bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Activation and Desensitization of Sensory Neurons by Resiniferatoxin
Authors: Acs Geza , Biro Tamas , Acs Peter , Modarres Shayan , Blumberg Peter M.
Published: April 2018

Recently, with use of rat dorsal root ganglion (DRG) neurons we have been able to dissociate the binding affinities of vanilloids from their potencies to induce45Ca uptake, which suggests the existence of distinct classes of the vanilloid receptor (Acs et al., 1996). In the present study, we have demonstrated that the ultrapotent capsaicin analog resiniferatoxin (RTX) desensitized rat DRG neurons to the subsequent induction of45Ca uptake by capsaicin and RTX with affinity and cooperativity similar to that found for [3H]RTX binding, contrasting with a ∼10-fold weaker potency and lack of cooperativity to induce45Ca uptake. Likewise, the competitive antagonist capsazepine inhibited RTX-induced desensitization with potency similar to that for inhibition of specific [3H]RTX binding, whereas the potency of capsazepine was ∼10-fold higher for inhibiting RTX-induced45Ca uptake. Finally, the noncompetitive antagonist ruthenium red inhibited both the RTX-induced desensitization and45Ca uptake but showed ∼60-fold selectivity for inhibiting RTX-induced desensitization. The RTX-induced desensitization was not associated with loss of specific [3H]RTX binding, suggesting lack of gross cell toxicity. In contrast to RTX, capsaicin caused desensitization with a potency corresponding to that for45Ca uptake and did so in a noncooperative manner. Unlike the RTX-induced desensitization, the desensitization by capsaicin was blocked by ruthenium red only at doses that blocked45Ca uptake and depended on external calcium. Our findings provide further support for the existence of vanilloid receptor subtypes on DRG neurons with distinct pharmacology and distinct patterns of desensitization.
Calcium Channel Density and Hippocampal Cell Death with Age in Long-Term Culture
Authors: Porter Nada M. , Thibault Olivier , Thibault Véronique , Chen Kuey-Chu , Landfield Philip W.
Published: April 2018

The expression of voltage-gated calcium (Ca2+) channel activity in brain cells is known to be important for several aspects of neuronal development. In addition, excessive Ca2+influx has been linked clearly to neurotoxicity bothin vivoandin vitro; however, the temporal relationship between the development of Ca2+channel activity and neuronal survival is not understood. Over a period spanning 28 din vitro, progressive increases in high voltage-activated whole-cell Ca2+current and L-type Ca2+channel activity were observed in cultured hippocampal neurons. On the basis of single-channel analyses, these increases seem to arise in part from a greater density of functionally available L-type Ca2+channels. An increase in mRNA for the α1subunit of L-type Ca2+channels occurred over a similar time course, which suggests that a change in gene expression may underlie the increased channel density. Parallel studies showed that hippocampal neuronal survival over 28 d was inversely related to increasing Ca2+current density. Chronic treatment of hippocampal neurons with the L-type Ca2+channel antagonist nimodipine significantly enhanced survival. Together, these results suggest that age-dependent increases in the density of Ca2+channels might contribute significantly to declining viability of hippocampal neurons. The results also are analogous to patterns seen in neurons of aged animals and therefore raise the possibility that long-term primary neuronal culture could serve as a model for some aspects of aging changes in hippocampal Ca2+channel function.
Nucleus Reuniens Thalami Modulates Activity in Hippocampal Field CA1 through Excitatory and Inhibitory Mechanisms
Authors: Dolleman-Van der Weel M. J. , Lopes da Silva F. H. , Witter M. P.
Published: April 2018

The nucleus reuniens thalami (RE) originates dense projections to CA1, forming asymmetrical synapses on spines (50%) and dendrites (50%). The hypothesis that RE input modulates transmission in CA1 through excitation of both pyramidal cells and interneurons was tested using electrophysiological methods in the anesthetized rat. The RE–CA1 afferents were selectively stimulated at their origin; evoked field potentials and unit activity were recorded in CA1. RE-evoked depth profiles showed a prominent negative deflection in the stratum lacunosum-moleculare and a positive one in the stratum radiatum. The lacunosum-moleculare sink–radiatum source configuration is compatible with RE-elicited depolarization of apical dendrites of pyramidal cells. Despite a consistent and robust paired pulse facilitation of RE-evoked field potentials, population spikes in the stratum pyramidale were not detected at any tested condition. This indicates the inability of RE–CA1 input to discharge pyramidal cells. However, stimulation of RE-elicited spiking of extracellularly recorded units in strata oriens/alveus and distal radiatum, indicative of the activation of local interneurons. Thus, RE seems to modulate transmission in CA1 through a (subthreshold) depolarization of pyramidal cells and a suprathreshold excitation of putative inhibitory oriens/alveus and radiatum interneurons.

RE-evoked monosynaptic or disynaptic field potentials were associated with stimulation of rostral or caudal RE, respectively. Anatomically, a projection from caudal to rostral RE was demonstrated that can account for the disynaptic RE–CA1 input. Because caudal RE receives input from the hippocampus via the subiculum, we propose the existence of a closed RE–hippocampal circuit that allows RE to modulate the activity in CA1, depending on hippocampal output.
Targeted Transduction of CNS Neurons with Adenoviral Vectors Carrying Neurotrophic Factor Genes Confers Neuroprotection That Exceeds the Transduced Population
Authors: Baumgartner Brian J. , Shine H. David
Published: April 2018

