bluuebunny/crossref_metadata_2025 huggingface dataset:
The Expression and Function ofNotchPathway Genes in the Developing Rat Eye
Authors: Bao Zheng-Zheng , Cepko Constance L.
Published: April 2018

TheNotchgene plays a role in the development of disparate tissues in multiple organisms. Because the vertebrate eye is an excellent model system for both patterning and cell fate determination, two processes that can involveNotch, we examined the expression patterns ofNotch 1andNotch 2, and their ligandsDeltaandJagged, in the developing rat eye.Notch 1andDeltawere found to be expressed in the neural retina during the period of cell fate determination and differentiation.Notch 2was found to be expressed in the non-neuronal derivatives of the optic cup, including the pigment epithelium, optic stalk, and ciliary body.Jaggedwas expressed in distinct regions within the optic vesicle, ciliary body, and lens, with patterns that changed over time. The potential function ofNotch 1in cell-type specification and differentiation was examined by introducing a constitutively active form of Notch 1in vivousing a replication-incompetent retrovirus. This form of Notch 1 was found to cause abnormal growth and interfere with the differentiation of multiple retinal cell types.
Phospholipase C-β1 Is Present in the Botrysome, an Intermediate Compartment-Like Organelle, and Is Regulated by Visual Experience in Cat Visual Cortex
Authors: Kind Peter C. , Kelly Gail M. , Fryer Hugh J. L. , Blakemore Colin , Hockfield Susan
Published: April 2018

Monoclonal antibody Cat-307 identifies a 165 kDa neuronal protein expressed in the cat visual cortex during the period of sensitivity to alterations in visual experience (Kind et al., 1994). Dark-rearing, which prolongs the sensitive period, also prolongs the expression of the Cat-307 protein. The Cat-307 protein localizes to an organelle, here called the botrysome (from the Greekbotrys, cluster of grapes), that is located between the endoplasmic reticulum (ER) and Golgi apparatus. The botrysome is composed of small ring-shaped profiles with electron-dense coats. The size and morphology of the rings and their coats are similar to those described for ER to Golgi transport vesicles. Biochemically, the Cat-307 protein cofractionates with microsomes and partitions with subunits of the coatomer proteins that coat ER-to-Golgi transport vesicles. Partial amino acid sequencing reveals that the Cat-307 protein is phospholipase C-β1, the G-protein-dependent phosphodiesterase that hydrolyses phosphatidylinositol 4,5 biphosphate into inositol 1,4,5 triphosphate and diacylglycerol after the stimulation of a variety of neurotransmitter receptors at the cell surface. These results suggest a role for phospholipase C-β1 and the botrysome in developmental plasticity and provide a possible link between receptor activation at the cell surface and protein transport during neuronal development.
Prism Adaptation of Reaching Movements: Specificity for the Velocity of Reaching
Authors: Kitazawa Shigeru , Kimura Tatsuya , Uka Takanori
Published: April 2018

Accurate reaching toward a visual target is disturbed after the visual field is displaced by prisms but recovers with practice. When the prisms are removed, subjects misreach in the direction opposite to the prism displacement (aftereffect). The present study demonstrated that the severity of the aftereffect depends on the velocity of the movements during and after the visual displacement. Trained subjects were required to reach with one of four movement durations (<300, ∼800, ∼2000, and ∼5000 msec) from a fixed starting point to a target that appeared at a random location on a tangent screen (400 mm away). The size of the aftereffect was largest when the movement after the removal was performed with the same duration as that performed with the prisms. It became smaller as the difference in velocity became larger. When the contralateral arm was used after visual displacement, the aftereffect was never significant. Because the adaptation does not generalize across velocities or to the other arm, we infer that the underlying changes occur at a later stage in the transformation from visual input to motor output, in which not only the direction but also the time-dependent parameters of movements, such as velocity, acceleration or force, are represented.
Presynaptic Recordings fromDrosophila: Correlatin of Macroscopic and Single-Channel K+Currents
Authors: Martı́nez-Padrón Manuel , Ferrús Alberto
Published: April 2018

We have performed direct electrophysiological recordings fromDrosophilapeptidergic synaptic boutonsin situ, taking advantage of a mutation,ecdysone, which causes an increase in size of these terminals. Using patch-clamp techniques, we have analyzed voltage-dependent potassium currents at the macroscopic and single-channel level. The synaptic membrane contained at least two distinct voltage-activated potassium currents with different kinetics and voltage sensitivity: anIA-like current with fast activation and inactivation kinetics and voltage-dependent steady-state inactivation; a complex delayed current that includes a slowly inactivating component, resembling theIKdescribed in other preparations; and a noninactivating component. TheIA-like current in these peptidergic boutons is not encoded by the geneShaker, because it is not affected by null mutations at this locus. Rather, synapticIAhas properties similar to those of theShal-encodedIA. Single-channel recordings revealed the presence in synaptic membranes of three different potassium channel types (A2, KD, KL), with biophysical properties that could account for the macroscopic currents and resemble those of theShal,Shab, andShawchannels described in heterologous expression systems andDrosophilaneuronal somata. A2channels (6–9 pS) have brief open times, and like the macroscopicIAthey exhibited voltage-dependent steady-state inactivation and a rapidly inactivating ensemble average current profile. KDchannels (13–16 pS) had longer open times, activate and inactivate with much slower kinetics, and may account for the slowly inactivating component of the macroscopic current. KL(44–54 pS) channels produced a noninactivating ensemble average and may contribute to the delayed macroscopic current observed.
Modulation of Actin Filament Behavior by GAP-43 (Neuromodulin) Is Dependent on the Phosphorylation Status of Serine 41, the Protein Kinase C Site
Authors: He Qin , Dent Erik W. , Meiri Karina F.
Published: April 2018

