bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunocytochemical localization of group III metabotropic glutamate receptors in the hippocampus with subtype-specific antibodies
Authors: Bradley SR , Levey AI , Hersch SM , Conn PJ
Published: April 2018

The metabotropic glutamate receptors (mGluRs) mGluR4 and mGluR7 have been postulated to serve as presynaptic autoreceptors in the hippocampal formation. The cellular and synaptic localization of these proteins in the hippocampus, however, is not known. Polyclonal antibodies that specifically react with mGluR4a or mGluR7 were produced and used for immunocytochemical localization of these receptor proteins. Immunoblot analysis with mGluRs expressed in stably transfected baby hamster kidney cells suggests that each of the antibodies reacts specifically with the appropriate mGluR subtype but not with other mGluRs. Both antibodies recognized native proteins in rat brain with molecular weights similar to the molecular weights of the bands in mGluR-transfected cell lines. The distribution of mGluR4a immunoreactivity was fairly uniform in all brain regions. Immunoreactivity for mGluR7 was variable in different brain regions and closely paralleled the previously reported distribution of mGluR7 mRNA. Immunocytochemistry with light and electron microscopy (EM) revealed that immunoreactivities for mGluR4a and mGluR7 are widely but differentially distributed throughout the hippocampal formation. Staining with antibodies directed against mGluR4a was intense in cell bodies and dendrites of pyramidal cells, granule cells, and scattered interneurons. Analysis at the EM level revealed postsynaptic mGluR4a localization at asymmetrical (presumably glutamatergic) synapses and presynaptic localization at both asymmetrical and symmetrical synapses. Immunoreactivity for mGluR7 revealed largely presynaptic localization of this receptor at asymmetrical but not symmetrical synapses. These data are consistent with a largely presynaptic role of mGluR7 in the hippocampus and suggest that mGluR4 may have both presynaptic and postsynaptic functions.
A different form of long-lasting potentiation revealed in tissue plasminogen activator mutant mice
Authors: Frey U , Muller M , Kuhl D
Published: April 2018

The establishment of hippocampal long-term (LTP) requires protein and mRNA synthesis, suggesting that neuronal activity resulting in LTP initiates a cascade of gene expression. The expression of the gene for the extracellular serine protease tissue plasminogen activator (t-PA) is induced during LTP. Here we analyze long-lasting LTP (L-LTP,>4 hr)in CA1 hippocampal slices of mice homozygous for disrupted t-PA genes. Although mutant mice appear to exhibit long-term potentiation, we provide evidence that these mice are devoid of conventional homosynaptic L-LTP at the Schaffer collateral-CA1 pyramidal cell synapse. Most remarkably, t-PA-deficient mice exhibit a different form of long-lasting potentiation that is characterized by an NMDA receptor- dependent modification of GABA transmission in the CA1 region.
Glial cells are increased proportionally in transgenic optic nerves with increased numbers of axons
Authors: Burne JF , Staple JK , Raff MC
Published: April 2018

To study how an increase in axon number influences the number of glial cells in the mammalian optic nerve, we have analyzed a previously described transgenic mouse that expresses the human bcl-2 gene from a neuron-specific enolase promoter. In these mice, the normal postnatal loss of retinal ganglion cell axons is greatly decreased and, as a consequence, the number of axons in the optic nerve is increased by approximately 80% compared with wild-type mice. Remarkably, the numbers of oligodendrocytes, astrocytes, and microglial cells are all increased proportionally in the transgenic optic nerve. The increase in oligodendrocytes apparently results from both a decrease in normal oligodendrocyte death and an increase in oligodendrocyte precursor cell proliferation, whereas the increase in astrocytes apparently results from an increase in the proliferation of astrocyte lineage cells. Unexpectedly, the transgene is expressed in oligodendrocytes and astrocytes, but this does not seem to be responsible for the increased numbers of these cells. These findings indicate that developing neurons and glial cells can interact to adjust glial cell numbers appropriately when neuronal numbers are increased. We also show that the expression of the bcl-2 transgene in retinal ganglion cells protects the cell body from programmed cell death when the axon is cut, but it does not protect the isolated axon from Wallerian degeneration, even though the transgene-encoded protein is present in the axon.
Representation of spatial orientation by the intrinsic dynamics of the head-direction cell ensemble: a theory
Authors: Zhang K
Published: April 2018

The head-direction (HD) cells found in the limbic system in freely mov ing rats represent the instantaneous head direction of the animal in the horizontal plane regardless of the location of the animal. The internal direction represented by these cells uses both self-motion information for inertially based updating and familiar visual landmarks for calibration. Here, a model of the dynamics of the HD cell ensemble is presented. The stability of a localized static activity profile in the network and a dynamic shift mechanism are explained naturally by synaptic weight distribution components with even and odd symmetry, respectively. Under symmetric weights or symmetric reciprocal connections, a stable activity profile close to the known directional tuning curves will emerge. By adding a slight asymmetry to the weights, the activity profile will shift continuously without disturbances to its shape, and the shift speed can be controlled accurately by the strength of the odd-weight component. The generic formulation of the shift mechanism is determined uniquely within the current theoretical framework. The attractor dynamics of the system ensures modality- independence of the internal representation and facilitates the correction for cumulative error by the putative local-view detectors. The model offers a specific one-dimensional example of a computational mechanism in which a truly world-centered representation can be derived from observer-centered sensory inputs by integrating self-motion information.
Prepulses inhibit startle-induced reductions of extracellular dopamine in the nucleus accumbens of rat
Authors: Humby T , Wilkinson LS , Robbins TW , Geyer MA
Published: April 2018

