bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoregulation of phosphorylation of the nicotinic acetylcholine receptor
Authors: Miles K , Audigier SS , Greengard P , Huganir RL
Published: April 2018

We have investigated the regulation of phosphorylation of the nicotinic ACh receptor (nAChR) in rat myotubes by the agonist carbamylcholine. Treatment of primary rat myotube cultures with carbamylcholine resulted in a 100% increase in phosphorylation of the nAChR gamma- (52 kDa) subunit and a 30% increase in phosphorylation of the nAChR delta- (62 kDa) and delta'- (66 kDa) subunits. These responses to carbamylcholine were dose dependent, with a half-maximal response occurring at 10 microM and a maximum response achieved within 2 min. Pretreatment of myotubes with d-tubocurare, but not with atropine, inhibited carbamylcholine-stimulated phosphorylation of the nAChR. Preincubation with open-channel blockers of the nAChR also inhibited phosphorylation of the nAChR induced by carbamylcholine. Depletion of extracellular calcium from myotube cultures prevented carbamylcholine-stimulated increases in nAChR phosphorylation whereas application of a calcium ionophore mimicked the effect of carbamylcholine on nAChR phosphorylation. Pretreatment of myotubes with TTX did not inhibit carbamylcholine-stimulated nAChR phosphorylation and potassium depolarization of myotubes had no effect on nAChR phosphorylation. Carbamylcholine increased nAChR phosphorylation to the same extent and with the same time course and subunit specificity as that induced by phorbol esters. However, chronic treatment of myotubes with phorbol esters that eliminated any subsequent phorbol ester-stimulated nAChR phosphorylation did not diminish the increase in nAChR phosphorylation induced by carbamylcholine. The calmodulin antagonist W7 was similarly unable to inhibit carbamylcholine-stimulated nAChR phosphorylation. These results suggest that the nAChR is a substrate for an uncharacterized protein kinase in situ, and that activity of this protein kinase is stimulated by calcium ions that permeate through the activated nAChR ion channel.(ABSTRACT TRUNCATED AT 250 WORDS)
Dopaminergic Regulation of Progesterone Receptors: Brain D5 Dopamine Receptors Mediate Induction of Lordosis by D1-Like Agonists in Rats
Authors: Apostolakis Ede Marie , Garai Janos , Fox Charles , Smith Carolyn L. , Watson Stanley J. , Clark James H. , O’Malley Bert W.
Published: April 2018

To characterize the signaling pathway by which the neurotransmitter dopamine modulates progesterone receptor (PR) activation, the steroid-dependent behavior lordosis was used in estrogen-primed ovariectomized Sprague–Dawley rats with stereotaxic implanted third ventricle cannulas. Lordosis was observed in response to solicitous males in females after central administration of the D1-like agonist SKF38393 and three of its analogs (SKF77434, SKF75640, and SKF85174). In contrast, D1-like antagonist SCH23390 and D1-like/D2 repopulation inhibitor EEDQ blocked behavior inducible by the D1-like agonists. Further, antisense oligonucleotides to D5, but not D1, dopamine receptor mRNA suppressed reproductive behavior associated with D1-like stimulation. This finding provides strong evidence that dopaminergic modulation of lordosis is mediated by the novel D5 dopamine receptor. Although D1, but not D5, dopamine receptor mRNAs were detected in the ventromedial nucleus (VMN) byin situhybridization, agonists microinjected into the VMN, but not into the arcuate nucleus or preoptic area, induced lordosis, suggesting the functional presence of D5 dopamine receptors in the VMN. Also in support, D5 receptor mRNA antisense microinjected into the VMN blocked the subsequent induction of lordosis by D1-like agonists. Finally, facilitation of sex behavior by D1-like agonists was blocked by the antiprogestin RU38486 and PR antisense oligonucleotide. Collectively, the data provide strong evidence for dopaminergic modulation of reproductive behavior through D5 dopamine receptor-mediated modulation of PR-dependent behavior in rat CNS.
Modification of NMDA Receptor Channels and Synaptic Transmission by Targeted Disruption of the NR2C Gene
Authors: Ebralidze Alexander K. , Rossi David J. , Tonegawa Susumu , Slater N. Traverse
Published: April 2018

A novel strain of mutant mouse has been generated with a deletion of the gene encoding the NR2C subunit of the NMDA receptor, which is primarily expressed in cerebellar granule cells. Patch-clamp recordings from granule cells in thin cerebellar slices were used to assess the consequences of the gene deletion. In granule cells of wild-type animals, a wide range of single-channel conductances were observed (19–60 pS). The disruption of the NR2C gene results in the disappearance of low-conductance NMDA receptor channels (<37 pS) normally expressed in granule cells during developmental maturation. The NMDA receptor-mediated synaptic current is markedly potentiated in amplitude, but abbreviated in duration (with no net difference in total charge), and the non-NMDA component of the synaptic current was reduced. We conclude that the NR2C subunit contributes to functional heteromeric NMDA receptor-subunit assemblies at the mossy fiber synapse and extrasynaptic sites during maturation, and the conductance level exhibited by a given receptor macromolecule may reflect the stochiometry of subunit composition.
Ultrastructural Evidence for Prominent Distribution of the μ-Opioid Receptor at Extrasynaptic Sites on Noradrenergic Dendrites in the Rat Nucleus Locus Coeruleus
Authors: Van Bockstaele Elisabeth J. , Colago Eric E. O. , Cheng Peter , Moriwaki Akiyoshi , Uhl George R. , Pickel Virginia M.
Published: April 2018