Synthesis of GAP-43 (also known as neuromodulin) in neurons is induced during axon growth, and high concentrations (estimated between 50 and 100 μm) accumulate in the growth cone. GAP-43 is tightly associated with the growth cone membrane skeleton, the structure that transduces extracellular guidance cues into alterations in morphology by spatially regulating polymerization of actin filaments, thereby causing directional changes in axon growth. GAP-43 cosediments with actin filaments, and its phosphorylation on serine 41 by PKC, too, is spatially regulated so that phosphorylated GAP-43 is found in areas where growth cones make productive, stable contacts with other cells. In contrast, unphosphorylated GAP-43, which binds calmodulin, is always found in parts of the growth cone that are retracting. Here we have used a cell-free assay to investigate how the phosphorylation status of GAP-43 affects its interactions with actin and show that both phosphorylated and unphosphorylated GAP-43 have different, independent effects on actin filament structure. Phosphorylated GAP-43 stabilizes long actin filaments (Kd= 161 nm), and antibodies to phosphorylated GAP-43 inhibit binding of actin to phalloidin, implying a lateral interaction with filaments. In contrast, unphosphorylated GAP-43 reduces filament length distribution (Kd= 1.2 μm) and increases the critical concentration for polymerization. Prebinding calmodulin potentiates this effect. The results show that spatially regulated post-translational modifications of GAP-43 within the growth cone, which can be regulated in response to extracellular signals, have the ability to directly influence the structure of the actin cytoskeleton.
A Monosynaptic GABAergic Input from the Inferior Colliculus to the Medial Geniculate Body in Rat
Authors: Peruzzi Daniel , Bartlett Edward , Smith Philip H. , Oliver Douglas L.
Published: April 2018

The goal was to investigate possible monosynaptic GABAergic projections from the inferior colliculus (IC) to thalamocortical neurons of the medial geniculate body (MGB) in the rat. Although there is little evidence for such a projection in other sensory thalamic nuclei, a GABAergic, ascending auditory projection was reported recently in the cat. In the present study, immunohistochemical and tract-tracing methods were used to identify neurons in the IC that contain GABA and project to the MGB. GABA-positive projection neurons were most numerous in the central nucleus and less so in the dorsal and lateral cortex. They were rare in the lateral tegmental system and brachium of the IC. The dorsal nucleus of the lateral lemniscus also contained GABA-positive projection neurons. In brain slices, stimulation of the brachium produced monosynaptic inhibitory postsynaptic potentials in morphologically identified thalamocortical relay neurons. The inhibitory potentials cannot originate locally, because they persisted when ionotropic glutamatergic transmission was blocked. Typically, brachium stimulation elicited a GABAA-mediated inhibitory potential followed by an excitatory potential and a longer latency GABAB-mediated inhibitory potential.

We conclude that the GABA-containing neurons of the IC make short-latency, monosynaptic inputs to the thalamocortical projection neurons in the MGB. Such inputs may distinguish the main auditory pathway from indirect or tegmental auditory pathways as well as from other sensory systems. Monosynaptic inhibitory inputs to the medial geniculate may be important for the regulation of firing patterns in thalamocortical neurons.
Short-Term Plasticity during Intrathalamic Augmenting Responses in Decorticated Cats
Authors: Steriade Mircea , Timofeev Igor
Published: April 2018

The intrathalamic mechanisms of frequency-dependent augmenting responses were investigated in decorticated cats by means of intracellular recordings from thalamocortical (TC) neurons in ventrolateral (VL) nucleus, including simultaneous impalements from two TC neurons. Pulse trains (10 Hz) applied to VL nucleus elicited two types of augmenting responses: (1) in 68% of cells, the incremental responses occurred on a progressive depolarization associated with the decrease in IPSPs produced by preceding stimuli in the train; (2) in the remaining cells, progressively growing low-threshold (LT) responses resulted from the enhancement of Cl−-dependent IPSPs, giving rise to postinhibitory rebound bursts, followed by a self-sustained sequence of spindle waves. Although in some TC cells the augmenting responses developed from LT responses once the latter reached a given level of depolarization, other neurons displayed augmenting responses immediately after the early antidromic spike that depolarized the neuron to the required level, without an intermediate step of LT rebound. Repeated pulse trains led to a progressive and persistent increase in slow depolarizing responses of TC cells, as well as to a persistent and prolonged decrease in the amplitudes of the IPSPs. On the basis of parallel experiments, we propose that the two types of augmentation in TC cells are a result of contrasting responses of thalamic reticular neurons evoked by repetitive thalamic stimuli: decremental responses, which may account for disinhibition leading to depolarizing responses in TC cells, and incremental responses, explaining the progressive hyperpolarization of TC cells. These data demonstrate that frequency-dependent changes in neuronal excitability are present in the thalamus of a decorticated hemisphere and suggest that short-term plasticity processes in the gateway to the cerebral cortex may decisively influence cortical excitability during repetitive responses.
Behavioral and Neurochemical Recovery from Partial 6-Hydroxydopamine Lesions of the Substantia Nigra Is Blocked by Daily Treatment with D1/D5, But Not D2, Dopamine Receptor Antagonists
Authors: Emmi Adriana , Rajabi Heshmat , Stewart Jane
Published: April 2018

