bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative migration and development of astroglial and oligodendroglial cell populations from a brain xenograft
Authors: Jacque C , Quinonero J , Collins PV , Villarroya H , Suard I
Published: April 2018

In previous studies of brain transplantation, the fate of the implanted glial cells has been investigated separately; that is, the interest has been focused either on the astroglia or on the oligodendroglia. However, the two populations of implanted glial cells may interact with each other, for example by secreting species-specific factors or by inducing reactions by the host. We have used two different models of brain transplantation: one that allows the identification of the implanted astrocytes, and another that allows the identification of the implanted oligodendroglia. The present model is a combination of both; it consists of the grafting of embryonic rabbit brain fragments into the brains of neonatal Shiverer mice. The myelin made by the implanted oligodendrocytes is identified by anti-myelin basic protein immunohistochemistry. The implanted astrocytes are identified by a monoclonal antibody that combines with rabbit but not with mouse glial fibrillary acidic protein. This study shows that although they use the same major routes of migration, both populations of glial cells tend to move differently. They demonstrate areas of common settlement but also areas where only one population of implanted glia is present. From the site of implantation in the dorsal striatum, the major routes of migration are the corpus callosum, the white matter fascicles in the striatum, and the internal capsule. After a delay of 6 weeks, no significant prevalence of one population of implanted glial cells over the other was observed.
Regulation of neuronal growth cone filopodia by intracellular calcium
Authors: Rehder V , Kater SB
Published: April 2018

Filopodia have been regarded as the sensory extensions of neuronal growth cones. As such, filopodia assay distant environments and are important for directing growth cones toward their targets. Since the territory encountered by a growth cone depends on the area spanned by the filopodia, changes in filopodial length or number result in the “exploration” of different-sized regions of the environment. The present study tests the potential regulatory role of intracellular calcium levels ([Ca2+]i) on filopodial morphology in identified neurons from the snail Helisoma. Experimentally evoked changes in [Ca2+]i were measured with the fluorescent calcium indicator fura-2 and directly correlated with growth cone filopodial morphology. A rise in [Ca2+]i caused two distinct, concentration-dependent effects separable by their different time courses: within the first 10 min, filopodia underwent significant elongation, while the second phase was characterized by a massive loss of filopodia. Both of these behaviors were increased in a calcium-dependent fashion. The magnitude of both filopodial elongation and filopodial loss correlated well with the transient peak values of [Ca2+]i reached during a given experimental treatment (r less than or equal to 0.98). In addition to the direct effect of the initial transient rise in [Ca2+]i, there is evidence for a form of adaptation of filopodial behavior to sustained calcium levels. A transient change in [Ca2+]i of as little as 30-50 nM reliably altered filopodial morphology. These results indicate that even small changes in intrinsic calcium homeostatic properties or extrinsic signals that alter intracellular calcium levels can act as regulators of the size of the environment sampled by an elongating growth cone.
Postsynaptic, but not presynaptic, activity controls the early time course of long-term potentiation in the dentate gyrus
Authors: Hanse E , Gustafsson B
Published: April 2018

The early time course (less than 1 hr) of long-term potentiation (LTP) in the dentate gyrus of the guinea pig hippocampal slice was examined using extracellular recordings from the outer two-thirds of the dendritic layer. LTP was induced by a single brief (2-40 impulses) high- frequency (20-400 Hz) train, or by pairing a single test stimulus with a brief heterosynaptic high-frequency train. The induction of LTP was facilitated by blockade of fast GABAergic postsynaptic inhibition. It was found that, irrespective of induction conditions and the amount of LTP induced, the onset of LTP was characterized by a latency of a few seconds following the induction event, and a rapid 30 sec growth phase. After a 1-2 min period of little or no further growth, LTP decayed but in a highly variable manner, from cases in which more than 60% of the peak value remained 1 hr after the induction to cases in which LTP decayed completely within 10 min. Factors increasing presynaptic activity (frequency or number of afferent stimulations) during the induction event did not affect the relative amount of LTP decay. Repetitive presynaptic activity was found not to be a necessary condition for eliciting long-lasting LTP (greater than 1 hr), as shown by experiments in which a single presynaptic impulse was paired with a brief heterosynaptic train. Factors increasing postsynaptic activity during the induction event, such as increased stimulus intensity, temporal pairing of two weak trains, or reduced postsynaptic inhibition, all reduced the relative amount of LTP decay. Moreover, partial pharmacological blockade of NMDA receptor channels increased the relative amount of decay. In conclusion, the amount of postsynaptic activity and associated NMDA receptor activation during the induction event appeared to be the main factor governing the early stability of LTP in the dentate gyrus.
Lesions of the perirhinal and parahippocampal cortices in the monkey produce long-lasting memory impairment in the visual and tactual modalities
Authors: Suzuki WA , Zola-Morgan S , Squire LR , Amaral DG
Published: April 2018

Compared to normal animals, monkeys with bilateral lesions of the perirhinal and parahippocampal cortices (PRPH lesion) were impaired on both a visual and a tactual version of the delayed nonmatching to sample task. In addition, the memory deficit was long-lasting, as indicated by the finding of a significant deficit when the visual version of the delayed nonmatching to sample task was readministered approximately 2 years after surgery. Animals with PRPH lesions performed normally on discrimination tasks in the visual and tactual modalities. Multimodal and long-lasting memory impairments are defining characteristics of human medial temporal lobe amnesia. Accordingly, these results demonstrate important parallels between the memory deficit associated with PRPH lesions and human medial temporal lobe amnesia. These data, taken together with previous findings, suggest that the perirhinal and parahippocampal cortices play an important role in memory function and that these cortical areas are critical components of the medial temporal lobe memory system.
Semisynthetic sphingolipids prevent protein kinase C translocation and neuronal damage in the perifocal area following a photochemically induced thrombotic brain cortical lesion
Authors: Kharlamov A , Guidotti A , Costa E , Hayes R , Armstrong D
Published: April 2018