In vivo brain microdialysis was used to monitor extracellular levels of dopamine (DA) in the nucleus accumbens (NAc) of rats during exposure to startling acoustic stimuli. Ten rats were prepared with guide cannulae into which dialysis probes were inserted 1 d before testing. Two to three hours after the start of perfusion, rats were placed into the startle chamber and exposed to a continuous 70 dB(A) background noise. Dialysis samples (2.0 microliter/min) were collected at 6 min intervals. Startle pulses (120 dB[A] noise) were presented in 20-trial blocks lasting 5 min. In some blocks, an 86 dB[A] prepulse preceded each of the 20 pulses by 100 msec, with the order of presentation of pulse-alone or prepulse + pulse blocks being counterbalanced between animals. Three to six sample periods intervened between stimulus- containing blocks. Monoamine and metabolite levels were measured using HPLC with electro-chemical detection. During the presentations of startling stimuli, DA levels in the NAc decreased relative to the immediately preceding 12 min baseline. This decrease in DA was maintained for only one additional sample period. By contrast, the presentation of prepulse + pulse trials failed to affect dialysate levels of NAc DA during or immediately after the stimulation. Thus, startling acoustic stimuli produce significant and transient decreases in dialysate levels of DA in the NAc. Furthermore, prepulse stimuli effectively inhibit these neurochemical effects of starting stimuli, in parallel with their establish ability to inhibit the amplitude the behavioral startle response.
Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles
Authors: Gilmor ML , Nash NR , Roghani A , Edwards RH , Yi H , Hersch SM , Levey AI
Published: April 2018

A cholinergic locus has recently been identified consisting of a unique mammalian genomic arrangement containing the genes for choline acetyltransferase (ChAT) and a putative vesicular acetylcholine transporter (VAChT). Although transcripts for ChAT and VAChT protein have been localized in cholinergic neurons, little is known about the encoded VAChT protein. Here we describe production of highly specific rabbit polyclonal antibodies, generated using a VAChT C- terminus/glutathione-S-transferase fusion protein, and immunological characterization of the native VAChT protein. These antibodies specifically recognized full-length recombinant VAChT expressed in transfected HeLa cells by Western blotting, with the prominent immunoreactive band at 55 kDa. In rat brain homogenates, a single VAChT- immunoreactive band of approximately 70 kDa was predominant in known areas of cholinergic innervation, including striatum, cortex, hippocampus,and amygdala. Light microscopic immunocytochemistry revealed reaction product in cholinergic cell groups but not in noncholinergic areas. More significantly, immunoreactivity was also concentrated in axonal fibers in many regions known to receive prominent cholinergic innervation, such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem regions. Electron microscopy using immunoperoxidase revealed that VAChT was localized in axon terminals, and using more precise immunogold techniques, to synaptic vesicles. In VAChT-positive perikarya, the immunogold particles were localized to the cytoplasmic face of the Golgi complex. These findings confirm that VAChT protein is expressed uniquely in cholinergic neurons, concentrated in synaptic vesicles, and at least for the C terminus, topologically oriented as predicted by models.
Membrane depolarization induces calcium-dependent secretion of tissue plasminogen activator
Authors: Gualandris A , Jones TE , Strickland S , Tsirka SE
Published: April 2018

Tissue plasminogen activator (tPA), a serine protease that converts inactive plasminogen to active plasmin, is produced in the rat and mouse hippocampus and participates in neuronal plasticity. To help define the role of tPA in the nervous system, we have analyzed the regulation of its expression in the neuronal cell line PC12. In control cultures, tPA activity is exclusively cell-associated, and no activity is measurable in the culture medium. When the cells are treated with depolarizing agents, such as KCI, tPA activity becomes detectable in the medium. The increased secreted tPA activity is not accompanied by an increase in tPA mRNA levels, and it is not blocked by protein synthesis inhibitors. In contrast, tPA release is abolished by Ca2+ channel blockers, suggesting that chemically induced membrane depolarization stimulates the secretion of preformed enzyme. Moreover, KCI has a similar effect in vivo when administered to the murine brain via an osmotic pump: tPA activity increases along the CA2-CA3 regions and dentate gyrus of the hippocampal formation. These results demonstrate a neuronal activity-dependent secretory mechanism that can rapidly increase the amount of tPA in neuronal tissue.
DNA strand breaks induced by sustained glutamate excitotoxicity in primary neuronal cultures
Authors: Didier M , Bursztajn S , Adamec E , Passani L , Nixon RA , Coyle JT , Wei JY , Berman SA
Published: April 2018