To determine whether D1/D5 dopamine (DA) receptors play a role in normalization of DA extracellular levels of striatal DA and behavioral recovery after partial 6-OHDA lesions of the substantia nigra, animals were treated on days 1–8 after lesioning with the D1/D5 DA receptor antagonists SCH 23390 (0.1 mg/kg, s.c.) and SCH 39166 (1.0 mg/kg, s.c.), the inactive enantiomer SCH 23388 (0.1 mg/kg, s.c.), the D2 antagonist eticlopride (0.1 mg/kg, i.p.), or saline. Spontaneous turning behavior was assessed on days 3 and 15. Basal extracellular DA and metabolites were measured in both striata using microdialysis on days 16 and 17, 8–9 d after termination of drug treatments. On day 3, all animals turned ipsilateral to the lesion. On day 15, animals previously treated with either saline, eticlopride, or SCH 23388 showed no behavioral asymmetries, whereas animals treated with SCH 23390 or SCH 39166 turned ipsilaterally. On days 16 and 17, extracellular DA did not differ on the two sides in animals treated with saline or eticlopride and were higher on the lesioned side after SCH 23388. In animals treated with the D1/D5 receptor antagonists, however, basal levels of DA were lower on the lesioned side, showing no evidence of normalization. These results suggest a role for the D1/D5 DA receptor in the development of compensatory changes in the DA neurons that accompany behavioral recovery from partial lesions of nigrostriatal DA system.
Differential Activation of the Caudate Nucleus in Primates Performing Spatial and Nonspatial Working Memory Tasks
Authors: Levy Richard , Friedman Harriet R. , Davachi Lila , Goldman-Rakic Patricia S.
Published: April 2018

The caudate nucleus is part of an anatomical network subserving functions associated with the dorsolateral prefrontal cortex (DLPFC). The aim of the present study was to investigate whether the metabolic activity in the striatum reflects specific changes in working memory tasks, which are known to be dependent on the DLPFC, and whether these changes reflect the topographic ordering of prefrontal connections within the striatum. Local cerebral glucose utilization (LCGU) rates were assessed in the striatum by the14C-2-deoxyglucose method in monkeys that performed a spatial (delayed spatial alternation), a nonspatial (delayed object alternation) visual working memory task, or tasks that did not involve working memory, i.e., a visual pattern discrimination or sensorimotor paradigm.

The results show a topographic segregation of activation related to spatial and nonspatial working memory, respectively. The delayed spatial alternation task increases LCGU rates bilaterally by 33–43% in the head of the caudate nucleus, where efferents from the dorsolateral prefrontal cortex project most densely. The delayed object alternation task enhances LCGU rates bilaterally by 32–37% in the body of the caudate nucleus, which is innervated by the temporal cortex. The visual pattern discrimination task similarly activated the body of the caudate, but in a smaller region and only in the right hemisphere.

These findings provide the first evidence for metabolic activation of the caudate nuclei in working memory, supporting the role of this nucleus as a node in a neural network mediating DLPFC-dependent working memory processes. The double dissociation of activation observed suggests an anatomical and functional segregation of cortico-striatal circuits subserving spatial and nonspatial cognitive operations.
Response Modulation in the Zebra Finch Neostriatum: Relationship to Nuclear Gene Regulation
Authors: Stripling Roy , Volman Susan F. , Clayton David F.
Published: April 2018

The sound of birdsong activates robust gene expression in the caudomedial neostriatum (NCM) of songbirds. To assess the function of this genomic response, we analyzed the temporal and quantitative relationships between electrophysiological activity and gene induction. Single units in zebra finch NCM showed large increases in firing in response to birdsong, whereas simple auditory tones tended to inhibit firing. Most cells showed little selectivity for individual songs based on total number of spikes produced. When a novel song stimulus was repeated, the cells rapidly modulated their firing rates so that the first response to a stimulus was markedly higher than consecutive responses. Even after many repetitions of a particular song, cells continued to fire in response to that stimulus, unlike the complete “habituation” observed previously for genomic activity. The initial modulation of the response to a particular song disappeared, however, once that song was repeated for 200 trials (∼34 min). These results indicate a dissociation between gross physiological activity and “immediate early” gene expression: genomic activity occurs only during a subset of electrophysiological responses. We propose a model in which nuclear responses in NCM are modulated by pathways distinct from the primary auditory inputs to NCM. This would account for the changing selectivity of the genomic response and implies an active role for the cell nucleus as an integrating agent in the physiological operation of neural circuits.
Modulation of a cAMP/Protein Kinase A Cascade by Protein Kinase C in Sensory Neurons ofAplysia
Authors: Sugita Shuzo , Baxter Douglas A. , Byrne John H.
Published: April 2018