A vascular thrombotic lesion localized to the rat sensorimotor cortex was produced following intravenous injection of the photosensitive dye rose bengal, and its activation with a small beam of high-intensity white light focused to the skull overlaying the sensorimotor cortex. In the sensorimotor cortex at various times after the triggering event, two contiguous brain regions with different degree(s) of neuronal damage can be distinguished: (1) a primary thrombotic ischemic core where the majority of cells are dead and (2) a penumbra region surrounding the core lesion in which a slower progressive neuronal degeneration is occurring. Importantly, in both brain regions the neuronal degeneration is associated with the activation and persistent translocation of protein kinase C (PKC) as indicated by an increase in 4-beta-3H-phorbol-12,13-dibutyrate (3H-PDBu) binding. Moreover, the demonstration that in the area penumbra the neuronal degeneration and the persistent translocation of PKC can be inhibited by a pretreatment with dizocilpine (i.e., MK-801) indicates that the dynamics of the progression of the neuronal degeneration are maintained by glutamate accumulating in the extraneuronal fluids. MK-801 additionally prevents the transcriptional activation of several immediate-early genes (IEGs) (e.g., c-fos) and their cognate third nuclear messenger (i.e., c-Fos) expression present in the hemisphere ipsilateral to the lesion. On the other hand, LIGA4 and LIGA20 derivatives of GM1 lysoganglioside reduce the membrane translocation of PKC and the neuronal damage in the penumbra area, but fail to change the increase of IEG expression in the cortex ipsilateral to the lesion.
The development of synaptic function and integration in the central auditory system
Authors: Sanes DH
Published: April 2018

The development of inhibitory synaptic transmission is difficult to assess because the afferents usually arise from intrinsic neurons that are difficult to stimulate independently. The postnatal maturation of excitatory and inhibitory synaptic function was compared in the gerbil lateral superior olive (LSO), where it is possible to stimulate physically discrete afferent projections. Intracellular recordings obtained in a brain slice preparation revealed that transmission was prominent at birth. The EPSPs and IPSPs were up to 2 orders of magnitude longer than in more mature animals. Brief trains of electrical stimulus pulses led to a temporal summation of postsynaptic potentials (PSPs) in 1–14 d animals resulting in prolonged depolarizations or hyperpolarizations. In neonates, the depolarization could exceed 1 sec following a 70 msec stimulus train. The IPSPs in neonates were often of sufficient amplitude to evoke a rebound depolarization or action potential. The number of converging afferents was estimated from the quantized increases in PSP size. There was a significant decrease with age, suggesting that both inhibitory and excitatory afferents were eliminated during the first 3 postnatal weeks. The integration of action potentials with IPSPs was examined with conjoint stimuli to the two afferent pathways, and demonstrated that the effective IPSP duration decreased approximately 20-fold during the first 3 postnatal weeks. The magnitudes and durations of electrical stimulus-evoked PSPs suggest that spontaneous discharge of afferents to the LSO could have a substantial impact on their development, even prior to the response to airborne sound at 12 d. Furthermore, the synaptic responses obtained at 12–14 d postnatal indicated that both amplitude and temporal processing remain compromised. These immature synaptic properties would be expected to compound the inadequacies present in the cochlea and cochlear nucleus.
Atrophy but not death of adult septal cholinergic neurons after ablation of target capacity to produce mRNAs for NGF, BDNF, and NT3
Authors: Sofroniew MV , Cooper JD , Svendsen CN , Crossman P , Ip NY , Lindsay RM , Zafra F , Lindholm D
Published: April 2018

The effect of unilateral excitotoxic ablation of hippocampal neurons was investigated on (1) the local production of mRNA for NGF and related neurotrophins, (2) the amount of NGF protein in the septal region, and (3) the viability and appearance of afferent septal cholinergic neurons in adult rats. After near complete ablation of hippocampal neurons, total levels of NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3) mRNA measured by quantitative Northern blot analysis in the hippocampal remnant fell significantly, to less than 25% of control values by 28 d and to less than 9% by 300 d. In the septal region ipsilateral to such lesions, NGF protein levels measured by ELISA fell significantly, to about 35% of control values, but the number of immunohistochemically detected cholinergic neurons did not decline significantly for up to 500 d. Instead, the cholinergic neurons persisted in an atrophied state, exhibiting severe shrinkage and reduced staining for the transmitter-synthesizing enzyme ChAT. The parameters of cell size and ChAT staining intensity correlated significantly with the amount of hippocampal tissue present. These findings indicate that in adult rats, target-derived NGF, BDNF, and NT3 do not regulate the survival of septal cholinergic neurons in proportion to the number of target neurons present. Moreover, the findings suggest that one or more of these target-derived neurotrophins regulate the structural and chemical phenotype of these neurons in the adult.
Ionic flux contributions to neocortical slow waves and nucleus basalis- mediated activation: whole-cell recordings in vivo
Authors: Metherate R , Ashe JH
Published: April 2018