Application of neurotrophic factors (NFs) to the cut stump of motor nerves of neonatal rats confers neuroprotection from trauma-induced neuronal death. To test whether motoneurons are capable of responding to endogenously produced NFs, facial motoneurons were genetically modifiedin vivoto express several NFs and then tested for their response to peripheral nerve damage. Replication-defective adenoviral vectors [Adv.Rous sarcoma virus (RSV)-nf] representing three families of NFs were constructed that carried genes for brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor. Media from cultured cells transduced with Adv.RSV-nfcontained NFs that supported the survival of cultured chick sensory neurons in the same manner as recombinant NF standards. When Adv.RSV-nfor an adenoviral vector containing the β-galactosidase gene (Adv.RSV-β-gal) were injected into the facial muscles of neonatal rats the vectors were retrogradely transported to the facial nucleus where the NFs or β-gal were expressed. A fraction (∼10%) of the neurons were transduced as demonstrated by reverse transcriptase-PCR, histochemistry, and immunocytochemistry. In the case of Adv.RSV-BDNF, Adv.RSV-CNTF, and Adv.RSV-GDNF, a significant portion of the facial nucleus neurons was protected, 16.5, 18.2, and 53.3%, respectively, from death after axotomy, showing that neurons are capable of transporting the Adv.RSV-nf, expressing the recombinant NF genes, and responding to the NFs. In the case of Adv.RSV-GDNF, a greater number of facial nucleus motoneurons survived than were transduced, indicating that neighboring untransduced neurons were protected by the GDNF expressed by the transduced neurons by a paracrine mechanism.
Slow Recovery from Inactivation of Na+Channels Underlies the Activity-Dependent Attenuation of Dendritic Action Potentials in Hippocampal CA1 Pyramidal Neurons
Authors: Colbert Costa M. , Magee Jeffrey C. , Hoffman Dax A. , Johnston Daniel
Published: April 2018

Na+action potentials propagate into the dendrites of pyramidal neurons driving an influx of Ca2+that seems to be important for associative synaptic plasticity. During repetitive (10–50 Hz) firing, dendritic action potentials display a marked and prolonged voltage-dependent decrease in amplitude. Such a decrease is not apparent in somatic action potentials. We investigated the mechanisms of the different activity dependence of somatic and dendritic action potentials in CA1 pyramidal neurons of adult rats using whole-cell and cell-attached patch-clamp methods. There were three main findings. First, dendritic Na+currents decreased in amplitude when repeatedly activated by brief (2 msec) depolarizations. Recovery was slow and voltage-dependent. Second, Na+currents decreased much less in somatic than in dendritic patches. Third, although K+currents remained constant during trains, K+currents were necessary for dendritic action potential amplitude to decrease in whole-cell experiments. These results suggest that regional differences in Na+and K+channels determine the differences in the activity dependence of somatic and dendritic action potential amplitudes.
Murine Astrocytes Express a Functional Chemokine Receptor
Authors: Tanabe Shigeyuki , Heesen Michael , Berman Michael A. , Fischer Michael B. , Yoshizawa Izumi , Luo Yi , Dorf Martin E.
Published: April 2018

Elevated levels of chemokines have been observed in various diseases of the CNS. Little is known, however, about how these chemokines affect parenchymal cells of the CNS. The current studies examine astrocyte chemotaxis to the mouse chemokine macrophage inflammatory protein-1α (MIP-1α). Murine astrocytes demonstrate directed migration along a chemical gradient in response to 10−10–10−8mMIP-1α. Peak chemotactic responses are noted at 10−9m. MIP-1α-induced astrocyte migration is specifically inhibitable with pertussis toxin, suggesting a role for Gαiproteins in the signaling process.

RT-PCR andin situhybridization were used to identify expression of the murine CCR1 MIP-1α receptor on astrocytes. Astrocytes contain mRNA for CCR1, but messages for CCR4 and the orphan chemokine receptor MIP-1αR-like#1 were not detected. The combined results suggest that a functional chemokine receptor is expressed on resident cells of the CNS. We speculate that the interactions of chemokines with astrocytes are involved in inflammatory reactions of the CNS.
Agonist-Specific Coupling of a ClonedDrosophila melanogasterD1-Like Dopamine Receptor to Multiple Second Messenger Pathways by Synthetic Agonists
Authors: Reale Vincenzina , Hannan Frances , Hall Linda M. , Evans Peter D.
Published: April 2018

The mechanism of coupling of a clonedDrosophilaD1-like dopamine receptor, DopR99B, to multiple second messenger systems when expressed inXenopusoocytes is described. The receptor is coupled directly to the generation of a rapid, transient intracellular Ca2+signal, monitored as changes in inward current mediated by the oocyte endogenous Ca2+-activated chloride channel, by a pertussis toxin-insensitive G-protein-coupled pathway. The more prolonged receptor-mediated changes in adenylyl cyclase activity are generated by an independent G-protein-coupled pathway that is pertussis toxin-sensitive but calcium-independent, and Gβγ-subunits appear to be involved in the transduction of this response. This is the first evidence for the direct coupling of a cloned D1-like dopamine receptor both to the activation of adenylyl cyclase and to the initiation of an intracellular Ca2+signal. The pharmacological profile of both second messenger effects is identical for a range of naturally occurring catecholamine ligands (dopamine > norepinephrine > epinephrine) and for the blockade of dopamine responses by a range of synthetic antagonists. However, the pharmacological profiles of the two second messenger responses differ for a range of synthetic agonists. Thus, the receptor exhibits agonist-specific coupling to second messenger systems for synthetic agonists. This feature could provide a useful tool in the genetic analysis of the roles of the multiple second messenger pathways activated by this receptor, given the likely involvement of dopamine in the processes of learning and memory in the insect nervous system.
κ-Opioid Receptor Activation Modulates Ca2+Currents and Secretion in Isolated Neuroendocrine Nerve Terminals
Authors: Rusin K. I. , Giovannucci D. R. , Stuenkel E. L. , Moises H. C.
Published: April 2018