We developed a new approach to study single- and double-stranded DNA breaks during chronic, moderate excitotoxicity resulting from the inhibition of the glutamate transporter in cerebellar granule cell primary cultures. A 24 hr treatment of 2-week-old cultures with L-alpha- amino adipate (LAA), an inhibitor of the cerebellar glutamate uptake transporter, caused a gradual extracellular accumulation of endogenous glutamate that induced reversible morphological change of granule neurons but no neuronal cell death despite sustained, but moderate, elevations of the free intracellular calcium concentrations. Nick translation experiments on isolated nuclei or cells from cerebellar cultures chronically exposed to LAA revealed increased radioactive nucleotide incorporation indicative of DNA nicking. This LAA effect was dose-dependent and suppressed by NMDA receptor antagonists. Cultures treated for 24 hr with LAA and subjected to in situ nick translation showed an intense nuclear labeling of neurons but not glia, which could be abolished by MK801. A similar labeling was also observed in altered nuclei of granule neurons acutely exposed to high glutamate concentrations or undergoing an apoptotic cell death. Although the TUNEL labeling method detected no DNA double-strand breaks in LAA- treated cerebellar cultures, it displayed clear evidence of DNA damage during acute glutamate excitotoxicity or during apoptosis. However, Southern blot analysis of nuclear DNA revealed a DNA laddering only in apoptotic cell death. Our results demonstrate that DNA damage, characterized by DNA single-strand breaks, is an early event in chronic, moderate excitotoxicity. This type of DNA degradation, which appears before any nuclear morphological changes, is distinct from the massive DNA single- and/or double-strand damages observed during acute glutamate excitotoxicity or apoptosis.
Modulated expression of plasminogen activator system components in cultured cells from dissociated mouse dorsal root ganglia
Authors: Hayden SM , Seeds NW
Published: April 2018

The development and regeneration of the peripheral nervous system (PNS) is highly dependent on the migration of Schwann cells and the extension of axons toward their distant targets. Plasminogen activators (PAs) are associated with the surface of several cell types of neural origin where they are believed to mediate localized degradation of extracellular matrix, thus facilitating cell motility. In this study, we characterize the expression of tissue-type (tPA) and urokinase (uPA) PAs, as well as the urokinase cell surface receptor (uPAR) during differentiation of cultured cells from mouse dorsal root ganglia. During the first day in culture, the mRNA levels of all three components increase from 75- to 163-fold, as shown using a quantitative PCR method. By 72 hr, the mRNA levels decrease and approach basal levels. This transient increase is in direct correlation with the differentiation of neurons and Schwann cells and the formation of a neuritic network in these regenerating cultures. Densitometric analysis of gel zymographs demonstrates that the elevation in mRNA levels is accompanied by similar increases in the activity levels of tPA and uPA. Interestingly, in situ hybridization studies of the cultures show that tPA mRNA is restricted to small sensory neurons, whereas uPA mRNA is localized predominantly in large sensory neurons. uPAR mRNA is expressed by both neuronal subpopulations and, to a lesser extent, by Schwann cells and fibroblasts. Taken together, these results further support a role for the PA system in facilitating axon extension and cell migration during development and regeneration of the PNS.
Structure and Pharmacological Properties of a Molluscan Glutamate-Gated Cation Channel and its Likely Role in Feeding Behavior
Authors: Stühmer Thorsten , Amar Muriel , Harvey Robert J. , Bermudez Isabel , van Minnen Jan , Darlison Mark G.
Published: April 2018

We describe the isolation of a molluscan (Lymnaea stagnalis) full-length complementary DNA that encodes a mature polypeptide (which we have named Lym-eGluR2) with a predicted molecular weight of 105 kDa that exhibits 44–48% identity to the mammalian kainate-selective glutamate receptor GluR5, GluR6, and GluR7 subunits. Injection ofin vitro-transcribed RNA from this clone intoXenopus laevisoocytes results in the robust expression of homo-oligomeric cation channels that can be gated byl-glutamate (EC50 = 1.2 ± 0.3 μm) and several other glutamate receptor agonists; rank order of potency: glutamate ≫ kainate > ibotenate > AMPA. These currents can be blocked by the mammalian non-NMDA receptor antagonists 6,7-dinitroquinoxaline-2,3-dione, 6-cyano-7-nitroquinoxaline-2,3-dione, and 1-(4-chlorobenzoyl)piperazine-2,3-dicarboxylic acid. Ionic-replacement experiments have shown that the agonist-induced current is carried entirely by sodium and potassium ions.In situhybridization has revealed that the Lym-eGluR2 transcript is present in all 11 ganglia of theLymnaeaCNS, including the 4-cluster motorneurons within the paired buccal ganglia. The pharmacological properties and deduced location of Lym-eGluR2 are entirely consistent with it being (a component of) the receptor, which has been identified previously on buccal motorneurons, that mediates the excitatory effects of glutamate released from neurons within the feeding central pattern generator.
Inhibition of cGMP Breakdown Promotes the Induction of Cerebellar Long-Term Depression
Authors: Hartell Nick A.
Published: April 2018

