation in spatial cognition may be related to its participation in a neural process in which information from a directional reference, in this case the sun compass, is used to learn the directional relationship among stimuli in space.
Cholinergic neuromuscular synapses in Aplysia have low endogenous acetylcholinesterase activity and a high-affinity uptake system for acetylcholine
Authors: Lloyd PE , Church PJ
Published: April 2018

In the present study, we have demonstrated that ACh is the predominant fast excitatory transmitter used by identified motor neurons innervating feeding muscles in Aplysia. A detailed study of ACh metabolism was then carried out in a well-characterized neuromuscular preparation, intrinsic muscle 5 (15). This neuromuscular system has a high-affinity uptake system for choline. The rate of uptake of choline was increased by motor neuron stimulation, and this increased uptake appears to be selectively targeted to motor neuron terminals. These properties appear similar to those observed in vertebrate neuromuscular preparations. However, we have made two observations that are surprising in light of our knowledge concerning the vertebrate neuromuscular junction where released ACh is rapidly hydrolyzed by acetylcholinesterase (AChE) to choline, which is then taken up by a high-affinity uptake system. This Aplysia neuromuscular system has limited endogenous AChE activity and contains a separate high-affinity uptake system for ACh itself that actually has a higher velocity than that for choline uptake. It is possible that the uptake system for ACh is involved in terminating the action of released transmitter in a manner similar to that previously described for noncholinergic transmitters. Using this preparation, we have demonstrated release of labeled ACh in response to intracellular stimulation of identified motor neurons. The release per spike appears to be highly plastic,increasing markedly with stimulation frequency. This preparation is amendable to study the regulation of release of peptide and conventional transmitters from the terminals of individual neurons.
Distinct regulatory pathways control neurofilament expression and neurotransmitter synthesis in immortalized serotonergic neurons
Authors: White LA , Eaton MJ , Castro MC , Klose KJ , Globus MY , Shaw G , Whittemore SR
Published: April 2018

Following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T-antigen (T-ag), a neuronal cell line, RN46A, was cloned by serial dilution. At 33 degrees C, RN46A cells express nuclear T-ag immunoreactivity and divide with a doubling time of 9 hr. Undifferentiated RN46A cells express low levels of neuron-specific enolase (NSE) and low (NF-L)-and medium (NF-M)- but not high (NF-H)- molecular-weight neurofilament proteins. Under differentiation conditions, RN46A cells cease dividing, take on a neuronal morphology, and express enhanced levels of NSE and all three NF proteins. Elevation of intracellular cAMP levels increases neurofilament protein expression, whereas activators of various other intracellular second messenger systems have no effect. Differentiated RN46A cells express low-affinity nerve growth factor (NGF) receptor (p75NGFR) and are immunoreactive using an antibody that recognizes the carboxy-terminal 13 amino acids of all three trk proteins (pan-trk). Both immunoreactivities could be potentiated by treatment with brain-derived neurotrophic factor (BDNF), NGF, and adrenocorticotropic hormone, fragment 4–10 (ACTH4–10). Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) immunoreactivity, which could be enhanced by treatment with ACTH4–10, BDNF, or NGF. Low levels of serotonin immunoreactivity are detected in differentiated RN46A cells, and this was potentiated by differentiating RN46A cells with BDNF for 8 d and 40 mM KCl for days 4–8. HPLC analysis confirmed these immunohistochemical data. RN46A cells should prove useful to elucidate intracellular mechanisms that control neurofilament assembly and 5-HT expression in differentiating raphe neurons.
Effects of prolonged sleep deprivation on local rates of cerebral energy metabolism in freely moving rats
Authors: Everson CA , Smith CB , Sokoloff L
Published: April 2018