Physiological studies have indicated that agonists at the μ-opioid receptor (μOR), such as morphine or the endogenous peptide methionine5-enkephalin, can markedly decrease the spontaneous activity of noradrenergic neurons in the locus coeruleus (LC). Messenger RNA and protein for μOR are also densely expressed by LC neurons. During opiate withdrawal, increased discharge rates of LC neurons coincide with the expression of behavioral features associated with the opiate withdrawal syndrome. To better define the cellular sites for the physiological activation of μOR in the LC and its relation to afferent terminals, we examined the ultrastructural localization of μOR immunoreactivity in sections dually labeled for the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Immunogold–silver labeling for μOR (i-μOR) was localized to parasynaptic and extrasynaptic portions of the plasma membranes of perikarya and dendrites, many of which also contained immunolabeling for TH. The dendrites containing exclusively i-μOR were more numerous in the rostral pole of the LC. The i-μOR in dendrites with and without detectable TH immunoreactivity were usually postsynaptic to unlabeled axon terminals containing heterogeneous types of synaptic vesicles and forming asymmetric synaptic specializations characteristic of excitatory-type synapses. These results provide the first direct ultrastructural evidence that μOR is strategically localized to modulate the postsynaptic excitatory responses of catecholamine-containing neurons in the LC.
Retinal Ganglion Cell Axons Recognize Specific Guidance Cues Present in the Deafferented Adult Rat Superior Colliculus
Authors: Bähr Mathias , Wizenmann Andrea
Published: April 2018

During development, retinal ganglion cell axons establish a topographically ordered projection from the retina to the superior colliculus (SC). The putative guidance activities for retinal axons that operate during embryonic development are not detectable in the normal adult SC. However, these cues reappear upon transection of the optic nerve of adult rats. In the present study, we used a modified version of the “stripe assay,” in which membranes from either anterior or posterior SC alternated with laminin stripes. Temporal embryonic retinal axons consistently avoid membranes from embryonic posterior SC, but only rarely from adult deafferented SC. However, they are attracted to membranes from both embryonic and adult deafferented anterior SC. Nasal retinal axons only show a significant preference for membranes from posterior SC after deafferentation. When retinal axons were offered a choice to grow on membranes either from their embryonic or their deafferented target regions, they showed a preference for the deafferented SC. On carpets consisting of laminin and membranes from normal SC (not deafferented) or nontarget regions (inferior colliculus), temporal and nasal axons grow either in a random fashion or show preferences for the laminin stripes.

Our modified version of the classic stripe assay shows specific growth preferences of embryonic retinal axons for membrane lanes from their appropriate embryonic or deafferented adult target regions. These findings suggest that the deafferentation of the SC in adult rats triggers the reexpression of specific guidance activities for retinal axons. Those “attractive” guidance cues appear to be differentially expressed in the developing and deafferented SC.
Collateral Sprouting of Uninjured Primary Afferent A-Fibers into the Superficial Dorsal Horn of the Adult Rat Spinal Cord after Topical Capsaicin Treatment to the Sciatic Nerve
Authors: Mannion Richard J. , Doubell Tim P. , Coggeshall Richard E. , Woolf Clifford J.
Published: April 2018

That terminals of uninjured primary sensory neurons terminating in the dorsal horn of the spinal cord can collaterally sprout was first suggested by Liu and Chambers (1958), but this has since been disputed. Recently, horseradish peroxidase conjugated to the B subunit of cholera toxin (B-HRP) and intracellular HRP injections have shown that sciatic nerve section or crush produces a long-lasting rearrangement in the organization of primary afferent central terminals, with A-fibers sprouting into lamina II, a region that normally receives only C-fiber input (Woolf et al., 1992). The mechanism of this A-fiber sprouting has been thought to involve injury-induced C-fiber transganglionic degeneration combined with myelinated A-fibers being conditioned into a regenerative growth state.

In this study, we ask whether C-fiber degeneration and A-fiber conditioning are both necessary for the sprouting of A-fibers into lamina II. Local application of the C-fiber-specific neurotoxin capsaicin to the sciatic nerve has previously been shown to result in C-fiber damage and degenerative atrophy in lamina II. We have used B-HRP to transganglionically label A-fiber central terminals and have shown that 2 weeks after topical capsaicin treatment to the sciatic nerve, the pattern of B-HRP staining in the dorsal horn is indistinguishable from that seen after axotomy, with lamina II displaying novel staining in the identical region containing capsaicin-treated C-fiber central terminals.

These results suggest that after C-fiber injury,uninjuredA-fiber central terminals can collaterally sprout into lamina II of the dorsal horn. This phenomenon may help to explain the pain associated with C-fiber neuropathy.
Absence of p75NTRCauses Increased Basal Forebrain Cholinergic Neuron Size, Choline Acetyltransferase Activity, and Target Innervation
Authors: Yeo Tracy T. , Chua-Couzens Jane , Butcher Larry L. , Bredesen Dale E. , Cooper Jonathan D. , Valletta Janice S. , Mobley William C. , Longo Frank M.
Published: April 2018