The effects of the nonspecific cyclic nucleotide inhibitors 1-methyl-3-isobutylxanthine (IBMX) and dipyridamole, and the cGMP-specific phosphodiesterase inhibitor Zaprinast were studied on parallel fiber–Purkinje cell synaptic responses in rat cerebellar slices. Bath application of all three compounds, at concentrations shown to inhibit cGMP breakdown, led to stable and robust long-term depression of PF responses. Injections of dipyridamole directly into the Purkinje cell dendrites were similarly effective as bath applications, confirming a postsynaptic site of action. Inhibitors of both protein kinase G and C and also the metabotropic glutamate receptor antagonist MCPG completely prevented the induction of LTD by dipyridamole and Zaprinast. The extent of phosphodiesterase-induced synaptic depression was dependent on the frequency of parallel fiber stimulation, and this form of LTD both occluded and was occluded by LTD induced by pairing parallel and climbing fiber inputs. The degree of LTD induced by IBMX was dose-dependent, and also required PKC and PKG activity, but was preceded by a large, transient potentiation of parallel fiber responses occurring by a postsynaptic mechanism independent of cGMP. These data not only confirm that cGMP is capable of inducing cerebellar LTD when paired with parallel fiber stimulation but indicate that cGMP is an endogenous intermediate in this form of synaptic plasticity.
INDO-1 Measurements of Absolute Resting and Light-Induced Ca2+Concentration inDrosophilaPhotoreceptors
Authors: Hardie Roger C.
Published: April 2018

Absolute Ca2+levels in dissociatedDrosophilaphotoreceptors were measured using the ratiometric indicator dye INDO-1 loaded via patch pipettes, which simultaneously recorded whole-cell currents. In wild-type photoreceptors, the ultraviolet (UV) excitation light used to measure fluorescence elicited a massive Ca2+influx that saturated the dye (>10 μmCa2+), but lagged the electrical response by 2.8 msec. Resting Ca2+levels in the dark, measured during the latent period before the response, averaged 160 nmin normal Ringer’s (1.5 mmCa2+). Ca2+increases in response to weak illumination were estimated (1) by using a weak adapting stimulus before the UV excitation light and measuring Ca2+during the latent period; and (2) by usingninaEmutants with greatly reduced rhodopsin levels. Ca2+rose linearly as a function of the time integral of the light-sensitive current with a slope of 2.7 nm/pC. In the transient receptor potential (trp) mutant, which lacks a putative light-sensitive channel subunit, the slope was only 1.1 nm/pC, indicating a 2.5-fold reduction in the fractional Ca2+current. From these data, it can also be estimated that >99% of the Ca2+influx is effectively buffered by the cell. In Ca2+-free Ringer’s, resting cytosolic Ca2+was reduced (to 30–70 nm), but contrary to previous reports, significant light-induced increases (∼250 nm) could be elicited. This rise was reduced to <20 nmwhen extracellular Na+was replaced withN-methyl-d-glucamine, suggesting that it could be attributed to Na+influx altering the Na/Ca exchanger equilibrium. It is concluded that any light-induced release from internal stores amounts to <20 nm.
The Divergent Homeobox GenePBX1Is Expressed in the Postnatal Subventricular Zone and Interneurons of the Olfactory Bulb
Authors: Redmond Lori , Hockfield Susan , Morabito Maria A.
Published: April 2018

In the mammalian brain, an important phase of neurogenesis occurs postnatally in the subventricular zone (SVZ). This region consists of a heterogeneous population of cells, some mitotically active, others postmitotic. A subset of mitotically active SVZ precursor cells gives rise to a population of neurons that migrates over a long distance to their final destination, the olfactory bulb. Other SVZ precursor cells continue to proliferate or undergo cell death. The combination of genes that regulates proliferation and cell fate determination of SVZ precursor cells remains to be identified. We have used the rat homolog of the human homeobox genePBX1in Northern analysis andin situhybridization studies to determine the temporal and regional localization ofPBX1expression during embryonic and postnatal rat brain development.PBX1is expressed embryonically in the telencephalon. In addition, it is expressed at high levels postnatally in the SVZ, in the migratory pathway to the olfactory bulb, and in the layers of the olfactory bulb that are the targets of these migratory neurons. Combiningin situhybridization forPBX1with immunostaining for markers of cell proliferation (PCNA), postmitotic neurons (class III β-tubulin), and glia (GFAP), we show that SVZ proliferating cells and their neuronal progeny express ratPBX1mRNA, whereas glial cells do not express detectable levels ofPBX1. The expression ofPBX1in SVZ precursor cells and postmitotic neurons suggests a role forPBX1in the generation of olfactory bulb interneurons and in mammalian neurogenesis.
The Dynamics of Dendritic Structure in Developing Hippocampal Slices
Authors: Dailey Michael E. , Smith Stephen J
Published: April 2018