The synaptic connections between the sensory neurons ofAplysiaand their follower neurons have been used as a model system for examining the cellular mechanisms contributing to neuronal and synaptic plasticity. Recent studies suggest that at least two protein kinases, protein kinase A (PKA) and protein kinase C (PKC), contribute to serotonin (5-HT)-induced short-term facilitation. The interaction between these two kinase cascades has not been examined, however. Using electrophysiological and biochemical approaches, we examined possible interactions between PKA and PKC cascades. The results indicated that prolonged activation of PKC by preincubation with phorbol esters attenuated PKA-mediated actions of 5-HT, including increases in sensory neuron excitability and spike broadening in the presence of tetraethylammonium (TEA) and nifedipine. Although phorbol esters also attenuated increases in excitability by an analog of cAMP and small cardioactive peptide B (SCPB), the degree of attenuation was smaller. In addition, phorbol esters did not attenuate broadening of TEA spikes by the cAMP analog and SCPB. Thus, phorbol esters appeared specifically to attenuate aspects of the 5-HT activation of the cAMP/PKA cascade. Measurements of cAMP levels with radioimmunoassays revealed that phorbol esters did not attenuate 5-HT-induced cAMP synthesis, however. Finally, the results indicated that phorbol esters themselves induced a small but significant increase in excitability as well as an increase in the level of cAMP. Our results suggest that there is crosstalk between the PKC and PKA cascades. The mechanisms by which phorbol esters specifically attenuate 5-HT-induced activation of the cAMP/PKA cascade are not known, however.
The Development of Excitatory Synapses in Cultured Spinal Neurons
Authors: O’Brien Richard J. , Mammen Andrew L. , Blackshaw Seth , Ehlers Michael D. , Rothstein Jeffrey D. , Huganir Richard L.
Published: April 2018

Immunohistochemical studies of synapses in the CNS have demonstrated that glutamate receptors (GluRs) are concentrated at postsynaptic sitesin vivoandin vitro(Baude et al., 1995). The mechanisms leading to receptor clustering at excitatory synapses are far less understood than those governing acetylcholine receptor accumulation at the neuromuscular junction (Hall and Sanes,1993) or glycine receptor aggregation at central inhibitory synapses (Kirsch et al., 1993). Using cultured rat spinal cord neurons, we demonstrate that clustering of the AMPA receptor subunit GluR1 is among the earliest events in excitatory synapse formationin vitro, coincident with the onset of miniature EPSCs and in many cases preceding presynaptic vesicle accumulation. Postsynaptic receptor clustering is induced in a highly specific and reiterative pattern, independent of receptor activation, by contact with a subset of axons capable of inducing receptor clusters. The subunit composition of AMPA receptor clusters varied significantly between neurons but was invariant within a given neuron. The presence of either GluR2 or GluR3 was common to all receptor clusters. Neither high-affinity glutamate transporters nor NMDA receptors appeared to be concentrated with AMPA receptor subunits at these excitatory synapses.
Purkinje Cell Expression of a Mutant Allele ofSCA1in Transgenic Mice Leads to Disparate Effects on Motor Behaviors, Followed by a Progressive Cerebellar Dysfunction and Histological Alterations
Authors: Clark H. Brent , Burright Eric N. , Yunis Wael S. , Larson Seth , Wilcox Claire , Hartman Boyd , Matilla Antoni , Zoghbi Huda Y. , Orr Harry T.
Published: April 2018

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurological disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract. Work presented here describes the behavioral and neuropathological course seen in mutantSCA1transgenic mice. Behavioral tests indicate that at 5 weeks of age mutant mice have an impaired performance on the rotating rod in the absence of deficits in balance and coordination. In contrast, these mutantSCA1mice have an increased initial exploratory behavior. Thus, expression of the mutantSCA1allele within cerebellar Purkinje cells has divergent effects on the motor behavior of juvenile animals: a compromise of rotating rod performance and a simultaneous enhancement of initial exploratory activity. With age, these animals develop incoordination with concomitant progressive Purkinje neuron dendritic and somatic atrophy but relatively little cell loss. Therefore, the eventual development of ataxia caused by the expression of a mutantSCA1allele is not the result of cell death per se, but the result of cellular dysfunction and morphological alterations that occur before neuronal demise.
Parapinopsin, a Novel Catfish Opsin Localized to the Parapineal Organ, Defines a New Gene Family
Authors: Blackshaw Seth , Snyder Solomon H.
Published: April 2018

Multiple sites of extraretinal photoreception are present in vertebrates, but the molecular basis of extraretinal phototransduction is poorly understood. This study reports the cloning of the first opsin specifically expressed in the directly photosensitive pineal and parapineal of cold-blooded vertebrates. This opsin, identified in channel catfish and termed parapinopsin, defines a new gene family of vertebrate photopigments and is expressed in a majority of parapinealocytes and a subset of pineal photoreceptor cells. Parapinopsin shows a caudal–rostral gradient of expression within the pineal organ. This study also reports the cloning of partial cDNAs encoding the channel catfish orthologues of rhodopsin and the red cone pigment—the full complement of retinal opsins in the species.In situhybridization studies using probes derived from these retinal opsins, together with parapinopsin, reveal no expression of retinal opsins in pineal and parapineal organ and no expression of any opsin tested in the “deep brain,” iris, or dermal melanophores. These data imply that phototransduction in these sites of extraretinal photoreception must be mediated by novel opsins.
The Actin-Severing Protein Gelsolin Modulates Calcium Channel and NMDA Receptor Activities and Vulnerability to Excitotoxicity in Hippocampal Neurons
Authors: Furukawa Katsutoshi , Fu Weiming , Li Ying , Witke Walter , Kwiatkowski David J. , Mattson Mark P.
Published: April 2018