Slow, rhythmic membrane potential (Vm) fluctuations occur spontaneously in cortical neurons of urethane-anesthetized rats, and likely underlie EEG activity in the same low-frequency (1–4 Hz or delta) range. Nucleus basalis (NB) stimulation elicits neocortical activation, simultaneously modifying Vm and EEG fluctuations, by way of cortical muscarinic ACh receptors (Metherate et al., 1992). To investigate the nature of spontaneous fluctuations and their modification by NB stimulation, we have obtained intracellular recordings from auditory cortex using the whole-cell recording technique in vivo. Spontaneous Vm fluctuations appeared to contain three components whose polarity and time course resembled the EPSP, putative Cl(-)-mediated IPSP, and putative K(+)- mediated, long-lasting IPSP elicited by thalamic stimulation. The spontaneous, long-lasting hyperpolarization, whose rhythmic occurrence appeared to set the slow-wave rhythm, was associated with an increased conductance that could shunt the thalamocortical EPSP. We hypothesized that spontaneous Vm fluctuations arise from intermixed rapid depolarizations, rapid Cl(-)-mediated hyperpolarizations, and long- lasting, K(+)-mediated hyperpolarizations. NB-mediated cortical activation might then result from muscarinic suppression of K+ permeability, allowing the rapid depolarizations and Cl- fluxes to continue uninterrupted. Tests of this hypothesis showed that (1) intracellular blockade of K+ channels by rapid diffusion of Cs+ from the recording pipette resulted in suppression of spontaneous, long- lasting hyperpolarizations, mimicking the effect of NB stimulation, and reducing shunting of the thalamocortical EPSP; (2) effects of Cs+ and NB stimulation suggested overlapping, or converging, mechanisms of action; however, there were important differential effects on the spontaneous, long-lasting hyperpolarizations and the K(+)-mediated IPSP; and (3) modifying Cl- fluxes with intracellular picrotoxin or high intracellular Cl- concentrations resulted in spontaneous and NB- elicited large-amplitude depolarizations. We conclude that spontaneous, long-lasting hyperpolarizations are K+ fluxes, but are not “spontaneous” K(+)-mediated IPSPs. Since NB-mediated reduction of spontaneous hyperpolarizations implies muscarinic suppression of a K+ conductance, the spontaneous hyperpolarizations more likely result from the calcium-activated K+ current, IK(Ca). Finally, Cl- fluxes form an important component of activated Vm fluctuations that acts to restrain excessive depolarization.
Modulation of a voltage-activated potassium channel by peptide growth factor receptors
Authors: Timpe LC , Fantl WJ
Published: April 2018

Regulation of the activity of a cloned component of a voltage-activated K+ channel, Kv1.5, was studied by expressing the K+ channel and receptors for platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF) simultaneously in Xenopus oocytes. Receptor activation mediated a decline in the Kv1.5 current amplitude, with a half-time of about 20 min. The reduction in K+ current amplitude occurred with little change in the kinetics or voltage sensitivity of activation. A similar phenomenon was found when the human thrombin or rat 5-HT1c receptors, two receptors that increase phospholipase C activity, were tested in coexpression experiments. A mutant FGF receptor, which does not activate phospholipase C-gamma 1 but retains several of its other functions, did not modulate the Kv1.5 current. Simultaneous injection of inositol trisphosphate and superfusion of phorbol 12-myristate 13-acetate reproduced the modulation of the Kv1.5 current. These results demonstrate that the PDGF and FGF receptors can modulate a voltage-activated K+ channel by increasing phospholipase C activity, and suggest that PDGF or FGF may be able to alter rapidly the electrical excitability of neurons.
Consistency in nervous systems: trial-to-trial and animal-to-animal variations in the responses to repeated applications of a sensory stimulus in Aplysia
Authors: Wu JY , Tsau Y , Hopp HP , Cohen LB , Tang AC , Falk CX
Published: April 2018

What is the internal noise in a nervous system? We studied this question by determining the trial-to-trial consistency of the neuronal response in the abdominal ganglion of Aplysia californica. Because our voltage-sensitive dye recordings detected the spike activity from a large fraction of the neurons in the ganglion, these results provide a reasonably complete characterization of the consistency of the response to a sensory stimulus. The consistency of each neuron was evaluated by the number and timing of spikes in the response. The variability in the spike count was described using the coefficient of variation. The spike count variations follow a Poisson distribution, indicating that most of these variations were the result of a random process. For each neuron the reliability of the response to touch was measured in two ways; both measures indicated a broad distribution of reliabilities within the neuron population. The time of the maximum response also varied substantially in some animals. These timing variations were in part due to random processes and in part due to systematic effects (changes in activity of many neurons that were highly correlated). The time course of the activity of individual neurons was compared with the time course of the gill withdrawal. In some animals the activity of individual neurons was only poorly correlated with the behavior; in contrast, the summed activity of groups of neurons matched the behavior quite well. This implies that the behavioral output of the system may be a distributed combination of the activity of many neurons. The differences between animals were substantially larger than the trial-to- trial differences in one animal. The responses made by different preparations differed along many dimensions.
Neuronal activity within the nucleus basalis and conditioned neocortical electroencephalographic activation
Authors: Whalen PJ , Kapp BS , Pascoe JP
Published: April 2018