Time-lapse fluorescence confocal microscopy was used to directly visualize the formation and dynamics of postsynaptic target structures (i.e., dendritic branches and spines) on pyramidal neurons within developing hippocampal tissue slices. Within a 2 week period of time, pyramidal neurons in cultured slices derived from early postnatal rat (postnatal days 2–7) developed complex dendritic arbors bearing numerous postsynaptic spines. At early stages (1–2 din vitro), many fine filopodial protrusions on dendrite shafts rapidly extended (maximum rate ∼2.5 μm/min) and retracted (median filopodial lifetime, 10 min), but some filopodia transformed into growth cones and nascent dendrite branches. As dendritic arbors matured, the population of fleeting lateral filopodia was replaced by spine-like structures having a low rate of turnover. This developmental progression involved a transitional stage in which dendrites were dominated by persistent (up to 22 hr) but dynamic spiny protrusions (i.e., protospines) that showed substantial changes in length and shape on a timescale of minutes. These observations reveal a highly dynamic state of postsynaptic target structures that may actively contribute to the formation and plasticity of synaptic connections during CNS development.
High-Frequency Oscillations in the Output Networks of the Hippocampal–Entorhinal Axis of the Freely Behaving Rat
Authors: Chrobak James J. , Buzsáki Gyorgy
Published: April 2018

Population bursts of the CA3 network, which occur during eating, drinking, awake immobility, and slow-wave sleep, produce a large field excitatory postsynaptic potential throughout stratum radiatum of the CA1 field (sharp wave). The CA3 burst sets into motion a short-lived, dynamic interaction between CA1 pyramidal cells and interneurons, the product of which is a 200 Hz oscillatory field potential (ripple) and phase-related discharge of the CA1 network. Although many CA1 pyramidal neurons discharge during the time frame (50–100 msec) of each sharp wave, each wave of a ripple (∼5 msec) reflects the synchronization of more discrete subsets of CA1 neurons.

When we used multi-site recordings in freely behaving rats, we observed ripples throughout the longitudinal extent (∼4–5 mm) of the dorsal CA1 region that were coherent for multiple cycles of each ripple. High-frequency ripples were also observed throughout the hippocampal–entorhinal output pathway that were concurrent but less coherent on a cycle-by-cycle basis. Single and multiunit neuronal activity was phase-related to local ripples throughout the hippocampal–entorhinal output pathway. Entorhinal ripples occurred 5–30 msec after the CA1 ripples and were related to the occurrence of an entorhinal sharp wave. Thus, during each hippocampal sharp wave, there is a powerful synchronization among the neuronal networks that connect the hippocampus to the neocortex. We suggest that this population interaction (1) biologically constrains theoretical models of hippocampal function and dysfunction and (2) has the capacity to support an “off-line” memory consolidation process.
Mouse Model of Hyperkinesis Implicates SNAP-25 in Behavioral Regulation
Authors: Hess Ellen J. , Collins Katherine A. , Wilson Michael C.
Published: April 2018

Although hyperkinesis is expressed in several neurological disorders, the biological basis of this phenotype is unknown. The mouse mutant coloboma (Cm/+) exhibits profound spontaneous locomotor hyperactivity resulting from a deletion mutation. This deletion encompasses several genes includingSnap, which encodes SNAP-25, a nerve terminal protein involved in neurotransmitter release. Administration of amphetamine, a drug that acts presynaptically, markedly reduced the locomotor activity in coloboma mice but increased the activity of control mice implicating presynaptic function in the behavioral abnormality. In contrast, the psychostimulant methylphenidate increased locomotor activity in both coloboma and control mice. When a transgene encoding SNAP-25 was bred into the coloboma strain to complement theSnapdeletion, the hyperactivity expressed by these mice was rescued, returning these corrected mice to normal levels of locomotor activity. These results demonstrate that the hyperactivity exhibited by these mice is the result of abnormalities in presynaptic function specifically attributable to deficits in SNAP-25 expression.
Increased Expression of IL-1β Converting Enzyme in Hippocampus after Ischemia: Selective Localization in Microglia
Authors: Bhat Ratan V. , DiRocco Richard , Marcy Val R. , Flood Dorothy G. , Zhu Yuan , Dobrzanski Pawel , Siman Robert , Scott Richard , Contreras Patricia C. , Miller Matthew
Published: April 2018

Although the interleukin-1β converting enzyme (ICE)/CED-3 family of proteases has been implicated recently in neuronal cell deathin vitroandin ovo, the role of specific genes belonging to this family in cell death in the nervous system remains unknown. To address this question, we examined thein vivoexpression of one of these genes,Ice, after global forebrain ischemia in gerbils. Using RT-PCR and Western immunoblot techniques, we detected an increase in the mRNA and protein expression of ICE in hippocampus during a period of 4 d after ischemia. Chromatin condensation was observed in CA1 neurons within 2 d after ischemia. Internucleosomal DNA fragmentation and apoptotic bodies were observed between 3 and 4 d after ischemia, a period during which CA1 neuronal death is maximal. In nonischemic brains, ICE-like immunoreactivity was relatively low in CA1 pyramidal neurons but high in scattered hippocampal interneurons. After ischemia, ICE-like immunoreactivity was not altered in these neurons. ICE-like immunoreactivity, however, was observed in microglial cells in the regions adjacent to the CA1 layer as early as 2 d after ischemic insult. The increase in ICE-like immunoreactivity was robust at 4 d after ischemia, a period that correlates with the DNA fragmentation observed in hippocampal homogenates of ischemic brains. These results provide the first evidence for the localization and induction of ICE expressionin vivoafter ischemia and suggest an indirect role for ICE in ischemic damage through mediation of an inflammatory response.
Ultrastructural Immunocytochemical Localization of μ-Opioid Receptors in Rat Nucleus Accumbens: Extrasynaptic Plasmalemmal Distribution and Association with Leu5-Enkephalin
Authors: Svingos Adena L. , Moriwaki Akiyoshi , Wang Jia Bei , Uhl George R. , Pickel Virginia M.
Published: April 2018