Whole-cell patch-clamp recordings were performed together with time-resolved measurements of membrane capacitance (Cm) in nerve terminals acutely dissociated from neurohypophysis of adult rats to investigate modulation of Ca2+currents and secretion by activation of opioid receptors. Bath superfusion of the κ-opioid agonists U69,593 (0.3–1 μm), dynorphin A (1 μm), or U50,488H (1–3 μm) reversibly suppressed the peak amplitude of Ca2+currents 32.7 ± 2.7% (in 41 of 56 terminals), 37.4 ± 5.3% (in 5 of 8 terminals), and 33.5 ± 8.1% (in 5 of 10 terminals), respectively. In contrast, tests in 11 terminals revealed no effect of the μ-opioid agonist [d-Pen2,5]-enkephalin (1–3 μm;n= 7) or of the δ-agonist Tyr-d-Ala-Gly-N-Me-Phe-Gly-ol (1 μm;n= 4) on Ca2+currents. Three components of high-threshold current were distinguished on the basis of their sensitivity to blockade by ω-conotoxin GVIA, nicardipine, and ω-conotoxin MVIIC: N-, L-, and P/Q-type current, respectively. Administration of U69,593 inhibited N-type current in these nerve terminals on average 32%, whereas L-type current was reduced 64%, and P/Q-type current was inhibited 28%. Monitoring of changes inCmin response to brief depolarizing steps revealed that the κ-opioid-induced reductions in N-, L-, or P/Q-type currents were accompanied by attenuations in two kinetically distinct components of Ca2+-dependent exocytotic release. These data provide strong evidence of a functional linkage between blockade of Ca2+influx through voltage-dependent Ca2+channels and inhibitory modulation of release by presynaptic opioid receptors in mammalian central nerve endings.
Adenoviral Vector-Mediated Expression of B-50/GAP-43 Induces Alterations in the Membrane Organization of Olfactory Axon TerminalsIn Vivo
Authors: Holtmaat Anthony J. G. D. , Hermens Wim T. J. M. C. , Sonnemans Marc A. F. , Giger Roman J. , Van Leeuwen Fred W. , Kaplitt Michael G. , Oestreicher A. Beate , Gispen Willem Hendrik , Verhaagen Joost
Published: April 2018

B-50/GAP-43 is an intraneuronal membrane-associated growth cone protein with an important role in axonal growth and regeneration. By using adenoviral vector-directed expression of B-50/GAP-43 we studied the morphogenic action of B-50/GAP-43 in mature primary olfactory neurons that have established functional synaptic connections. B-50/GAP-43 induced gradual alterations in the morphology of olfactory synapses. In the first days after overexpression, small protrusions originating from the preterminal axon shaft and from the actual synaptic bouton were formed. With time the progressive formation of multiple ultraterminal branches resulted in axonal labyrinths composed of tightly packed sheaths of neuronal membrane. Thus, B-50/GAP-43 is a protein that can promote neuronal membrane expansion at synaptic boutons. This function of B-50/GAP-43 suggests that this protein may subserve an important role in ongoing structural synaptic plasticity in adult neurons and in neuronal membrane repair after injury to synaptic fields.
Assembly of GABAAReceptors Composed of α1 and β2 Subunits in Both Cultured Neurons and Fibroblasts
Authors: Gorrie George H. , Vallis Yvonne , Stephenson Anne , Whitfield Jonathan , Browning Brenda , Smart Trevor G. , Moss Stephen J.
Published: April 2018

GABAAreceptors are believed to be pentameric hetero-oligomers, which can be constructed from six subunits (α, β, γ, δ, ε, and ρ) with multiple members, generating a large potential for receptor heterogeneity. The mechanisms used by neurons to control the assembly of these receptors, however, remain unresolved. Using Semliki Forest virus expression we have analyzed the assembly of 9E10 epitope-tagged receptors comprising α1 and β2 subunits in baby hamster kidney cells and cultured superior cervical ganglia neurons. Homomeric subunits were retained within the endoplasmic reticulum, whereas heteromeric receptors were able to access the cell surface in both cell types. Sucrose density gradient fractionation demonstrated that the homomeric subunits were incapable of oligomerization, exhibiting 5 S sedimentation coefficients. Pulse–chase analysis revealed that homomers were degraded, with half-lives of ∼2 hr for both the α1(9E10)and β2(9E10)subunits. Oligomerization of the α1(9E10)and β2(9E10)subunits was evident, as demonstrated by the formation of a stable 9 S complex, but this process seemed inefficient. Interestingly the appearance of cell surface receptors was slow, lagging up to 6 hr after the formation of the 9 S receptor complex. Using metabolic labeling a ratio of α1(9E10):β2(9E10)of 1:1 was found in this 9 S fraction. Together the results suggest that GABAAreceptor assembly occurs by similar mechanisms in both cell types, with retention in the endoplasmic reticulum featuring as a major control mechanism to prevent unassembled receptor subunits accessing the cell surface.
Developing Neonatal Rat Sympathetic and Sensory Neurons Differ in Their Regulation of 5-HT3Receptor Expression
Authors: Rosenberg Madelaine , Pié Brigitte , Cooper Ellis
Published: April 2018