Emerging evidence suggests that the p75 neurotrophin receptor (p75NTR) mediates cell death; however, it is not known whether p75NTRnegatively regulates other neuronal phenotypes. We found that mice null for p75NTRdisplayed highly significant increases in the size of basal forebrain cholinergic neurons, including those that are TrkA-positive. Cholinergic hippocampal target innervation also was increased significantly. Activity of the cholinergic neurotransmitter synthetic enzyme choline acetyltransferase (ChAT) was increased in both the medial septum and hippocampus. Upregulation of these cholinergic features was not associated with increased basal forebrain or hippocampal target NGF levels. In contrast, striatal cholinergic neurons, which do not express p75NTR, showed no difference in neuronal number, size, or ChAT activity between wild-type and p75NTRnull mutant mice. These findings indicate that p75NTRnegatively regulates cholinergic neuronal phenotype of the basal forebrain cholinergic neurons, including cell size, target innervation, and neurotransmitter synthesis.
Estimating the Time Course of the Excitatory Synaptic Conductance in Neocortical Pyramidal Cells Using a Novel Voltage Jump Method
Authors: Häusser Michael , Roth Arnd
Published: April 2018

We introduce a method that permits faithful extraction of the decay time course of the synaptic conductance independent of dendritic geometry and the electrotonic location of the synapse. The method is based on the experimental procedure of Pearce (1993), consisting of a series of identical somatic voltage jumps repeated at various times relative to the onset of the synaptic conductance. The progression of synaptic charge recovered by successive jumps has a characteristic shape, which can be described by an analytical function consisting of sums of exponentials. The voltage jump method was tested with simulations using simple equivalent cylinder cable models as well as detailed compartmental models of pyramidal cells. The decay time course of the synaptic conductance could be estimated with high accuracy, even with high series resistances, low membrane resistances, and electrotonically remote, distributed synapses. The method also provides the time course of the voltage change at the synapse in response to a somatic voltage-clamp step and thus may be useful for constraining compartmental models and estimating the relative electrotonic distance of synapses. In conjunction with an estimate of the attenuation of synaptic charge, the method also permits recovery of the amplitude of the synaptic conductance. We use the method experimentally to determine the decay time course of excitatory synaptic conductances in neocortical pyramidal cells. The relatively rapid decay time constant we have estimated (τ ∼1.7 msec at 35°C) has important consequences for dendritic integration of synaptic input by these neurons.
Late Compartments of Amyloid Precursor Protein Transport in SY5Y Cells Are Involved in β-Amyloid Secretion
Authors: Peraus Gisela C. , Masters Colin L. , Beyreuther Konrad
Published: April 2018

Amyloid plaques, composed mainly of the 39–43 amino acid βA4 peptide, are a characteristic feature of Alzheimer’s disease. Generation of βA4 by proteolytic processing of the amyloid precursor protein (APP) is thought to occur in a pathway that includes the activity of two as yet unknown proteases, with β-secretase cleaving at the N terminus and γ-secretase releasing the C terminus of βA4. Inhibition studies and the finding that cell surface APP can serve as a direct precursor of βA4 suggest that the endosomal/lysosomal compartment is involved in the proteolysis of APP into βA4.

In this study we targeted APP695 chimeric proteins directly into the endosomal/lysosomal compartment. This decreased the amount of released βA4, while the generation of the βA4 N terminus continued. APP695 proteins were constructed also, which carried sorting signals responsible for recycling between thetrans-Golgi network (TGN) and the cell surface. These proteins were processed into secreted βA4 at even higher levels than wild-type APP695. Moreover, retention of APP695 proteins in the endoplasmic reticulum led to neither βA4 secretion nor to processing by β-secretase in human SH-SY5Y neuroblastoma cells.

These data suggest that a β-cleavage activity resides in a late endosomal compartment and that a γ-cleavage occurs in early endosomes, resulting in the generation of βA4 peptides with the majority ending at residue 40.
Neurons Promote the Translocation of Peripheral Myelin Protein 22 into Myelin
Authors: Pareek Sangeeta , Notterpek Lucia , Snipes G. Jackson , Naef Roland , Sossin Wayne , Laliberté Jacynthe , Iacampo Sandra , Suter Ueli , Shooter Eric M. , Murphy Richard A.
Published: April 2018

Schwann cells express low levels of myelin proteins in the absence of neurons. When Schwann cells and neurons are cultured together the production of myelin proteins is elevated, and myelin is formed. For peripheral myelin protein 22 (PMP22), the exact amount of protein produced is critical, because peripheral neuropathies result from its underexpression or overexpression. In this study we examined the effect of neurons on Schwann cell PMP22 production in culture and in peripheral nerve using metabolic labeling and pulse-chase studies as well as immunocytochemistry. Most of the newly synthesized PMP22 in Schwann cells is rapidly degraded in the endoplasmic reticulum. Only a small proportion of the total PMP22 acquires complex glycosylation and accumulates in the Golgi compartment. This material is translocated to the Schwann cell membrane in detectable amounts only when axonal contact and myelination occur. Myelination does not, however, alter the rapid turnover of PMP22 in Schwann cells. PMP22 may therefore be a unique myelin protein in that axonal contact promotes its insertion into the Schwann cell membrane and myelin without altering its rapid turnover rate within the cell.
Regulation of Neuroblast Cell-Cycle Kinetics Plays a Crucial Role in the Generation of Unique Features of Neocortical Areas
Authors: Polleux Franck , Dehay Colette , Moraillon Bétrice , Kennedy Henry
Published: April 2018