μ-Opioid receptors and their endogenous ligands, including Leu5-enkephalin (LE), are distributed abundantly in the nucleus accumbens (NAC), a region implicated in mechanisms of opiate reinforcement. We used immunoperoxidase and/or immunogold–silver methods to define ultrastructural sites for functions ascribed to μ-opioid receptors and potential sites for activation by LE in the NAC. An antipeptide antibody raised against an 18 amino acid sequence of the cloned μ-opioid receptor (MOR) C terminus showed that MOR-like immunoreactivity (MOR-LI) was localized predominantly to extrasynaptic sites along neuronal plasma membranes. The majority of neuronal profiles containing MOR-LI were dendrites and dendritic spines. The dendritic plasma membranes immunolabeled for MOR were near sites of synaptic input from LE-labeled terminals and other unlabeled terminals forming either inhibitory or excitatory type synapses. Unmyelinated axons and axon terminals were also intensely but less frequently immunoreactive for MOR. Observed sites for potential axonal associations with LE included coexistence of MOR and LE within the same terminal, as well as close appositions between differentially labeled axons. Astrocytic processes rarely contained detectable MOR-LI, but also were sometimes observed in apposition to LE-labeled terminals. We conclude that in the rat NAC, MOR is localized prominently to extrasynaptic neuronal and more rarely to glial plasma membranes that are readily accessible to released LE and possibly other opioid peptides and opiate drugs. The close affiliation of MOR with spines receiving excitatory synapses and dendrites receiving inhibitory synapses provides the first direct morphological evidence that MOR selectively modulates postsynaptic responses to cortical and other afferents.
Binaural Cross-Correlation Predicts the Responses of Neurons in the Owl’s Auditory Space Map under Conditions Simulating Summing Localization
Authors: Keller Clifford H. , Takahashi Terry T.
Published: April 2018

Summing localization describes the perceptions of human listeners to two identical sounds from different locations presented with delays of 0–1 msec. Usually a single source is perceived to be located between the two actual source locations, biased toward the earlier source. We studied neuronal responses within the space map of the barn owl to sounds presented with this same paradigm. The owl’s primary cue for localization along the azimuth, interaural time difference (ITD), is based on a cross-correlation-like treatment of the signals arriving at each ear. The output of this cross-correlation is displayed as neural activity across the auditory space map in the external nucleus of the owl’s inferior colliculus. Because the ear input signals reflect the physical summing of the signals generated by each speaker, we first recorded the sounds at each ear and computed their cross-correlations at various interstimulus delays. The resulting binaural cross-correlation surface strongly resembles the pattern of activity across the space map inferred from recordings of single space-specific neurons. Four peaks are observed in the cross-correlation surface for any nonzero delay. One peak occurs at the correlation delay equal to the ITD of each speaker. Two additional peaks reflect “phantom sources” occurring at correlation delays that match the signal of the left speaker in one ear with the signal of the right speaker in the other ear. At zero delay, the two phantom peaks coincide. The surface features are complicated further by the interactions of the various correlation peaks.
Visual Motion-Detection Circuits in Flies: Small-Field Retinotopic Elements Responding to Motion Are Evolutionarily Conserved across Taxa
Authors: Buschbeck Elke K. , Strausfeld Nicholas J.
Published: April 2018

The Hassenstein–Reichardt autocorrelation model for motion computation was derived originally from studies of optomotor turning reactions of beetles and further refined from studies of houseflies. Its application for explaining a variety of optokinetic behaviors in other insects assumes that neural correlates to the model are principally similar across taxa. This account examines whether this assumption is warranted. The results demonstrate that an evolutionarily conserved subset of neurons corresponds to small retinotopic neurons implicated in motion-detecting circuits that link the retina to motion-sensitive neuropils of the lobula plate. The occurrence of these neurons in basal groups suggests that they must have evolved at least 240 million years before the present time. Functional contiguity among the neurons is suggested by their having layer relationships that are independent of taxon-specific variations such as medulla stratification, the shape of terminals or dendrites, the presence of other taxon-specific neurons, or the absence of orientation-specific motion-sensitive levels in the lobula plate.
Major Histocompatibility Class II Molecules in the CNS: Increased Microglial Expression at the Onset of Narcolepsy in a Canine Model
Authors: Tafti Mehdi , Nishino Seiji , Aldrich Michael S. , Liao Wennie , Dement William C. , Mignot Emmanuel
Published: April 2018