Calcium influx through NMDA receptors and voltage-dependent calcium channels (VDCC) mediates an array of physiological processes in neurons and may also contribute to neuronal degeneration and death in neurodegenerative conditions such as stroke and severe epileptic seizures. Gelsolin is a Ca2+-activated actin-severing protein that is expressed in neurons, wherein it may mediate motility responses to Ca2+influx. Primary hippocampal neurons cultured from mice lacking gelsolin exhibited decreased actin filament depolymerization and enhanced Ca2+influx after exposure to glutamate. Whole-cell patch-clamp analyses showed that currents through NMDA receptors and VDCC were enhanced in hippocampal neurons lacking gelsolin, as a result of decreased current rundown; kainate-induced currents were similar in neurons containing and lacking gelsolin. Vulnerability of cultured hippocampal neurons to glutamate toxicity was greater in cells lacking gelsolin. Seizure-induced damage to hippocampal pyramidal neurons was exacerbated in adult gelsolin-deficient mice. These findings identify novel roles for gelsolin in controlling actin-mediated feedback regulation of Ca2+influx and in neuronal injury responses. The data further suggest roles for gelsolin and the actin cytoskeleton in both physiological and pathophysiological events that involve activation of NMDA receptors and VDCC.
Acetylcholine Receptors in Innervated Muscles of DystrophicmdxMice Degrade as after Denervation
Authors: Xu Rufeng , Salpeter Miriam M.
Published: April 2018

Acetylcholine receptors (AChRs) are present at the top of the postsynaptic membrane of the neuromuscular junction (NMJ) at very high density, possibly anchored to cytoskeletal elements. The present study investigated whether AChR degradation is affected in animals lacking dystrophin, a protein that is an integral part of the cytoskeletal complex and is missing in Duchenne muscular dystrophy. The animal model for Duchenne muscular dystrophy, the mutantmdxmouse, was used to determine whether disruption of the cytoskeleton, caused by the absence of dystrophin, affects AChR degradation. Of the two populations of junctional AChRs, Rs (expressed in innervated adult muscles) and Rr (expressed in embryonic or denervated muscles), only Rs are affected inmdxanimals. In innervatedmdxsoleus, diaphragm, and sternomastoid muscles, the AChRs have an accelerated degradation rate (t1/2of ∼3–5 d), similar to that acquired by Rs in control muscles after denervation. The Rs inmdxNMJs do not accelerate further when the muscles are denervated. The absence of dystrophin does not affect the degradation rate of the Rr AChRs (t1/2of 1 d), which are expressed after denervation inmdxas in control muscles. These results suggest that dystrophin or an intact cytoskeletal complex may be required for neuronal stabilization of Rs receptors at the adult neuromuscular junctions.
Neuronal α-Bungarotoxin Receptors Differ Structurally from Other Nicotinic Acetylcholine Receptors
Authors: Rangwala Fatima , Drisdel Renaldo C. , Rakhilin Sergey , Ko Elizabeth , Atluri Pramod , Harkins Amy B. , Fox Aaron P. , Salman, and William N. Green Suleiman B.
Published: April 2018

We have characterized the α-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of α7-containing receptors that bind α-Bgt with high affinity (Kd= 94 pm). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and α-Bgt block ∼90% of the whole-cell current. The binding of nicotinic agonists to cell-surface BgtRs was highly cooperative with four different agonists showing Hill coefficients in the range of 2.3–2.4. A similar agonist binding cooperativity was observed for BgtR homomers formed from chimeric α7/5HT3 subunits expressed in tsA 201 cells. Two classes of agonist binding sites, in the ratio of 4:1 for PC12 cell BgtRs and 3:1 for α7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the reduced sites on both PC12 BgtRs and α7/5HT3 BgtRs. We conclude from this data that PC12 BgtRs and α7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other nicotinic receptors.
Alternative Splicing in the Pore-Forming Region ofshakerPotassium Channels
Authors: Kim Marshall , Baro Deborah J. , Lanning Cathy C. , Doshi Mehul , Farnham Jeremy , Moskowitz Howard S. , Peck Jack H. , Olivera Baldomero M. , Harris-Warrick Ronald M.
Published: April 2018

We have cloned cDNAs for theshakerpotassium channel gene from the spiny lobsterPanulirus interruptus. As previously found inDrosophila, there is alternative splicing at the 5′ and 3′ ends of the coding region. However, inPanulirus shaker, alternative splicing also occurs within the pore-forming region of the protein. Three different splice variants were found within the P region, two of which bestow unique electrophysiological characteristics to channel function. Pore I and pore II variants differ in voltage dependence for activation, kinetics of inactivation, current rectification, and drug resistance. The pore 0 variant lacks a P region exon and does not produce a functional channel. This is the first example of alternative splicing within the pore-forming region of a voltage-dependent ion channel. We used a recently identified potassium channel blocker, κ-conotoxin PVIIA, to study the physiological role of the two pore forms. The toxin selectively blocked one pore form, whereas the other form, heteromers between the two pore forms, andPanulirus shalwere not blocked. When it was tested in thePanulirusstomatogastric ganglion, the toxin produced no effects on transient K+currents or synaptic transmission between neurons.
Association and Colocalization of the Kvβ1 and Kvβ2 β-Subunits with Kv1 α-Subunits in Mammalian Brain K+Channel Complexes
Authors: Rhodes Kenneth J. , Strassle Brian W. , Monaghan Michael M. , Bekele-Arcuri Zewditu , Matos Maria F. , Trimmer James S.
Published: April 2018