The relationship between neuronal activity within the nucleus basalis (NB) and conditioned neocortical EEG activation was investigated in New Zealand rabbits during Pavlovian differential conditioning. Twenty- seven of 56 neurons recorded in conditioned animals demonstrated a significantly greater change in activity to a tone (CS+) that predicted the occurrence of a mildly aversive unconditioned stimulus when compared to a tone (CS-) that did not. Twenty-four of these 27 neurons demonstrated a significant increase in activity to the CS+ compared to the CS-, while the remaining three neurons demonstrated a significant decrease in activity to the CS+ compared to the CS-. In 24 of these 27 neurons (89%) these changes in neuronal activity during CS presentations correlated significantly with a decrease in the power of delta activity in the EEG. In addition, 13 of these 24 neurons (54%) demonstrated significant correlations between neuronal activity and the power of delta activity during CS-free periods. In experimentally naive animals, the activity of 10 of 22 neurons (45%) recorded within the region of the NB correlated with the power of delta activity in the EEG during stimulus-free periods. These results complement a growing body of evidence and provide strong support for the hypothesis that the NB contributes to neocortical activation in the conscious animal.
Differential modulation of alpha 3 beta 2 and alpha 3 beta 4 neuronal nicotinic receptors expressed in Xenopus oocytes by flufenamic acid and niflumic acid
Authors: Zwart R , Oortgiesen M , Vijverberg HP
Published: April 2018

Effects of flufenamic acid (FFA) and niflumic acid (NFA), which are often used to block Ca(2+)-activated Cl- current, have been investigated in voltage-clamped Xenopus oocytes expressing alpha 3 beta 2 and alpha 3 beta 4 nicotinic ACh receptors (nAChRs). NFA and FFA inhibit alpha 3 beta 2 nAChR-mediated inward currents and potentiate alpha 3 beta 4 nAChR-mediated inward currents in normal, Cl(-)-free and Ca(2+)-free solutions to a similar extent. The concentration-dependence of the inhibition of alpha 3 beta 2 nAChR-mediated ion current yields IC50 values of 90 microM for FFA and of 260 microM for NFA. The potentiation of alpha 3 beta 4 nAChR-mediated ion current by NFA yields an EC50 value of 30 microM, whereas the effect of FFA does not saturate for concentrations of up to 1 mM. At 100 microM, FFA reduces the maximum of the concentration-effect curve of ACh for alpha 3 beta 2 nAChRs, but leaves the EC50 of ACh unaffected. The same concentration of FFA potentiates alpha 3 beta 4 nAChR-mediated ion currents for all ACh concentrations and causes a small shift of the concentration-effect curve of ACh to lower agonist concentrations. The potentiation, like the inhibition, is most likely due to a noncompetitive effect of FFA. Increasing ACh-induced inward current either by raising the agonist concentration from 10 microM to 200 microM or by coapplication of 10 microM ACh and 200 microM FFA causes a similar enhancement of block of the alpha 3 beta 4 nAChR-mediated ion current by Mg2+. This suggests that the effects of FFA and of an increased agonist concentration result in a similar functional modification of the alpha 3 beta 4 nAChR- operated ion channel. It is concluded that alpha 3 beta 4 and alpha 3 beta 2 nAChRs are oppositely modulated by FFA and NFA through a direct beta-subunit-dependent effect.
Molecular characterization and spatial distribution of SAP97, a novel presynaptic protein homologous to SAP90 and the Drosophila discs-large tumor suppressor protein
Authors: Muller BM , Kistner U , Veh RW , Cases-Langhoff C , Becker B , Gundelfinger ED , Garner CC
Published: April 2018

Synapses are highly specialized sites of cell-cell contact involved in signal transfer. The molecular mechanisms modulating the assembly and stability of synapses are unknown. We previously reported the identification of a 90 kDa synapse-associated protein, SAP90, that is localized at the presynaptic termini of inhibitory GABAergic synapses. SAP90 is a mosaic protein composed of three 90 amino acid residue repeats, an SH3 domain and a region homologous to guanylate kinases. SAP90 shares domain specific homology with a family of proteins involved in the assembly and possibly stability of sites of cell contact. These include the product of the lethal(1) discs-large-1 (dlgA) tumor suppressor gene and the zonula occludens proteins ZO-1, ZO- 2. The further characterization of cDNA clones encoding components of synaptic junctions has lead to the identification of a 97 kDa protein, called SAP97, that exhibits a strong overall sequence similarity to SAP90. The present study was undertaken to determine the spatial distribution of SAP97, and to reveal further clues to the possible roles of these proteins in synapses. Light and immunoelectron microscopic analysis of the rat hippocampal formation revealed that SAP97 is localized in the presynaptic nerve termini of excitatory synapses. In other brain regions, SAP97 is found in and along bundles of unmyelinated axons. SAP97 is not restricted to the CNS, but is also present at the basal lateral membrane between a variety of epithelial cells. In cultured T84 cells, it is restricted to the cytoplasmic surface of the plasma membranes between adjacent cells, but not at the edges of cells lacking cell-cell contact suggesting a role for SAP97 in cell adhesion. These data suggest that members of the SAP90/SAP97 subfamily may be involved in the site specific assembly, stability or functions of membrane specialization at sites of cell-cell contact.
Astrocytes protect neurons from hydrogen peroxide toxicity
Authors: Desagher S , Glowinski J , Premont J
Published: April 2018