Human narcolepsy is a neurological disorder known to be closely associated with HLA-DR2 and DQB1*0602. Because most autoimmune diseases are HLA-associated, a similar mechanism has been proposed for narcolepsy. However, neither systemic nor CNS evidence of an autoimmune abnormality has ever been reported. In this study, major histocompatibility (MHC) class I and class II expression was studied in the CNS of human and canine narcoleptics using immunohistochemistry and Northern analysis. Results indicate that canine narcolepsy is associated with a significant increase of MHC class II expression by the microglia. Moreover, the highest values were found between 3 and 8 months of age, strikingly concomitant to the development of narcolepsy in the canine model. In humans, class II expression was not found significantly different between control and narcoleptic subjects. This result could be explained by the old age of the subjects (69.86 ± 5.31 and 68.36 ± 4.74 years in narcoleptics and controls, respectively), because class II expression is significantly correlated with age in both humans and dogs. For the first time, this study demonstrated that the expression of MHC class II molecules in the CNS is age-dependent and that a consistent increase of their expression by the microglia might be critically involved in the development of narcolepsy.
Protection of the Neostriatum against Excitotoxic Damage by Neurotrophin-Producing, Genetically Modified Neural Stem Cells
Authors: Martı́nez-Serrano Alberto , Björklund Anders
Published: April 2018

Huntington’s disease is a progressive neurodegenerative disease that affects the striatum, above all, the GABAergic striatal projection neurons. In the present study, we have explored the use of genetically modified neural stem cell lines producing nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) as a means to protect the striatal neurons against excitotoxic damage after transplantation to the striatum, 1 week before the injection of quinolinic acid into the same area. One month after the lesion, striatal degeneration, lesion size, and loss of DARPP-32-positive projection neurons were only slightly affected by the BDNF-secreting cells, but substantially prevented when NGF-producing stem cells were used as a source of exogenous trophic factor; innervation of the target fields (pars reticulata of the substantia nigra and the globus pallidus) was preserved as well. Cholinergic striatal interneurons (choline acetyltransferase-immunoreactive) were affected by the lesion and completely rescued by the NGF-transduced cells. The astroglial and microglial reactions to the excitotoxic lesion were substantially reduced in the striata, which had received transplants of NGF-producing cells. The generalized protective effects of the NGF-producing cell grafts in this model are discussed in the context of an indirect action preventing the development of toxicity mediated by cellular elements in the host striatum in response to the excitotoxin. We conclude that continuous supply of trophic factors by means of genetically modified neural stem cells represents a highly effective procedure to counteract neuronal degeneration in the excitotoxically lesioned striatum.
Functional NMDA Receptors Are Transiently Active and Support the Survival of Purkinje Cells in Culture
Authors: Yuzaki Michisuke , Forrest Douglas , Verselis Lynne M. , Sun Shaiu C. , Curran Tom , Connor John A.
Published: April 2018

Conflicting evidence exists concerning the activity of NMDA receptors (NMDARs) in cerebellar Purkinje cells and their possible functions. To investigate the activity of NMDARs, we used whole-cell recording on immunocytochemically identified Purkinje cells in primary culture. In addition, we used mice with a disrupted NMDAR1 gene that lack functional NMDARs (NR1−/−) to assess the physiological role of NMDARs. In cultures from normal mice, NMDA-mediated currents were detected in all identified Purkinje cells at 4 din vitro(div). After 14 d, however, NMDA responses were reduced in amplitude, whereas the responses to kainate and glutamate increased steadily in amplitude. In addition, the NMDA-induced current displayed a pronounced desensitization at these later stages; peak current declined to zero during steady application of NMDA. At 7 div, the number of surviving Purkinje cells was less in cultures treated with NMDA antagonists, and their survival was dose-dependent. Purkinje cell survival was correspondingly poorer in cultures from the NR1−/− mice than in wild-type controls, suggesting that NMDAR activity enhances the survival of Purkinje cellsin vitro. The addition of moderate doses of NMDA promoted the survival of wild-type Purkinje cells in the presence of tetrodotoxin. Feeder layers of cerebellar granule cells derived from wild-type or NR1−/− mice promoted survival of Purkinje cells to a similar degree, suggesting that the NMDAR in Purkinje cells, but not in other cells, is directly involved in Purkinje cell viability. The results demonstrate that NMDARs transiently produce membrane current in Purkinje cells and may serve as one of the epigenetic factors that support the survival of Purkinje cellsin vitro.
Synchronous Onset of NGF and TrkA Survival Dependence in Developing Dorsal Root Ganglia
Authors: White Fletcher A. , Silos-Santiago Inmaculada , Molliver Derek C. , Nishimura Merry , Phillips Heidi , Barbacid Mariano , Snider William D.
Published: April 2018