Cortical neurons are generated in the germinal zones lining the ventricles before migrating predominantly radially. To investigate regional differences in the cell-cycle kinetics of neuroblasts, pulse [3H]-thymidine injections were made throughout corticogenesis, and labeled neuron counts were compared in areas 3, 6, 17, and 18a in the adult mouse. The relationship between height in the cortex and intensity of autoradiographic signal distinguishes first generation and subsequent generations of neurons. This provides the mitotic history of defined sets of neurons and is a powerful tool for analyzing areal differences in cell-cycle kinetics. The infragranular laminar labeling indices of different generations show significant differences in areas 3 and 6. The labeling index of first generation neurons shows that the rate of neuron production is higher in area 3 than in area 6. This increased generation rate in area 3 was accompanied by two major changes. First, computation of the labeling index of the subsequent generation neurons (which reflects percentages of precursors in S-phase at the moment of the pulse) indicates a shorter cell cycle in area 3. Second, the total population of labeled neurons contains a higher proportion of first generation neurons in area 3, implying a higher leaving fraction in this area. Computer simulations of these areal differences of cell-cycle kinetics generate neuron numbers that are in close agreement with published data. Altogether these findings reveal an early regionalization of the ventricular zone that serves to generate unique features of future cortical areas.
Serotonin Promotes the Differentiation of Glutamate Neurons in Organotypic Slice Cultures of the Developing Cerebral Cortex
Authors: Lavdas Alexandros A. , Blue Mary E. , Lincoln Jill , Parnavelas John G.
Published: April 2018

The monoamines serotonin (5-HT), noradrenaline (NA), and dopamine (DA), which are present in the developing brain apparently before they assume their neurotransmitter functions, are regarded as strong candidates for a role in the maturation of the cerebral cortex. Here we sought to investigate their effects on the generation and differentiation of cortical cell types. Slice cultures, prepared from the cortices of embryonic day (E) 14, E16, and E19 rat fetuses, were kept in defined medium or in defined medium plus 5-HT for 7 d. E16 cortices were also exposed to NA or DA for the same period. At the end of this period, the proportions of the neuronal [glutamate (Glu)-, GABA-, calbindin-, calretinin-labeled], glial (GFAP), and neuroepithelial (nestin) cell types were estimated for all conditions. We found that in E16 cultures, application of 5-HT, but not of NA or DA, significantly increased the proportion of Glu-containing neurons without affecting the overall neuronal population or the proportions of any other cell types. A similar effect was observed in co-cultures of E16 cortex with slices through the midbrain raphe nuclei of E19 rats. The total amount of cortical Glu, as measured with HPLC, was also increased in these co-cultures. To investigate whether the effect of 5-HT was the result of changes in cell proliferation, we exposed slices to bromodeoxyuridine (BrdU) and found that the proportion of BrdU-labeled cells was similar in the 5-HT-treated and control slices. These results indicate that 5-HT promotes the differentiation of cortical Glu-containing neurons without affecting neuroepithelial cell proliferation.
Pattern Deformities and Cell Loss inEngrailed-2Mutant Mice Suggest Two Separate Patterning Events during Cerebellar Development
Authors: Kuemerle Barbara , Zanjani Hadi , Joyner Alexandra , Herrup Karl
Published: April 2018

Null alleles of the mouseEngrailed-2gene, a molecular homolog of the fly geneengrailed, have demonstrable effects on the anteroposterior (A/P) patterning of cerebellum as reflected in the disruption of the normal process of foliation of the cerebellar cortex and the alteration of transgene expression boundaries in the adult.Engrailed-2also affects the transient mediolateral (M/L) pattern ofEn-1andWnt-7bexpression seen in late embryogenesis. We have examined three markers of cerebellar compartmentation inEn-2mutant mice: the Zebrin II and Ppath monoclonal antibodies and the transgeneL7lacZ. InEn-2mutants, the normal temporal pattern of expression is preserved for all three markers, although the size and spatial location of various bands differ from those of the wild type. Unlike the foliation abnormalities, the M/L pattern disturbances we have found occur in nearly all cerebellar regions. Cell counts reveal that all major cell types of the olivocerebellar circuit are reduced by 30–40%. We propose that these results are best explained by a model in which theEngrailed-2gene is involved in the early specification of the cerebellar field including the number of progenitors. Because each of these progenitors gives rise to a clone of defined size,Engrailed-2helps specify adult cell number. We further postulate that the configuration of the seven Zebrin bands as well as the shapes and locations of the cerebellar lobules are set up by a second patterning event that occurs after neurogenesis is complete.
CREB (cAMP Response Element-Binding Protein) in the Locus Coeruleus: Biochemical, Physiological, and Behavioral Evidence for a Role in Opiate Dependence
Authors: Lane-Ladd Sarah B. , Pineda Joseba , Boundy Virginia A. , Pfeuffer T. , Krupinski John , Aghajanian George K. , Nestler Eric J.
Published: April 2018