The differential expression and association of cytoplasmic β-subunits with pore-forming α-subunits may contribute significantly to the complexity and heterogeneity of voltage-gated K+channels in excitable cells. Here we examined the association and colocalization of two mammalian β-subunits, Kvβ1 and Kvβ2, with the K+channel α-subunits Kv1.1, Kv1.2, Kv1.4, Kv1.6, and Kv2.1 in adult rat brain. Reciprocal coimmunoprecipitation experiments using subunit-specific antibodies indicated that Kvβ1 and Kvβ2 associate with all the Kv1 α-subunits examined, and with each other, but not with Kv2.1. A much larger portion of the total brain pool of Kv1-containing channel complexes was found associated with Kvβ2 than with Kvβ1. Single- and multiple-label immunohistochemical staining indicated that Kvβ1 codistributes extensively with Kv1.1 and Kv1.4 in cortical interneurons, in the hippocampal perforant path and mossy fiber pathways, and in the globus pallidus and substantia nigra. Kvβ2 codistributes extensively with Kv1.1 and Kv1.2 in all brain regions examined and was strikingly colocalized with these α-subunits in the juxtaparanodal region of nodes of Ranvier as well as in the axons and terminals of cerebellar basket cells. Taken together, these data provide a direct demonstration that Kvβ1 and Kvβ2 associate and colocalize with Kv1 α-subunits in native tissues and provide a biochemical and neuroanatomical basis for the differential contribution of Kv1 α- and β-subunits to electrophysiologically diverse neuronal K+currents.
Analysis of Rat Vestibular Hair Cell Development and Regeneration Using Calretinin as an Early Marker
Authors: Zheng J. Lisa , Gao Wei-Qiang
Published: April 2018

Despite increased interest in inner ear hair cell regeneration, it is still unclear what exact mechanisms underlie hair cell regeneration in mammals because of our limited understanding of hair cell development and the lack of specific hair cell markers. In this report, we studied hair cell development using immunohistochemistry on sections prepared from embryonic day (E) 13 to postnatal day 7 rat inner ear tissues. Of many epithelial, neuronal, and glial markers, we found that calcium-binding protein antibodies recognizing calretinin, calmodulin, or parvalbumin labeled immature hair cells in rat vestibular end organs. In particular, calretinin antiserum labeled the initial differentiating hair cells at E15, a stage immediately after the terminal mitosis of hair cell progenitors. The selective immunoreactivity of postmitotic presumptive hair cells, but not supporting cells or peripheral epithelial cells, was confirmed in utricular epithelial sheet cultures. Double labeling with calretinin and bromodeoxyuridine antibodies in long-term cultures showed that only a few mitotic utricular supporting cells became calretinin positive. Thus, although proliferation-mediated regeneration of new hair cells might directly contribute to hair cell regeneration in rat utricles after injury, it is very limited. In addition, double labeling with calretinin and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) revealed that differentiated hair cells underwent apoptosis during normal development at late embryonic and early postnatal stagesin vivoandin vitro. Therefore, these experiments lay the groundwork for the time course of differentiation, regeneration, and apoptosis of mammalian vestibular hair cells. This work also suggests that calcium-binding proteins are useful markers for studies on inner ear hair cell differentiation and regeneration.
Modulation of Intrinsic Circuits by Serotonin 5-HT3Receptors in Developing Ferret Visual Cortex
Authors: Roerig Birgit , Katz Lawrence C.
Published: April 2018

Serotonergic projections are widespread in the developing neocortex, but their functions are obscure. The effects of 5-HT3receptor agonists on cortical circuit response properties were studied in slices of ferret primary visual cortex using high-speed optical imaging of voltage-sensitive dye signals and whole-cell patch-clamp recording. Activation of the 5-HT3receptor decreased the amplitude and lateral extent of excitation throughout postnatal development. This effect peaks after eye opening, which indicates a function for serotonergic modulation of circuit responses during the period of refinement of cortical connections. Whole-cell patch-clamp recordings from single neurons revealed that synaptic responses evoked by white matter stimulation were reduced by 5-HT3receptor agonists, whereas the frequency of spontaneous GABAergic synaptic currents was enhanced dramatically. This indicates that the modulation of spontaneous synaptic activity by fast-acting serotonin receptors is reflected in an inhibition of the circuit response, in line with the notion of background synaptic activity altering the spatiotemporal integration properties of cortical cells by changing their membrane potential and their electrotonic structure. These mechanisms may regulate the response properties of intrinsic circuits in both the adult and developing neocortex.
Nerve Growth Factor- and Neurotrophin-3-Induced Changes in Nociceptive Threshold and the Release of Substance P from the Rat Isolated Spinal Cord
Authors: Malcangio Marzia , Garrett Neil E. , Cruwys Simon , Tomlinson David R.
Published: April 2018