Recent reports indicate that neurons are particularly sensitive to hydrogen peroxide (H2O2). The present study was undertaken to investigate the putative role of astrocytes in the modulation of the neurotoxic effect of H2O2. The exposure to H2O2 of cultured striatal neurons from mouse embryos induced a concentration-dependent (10–1000 microM) cell death as estimated 24 hr later. Two methods were used to estimate neuronal survival: the 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide assay or an enzyme-linked immunosorbent assay with antibodies directed against an antigen located in neurons (microtubule-associated protein-2). The neurotoxic effect of H2O2 on neurons cocultured with astrocytes was strongly attenuated compared with that observed on a pure population of neurons seeded at the same density. Moreover, the protective effect of astrocytes depended on the astrocytes/neurons ratio, a significant neuroprotection being detectable for 1 astrocyte to 20 neurons. Catalase seems to be the main hydrogen peroxidase activity involved in the neuroprotective effect of astrocytes. Indeed, in the culture conditions used, this enzymatic activity was enriched in this cell type compared with neurons; its inhibition, and not that of glutathione peroxidase, reduced the disappearance rate of the oxidant. On the contrary, glutathione peroxidase appeared to be the main enzymatic activity involved in the neuronal defense against H2O2 toxicity. Therefore, astrocytes could delay neuronal death in pathological situations in which H2O2 has been, at least partially, demonstrated to be involved.
Oligodendrocyte progenitor cell proliferation and lineage progression are regulated by glutamate receptor-mediated K+ channel block
Authors: Gallo V , Zhou JM , McBain CJ , Wright P , Knutson PL , Armstrong RC
Published: April 2018

We have analyzed the role of glutamate and its receptors (GluRs) in regulating the development of oligodendrocytes. Activation of AMPA- preferring GluRs with selective agonists inhibited proliferation of purified cortical oligodendrocyte progenitor (O-2A) cells cultured with different mitogens, as measured by [3H]thymidine incorporation or bromodeoxyuridine staining. In contrast, activation of GABA or muscarinic receptors did not affect O-2A proliferation. Cell viability and apoptosis assays demonstrated that the inhibition of O-2A proliferation was not attributable to a cytotoxic action of GluR agonists, and was reversible. Activation of GluRs prevented lineage progression from the O-2A (GD3+/nestin+) stage to the prooligodendroblast (O4+) stage, but did not affect O-2A migration. Additional experiments examined the membrane ionic channels mediating these GluR activation effects. We found that proliferating O-2A cells expressed functional delayed rectifier K+ channels, which were absent in pro-oligodendroblasts. GluR agonists and the K+ channel blocker tetraethylammonium (TEA) strongly inhibited delayed rectifier K+ currents in O-2A cells. TEA reproduced the effects of GluR activation on O-2A proliferation and lineage progression in the same concentration range that blocked delayed rectifier K+ currents. These results indicate that glutamate regulates oligodendrogenesis specifically at the O-2A stage by modulating K+ channel activity.
Effects of chronic morphine administration on mu opioid receptor- stimulated [35S]GTPgammaS autoradiography in rat brain
Authors: Sim LJ , Selley DE , Dworkin SI , Childers SR
Published: April 2018

Chronic opiate administration results in the development of tolerance and dependence, but the regulation of mu opioid receptor function during this process is not clearly understood. To localize changes in mu opioid receptor-coupled G-protein activity in various brain regions after chronic morphine treatment, the present study examined mu opioid agonist-stimulated [35S]GTPgammaS binding to brain sections by in vitro autoradiography. Rats were treated for 12 d with increasing doses (10– 320mg . kg-1 . d-1) of morphine. Control rats were injected with either saline or a single acute injection of morphine (20 mg/kg). mu opioid- stimulated [35S]GTPgammaS binding was measured by autoradiography of brain sections in the presence and absence of the mu opioid-selective agonist DAMGO. In rats injected with a single acute dose of morphine, no significant changes were detected in basal or agonist-stimulated [35S]GTPgammaS binding in any region. In sections from chronic morphine- treated rats, however, DAMGO-stimulated [35S]GTPgammaS binding was reduced significantly compared with control rats in the following brain- stem nuclei: dorsal raphe nucleus, locus coeruleus, lateral and medial parabrachial nuclei, and commissural nucleus tractus solitarius. No significant changes were observed in several other brain regions, including the nucleus accumbens, amygdala, thalamus, and substantia nigra. These data indicate that chronic morphine administration results in reductions in mu opioid activation of G-proteins in specific brainstem nuclei involved in physiological homeostasis and autonomic function, which may have implications in the development of opiate tolerance and physical dependence.
Impaired classical eyeblink conditioning in cerebellar-lesioned and Purkinje cell degeneration (pcd) mutant mice
Authors: Chen L , Bao S , Lockard JM , Kim JK , Thompson RF
Published: April 2018

Converging lines of evidence from rabbits, rats, and humans argue for the crucial involvement of the cerebellum in classical conditioning of the eyeblink/nictitating membrane response in mammals. For example, selective lesions (permanent or reversible) of the cerebellum block both acquisition and retention of eyeblink conditioning. Correspondingly, electrophysiological and brain-imaging studies indicate learning-related plasticity in the cerebellum. The involvement of the cerebellum in eyeblink conditioning is also supported by stimulation studies showing that direct stimulation of the two major afferents to the cerebellum (the mossy fibers emanating from the pontine nucleus and climbing fibers originating from the inferior olive) can substitute for the peripheral conditioned stimulus (CS) and unconditioned stimulus (US), respectively, to yield normal behavioral learning. In the present study, we examined the relative contribution of the cerebellar cortex versus deep nuclei (specifically the interpositus nucleus) in eyeblink learning by using mutant mice deficient of Purkinje cells, the exclusive output neurons of the cerebellar cortex. We report that Purkinje cell degeneration (pcd) mice exhibit a profound impairment in the acquisition of delay eyeblink conditioning in comparison with their wild-type littermates. Nevertheless, the pcd animals did acquire a subnormal level of conditioned eyeblink responses. In contrast, wild-type mice with lesions of the interpositus nucleus were completely unable to learn the conditioned eyeblink response. These results suggest that both cerebellar cortex and deep nuclei are important for normal eyeblink conditioning.
Synaptogenesis and FOS Expression in the Motor Cortex of the Adult Rat after Motor Skill Learning
Authors: Kleim Jeffrey A. , Lussnig Erich , Schwarz Edward R. , Comery Thomas A. , Greenough William T.
Published: April 2018