Chronic morphine administration increases levels of adenylyl cyclase and cAMP-dependent protein kinase (PKA) activity in the locus coeruleus (LC), which contributes to the severalfold activation of LC neurons that occurs during opiate withdrawal. A role for the transcription factor cAMP response element-binding protein (CREB) in mediating the opiate-induced upregulation of the cAMP pathway has been suggested, but direct evidence is lacking. In the present study, we first demonstrated that the morphine-induced increases in adenylyl cyclase and PKA activity in the LC are associated with selective increases in levels of immunoreactivity of types I and VIII adenylyl cyclase and of the catalytic and type II regulatory subunits of PKA. We next used antisense oligonucleotides directed against CREB to study the role of this transcription factor in mediating these effects. Infusion (5 d) of CREB antisense oligonucleotide directly into the LC significantly reduced levels of CREB immunoreactivity. This effect was sequence-specific and not associated with detectable toxicity. CREB antisense oligonucleotide infusions completely blocked the morphine-induced upregulation of type VIII adenylyl cyclase but not of PKA. The infusions also blocked the morphine-induced upregulation of tyrosine hydroxylase but not of Giα, two other proteins induced in the LC by chronic morphine treatment. Electrophysiological studies revealed that intra-LC antisense oligonucleotide infusions completely prevented the morphine-induced increase in spontaneous firing rates of LC neurons in brain slices. This blockade was completely reversed by addition of 8-bromo-cAMP (which activates PKA) but not by addition of forskolin (which activates adenylyl cyclase). Intra-LC infusions of CREB antisense oligonucleotide also reduced the development of physical dependence to opiates, based on attenuation of opiate withdrawal. Together, these findings provide the first direct evidence that CREB mediates the morphine-induced upregulation of specific components of the cAMP pathway in the LC that contribute to physical opiate dependence.
Regulators of G-Protein Signaling (RGS) Proteins: Region-Specific Expression of Nine Subtypes in Rat Brain
Authors: Gold Stephen J. , Ni Yan G. , Dohlman Henrik G. , Nestler Eric J.
Published: April 2018

The recently discovered regulators of G-protein signaling (RGS) proteins potently modulate the functioning of heterotrimeric G-proteins by stimulating the GTPase activity of G-protein α subunits. The mRNAs for numerous subtypes of putative RGS proteins have been identified in mammalian tissues, but little is known about their expression in brain. We performed a systematic survey of the localization of mRNAs encoding nine of these RGSs, RGS3–RGS11, in brain byin situhybridization. Striking region-specific patterns of expression were observed. Five subtypes, RGS4, RGS7, RGS8, RGS9, and RGS10 mRNAs, are densely expressed in brain, whereas the other subtypes (RGS3, RGS5, RGS6, and RGS11) are expressed at lower density and in more restricted regions. RGS4 mRNA is notable for its dense expression in neocortex, piriform cortex, caudoputamen, and ventrobasal thalamus. RGS8 mRNA is highly expressed in the cerebellar Purkinje cell layer as well as in several midbrain nuclei. RGS9 mRNA is remarkable for its nearly exclusive enrichment in striatal regions. RGS10 mRNA is densely expressed in dentate gyrus granule cells, superficial layers of neocortex, and dorsal raphe. To assess whether the expression of RGS mRNAs can be regulated, we examined the effect of an acute seizure on levels of RGS7, RGS8, and RGS10 mRNAs in hippocampus. Of the three subtypes, changes in RGS10 levels were most pronounced, decreasing by ∼40% in a time-dependent manner in response to a single seizure. These results, which document highly specific patterns of RGS mRNA expression in brain and their ability to be regulated in a dynamic manner, support the view that RGS proteins may play an important role in determining the intensity and specificity of signaling pathways in brain as well as their adaptations to synaptic activity.
Inflammation Increases the Distribution of Dorsal Horn Neurons That Internalize the Neurokinin-1 Receptor in Response to Noxious and Non-Noxious Stimulation
Authors: Abbadie Catherine , Trafton Jodie , Liu Hantao , Mantyh Patrick W. , Basbaum Allan I.
Published: April 2018

Although the neurokinin-1 (NK-1)/substance P (SP) receptor is expressed by neurons throughout the spinal dorsal horn, noxious chemical stimulation in the normal rat only induces internalization of the receptor in cell bodies and dendrites of lamina I. Here we compared the effects of mechanical and thermal stimulation in normal rats and in rats with persistent hindpaw inflammation. Electron microscopic analysis confirmed the upregulation of receptor that occurs with inflammation and demonstrated that in the absence of superimposed stimulation, the increased receptor was, as in normal rats, concentrated on the plasma membrane. In general, noxious mechanical was more effective than noxious thermal stimulation in inducing NK-1 receptor internalization, and this was increased in the setting of inflammation. Although a 5 sec noxious mechanical stimulus only induced internalization in 22% of lamina I neurons in normal rats, after inflammation, it evoked near-maximal (98%) internalization in lamina I, produced significant changes in laminae III–VI, and expanded the rostrocaudal distribution of neurons with internalized receptor. Even non-noxious (brush) stimulation of the inflamed hindpaw induced internalization in large numbers of superficial and deep neurons. For thermal stimulation, the percentage of cells with internalized receptor increased linearly at >45°C, but in normal rats, these were restricted to lamina I. After inflammation, however, the 52°C stimulus also induced internalization in 25% of laminae III–IV cells. These studies provide a new perspective on the reorganization of dorsal horn circuits in the setting of persistent injury and demonstrate a critical contribution of SP.
Molecular Analysis of the X11–mLin-2/CASK Complex in Brain
Authors: Borg Jean-Paul , Lõpez-Figueroa Manuel O. , de Taddèo-Borg Mylène , Kroon Dallas E. , Turner R. Scott , Watson Stanley J. , Margolis Ben
Published: April 2018