Although sleep deprivation interferes with biological processes essential for performance, health, and longevity, previous studies have failed to reveal any structural or functional changes in brain. We have therefore measured local rates of cerebral glucose utilization (ICMRglc) with the quantitative autoradiographic 2–14C-deoxyglucose method in an effort to determine if and, if so, where sleep deprivation might affect function in sleep-deprived rats. Sleep deprivation was maintained for 11–12 d, long enough to increase whole body energy metabolism, thus confirming that pathophysiological processes that might involve brain functions were evolving. Deep brain temperature was also measured in similarly treated rats and found to be mildly elevated relative to core body temperature. Despite the increased deep brain temperature, systemic hypermetabolism, and sympathetic activation, ICMRglc was not elevated in any of the 60 brain structures examined. Average glucose utilization in the brain as a whole was unchanged in the sleep-deprived rats, but regional decreases were found. The most marked decreases in ICMRglc were in regions of the hypothalamus, thalamus, and limbic system. Mesencephalic and pontine regions were relatively unaffected except for the central gray area. The medulla was entirely normal. The effects of sleep deprivation on brain tended, therefore, to be unidirectional toward decreased energy metabolism, primarily in regions associated with mechanisms of thermoregulation, endocrine regulation, and sleep. Correspondence was found between the hypometabolic brain regions and some aspects of peripheral symptoms.
Squirrel monkey lateral thalamus. I. Somatic nociresponsive neurons and their relation to spinothalamic terminals
Authors: Apkarian AV , Shi T
Published: April 2018

The incidence and response properties of nociresponsive neurons, their locations relative to spinothalamic terminals, and their relations to cytoarchitectonic borders were studied in the lateral thalamus of the squirrel monkey. Nociceptive neurons were found in ventral posterior inferior nucleus (VPI), in the lateral and medial nuclei (VPL and VPM) of the ventral posterior complex (VP = VPL + VPM), as well as the posterior complex (PO). The overall incidence of nociresponsive cells was 19% (50 of 270 cells). The proportion of nociresponsive neurons within VPI was 50% (23 of 46), 38% in PO (8 of 21), and 10% in VP (19 of 203). Most nociresponsive cells (90%) in VP were of wide-dynamic- range type, while within VPI 43% of nociresponsive cells were nociceptive-specific type. Most of these nociresponsive cells had thermal and mechanical responses, and a small number also responded to cooling. The receptive fields of nociresponsive cells in VPL were in continuity, in both size and body location, with surrounding low- threshold units. The receptive fields of VPI and PO nociresponsive cells were larger than those in VPL. The probability of encountering nociresponsive cells located within 100 microns of spinothalamic terminations was high in VPI (73%) and low in VPL (33%). On the other hand, the probability of encountering non-nociceptive cells located within 100 microns of spinothalamic terminals was low in both VPI (20%) and VPL (26%). The results indicate segregation of nociresponsive cell types across VP, VPI, and PO and suggest that VPI, and perhaps PO, is an important region for discriminative processing and perception of painful stimuli.
Squirrel monkey lateral thalamus. II. Viscerosomatic convergent representation of urinary bladder, colon, and esophagus
Authors: Bruggemann J , Shi T , Apkarian AV
Published: April 2018

The response properties of 106 visceroceptive lateral thalamic neurons were investigated in anesthetized squirrel monkeys. Most neurons were located in the ventral posterior lateral nucleus (VPL), and a smaller number of cells was also found in a variety of thalamic nuclei around VPL. Ninety (85%) of these cells responded to distension of the urinary bladder, the distal colon, and/or the lower esophagus. The majority of the visceral-responsive cells also had convergent somatic and multivisceral responses (71% of the 85%). A small population (6%) was visceral specific; that is, these neurons were not activated with somatic stimuli. Visceral responses were excitatory, inhibitory, or mixed, and most were either visceral nociceptive specific (65%) or visceral wide-dynamic-range type (34%). Very few visceral responses (1%) could be classified as low threshold. The incidence of these response types were highly dependent on the specific viscus stimulated. Most visceral responses were able to code stimulus duration and intensity. The majority (69%) of the visceroceptive neurons had somatic low-threshold convergent input mainly from the surface of the lower body. The somatic receptive field locations and the somatic response properties seem unrelated to the convergent visceral input and the visceral response properties, although there were some exceptions. No obvious viscerotopical organization was found in VPL. The results lead us to propose two different modes of representation for processing of and distinguishing between visceral and somatic inputs: a distributed population code for visceral inputs, and a local code for somatic inputs. Based on these codes, we discuss a new hypothesis for referred pain.
Comparison of innervation and agrin-induced tyrosine phosphorylation of the nicotinic acetylcholine receptor
Authors: Qu Z , Huganir RL
Published: April 2018