Recent work has suggested that changes in synapse number as well as changes in the expression of the Fos protein may occur within the motor cortex in association with motor learning. The number of synapses per neuron and the percentage of Fos-positive neurons within layer II/III of the rat motor cortex was measured after training on a complex motor learning task. Adult female rats were allocated randomly to either an acrobatic condition (AC), a motor control condition (MC), or an inactive control condition (IC). AC animals were trained to traverse a complex series of obstacles, and each AC animal was pair matched with an MC animal that traversed an obstacle-free runway. IC animals received no motor training. Animals from each condition were killed at various points during training, and unbiased stereological techniques were used to estimate the number of synapses per neuron and the percentage of Fos-positive cells within layer II/III of the motor cortex. AC animals exhibited an overall increase in the number of synapses per neuron in comparison to MC and IC animals at later stages of training. AC animals also had a significantly higher overall percentage of Fos-positive cells in comparison to both controls, with a trend for the increase to be greater during the acquisition versus the maintenance phase. These data suggest that Fos may be involved in the biochemical processes underlying skill acquisition and that motor learning, as opposed to motor activity, leads to increases in synapse number in the motor cortex.
Peptidergic Modulation of Intrathalamic Circuit ActivityIn Vitro: Actions of Cholecystokinin
Authors: Cox Charles L. , Huguenard John R. , Prince David A.
Published: April 2018

Cholecystokinin (CCK)-mediated actions on intrathalamic rhythmic activities were examined in anin vitrorat thalamic slice preparation. Single electrical stimuli in the thalamic reticular nucleus (nRt) evoked rhythmic activity (1–15 sec duration) in nRt and the adjacent ventrobasal nucleus (VB). Low CCK concentrations (20–50 nm) suppressed rhythmic oscillations in 43% of experiments but prolonged such activities in the remaining slices. Higher CCK concentrations (100–400 nm) had a predominantly antioscillatory effect.

Suppression of oscillations was associated with a relatively large membrane depolarization of nRt neurons that changed their firing mode from phasic (burst) to tonic (single-spike) output. This decreased burst discharge of nRt neurons during CCK application reduced inhibitory drive onto VB neurons from multiple peaked inhibitory postsynaptic currents (IPSCs) to single peaked inhibitory events. We hypothesize that suppression of inhibitory drive onto VB neurons decreases their probability of burst output, which, together with a reduction of nRt burst output, dampens the oscillatory activity.

Low CCK concentrations, which produced little or no depolarization of nRt neurons, did not alter the firing mode of the nRt neurons. However, the probability of burst output from nRt neurons in response to subthreshold stimuli was increased in low CCK concentrations, presumably leading to an increase in the number of nRt neurons participating in the rhythmic activity. Our findings suggest that the neuropeptide CCK, by altering the firing characteristics of nRt neurons, has powerful modulatory effects on intrathalamic rhythms; the ultimate action was dependent on CCK concentration and resting state of these cells.
Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP-38) Protects Cerebellar Granule Neurons from Apoptosis by Activating the Mitogen-Activated Protein Kinase (MAP Kinase) Pathway
Authors: Villalba Martin , Bockaert Joël , Journot Laurent
Published: April 2018

Pituitary adenylate cyclase-activating polypeptides (PACAP-27 and PACAP-38) are neuropeptides of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. PACAP receptors are expressed in different brain regions, including cerebellum. We used primary culture of rat cerebellar granule neurons to study the effect of PACAP-38 on apoptosis induced by potassium deprivation. We demonstrated that PACAP-38 increased survival of cerebellar neurons in a dose-dependent manner by decreasing the extent of apoptosis estimated by DNA fragmentation. PACAP-38 induced activation of the extracellular signal-regulated kinase (ERK)-type of mitogen-activated protein (MAP) kinase through a cAMP-dependent pathway. PD98059, an inhibitor of MEK (MAP kinase kinase), completely abolished the antiapoptotic effect of PACAP-38, suggesting that MAP kinase pathway activation is necessary for PACAP-38 action.
Suppression of Cathepsins B and L Causes a Proliferation of Lysosomes and the Formation of Meganeurites in Hippocampus
Authors: Bednarski Eric , Ribak Charles E. , Lynch Gary
Published: April 2018