A heterotrimeric complex containing Lin-10/X11α, Lin-2/CASK, and Lin-7 is evolutionarily conserved from worms to mammals. InCaenorhabditis elegans, it localizes Let-23, a receptor tyrosine kinase, to the basolateral side of vulval epithelium, a step crucial for proper vulva development. In mammals, the complex may also participate in receptor targeting in neurons. Accordingly, phosphotyrosine binding (PTB) and postsynaptic density-95/Discs large/Zona Occludens-1 domains found in X11α and mLin-2/CASK bind to cell-surface proteins, including amyloid precursor protein, neurexins, and syndecans. In this paper, we have further analyzed the X11α–mLin-2/CASK association that is mediated by a novel protein–protein interaction. We show that the mLin-2/CASK calmodulin kinase II (CKII) domain directly binds to a 63 amino acids peptide located between the Munc-18-1 binding site and the PTB domain in X11α. Ca2+/calmodulin association with mLin-2/CASK does not modify the X11α–mLin-2 interaction. A region containing the mLin-2/CASK guanylate kinase domain also interacts with X11α but with a lower affinity than the CKII domain. Immunostaining of X11α in the brain shows that the protein is expressed in areas shown previously to be positive for mLin-2/CASK staining. Together, our data demonstrate that the X11α–mLin-2 complex contacts many partners, creating a macrocomplex suitable for receptor targeting at the neuronal plasma membrane.
Organization of Disparity-Selective Neurons in Macaque Area MT
Authors: DeAngelis Gregory C. , Newsome William T.
Published: April 2018

Neurons selective for binocular disparity are found in a number of visual cortical areas in primates, but there is little evidence that any of these areas are specialized for disparity processing. We have examined the organization of disparity-selective neurons in the middle temporal visual area (MT), an area shown previously to contain an abundance of disparity-sensitive neurons. We recorded extracellularly from MT neurons at regularly spaced intervals along electrode penetrations that passed through MT either normal to the cortical surface or at a shallow oblique angle. Comparison of multiunit and single-unit recordings shows that neurons are clustered in MT according to their disparity selectivity. Across the surface of MT, disparity-selective neurons are found in discrete patches that are separated by regions of MT that exhibit poor disparity tuning. Within disparity-selective patches of MT, we typically observe a smooth progression of preferred disparities (e.g., near to far) as our electrode travels parallel to the cortical surface. In electrode penetrations normal to the cortical surface, on the other hand, MT neurons generally have similar disparity tuning, with little variation from one recording site to the next. Thus disparity-tuned neurons are organized into cortical columns by preferred disparity, and preferred disparity is mapped systematically within larger, disparity-tuned patches of MT. Combined with other recent findings, the data suggest that MT plays an important role in stereoscopic depth perception in addition to its well known role in motion perception.
Effects of Ibotenate Hippocampal and Extrahippocampal Destruction on Delayed-Match and -Nonmatch-to-Sample Behavior in Rats
Authors: Hampson Robert E. , Jarrard Leonard E. , Deadwyler Sam A.
Published: April 2018

The effects of ibotenate lesions of the hippocampus (HIPP) or hippocampus plus collateral damage to extrahippocampal structures (HCX) were investigated in rats trained to criterion on spatial versions of either a delayed-match (DMS) or delayed-nonmatch-to-sample (DNMS) task. After recovery from surgery, animals were retrained at “0” sec delays, then assessed at 0–30 sec delays for 15 d, retrained again at 0 sec delays, and retested for another 25 d on 0–30 sec delays. Pretrained HIPP-lesioned animals showed marked delay-dependent deficits in both tasks that never recovered. Detailed examination of within- and between-trial performance factors, including changes in response preferences, length of previous trial delay, and sequential dependencies, revealed important factors operating in lesioned animals that were either absent or insignificant before the lesion. Pretrained HCX-lesioned animals showed deficits similar to those of HIPP animals, with the noticeable exception of a strong “recency” influence of the previous trial. Another group of HIPP- and HCX-lesioned animals trained on the tasks after the lesion showed reduced impairments of the type described above, suggesting that extrahippocampal structures trained after the lesion can assume the role of the hippocampus to some degree. The findings indicate that both the type of lesion and the previous history of the animal determine the postlesion DMS and DNMS performance of animals suffering damage to the hippocampus and/or related structures.
The Sexually Dimorphic Expression of Androgen Receptors in the Song Nucleus Hyperstriatalis Ventrale Pars Caudale of the Zebra Finch Develops Independently of Gonadal Steroids
Authors: Gahr Manfred , Metzdorf Reinhold
Published: April 2018

The development of sex differences in brain structure and brain chemistry (“brain sex”) of vertebrates is frequently thought to depend entirely on gonadal steroids such as androgens and estrogens, which act on the brain at the genomic level by binding to intracellular transcription factors, the androgen receptors (ARs) and estrogen receptors (ERs). These hormone actions are thought to shift the brain from a monomorphic to a dimorphic phenotype. One prominent such example is the nucleus hyperstriatalis ventrale pars caudale (HVc) of the zebra finch (Poephila guttata), a set of cells in the caudal forebrain involved in the control of singing. In contrast with previous studies using nonspecific cell staining techniques, the size and neuron number of the HVc measured by the distribution of AR mRNA is already sexually dimorphic on posthatching day (P)9. No ARs or ERs are expressed in the HVc before day 9. Slice cultures of the caudal forebrain of P5 animals show that the sexually dimorphic expression of AR mRNA in HVc is independent of the direct action of steroids on this nucleus or any of its immediate presynaptic or postsynaptic partners. Therefore, gonadal steroids do not appear to be directly involved in the initial sex difference in the expression pattern of AR mRNA, size, and neuron number of the HVc. Furthermore, we demonstrate that the initial steroid-independent size and its subsequent steroid-independent growth by extension linearly with the extension of the forebrain explains 60–70% of the masculine development of the HVc. Thus, we suggest that epigenetic factors such as the gonadal steroids modify but cannot overwrite the sex difference in HVc volume determined autonomously in the brain.
Mapping Quantitative Trait Loci for Seizure Response to a GABAAReceptor Inverse Agonist in Mice
Authors: Gershenfeld Howard K. , Neumann Paul E. , Li Xiaohua , St. Jean Pamela L. , Paul Steven M.
Published: April 2018