Serotonin 5-HT3receptors (5-HT3Rs) are ligand-gated ion channels expressed by many peripheral neurons and are involved in several physiological processes. To learn more about the developmental regulation of 5-HT3R expression, we investigated rat sympathetic and vagal sensory neurons. We found that sympathetic and sensory neurons differ in their regulation of 5-HT3R expression during early postnatal life and as these neurons develop in culture. In SCG neurons 5-HT3R transcript levels are low at postnatal day 1 (P1) and increase 7.5-fold by P21; this increase occurs even after elimination of preganglionic innervation. In comparison, 5-HT3R mRNA levels in P1 nodose neurons are over 14-fold greater than in P1 SCG and change little by P21. We show that 5-HT3R transcript levels in nodose neurons depend on intact target innervation and drop by 60% after axotomy. When P1 SCG neurons develop in culture, we observed a significant increase in 5-HT3R expression: after 7 d in culture, transcript levels increase ninefold versus a threefold increase for neurons developing for 7 din vivo. In contrast, 5-HT3R mRNA levels in cultured nodose neurons drop by 70% within 24 hr; however, this drop is transient. After 2 d, transcript levels begin to increase, and after 7 d, they are above initial values. We show that this delayed increase in 5-HT3R expression depends on neurotrophins. In both nodose and sympathetic neurons we found that the changes in 5-HT3R gene expression correlate directly with the appearance of 5-HT-evoked current densities.
Prolonged Sodium Channel Inactivation Contributes to Dendritic Action Potential Attenuation in Hippocampal Pyramidal Neurons
Authors: Jung Hae-Yoon , Mickus Timothy , Spruston Nelson
Published: April 2018

During low-frequency firing, action potentials actively invade the dendrites of CA1 pyramidal neurons. At higher firing rates, however, activity-dependent processes result in the attenuation of back-propagating action potentials, and propagation failures occur at some dendritic branch points. We tested two major hypotheses related to this activity-dependent attenuation of back-propagating action potentials: (1) that it is mediated by a prolonged form of sodium channel inactivation and (2) that it is mediated by a persistent dendritic shunt activated by back-propagating action potentials. We found no evidence for a persistent shunt, but we did find that cumulative, prolonged inactivation of sodium channels develops during repetitive action potential firing. This inactivation is significant after a single action potential and continues to develop during several action potentials thereafter, until a steady-state sodium current is established. Recovery from this form of inactivation is much slower than its induction, but recovery can be accelerated by hyperpolarization. The similarity of these properties to the time and voltage dependence of attenuation and recovery of dendritic action potentials suggests that dendritic sodium channel inactivation contributes to the activity dependence of action potential back-propagation in CA1 neurons. Hence, the biophysical properties of dendritic sodium channels will be important determinants of action potential-mediated effects on synaptic integration and plasticity in hippocampal neurons.
Alteration of Ca2+Dependence of Neurotransmitter Release by Disruption of Ca2+Channel/Syntaxin Interaction
Authors: Rettig Jens , Heinemann Christian , Ashery Uri , Sheng Zu-Hang , Yokoyama Charles T. , Catterall William A. , Neher Erwin
Published: April 2018

Presynaptic N-type calcium channels interact with syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) through a binding site in the intracellular loop connecting domains II and III of the α1subunit. This binding region was loaded into embryonic spinal neurons ofXenopusby early blastomere injection. After culturing, synaptic transmission of peptide-loaded and control cells was compared by measuring postsynaptic responses under different external Ca2+concentrations. The relative transmitter release of injected neurons was reduced by ∼25% at physiological Ca2+concentration, whereas injection of the corresponding region of the L-type Ca2+channel had virtually no effect. When applied to a theoretical model, these results imply that 70% of the formerly linked vesicles have been uncoupled after action of the peptide. Our data suggest that severing the physical interaction between presynaptic calcium channels and synaptic proteins will not prevent synaptic transmission at this synapse but will make it less efficient by shifting its Ca2+dependence to higher values.
Expression of Zinc Transporter Gene, ZnT-1, Is Induced after Transient Forebrain Ischemia in the Gerbil
Authors: Tsuda Manabu , Imaizumi Kazunori , Katayama Taiichi , Kitagawa Kazuo , Wanaka Akio , Tohyama Masaya , Takagi Tsutomu
Published: April 2018

To elucidate the molecular mechanisms underlying neuronal death after transient forebrain ischemia, we cloned genes expressed after transient forebrain ischemia in the Mongolian gerbil by a differential display method. A gerbil homolog of rat zinc transporter, ZnT-1, which transports intracellular Zn2+out of cells, was isolated. Its expression became detectable exclusively in pyramidal neurons of the CA1 region 12 hr after ischemia and reached a maximum from day 1 to day 2 as shown byin situhybridization. By day 7, expression had disappeared entirely from the cells in the CA1 region, because the neurons had died. No other brain regions exhibited such a significant level of ZnT-1 mRNA expression during this period. Zn2+was shown to accumulate in CA1 pyramidal neurons expressing ZnT-1 mRNA after the ischemia by using zinquin, a zinc-specific fluorescent dye. When primary hippocampal neurons were exposed to a high dose of Zn2+, ZnT-1 mRNA accumulated. These results suggest that the induction of ZnT-1 mRNA observed in CA1 neurons was caused by an increase in the intracellular Zn2+concentration. It was reported recently that Zn2+chelator blocked neuronal death after ischemia and that the influx of Zn2+might be a key mechanism underlying neuronal death. The induction of ZnT-1 mRNA in CA1 pyramidal neurons fated to die after transient ischemia is of interest to the study of postischemic events and the molecular mechanisms underlying delayed neuronal death.
Glial Growth Factor Rescues Schwann Cells of Mechanoreceptors from Denervation-Induced Apoptosis
Authors: Kopp Diane M. , Trachtenberg Joshua T. , Thompson Wesley J.
Published: April 2018