Cultured hippocampal slices exhibited prominent ultrastructural features of brain aging after exposure to an inhibitor of cathepsins B and L. Six days of treatment withN-CBZ-L-phenylalanyl-l-alanine-diazomethylketone (ZPAD) resulted in a dramatic increase in the number of lysosomes in the perikarya of neurons and glial cells throughout the slices. Furthermore, lysosomes in CA1 and CA3 pyramidal cells were not restricted to the soma but instead were located throughout dendritic processes. Clusters of lysosomes were commonly found within bulging segments of proximal dendrites that were notable for an absence of microtubules and neurofilaments. Although pyknotic nuclei were sometimes encountered, most of the cells in slices exposed to ZPAD for 6 d appeared relatively normal. Slices given 7 d of recovery contained several unique features, compared with those processed immediately after incubation with the inhibitor. Cell bodies of CA1 neurons were largely cleared of the excess lysosomes but had gained fusiform, somatic extensions that were filled with fused lysosomes and related complex, dense bodies. These appendages, similar in form and content to structures previously referred to as “meganeurites,” were not observed in CA3 neurons or granule cells. Because meganeurites were often interposed between cell body and axon, they have the potential to interfere with processes requiring axonal transport. It is suggested that inactivation of cathepsins B and L results in a proliferation of lysosomes and that meganeurite generation provides a means of storing residual catabolic organelles. The accumulated material could be eliminated by pinching off the meganeurite but, at least in some cases, this action would result in axotomy. Reduced cathepsin L activity, increased numbers of lysosomes, and the formation of meganeurites are all reported to occur during brain aging; thus, it is possible that the infusion of ZPAD into cultured slices sets in motion a greatly accelerated gerontological sequence.
Alzheimer’s Presenilin Mutation Sensitizes Neural Cells to Apoptosis Induced by Trophic Factor Withdrawal and Amyloid β-Peptide: Involvement of Calcium and Oxyradicals
Authors: Guo Qing , Sopher Bryce L. , Furukawa Katsutoshi , Pham Dao G. , Robinson Nic , Martin George M. , Mattson Mark P.
Published: April 2018

Most autosomal dominant inherited forms of early onset Alzheimer’s disease (AD) are caused by mutations in the presenilin-1 (PS-1) gene on chromosome 14. PS-1 is an integral membrane protein with six to nine membrane-spanning domains and is expressed in neurons throughout the brain wherein it is localized mainly in endoplasmic reticulum (ER). The mechanism or mechanisms whereby PS-1 mutations promote neuron degeneration in AD are unknown. Recent findings suggest links among deposition of amyloid β-peptide (Aβ), oxidative stress, disruption of ion homeostasis, and an apoptotic form of neuron death in AD. We now report that expression of the human PS-1 L286V mutation in PC12 cells increases their susceptibility to apoptosis induced by trophic factor withdrawal and Aβ. Increases in oxidative stress and intracellular calcium levels induced by the apoptotic stimuli were exacerbated greatly in cells expressing the PS-1 mutation, as compared with control cell lines and lines overexpressing wild-type PS-1. The antiapoptotic gene product Bcl-2 prevented apoptosis after NGF withdrawal from differentiated PC12 cells expressing mutant PS-1. Elevations of [Ca2+]iin response to thapsigargin, an inhibitor of the ER Ca2+-ATPase, were increased in cells expressing mutant PS-1, and this adverse effect was abolished in cells expressing Bcl-2. Antioxidants and blockers of calcium influx and release from ER protected cells against the adverse consequences of the PS-1 mutation. By perturbing cellular calcium regulation and promoting oxidative stress, PS-1 mutations may sensitize neurons to apoptotic death in AD.
Quantitative Ultrastructural Analysis of Hippocampal Excitatory Synapses
Authors: Schikorski Thomas , Stevens Charles F.
Published: April 2018

From three-dimensional reconstructions of CA1 excitatory synapses in the rodent hippocampus and in culture, we have estimated statistical distributions of active zone and postsynaptic density (PSD) sizes (average area ∼0.04 μm2), the number of active zones per bouton (usually one), the number of docked vesicles per active zone (∼10), and the total number of vesicles per bouton (∼200), and we have determined relationships between these quantities, all of which vary from synapse to synapse but are highly correlated. These measurements have been related to synaptic physiology. In particular, we propose that the distribution of active zone areas can account for the distribution of synaptic release probabilities and that each active zone constitutes a release site as identified in the standard quantal theory attributable to Katz (1969).
Nitric Oxide Depresses GABAAReceptor Function via Coactivation of cGMP-Dependent Kinase and Phosphodiesterase
Authors: Wexler Eric M. , Stanton Patric K. , Nawy Scott
Published: April 2018

Nitric oxide (NO) is thought to play an essential role in neuronal processing, but the downstream mechanisms of its action remain unclear. We report here that NO analogs reduce GABA-gated currents in cultured retinal amacrine cells via two distinct, but convergent, cGMP-dependent pathways. Either extracellular application of the NO-mimeticS-nitroso-N-acetyl-penicillamine (SNAP) or intracellular perfusion with cGMP depressed GABA currents. This depression was partially blocked by a pseudosubstrate peptide inhibitor of cGMP-dependent protein kinase (PKG), suggesting both PKG-dependent and independent actions of cGMP. cAMP-dependent protein kinase (PKA) is known to enhance retinal GABA responses. 8-Bromoinosine 3′,5′-cyclic monophosphate (8Br-cIMP), which activates a type of cGMP-stimulated phosphodiesterase that hydrolyzes cAMP, also significantly reduced GABA currents. 1-Methyl-3-isobutylxanthine (IBMX), a nonspecific phosphodiesterase (PDE) inhibitor, blocked both the action of 8Br-cIMP and the portion of SNAP-induced depression that was not blocked by PKG inhibition. Our results suggest that NO depresses retinal GABAAreceptor function by simultaneously upregulating PKG and downregulating PKA.
Oxytocin Modulates Glutamatergic Synaptic Transmission between Cultured Neonatal Spinal Cord Dorsal Horn Neurons
Authors: Jo Young-Hwan , Stoeckel Marie-Elisabeth , Freund-Mercier Marie-José , Schlichter Rémy
Published: April 2018