To define the genetic contributions affecting individual differences in seizure threshold, a β carboline [methyl-β-carboline-3-carboxylate (β-CCM)]-induced model of generalized seizures was genetically dissected in mice. β-CCM is a GABAAreceptor inverse agonist and convulsant. By measuring the latency to generalized seizures after β-CCM administration to A/J and C57BL6/J mice and their progeny, we estimated a heritability of 0.28 ± 0.10. A genome wide screen in an F2 population of these parental strains (n= 273) mapped quantitative trait loci (QTLs) on proximal chromosome 7 [logarithm of the likelihood for linkage (LOD) = 3.71] and distal chromosome 10 (LOD = 4.29) for seizure susceptibility, explaining ∼22 and 25%, respectively, of the genetic variance for this seizure trait. The best fitting logistic regression model suggests that the A/J allele at each locus increases the likelihood of seizures approximately threefold. In a subsequent backcross population (n= 223), we mapped QTLs on distal chromosome 4 (LOD = 2.88) and confirmed the distal chromosome 10 QTLs (LOD = 4.36). In the backcross, the C57BL/6J allele of the chromosome 10 QTL decreases the risk of seizures approximately twofold. These QTLs may ultimately lead to the identification of genes influencing individual differences in seizure threshold in mice and the discovery of novel anticonvulsant agents. The colocalization on distal chromosome 10 of a β-CCM susceptibility QTL and a QTL for open field ambulation and vertical movement suggests the existence of a single, pleiotropic locus, which we have namedExq1.
Roles of G-Protein βγ, Arachidonic Acid, and Phosphorylation in Convergent Activation of an S-Like Potassium Conductance by Dopamine, Ala-Pro-Gly-Trp-NH2, and Phe-Met-Arg-Phe-NH2
Authors: van Tol-Steye Hind , Lodder Johannes C. , Mansvelder Huibert D. , Planta Rudi J. , van Heerikhuizen Harm , Kits Karel S.
Published: April 2018

Dopamine and the neuropeptides Ala-Pro-Gly-Trp-NH2(APGWamide or APGWa) and Phe-Met-Arg-Phe-NH2(FMRFamide or FMRFa) all activate an S-like potassium channel in the light green cells of the molluscLymnaea stagnalis, neuroendocrine cells that release insulin-related peptides. We studied the signaling pathways underlying the responses, the role of the G-protein βγ subunit, and the interference by phosphorylation pathways. All responses are blocked by an inhibitor of arachidonic acid (AA) release, 4-bromophenacylbromide, and by inhibitors of lipoxygenases (nordihydroguaiaretic acid and AA-861) but not by indomethacin, a cyclooxygenase inhibitor. AA and phospholipase A2(PLA2) induced currents with similarI–Vcharacteristics and potassium selectivity as dopamine, APGWa, and FMRFa. PLA2occluded the response to FMRFa. We conclude that convergence of the actions of dopamine, APGWa, and FMRFa onto the S-like channel occurs at or upstream of the level of AA and that formation of lipoxygenase metabolites of AA is necessary to activate the channel. Injection of a synthetic peptide, which interferes with G-protein βγ subunits, inhibited the agonist-induced potassium current. This suggests that βγ subunits mediate the response, possibly by directly coupling to a phospholipase. Finally, the responses to dopamine, APGWa, and FMRFa were inhibited by activation of PKA and PKC, suggesting that the responses are counteracted by PKA- and PKC-dependent phosphorylation. The PLA2-activated potassium current was inhibited by 8-chlorophenylthio-cAMP but not by 12-O-tetradecanoylphorbol 13-acetate (TPA). However, TPA did inhibit the potassium current induced by irreversible activation of the G-protein using GTP-γ-S. Thus, it appears that PKA targets a site downstream of AA formation, e.g., the potassium channel, whereas PKC acts at the active G-protein or the phospholipase.
Distinct Domains of the CB1 Cannabinoid Receptor Mediate Desensitization and Internalization
Authors: Jin Wenzhen , Brown Sean , Roche John P. , Hsieh Candace , Celver Jeremy P. , Kovoor Abraham , Chavkin Charles , Mackie Ken
Published: April 2018

Desensitization of cannabinoid receptor signaling by a G-protein coupled receptor kinase (GRK) was examined using theXenopusoocyte expression system. Application of a CB1 agonist, WIN 55,212-2, evoked a concentration-dependent increase in K+conductance (Kir3) in oocytes coexpressing rat CB1 with the G-protein-gated, inwardly rectifying K+channels Kir3.1 and Kir3.4. Desensitization was slight during continuous agonist application in the absence of GRK and arrestin. However, coexpression of GRK3 and β-arrestin 2 (β-arr2) caused profound homologous CB1 receptor desensitization, supporting the hypothesis that GRK3 and β-arr2 effectively produce CB1 receptor desensitization. To identify the regions of the CB1 receptor responsible for GRK3- and β-arr2-mediated desensitization, we constructed several CB1 receptor mutants. Truncation of the C-terminal tail of CB1 receptor at residue 418 (Δ418) almost completely abolished desensitization but did not affect agonist activation of Kir3. In contrast, truncation at residues 439 and 460 did not significantly affect GRK3- and β-arr2-dependent desensitization. A deletion mutant (Δ418–439) did not desensitize, indicating that residues within this region are important for GRK3- and β-arr2-mediated desensitization. Phosphorylation in this region was likely involved in desensitization, because mutation of either of two putative phosphorylation sites (S426A or S430A) significantly attenuated desensitization. CB1 receptors rapidly internalize after activation by agonist. Phosphorylation of S426 or S430 was not necessary for internalization, because the S426A/S430A CB1 mutant internalized when stably expressed in AtT20 cells. These studies establish that CB1 desensitization can be regulated by a GRK and that different receptor domains are involved in GRK- and β-arrestin-dependent desensitization and CB1 internalization.
The Stimulatory Action and the Development of Tolerance to Caffeine Is Associated with Alterations in Gene Expression in Specific Brain Regions
Authors: Svenningsson Per , Nomikos George G. , Fredholm Bertil B.
Published: April 2018