Acute superfusion of nerve growth factor (NGF; 1–100 ng/ml) through a naive rat spinal cord preparation did not alter basal or electrically evoked release of substance P-like immunoreactivity (SP-LI). In contrast, neurotrophin-3 (NT-3; 1–100 ng/ml), although not modifying SP-LI basal outflow, dose-dependently inhibited the electrically evoked, but not capsaicin (10 nm)-induced, release of the peptide. This NT-3 (10 ng/ml)-induced inhibition persisted even in the presence of 100 ng/ml NGF in the perfusion fluid and was still significant when the evoked release of SP-LI was enhanced by a prolongedin vivotreatment with NGF. Co-superfusion with naloxone (0.1 μm), but not CGP 36742 (100 μm), a GABABantagonist, prevented NT-3 (10 ng/ml) inhibition of SP-LI release. Basal and electrically evoked release of SP-LI from the rat spinal cordin vitrowas not modified 24 hr after single systemic injection of either NGF (1 mg/kg) or NT-3 (10 mg/kg). At these time intervals from administration, NGF had induced thermal and mechanical hyperalgesia in the rat hindpaw, and NT-3 had induced mechanical, but not thermal, hypoalgesia. NT-3 administered six times over a 2 week period (at 1 mg/kg) did not alter thermal threshold but significantly reduced electrically evoked release of SP-LI from the spinal cord. An identical treatment regimen with 1 mg/kg NGF induced a significant increase in evoked release of SP-LI. However, this was not associated with a significant hyperalgesia. Although finding that NGF-induced hyperalgesia does not clearly correlate with changes in the release of SP-LI in the spinal cord, this study shows that NT-3 is an inhibitor of SP-LI release and suggests that this mechanism may be responsible for NT-3-induced antinociception.
Opposite Modulation of Opiate Withdrawal Behaviors on Microinfusion of a Protein Kinase A Inhibitor Versus Activator into the Locus Coeruleus or Periaqueductal Gray
Authors: Punch Laurie J. , Self David W. , Nestler Eric J. , Taylor Jane R.
Published: April 2018

Chronic opiate administration upregulates the cAMP pathway in the locus coeruleus (LC). This adaptation is thought to increase the electrical excitability of LC neurons and contribute to the dramatic increase in LC firing induced by opioid receptor antagonists in opiate-dependent animals. The goal of the present study was to evaluate directly a role of the cAMP pathway in opiate withdrawal behaviors by studying,in vivo, whether withdrawal is influenced by intra-LC infusion of compounds known to activate or inhibit protein kinase A (PKA). Infusions into amygdala or periaqueductal gray (PAG) were studied for comparison. In one series of experiments the effect of intra-LC, intra-amygdala, or intra-PAG infusions of the PKA inhibitor Rp-cAMPS on naloxone-precipitated withdrawal from morphine was examined. Intra-LC infusions of Rp-cAMPS significantly attenuated several prominent behavioral signs of morphine withdrawal. Intra-PAG infusions of Rp-cAMPS also significantly attenuated opiate withdrawal behaviors, although different behaviors were affected. In contrast, intra-amygdala infusions of Rp-cAMPS were without significant effect. In a second series of experiments the effect of intra-LC or intra-PAG infusions of the PKA activator Sp-cAMPS on behavior in nondependent drug-naive animals was determined. Sp-cAMPS infusions into either brain region induced a quasi-withdrawal syndrome, but the observed behaviors differed between the two groups. Analysis of the phosphorylation state of tyrosine hydroxylase, a well characterized substrate for PKA, confirmed the ability of Rp-cAMPS and Sp-cAMPS to inhibit and activate, respectively, PKA activityin vivo. Together, these data provide direct evidence for involvement of the cAMP–PKA system in the LC, as well as in the PAG, in opiate withdrawal and withdrawal-related behaviors.
Rhinal Cortex Removal Produces Amnesia for Preoperatively Learned Discrimination Problems But Fails to Disrupt Postoperative Acquisition and Retention in Rhesus Monkeys
Authors: Thornton Jennifer A. , Rothblat Lawrence A. , Murray Elisabeth A.
Published: April 2018

To test whether the rhinal cortex (i.e., entorhinal and perirhinal cortex) plays a time-limited role in information storage, eight rhesus monkeys were trained to criterion on two sets of 60 object discrimination problems, one set at each of two different time periods separated by 15 weeks. After the monkeys had learned both sets, two groups balanced for preoperative acquisition rates were formed. One group received bilateral ablation of the rhinal cortex (n= 4), and the other was retained as an unoperated control group (n= 4). After a 2 week rest period, monkeys were assessed for retention of the object discrimination problems. Retention was significantly poorer in monkeys with removals of the rhinal cortex relative to the controls (68 vs 91%). Although both groups showed slightly better retention of problems from the more recently learned set, there was no evidence of a differential effect of the cortical removal across sets (i.e., no temporal gradient). In addition, the monkeys with rhinal cortex lesions subsequently learned three new sets of 10 object discrimination problems as quickly as the controls did, thus ruling out the possibility of a gross impairment in visual perception or discrimination abilities. Furthermore, they retained these postoperatively learned object discriminations as well as the controls did. The findings indicate that the rhinal cortex is critical for the storage and/or retrieval of object discrimination problems that were learned up to 16 weeks before rhinal cortex ablation; however, in the absence of the rhinal cortex, efficient learning and retention of new discrimination problems can still occur.
Cloning and Characterization of Murine Glial Cell-Derived Neurotrophic Factor Inducible Transcription Factor (MGIF)
Authors: Yajima Shunsuke , Lammers Claas-Hinrich , Lee Sang-Hyeon , Hara Yoshinobu , Mizuno Keiko , Mouradian M. Maral
Published: April 2018