Golgi tendon organs and Pacinian corpuscles are peripheral mechanoreceptors that disappear after denervation during a critical period in early postnatal development. Even if regeneration is allowed to occur, Golgi tendon organs do not reform, and the reformation of Pacinian corpuscles is greatly impaired. The sensory nerve terminals of both types of mechanoreceptors are closely associated with Schwann cells. Here we investigate the changes in the Schwann cells found in Golgi tendon organs and Pacinian corpuscles after nerve resection in the early neonatal period. We report that denervation induces the apoptotic death of these Schwann cells and that this apoptosis can be prevented by administration of a soluble form of neuregulin, glial growth factor 2. Schwann cells associated with these mechanoreceptors are immunoreactive for the neuregulin receptors erbB2, erbB3, and erbB4, and the sensory nerve terminals are immunoreactive for neuregulin. Our results suggest that Schwann cells in developing sensory end organs are trophically dependent on sensory axon terminals and that an axon-derived neuregulin mediates this trophic interaction. The denervation-induced death of mechanoreceptor Schwann cells is correlated with deficiencies in the re-establishment of these sensory end organs by regenerating axons.
Brain-Derived Neurotrophic Factor Enhances Long-Term Potentiation in Rat Visual Cortex
Authors: Akaneya Yukio , Tsumoto Tadaharu , Kinoshita Shuichiro , Hatanaka Hiroshi
Published: April 2018

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the nerve growth factor (NGF) gene family, have been suggested to play a role in experience-dependent modification of neural networks in the developing nervous system. In this study we addressed the question of whether these neurotrophins are involved in long-term potentiation (LTP) in developing visual cortex. We recorded layer II/III field potentials and whole-cell currents evoked by test stimulation of layer IV at 0.1 Hz in visual cortical slices prepared from young rats (postnatal day 15–25) and observed effects of BDNF, NT-3, and NGF on these responses. Then we analyzed the effects of these neurotrophins on LTP induced by tetanic (Θ-burst type) stimulation of layer IV. We found that BDNF at 200 ng/ml potentiated field potentials and EPSCs in most cases and that this potentiation lasted after cessation of the BDNF application. At the concentration of 20 ng/ml, BDNF did not show such an effect, but it enhanced the magnitude of expressed LTP. On the other hand, NT-3 and NGF had none of these effects. Immunohistochemical staining of slices with antibody against BDNF showed that exogenous BDNF penetrated into the whole slice within ∼5 min of its application. The actions of BDNF were blocked by preincubation of slices with TrkB-IgG fusion protein, a BDNF scavenger, or coapplication of K252a, an inhibitor for receptor tyrosine kinases. TrkB-IgG or K252a itself completely blocked LTP, suggesting that endogenous BDNF or another TrkB ligand plays a role in LTP in the developing visual cortex.
Spatial and Temporal Expression of theperiodandtimelessGenes in the Developing Nervous System ofDrosophila: Newly Identified Pacemaker Candidates and Novel Features of Clock Gene Product Cycling
Authors: Kaneko Maki , Helfrich-Förster Charlotte , Hall Jeffrey C.
Published: April 2018

The circadian timekeeping system ofDrosophilafunctions from the first larval instar (L1) onward but is not known to require the expression of clock genes in larvae. We show thatperiod(per) andtimeless(tim) are rhythmically expressed in several groups of neurons in the larval CNS both in light/dark cycles and in constant dark conditions. Among the clock gene-expressing cells there is a subset of the putative pacemaker neurons, the “lateral neurons” (LNs), that have been analyzed mainly in adult flies. Like the adult LNs, the larval ones are also immunoreactive to a peptide called pigment-dispersing hormone. Their putative dendritic trees were found to be in close proximity to the terminals of the larval optic nerve Bolwig’s nerve, possibly receiving photic input from the larval eyes. The LNs are the only larval cells that maintain a strong cycling in PER fromL1onward, throughout metamorphosis and into adulthood. Therefore, they are the best candidates for being pacemaker neurons responsible for the larval “time memory” (inferred from previous experiments). In addition to the LNs, a subset of the larval dorsal neurons (DNLs) expressesperandtim. Intriguingly, two neurons of this DNLgroup cycle in PER and TIM immunoreactivity almost in antiphase to the other DNLs and to the LNs. Thus, the temporal expression ofperandtimare regulated differentially in different cells. Furthermore, the light sensitivity associated with levels of the TIM protein is different from that in the heads of adultDrosophila.
Dopaminergic Neurons Intrinsic to the Primate Striatum
Authors: Betarbet Ranjita , Turner. Robert , Chockkan Vijay , DeLong Mahlon R. , Allers Kelly A. , Walters Judith , Levey Allan I. , Greenamyre J. Timothy
Published: April 2018