Studies of the regulation of tyrosine phosphorylation at the neuromuscular junction during development and following denervation suggest that tyrosine phosphorylation of the nicotinic acetylcholine receptor is regulated by neuronal innervation of muscle. The finding that agrin, a neuronally derived extracellular matrix protein also induces tyrosine phosphorylation of the nicotinic receptor, suggests that nerve-induced tyrosine phosphorylation may be mediated by agrin. To study this further, we have examined the regulation of tyrosine phosphorylation of the nicotinic receptor by innervation in vitro using muscle-neuron cocultures. Innervation of chick myotubes by chick ciliary ganglia neurons induced tyrosine phosphorylation of the nicotinic receptor with the same subunit specificity seen with bath applied purified agrin. Both innervation and agrin-induced phosphorylation of the nicotinic receptor resulted in an increase in tyrosine and serine phosphorylation. In addition, thermolysin phosphopeptide maps of the subunits after innervation or agrin- treatment were identical. The similarity in the agrin- and nerve- induced phosphorylation of the acetylcholine receptor suggests that agrin mediates the nerve-induced phosphorylation during development in vivo and that phosphorylation of the acetylcholine receptor may play an important role in the development of the neuromuscular junction.
Perforant path damage results in progressive neuronal death and somal atrophy in layer II of entorhinal cortex and functional impairment with increasing postdamage age
Authors: Peterson DA , Lucidi-Phillipi CA , Eagle KL , Gage FH
Published: April 2018

In vivo model systems that can evaluate neuronal death, survival, and regeneration are critical to revealing basic mechanisms of neuronal response and developing strategies for CNS repair. We propose a distinct experimental model of CNS degeneration following lesions to the perforant path connecting the hippocampus and the entorhinal cortex. Within 2 weeks of a unilateral aspirative perforant path lesion, 30% of the ipsilateral entorhinal cortex layer II (ECL II) projection neurons had died with no change in the contralateral ECL II population. Although there was no loss of ECL II neurons with normal aging, animals that survived for 15 months postlesion experienced an almost 50% loss of ipsilateral neurons compared to unlesioned controls. This progressive neuronal death was bilateral, with the contralateral ECL II experiencing a 30% decline in neuronal number relative to unlesioned controls. The use of unbiased stereology ensured that estimates of total number were not distorted by changes in the reference volume. The documented progressive neuronal death resulted in delayed behavioral impairment in spatial learning and performance (latency nearly 200% of controls). We propose, therefore, that the perforant path model is suitable for experimental investigation of neuronal survival and regeneration following CNS trauma.
A population of SCP-containing neurons in the buccal ganglion of Aplysia are radula mechanoafferents and receive excitation of central origin
Authors: Miller MW , Rosen SC , Schissel SL , Cropper EC , Kupfermann I , Weiss KR
Published: April 2018

The rostral cluster of SCP-immunoreactive cells, originally identified in each buccal hemiganglion of juvenile Aplysia, was examined in mature specimens. Immunohistochemical and dye-fill experiments showed that each rostral cluster consists of approximately 40 cells. Although these neurons exhibit heterogeneity of size and shape, all cells project an axon into the radula nerve. Tracing of dye-filled cells showed that they project to the layer of tissue that lines the inner surface of the food-grasping portion of the chitinous radula. This tissue contains SCP- immunoreactive nerve fibers and varicosities in regions corresponding to the projections of dye-filled neurons. Several observations indicate that rostral cluster neurons transduce tactile stimuli applied to the radula surface: (1) each cell responds to touch of a circumscribed receptive field with a rapidly adapting burst of action potentials, (2) the evoked spikes arise abruptly from the resting potential without prepotentials, and (3) the responses persist when central and peripheral synaptic transmission is blocked in high Mg2+, low Ca2+ artificial seawater solutions. These cells, designated radula mechanoafferent (RM) neurons, do not respond to chemical stimuli including NaCl, glutamate, and seaweed extract. The highest density of receptive fields is found on the posterodorsal edges of the radula halves, areas most directly involved in grasping food. The RM neurons are electrically coupled cells, with coupling coefficients ranging from 0.006 to 0.22. They fire phasically during buccal motor programs, even in the absence of peripheral feedback from the radula or other portions of the buccal mass. In radiolabeling studies the RM cells were found to synthesize authentic SCPA and SCPB. Sensorin-A, a peptide that is localized to other Aplysia mechanoafferent neurons, was not detected immunohistochemically in these cells.
The metabotropic glutamate receptor types 2/3 inhibit L-type calcium channels via a pertussis toxin-sensitive G-protein in cultured cerebellar granule cells
Authors: Chavis P , Shinozaki H , Bockaert J , Fagni L
Published: April 2018