The potent neurotrophic factor glial cell-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor-β (TGF-β) superfamily of proteins. We report a transcription factor that is the first nuclear protein known to be induced by GDNF, thus designated murine GDNF inducible factor (mGIF). The cDNA was cloned in the course of investigating transcription factors that bind to Sp1 consensus sequences, using thein situfilter detection method, and it was found to encode a protein having the same C2–H2zinc finger motif as Sp1. Sequence analysis indicated that mGIF is homologous to the human TGF-β inducible early gene (TIEG) and human early growth response gene-α (EGR-α). mGIF is widely distributed in the adult mouse with high mRNA levels in kidney, lung, brain, liver, heart, and testis. In the adult brain, mGIF is abundantly expressed in hippocampus, cerebral cortex, cerebellum, and amygdala with lower amounts in striatum, nucleus accumbens, olfactory tubercle, thalamus, and substantia nigra. During development, mGIF mRNA also has a wide distribution, including in cerebral cortex, cerebellar primordium, kidney, intestine, liver, and lung. GDNF induces the expression of mGIF rapidly and transiently both in a neuroblastoma cell line and in primary cultures of rat embryonic cortical neurons. Co-transfection of theDrosophilaSL2 cells using mGIF expression plasmid and reporter constructs having Sp1 binding sites indicated that mGIF represses transcription from a TATA-containing as well as from a TATA-less promoter. These observations suggest that the zinc finger transcription factor mGIF could be important in mediating some of the biological effects of GDNF.
Peroxide Modulation of Slow Onset Potentiation in Rat Hippocampus
Authors: Auerbach Jonathan M. , Segal Menahem
Published: April 2018

Exposure of rat hippocampal slices to low concentrations of the muscarinic agonist carbachol (CCh) has been shown to produce a slow onset long-term potentiation (LTP) of reactivity to afferent stimulation in CA1 neurons. Although this potentiation shares a number of properties with tetanic LTP, muscarinic LTP (LTPm) is independent of activation of the NMDA receptor. We now demonstrate that low levels of hydrogen peroxide (H2O2) cause hippocampal slices to lose the ability to express LTPm. This powerful effect of H2O2is selective in that it does not affect the reactivity of hippocampal neurons to higher concentrations of CCh. In fact, H2O2also blocks induction of a slow onset, non-NMDA-dependent tetanic LTP (NN-LTP). The functional relevance of this action of H2O2is exemplified by the fact that the hippocampus of aged rats, which produces higher levels of endogenous H2O2than that of young rats, lacks LTPmand expresses a markedly reduced NN-LTP. In aged rats, the lack of LTPmcontrasts with an apparently normal muscarinic suppression of the EPSP slope induced by higher concentrations of CCh. When hippocampal slices from aged animals are treated with catalase, an enzyme that breaks down H2O2, LTPmis restored, and NN-LTP is enhanced. Thus, our study proposes a unique and novel age-dependent peroxide regulation of LTPmin the brain and provides a link between the cholinergic system, aging, and memory functions.
Critical Role of TrkB and Brain-Derived Neurotrophic Factor in the Differentiation and Survival of Retinal Pigment Epithelium
Authors: Liu Zheng Z. , Zhu Ling Q. , Eide Fernette F.
Published: April 2018

In the vertebrate eye, the retinal pigment epithelium (RPE) and the neural retina arise from a single layer of neuroectoderm. Factors influencing the differentiation of retinal neurons have been identified; however, little is known about molecules directing the differentiation of the RPE. Here we have found that the neurotrophin brain-derived neurotrophic factor (BDNF) plays an autocrine role in the differentiation and survival ofXenopus laevisRPE. Fluorescentin situhybridization studies showed a precise co-expression of BDNF and its receptor trkB in the retinal neuroepithelium and actively differentiating RPE;in vitrostudies demonstrated survival- and differentiation-promoting effects in serum-free explants and dissociated cultures. When a dominant negative mutant of the trkB receptor was expressed in developing embryos, severe arrest of RPE differentiation was seen with persistence of nestin- andNotch-positive neuroblasts.
Social Stress in Hamsters: Defeat Activates Specific Neurocircuits within the Brain
Authors: Kollack-Walker S. , Watson S. J. , Akil H.
Published: April 2018

During an agonistic encounter, subordinate male hamsters display defensive and submissive postures and show increased secretion of glucocorticoids, whereas dominant males do not. To determine whether specific neuronal pathways are activated during the behavioral and neuroendocrine responses of subordinate males, expression of c-fosmRNA within the brains of subordinate males was compared with the pattern in dominant males after fighting. After 1 week of handling, pairs of hamsters were either swapped between cages (handled control males), or were allowed to interact for 30 min [dominant (DOM) males and subordinate (SUB) males]. A second group of control animals that received no handling or social stimulation (unhandled control males) were also included. After testing, all animals were killed by decapitation, their brains were removed for c-fos in situhybridization, and trunk blood was collected for analysis of plasma cortisol and corticosterone levels. Exposure of males to their partner’s cage for 30 min resulted in increased expression ofc-fosmRNA in multiple brain regions. In addition, fighting increased c-fosexpression in the medial amygdaloid nucleus of both DOM and SUB males as well as having more selective effects. In DOM males, c-fosexpression was elevated within the supraoptic nucleus of the hypothalamus. In SUB males, c-fosexpression increased within a multitude of brain areas, including cingulate cortex, lateral septum, bed nucleus of the stria terminalis, medial preoptic area, several hypothalamic nuclei, central amygdaloid nucleus, amygdalohippocampal area, dorsal periaqueductal gray, dorsal raphe, cuneiform nucleus, and locus coeruleus. These findings are discussed in relation to neurocircuits associated with behavioral arousal and stress.