The functional characteristics of binding sites for the neuropeptide oxytocin (OT) detected by radioautography in laminae I and II of the dorsal horn (DH) and on cultured neonatal DH neurons were studied on the latter using perforated patch-clamp recordings. The neurons were identified by their spike discharge properties and on the basis of the presence of met-enkephalin-like and glutamate decarboxylase-like immunoreactivities. OT (100 nm) never induced any membrane current at a holding potential of −60 mV but increased the frequency of spontaneously occurring AMPA receptor-mediated EPSCs or the mean amplitude of electrically evoked EPSCs in a subset (35%) of neurons. The frequency of miniature EPSCs (m-EPSCs) recorded in the presence of 0.5 μmtetrodotoxin was also increased by OT (100 nm) without any change in their mean amplitude, indicating an action at a site close to the presynaptic terminal. The decay kinetics of any type of EPSC were never modified by OT. The effect of OT was reproduced by [Thr4,Gly7]-OT (100 nm), a selective OT receptor agonist, and blocked by d(CH2)5-[Tyr(Me)2,Thr4,Tyr-NH29]-ornithine vasotocin (100 nm), a specific OT receptor antagonist. Reducing the extracellular Ca2+concentration from 2.5 to 0.3 mmin the presence of Cd2+(100 μm) reversibly blocked the effect of OT on m-EPSCs. The OT receptors described here may represent the substrate for modulatory actions of descending hypothalamo-spinal OT-containing pathways on the nociceptive system.
Role of Actin in Anchoring Postsynaptic Receptors in Cultured Hippocampal Neurons: Differential Attachment of NMDA versus AMPA Receptors
Authors: Allison Daniel W. , Gelfand Vladimir I. , Spector Ilan , Craig Ann Marie
Published: April 2018

We used actin-perturbing agents and detergent extraction of primary hippocampal cultures to test directly the role of the actin cytoskeleton in localizing GABAAreceptors, AMPA- and NMDA-type glutamate receptors, and potential anchoring proteins at postsynaptic sites. Excitatory postsynaptic sites on dendritic spines contained a high concentration of F-actin that was resistant to cytochalasin D but could be depolymerized using the novel compound latrunculin A. Depolymerization of F-actin led to a 40% decrease in both the number of synaptic NMDA receptor (NMDAR1) clusters and the number of AMPA receptor (GluR1)-labeled spines. The nonsynaptic NMDA receptors appeared to remain clustered and to coalesce in cell bodies. α-Actinin-2, which binds both actin and NMDA receptors, dissociated from the receptor clusters, but PSD-95 remained associated with both the synaptic and nonsynaptic receptor clusters, consistent with a proposed cross-linking function. AMPA receptors behaved differently; on GABAergic neurons, the clusters redistributed to nonsynaptic sites, whereas on pyramidal neurons, many of the clusters appeared to disperse. Furthermore, in control neurons, AMPA receptors were detergent extractable from pyramidal cell spines, whereas AMPA receptors on GABAergic neurons and NMDA receptors were unextractable. GABAAreceptors were not dependent on F-actin for the maintenance or synaptic localization of clusters. These results indicate fundamental differences in the mechanisms of receptor anchoring at postsynaptic sites, both regarding the anchoring of a single receptor (the AMPA receptor) in pyramidal cells versus GABAergic interneurons and regarding the anchoring of different receptors (AMPA vs NMDA receptors) at a single class of postsynaptic sites on pyramidal cell dendritic spines.
Acceleration in the Rate of CNS Remyelination in Lysolecithin-Induced Demyelination
Authors: Pavelko Kevin D. , van Engelen Baziel G. M. , Rodriguez Moses
Published: April 2018

One important therapeutic goal during CNS injury from trauma or demyelinating diseases such as multiple sclerosis is to develop methods to promote remyelination. We tested the hypothesis that spontaneous remyelination in the toxic nonimmune model of lysolecithin-induced demyelination can be enhanced by manipulating the inflammatory response. In PBS-treated SJL/J mice, the number of remyelinating axons per square millimeter of lesion area increased significantly 3 and 5 weeks after lysolecithin injection in the spinal cord. However, methylprednisolone or a monoclonal antibody (mAb), SCH94.03, developed for its ability to promote remyelination in the Theiler’s virus murine model of demyelination, further increased the number of remyelinating axons per lesion area at 3 weeks by a factor of 2.6 and 1.9, respectively, but did not increase the ratio of myelin sheath thickness to axon diameter or the number of cells incorporating tritiated thymidine in the lesion. After 3 weeks, the number of remyelinating axons in the methylprednisolone or mAb SCH94.03 treatment groups was similar to the spontaneous remyelination in the 5 week PBS control-treated group, indicating that these treatments promoted remyelination by increasing its rate rather than its extent. To address a mechanism for promoting remyelination, through an effect on scavenger function, we assessed morphometrically the number of macrophages in lesions after methylprednisolone and mAb SCH94.03 treatment. Methylprednisolone reduced the number of macrophages, but SCH94.03 did not, although both enhanced remyelination. This study supports the hypothesis that even in toxic nonprimary immune demyelination, manipulating the inflammatory response is a benefit in myelin repair.