Intrinsic, striatal tyrosine hydroxylase-immunoreactive (TH-i) cells have received little consideration. In this study we have characterized these neurons and their regulatory response to nigrostriatal dopaminergic deafferentation. TH-i cells were observed in the striatum of both control and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys; TH-i cell counts, however, were 3.5-fold higher in the striatum of MPTP-lesioned monkeys. To establish the dopaminergic nature of the TH-i cells, sections were double-labeled with antibodies to dopamine transporter (DAT). Immunofluorescence studies demonstrated that nearly all TH-i cells were double-labeled with DAT, suggesting that they contain the machinery to be functional dopaminergic neurons. Two types of TH-i cells were identified in the striatum: small, aspiny, bipolar cells with varicose dendrites and larger spiny, multipolar cells. The aspiny cells, which were more prevalent, corresponded morphologically to the GABAergic interneurons of the striatum. Double-label immunofluorescence studies using antibodies to TH and glutamate decarboxylase (GAD67), the synthetic enzyme for GABA, showed that 99% of the TH-i cells were GAD67-positive. Very few (<1%) of the TH-i cells, however, were immunoreactive for the calcium-binding proteins calbindin and parvalbumin. In summary, these results demonstrate that the dopaminergic cell population of the striatum responds to dopamine denervation by increasing in number, apparently to compensate for loss of extrinsic dopaminergic innervation. Moreover, this population of cells corresponds largely with the intrinsic GABAergic cells of the striatum. This study also suggests that the adult primate striatum does retain some intrinsic capacity to compensate for dopaminergic cell loss.
Cellular–Synaptic Generation of Sleep Spindles, Spike-and-Wave Discharges, and Evoked Thalamocortical Responses in the Neocortex of the Rat
Authors: Kandel Adam , Buzsáki György
Published: April 2018

Thalamocortical neuronal oscillations underlie various field potentials that are expressed in the neocortex, including sleep spindles and high voltage spike-and-wave patterns (HVSs). The mechanism of extracellular current generation in the neocortex was studied in the anesthetized and awake rat. Field potentials and unit activity were recorded simultaneously along trajectories perpendicular to the cortical layers at spatial intervals of 100 μm by multiple-site recording silicon probes. Current source density (CSD) analysis revealed that the spatial positions of sinks in layers IV, V–VI, and II–III and of the accompanying sources were similar during sleep spindles, HVSs, and thalamic-evoked responses, although their relative strengths and timings differed. The magnitude and relative timing of the multiple pairs of sinks and sources determined the amplitude variability of HVSs and sleep spindles. The presence of temporally shifted dipoles was also supported by the time distribution of unit discharges in different layers. Putative interneurons discharged with repetitive bursts of 300–500 Hz. The spike component of HVSs was associated with fast field oscillations (400–600 Hz “ripples”). Discharges of pyramidal cells were phase-locked to the ripples. These findings indicate that the major extracellular currents underlying sleep spindles, HVSs, and evoked responses result from activation of intracortical circuitries. We hypothesize that the fast field ripples reflect summed IPSPs in pyramidal cells resulting from the high frequency barrage of interneurons.
Granule Cell Activation of Complex-Spiking Neurons in Dorsal Cochlear Nucleus
Authors: Davis Kevin A. , Young Eric D.
Published: April 2018

Dorsal cochlear nucleus (DCN) principal cells receive, in addition to their well known auditory inputs, various nonauditory inputs via a cerebellar-like granule cell circuit located in the superficial layers of the DCN. Activation of this circuit (granule cell axons make excitatory synapses on the principal cells but also contact inhibitory interneurons that project to the principal cells) produces strong inhibition of the principal cells. Here we investigate the role of cartwheel cells, homologs of cerebellar Purkinje cells, in producing this inhibition. The responses of type IV units (one type of principal cells) and of cartwheel cells were recorded to ortho- and antidromic activation of the granule cells (i.e., by stimulation of their inputs from the somatosensory cuneate and spinal trigeminal nuclei and by direct stimulation of their parallel fiber axons). Cartwheel cells were identified on the basis of recording depth and complex action potential shape. A four-pulse facilitation paradigm (four pulses at 50 msec intervals) was used; this stimulus allows separation of the apparently simple inhibitory somatosensory response of type IV units into a three-component (inhibition–excitation–inhibition) response. As expected, cartwheel cells are excited by granule cell activation; the latencies and four-pulse amplitudes of these responses correspond to the properties of the second, long-latency inhibitory component of type IV responses. The source of the first, short-latency inhibitory response is still unknown. Nevertheless, these results show that cartwheel cells convey inhibitory polysensory information to DCN principal cells.
Heterogeneity of Astrocyte Resting Membrane Potentials and Intercellular Coupling Revealed by Whole-Cell and Gramicidin-Perforated Patch Recordings from Cultured Neocortical and Hippocampal Slice Astrocytes
Authors: McKhann Guy M. , D’Ambrosio Raimondo , Janigro Damir
Published: April 2018

Astrocytes are thought to regulate the extracellular potassium concentration by mechanisms involving both voltage-dependent and transport-mediated ion fluxes combined with intercellular communication via gap junctions. Mechanisms regulating resting membrane potential (RMP) play a fundamental role in determining glial contribution to buffering of extracellular potassium and uptake of potentially toxic neurotransmitters. We have investigated the passive electrophysiological properties of cultured neocortical astrocytes and astrocytes recorded in hippocampal slices from 18–25 d postnatal rats. These experiments revealed a wide range of astrocyte RMPs that were independent of developmental factors, length of culturing, cellular morphology, the electrophysiological techniques used (whole-cell vs perforated recording), cell-specific expression of Na+/2HCO3−co-transporters, or voltage-dependent Na+channels. Exposure of cultured astrocytes to differentiation-inducing factors (such as cAMP) or inhibition of proliferation (by serum deprivation) did not significantly influence RMP. Expression of ATP-sensitive potassium channels was absent in these glia; thus, K(ATP)-related mechanisms did not contribute to cell resting potential. In both cultured and slice astrocytes, spontaneous electrophysiological changes were commonly observed. These reversible events, which resulted in differential sensitivity to potassium channel blockers (cesium and barium) and sudden current–voltage profile changes, were attributable to dynamic changes in cell-to-cell coupling, as confirmed by recordings from isolated pairs of cells. We conclude that the heterogeneity of astrocytic RMP and intercellular coupling both in culture andin situare intrinsic properties of glia that may contribute to transcellular transport of potassium. We propose a model in which spatial buffering may be facilitated by heterogeneous mechanisms controlling glial RMP in combination with dynamic changes in intercellular coupling.
Mechanisms of Reduced Striatal NMDA Excitotoxicity in Type I Nitric Oxide Synthase Knock-Out Mice
Authors: Ayata Cenk , Ayata Gamze , Hara Hideaki , Matthews Russel T. , Beal M. Flint , Ferrante Robert J. , Endres Matthias , Kim Albert , Christie Richard H. , Waeber Christian , Huang Paul L. , Hyman Bradley T. , Moskowitz Michael A.
Published: April 2018