Modulation of Ca2+ channels by metabotropic glutamate receptors (mGluRs) was investigated in cerebellar granule cells using the cell- attached configuration of the patch-clamp technique. Experiments were performed in the absence of external Ca2+ and Ba2+ was used as charge carrier. Bath applied glutamate or (1S,3R) trans-1-aminocyclopentane- 1,3-dicarboxylic acid (1S,3R t-ACPD) inhibited Ca2+ channels activated by depolarizing pulses. These channels were sensitive to dihydropyridines and displayed a 23 pS conductance. This effect was mimicked by (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I), a selective agonist of mGluR2/R3 receptors, but not by quisqualate at a concentration that stimulated inositol phosphate (InsP) synthesis, showing that mGluR1 and mGluR5 did not participate to this mechanism. The phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX), did not alter the action of the mGluR agonists and biochemical measurements showed that 1S,3R t-ACPD, in the presence of IBMX, decreased cAMP formation in such a small amount that this change could not explain the almost complete inhibition of the channel activity observed under similar experimental conditions. Moreover, whole-cell recorded L-type Ca2+ currents were inhibited by L-CCG-I, in the presence of 1 mM intracellular cAMP. These observations were consistent with the hypothesis that cyclic nucleotide second messengers were not involved in this effect. Neither the protein kinase C activator phorbol-12,13- dibutyrate (PDBU) nor the phosphatase inhibitor okadaic acid affected the action of 1S,3R t-ACPD. The inhibitory action of 1S,3R t-ACPD was abolished by pertussis toxin (PTX). These results suggest that mGluR2 or mGluR3 receptors suppress the activity of L-type Ca2+ channels by a mechanism involving Gi or G(o) proteins. A likely direct effect of G- proteins on the channels is discussed.
Role of neuronal synchronizing mechanisms in the propagation of spreading depression in the in vivo hippocampus
Authors: Herreras O , Largo C , Ibarz JM , Somjen GG , Martin del Rio R
Published: April 2018

To detect what initiates spreading depression (SD), the early prodromal events were investigated in hippocampal CA1 of urethane-anesthetized rats. SD was provoked by microdialysis or focal microinjection of high- K+ solution. Extracellular DC potentials and extracellular potassium concentration ([K+]o) were recorded, and spontaneous and evoked potentials analyzed for current source-density (CSD). In the front of an approaching SD wave, several seconds before the onset of the typical sustained negative potential shift (delta Vo) and the increased [K+]o, fast electrical activity was detected. This consisted initially of small rhythmic (60–70 Hz) sawtooth wavelets, which then gave way to a shower of population spikes (PSs) of identical frequency. Prodromal wavelets and PSs were synchronized over considerable distances in the tissue. Sawtooth wavelets were identified as pacemakers of the prodromal PS burst. Simultaneous recording at three depths revealed that the spontaneous prodromal PSs occurred exactly in phase in dendrites and somata whereas synaptically transmitted PSs arose first in the proximal dendrites and were conducted from there into the soma membrane. During a spike burst, stratum (st.) pyramidale served as current sink, while in the proximal sublayer of st. radiatum spike- sinks gave way to spike sources that grew larger as the sinks in st. pyramidale began to subside. Blocking synaptic transmission did not abolish the prodromal spike burst, yet repetitive orthodromic activation inhibited it without altering the subsequent SD waveform. Complex changes in cell excitability were detected even before fast spontaneous activities. We concluded that, in the initial evolution of SD, changes in neuron function precede the regenerating depolarization by several seconds. We propose that the opening of normally closed electric junctions among neurons can best explain the long-distance synchronization and the flow current that occurs in the leading edge of a propagating wave of SD.