We sought neurochemical correlates to the stimulatory action of caffeine in rats and to adaptations during development of tolerance. Acute intraperitoneal injections of caffeine (7.5 mg/kg) increased locomotion and NGFI-A mRNA, a marker of neuronal activity, in the hippocampal area CA1, but decreased NGFI-A mRNA in rostral striatum and nucleus accumbens. Rats that received caffeine (0.3 gm/l) in their drinking water for 14 d developed tolerance to the stimulatory effect of a challenge with caffeine (7.5 mg/kg) and responded with a less pronounced decrease of NGFI-A mRNA in rostral striatum and nucleus accumbens. Metabolism of caffeine to its active metabolites was increased in tolerant animals, but the total level of active metabolites in brain was not significantly altered. Thus, there are changes in caffeine metabolism after long-term caffeine treatment, but they cannot explain development of tolerance.

Caffeine-tolerant animals had downregulated levels of adenosine A2Areceptors and the corresponding mRNA in rostral parts of striatum, but an increased expression of adenosine A1receptor mRNA in the lateral amygdala. No changes in mesencephalic tyrosine hydroxylase mRNA were found in caffeine-tolerant rats.

Thus, we have identified neuronal pathways that are regulated by adenosine A1and/or A2Areceptors and are targets for the stimulatory action of caffeine. Furthermore, adaptive changes in gene expression in these brain areas were associated with the development of locomotor tolerance to caffeine.
Electron Microscopic Evidence against Apoptosis as the Mechanism of Neuronal Death in Global Ischemia
Authors: Colbourne Frederick , Sutherland Garnette R. , Auer Roland N.
Published: April 2018

It has been repeatedly claimed that neuronal death in the hippocampal CA1 sector after untreated global ischemia occurs via apoptosis. This is based largely on DNA laddering, nick end labeling, and light microscopy. Delineation of apoptosis requires fine structural examination to detect morphological events of cell death. We studied the light and ultrastructural characteristics of CA1 injury after 5 min of untreated global ischemia in gerbils. To increase the likelihood of apoptosis, some ischemic gerbils were subjected to delayed postischemic hypothermia, a treatment that mitigates injury and delays the death of some neurons. In these gerbils, 2 d of mild hypothermia was initiated 1, 6, or 12 hr after ischemia, and gerbils were killed 4, 14, or 60 d later. Ischemia without subsequent cooling killed 96% of CA1 neurons by day 4, whereas all hypothermia-treated groups had significantly reduced injury at all survival times (2–67% loss). Electron microscopy of ischemic neurons with or without postischemic hypothermia revealed features of necrotic, not apoptotic, neuronal death even in cells that died 2 months after ischemia. Dilated organelles and intranuclear vacuoles preceded necrosis. Unique to the hypothermia-treated ischemic groups, some salvaged neurons were persistently abnormal and showed accumulation of unusual, morphologically complex secondary lysosomes. These indicate selective mitochondrial injury, because they were closely associated with normal and degenerate mitochondria, and transitional forms between mitochondria and lysosomes occurred. The results show that untreated global ischemic injury has necrotic, not apoptotic, morphology but do not rule out programmed biochemical events of the apoptotic pathway occurring before neuronal necrosis.
Mice Deficient for Tenascin-R Display Alterations of the Extracellular Matrix and Decreased Axonal Conduction Velocities in the CNS
Authors: Weber Philipp , Bartsch Udo , Rasband Matthew N. , Czaniera Reiner , Lang Yolande , Bluethmann Horst , Margolis Richard U. , Levinson S. Rock , Shrager Peter , Montag Dirk , Schachner Melitta
Published: April 2018

Tenascin-R (TN-R), an extracellular matrix glycoprotein of the CNS, localizes to nodes of Ranvier and perineuronal nets and interactsin vitrowith other extracellular matrix components and recognition molecules of the immunoglobulin superfamily. To characterize the functional roles of TN-Rin vivo, we have generated mice deficient for TN-R by homologous recombination using embryonic stem cells. TN-R-deficient mice are viable and fertile. The anatomy of all major brain areas and the formation and structure of myelin appear normal. However, immunostaining for the chondroitin sulfate proteoglycan phosphacan, a high-affinity ligand for TN-R, is weak and diffuse in the mutant when compared with wild-type mice. Compound action potential recordings from optic nerves of mutant mice show a significant decrease in conduction velocity as compared with controls. However, at nodes of Ranvier there is no apparent change in expression and distribution of Na+channels, which are thought to bind to TN-R via their β2 subunit. The distribution of carbohydrate epitopes of perineuronal nets recognized by the lectinWisteria floribundaor antibodies to the HNK-1 carbohydrate on somata and dendrites of cortical and hippocampal interneurons is abnormal. These observations indicate an essential role for TN-R in the formation of perineuronal nets and in normal conduction velocity of optic nerve.