We investigated the role of neuronal (type I) nitric oxide synthase (nNOS) in NMDA-mediated excitotoxicity in wild-type (SV129 and C57BL/6J) and type I NOS knock-out (nNOS−/−) mice and examined its relationship to apoptosis. Excitotoxic lesions were produced by intrastriatal stereotactic NMDA microinjections (10–20 nmol). Lesion size was dose- and time-dependent, completely blocked by MK-801 pretreatment, and smaller in nNOS knock-out mice compared with wild-type littermates (nNOS+/+, 11.7 ± 1.7 mm3;n= 8; nNOS−/−, 6.4 ± 1.8 mm3;n= 7). The density and distribution of striatal NMDA binding sites, determined by NMDA receptor autoradiography, did not differ between strains. Pharmacological inhibition of nNOS by 7-nitroindazole (50 mg/kg, i.p.) decreased NMDA lesion size by 32% in wild-type mice (n= 7). Neurochemical and immunohistochemical measurements of brain nitrotyrosine, a product of peroxynitrite formation, were increased markedly in wild-type but not in the nNOS−/−mice. Moreover, elevations in 2,3- and 2,5-dihydroxybenzoic acid levels were significantly reduced in the mutant striatum, as a measure of hydroxyl radical production.

The importance of apoptosis to NMDA receptor-mediated toxicity was evaluated by DNA laddering and by quantitative histochemistry [terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate–biotin nick end-labeling (TUNEL) staining]. DNA laddering was first detected within lesioned tissue after 12–24 hr. TUNEL-positive cells were first observed at 12 hr, increased in number at 48 hr and 7 d, and were located predominantly in proximity to the lesion border. The density was significantly lower in nNOS−/−mice. Hence, oligonucleosomal DNA breakdown suggesting apoptosis develops as a late consequence of NMDA microinjection and is reduced in nNOS mutants. The mechanism of protection in nNOS−/−mice may relate to decreased oxygen free radical production and related NO reaction products and, in part, involves mechanisms of neuronal death associated with the delayed appearance of apoptosis.
Regulation of Mouse Skeletal Muscle L-Type Ca2+Channel by Activation of the Insulin-Like Growth Factor-1 Receptor
Authors: Delbono Osvaldo , Renganathan Muthukrishnan , Messi Marı́a Laura
Published: April 2018

We investigated the modulation of the skeletal muscle L-type Ca2+channel/dihydropyridine receptor in response to insulin-like growth factor-1 receptor (IGF-1R) activation in single extensor digitorum longus muscle fibers from adult C57BL/6 mice. The L-type Ca2+channel activity in its dual role as a voltage sensor and a selective Ca2+-conducting pore was recorded in voltage-clamp conditions. Peak Ca2+current amplitude consistently increased after exposure to 20 ng/ml IGF-1 (EC50= 5.6 ± 1.8 nm). Peak IGF-1 effect on current amplitude at −20 mV was 210 ± 18% of the control. Ca2+current potentiation resulted from a shift in 13 mV of the Ca2+current–voltage relationship toward more negative potentials. The IGF-1-induced facilitation of the Ca2+current was not associated with an effect on charge movement amplitude and/or voltage distribution. These phenomena suggest that the L-type Ca2+channel structures involved in voltage sensing are not involved in the response to the growth factor. The modulatory effect of IGF-1 on L-type Ca2+channel was blocked by tyrosine kinase and PKC inhibitors, but not by a cAMP-dependent protein kinase inhibitor. IGF-1-dependent phosphorylation of the L-type Ca2+channel α1 subunit was demonstrated by incorporation of [γ-32P]ATP to monolayers of adult fast-twitch skeletal muscles. IGF-1 induced phosphorylation of a protein at the 165 kDa band, corresponding to the L-type Ca2+channel α1subunit. These results show that the activation of the IGF-1R facilitates skeletal muscle L-type Ca2+channel activity via a PKC-dependent phosphorylation mechanism.
Phosphorylation of the Synaptic Protein Interaction Site on N-type Calcium Channels Inhibits Interactions with SNARE Proteins
Authors: Yokoyama Charles T. , Sheng Zu-Hang , Catterall William A.
Published: April 2018

The synaptic protein interaction (synprint) site on the N-type calcium channel α1Bsubunit binds to the solubleN-ethylmaleimide-sensitive attachment factor receptor (SNARE) proteins syntaxin and synaptosomal protein of 25 kDa (SNAP-25), and this association may be required for efficient fast synaptic transmission. Protein kinase C (PKC) and calcium and calmodulin-dependent protein kinase type II (CaM KII) phosphorylated a recombinant his-tagged synprint site polypeptide rapidly to a stoichiometry of 3–4 mol of phosphate/mol, whereas cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) phosphorylated the synprint peptide more slowly to a stoichiometry of <1 mol/mol. Two-dimensional phosphopeptide mapping revealed similar patterns of phosphorylation of synprint polypeptides and native rat brain N-type calcium channel α1Bsubunits by PKC and Cam KII. Phosphorylation of the synprint peptide with PKC or CaM KII, but not PKA or PKG, strongly inhibited binding of recombinant syntaxin or SNAP-25, even at a level of free calcium (15 μm) that stimulates maximal binding. In contrast, phosphorylation of syntaxin and SNAP-25 with PKC and CaM KII did not affect interactions with the synprint site. Binding assays with polypeptides representing the N- and C-terminal halves of the synprint site indicate that the PKC- and CaM KII-mediated inhibition of binding involves multiple, disperse phosphorylation sites. PKC or CaM KII phosphorylation of the synprint peptide also inhibited its interactions with native rat brain SNARE complexes containing syntaxin and SNAP-25. These results suggest that phosphorylation of the synprint site by PKC or CaM KII may serve as a biochemical switch for interactions between N-type calcium channels and SNARE protein complexes.
Neuroprotective Actions of FK506 in Experimental Stroke:In VivoEvidence against an Antiexcitotoxic Mechanism
Authors: Butcher Steven P. , Henshall David C. , Teramura Yoshinori , Iwasaki Kazuhide , Sharkey John
Published: April 2018

The cellular mechanisms underlying the neuroprotective action of the immunosuppressant FK506 in experimental stroke remain uncertain, althoughin vitrostudies have implicated an antiexcitotoxic action involving nitric oxide and calcineurin. The presentin vivostudy demonstrates that intraperitoneal pretreatment with 1 and 10 mg/kg FK506, doses that reduced the volume of ischemic cortical damage by 56–58%, did not decrease excitotoxic damage induced by quinolinate, NMDA, and AMPA. Similarly, intravenous FK506 did not reduce the volume of striatal quinolinate lesions at a dose (1 mg/kg) that decreased ischemic cortical damage by 63%. The temporal window for FK506 neuroprotection was defined in studies demonstrating efficacy using intravenous administration at 120 min, but not 180 min, after middle cerebral artery occlusion. The noncompetitive NMDA receptor antagonist MK801 reduced both ischemic and excitotoxic damage. Histopathological data concerning striatal quinolinate lesions were replicated in neurochemical experiments. MK801, but not FK506, attenuated the loss of glutamate decarboxylase and choline acetyltransferase activity induced by intrastriatal injection of quinolinate. The contrasting efficacy of FK506 in ischemic and excitotoxic lesion models cannot be explained by drug pharmacokinetics, because brain FK506 content rose rapidly using both treatment protocols and was sustained at a neuroprotective level for 3 d. Although these data indicate that an antiexcitotoxic mechanism is unlikely to mediate the neuroprotective action of FK506 in focal cerebral ischemia, the finding that intravenous cyclosporin A (20 mg/kg) reduced ischemic cortical damage is consistent with the proposed role of calcineurin.
Protein Phosphorylation and Taurine BiosynthesisIn VivoandIn Vitro
Authors: Tang Xiao Wen , Hsu Che-Chang , Schloss John V. , Faiman Morris D. , Wu Elliott , Yang Chao-Yuh , Wu Jang-Yen
Published: April 2018

Taurine is known to be involved in many important physiological functions. Here we report that bothin vivoandin vitrothe taurine-synthesizing enzyme in the brain, namely cysteine sulfinic acid decarboxylase (CSAD), is activated when phosphorylated and inhibited when dephosphorylated. Furthermore, protein kinase C and protein phosphatase 2C have been identified as the enzymes responsible for phosphorylation and dephosphorylation of CSAD, respectively. In addition, the effect of neuronal depolarization on CSAD activity and32P incorporation into CSAD in neuronal cultures is also included. A model to link neuronal excitation and CSAD activation by a Ca2+-dependent protein kinase is proposed.
Involvement of Sphingosine 1-Phosphate in Nerve Growth Factor-Mediated Neuronal Survival and Differentiation
Authors: Edsall Lisa Cseh , Pirianov Grisha G. , Spiegel Sarah
Published: April 2018

Sphingolipid metabolites, such as ceramide and sphingosine-1-phosphate (SPP), are emerging as a new class of second messengers involved in cellular proliferation, differentiation, and apoptosis. Nerve growth factor (NGF), a neurotrophic factor for pheochromocytoma PC12 cells, induced a biphasic increase in the activity of sphingosine kinase, the enzyme that catalyzes the formation of SPP. This activation was blocked by K252a, an inhibitor of tyrosine kinase A (trkA). A rapid 1.7-fold increase was followed by a marked prolonged increase reaching a maximum of fourfold to fivefold stimulation with a concomitant increase in SPP levels and a corresponding decrease in endogenous sphingosine levels. Levels of ceramide, the precursor of sphingosine, were only slightly decreased by NGF in serum-containing medium. However, NGF decreased the elevation of ceramide induced by serum withdrawal. Treatment of PC12 cells with SPP did not induce neurite outgrowth or neurofilament expression, yet it enhanced neurofilament expression elicited by suboptimal doses of NGF. Moreover, SPP also protected PC12 cells from apoptosis induced by serum withdrawal. To further substantiate a role for SPP in the cytoprotective actions of NGF, we found thatN,N-dimethylsphingosine, a competitive inhibitor of sphingosine kinase, also induced apoptosis and interfered with the survival effect of NGF. These effects were counteracted by exogenous SPP. Moreover, other structurally related compounds, such as dihydrosphingosine 1-phosphate and lysophosphatidic acid, had no significant protective effects. Our results suggest that activation of sphingosine kinase and subsequent formation of SPP may play an important role in the differentiation and survival effects induced by NGF.