Determinations of dorsal root ganglion (DRG) neuron loss in nerve growth factor (NGF) and neurotrophin-3 (NT-3) null mutant mice have supported the concept that neurons can switch neurotrophin dependence by revealing that many neurons must require both of these factors acting either sequentially or simultaneously during development. The situation is complex, however, in thatNT-3(−/−)mutant mice show far greater neuron loss than mice deficient in the NT-3 receptor TrkC, suggesting that NT-3 may support many DRG neurons via actions on the NGF receptor TrkA. To assess the possibility of ligand-receptor cross-talk as a developmental mechanism, we have compared the onset of survival dependence of lumbar DRG neurons on NT-3, TrkC, NGF, and TrkA signaling in mice deficient in these molecules as a result of gene targeting. At embryonic day 11.5 (E11.5), virtually all lumbar DRG cells express TrkC mRNA and many require NT-3 and TrkC signaling for survival. In contrast, although many lumbar DRG cells also express TrkA at E11.5, there is little survival dependence on TrkA signaling. By E13.5, most lumbar DRG cells have downregulated TrkC mRNA. The onset of survival dependence on NGF and TrkA-signaling is concurrent and of equal magnitude at E13.5, demonstrating that NT-3 alone does not support DRG neurons via TrkA, nor can NT-3 compensate for the loss of NGF. We conclude that many murine DRG cells require NT-3 for survival before exhibiting NGF dependence and that NT-3 activation of TrkA is unimportant to these early NT-3 survival-promoting actions. We suggest that the discrepancy in cell loss betweenNT-3(−/−)andtrkC(−/−)mutants is attributable to the ability of NT-3 to support DRG neurons via TrkA in the artificial situation where TrkC is absent.
Functional GABAergic Synaptic Connection in Neonatal Mouse Barrel Cortex
Authors: Agmon Ariel , Hollrigel Greg , O’Dowd Diane K.
Published: April 2018

Intracortical inhibition is crucial to proper functioning of the mature neocortex, yet, paradoxically, is reported to be rare or absent in the neonatal animal. We reexamined this issue by recording whole-cell postsynaptic currents (PSCs) of barrel cortex neurons in thalamocortical brain slices from neonatal mice. Monosynaptic, excitatory thalamocortical responses were elicited in layers V/VI neurons as early as postnatal day 0 (P0, the first 24 hr after birth) and in presumptive layer IV as early as P2. At very low stimulation frequencies, the monosynaptic response was invariably followed by a prolonged (up to 1 sec) synaptic barrage, which fatigued at stimulus repetition rates of 2/min or higher. This barrage consisted of postsynaptic responses to spiking activity in neighboring cortical cells, because (1) it could also be evoked by intracortical stimulation in coronal slices and (2) it was abolished by antagonists to NMDA receptors (NMDARs), even when NMDARs on the recorded cell were under a voltage-dependent block. Some of the larger polysynaptic events changed polarity at a negative reversal potential and were blocked by GABAAreceptor (GABAAR) antagonists, with a concurrent enhancement of the extracellular field potential, indicating that they were GABAAR- mediated, Cl-dependent inhibitory PSCs (IPSCs). We conclude that a network of functional intracortical GABAAR-mediated synaptic connections exists from the earliest postnatal ages, although it gives rise to responses that differ from mature IPSCs in reversal potential and latency.
Potassium Deprivation-Induced Apoptosis of Cerebellar Granule Neurons: A Sequential Requirement for New mRNA and Protein Synthesis, ICE-Like Protease Activity, and Reactive Oxygen Species
Authors: Schulz Jörg B. , Weller Michael , Klockgether Thomas
Published: April 2018

Potassium (K+) deprivation-induced apoptosis of cerebellar granule neurons requires new mRNA and protein synthesis. Using a fluorogenic substrate for interleukin-1β converting enzyme (ICE), we show that K+deprivation of cerebellar granule neurons induces cycloheximide-sensitive ICE-like protease activity. A peptide inhibitor of ICE-like protease activity, Ac-YVAD-chloromethylketone (Ac-YVAD-CMK), prevents K+deprivation-induced apoptosis. Further, reactive oxygen species (ROS) are essential mediators of K+deprivation-induced apoptosis of cerebellar granule neurons because neuronal death is also blocked by superoxide dismutase,N-acetyl-l-cysteine, and free radical spin traps. Using fluorescent assays, we show that ROS production after K+deprivation is blocked by actinomycin D, cycloheximide, and Ac-YVAD-CMK, suggesting that ROS act downstream of gene transcription, mRNA translation, and ICE activation. Taken together, we show that new mRNA and protein synthesis, activation of ICE-like proteases, and ROS production are sequential events in K+deprivation-induced apoptosis of cerebellar granule neurons.
Compartmental Localization of a Metabotropic Glutamate Receptor (mGluR7): Two Different Active Sites at a Retinal Synapse
Authors: Brandstätter Johann Helmut , Koulen Peter , Kuhn Rainer , van der Putten Herman , Wässle Heinz
Published: April 2018

The distribution of the metabotropic glutamate receptor 7 (mGluR7) was studied in the rat retina using a specific antiserum. Punctate immunofluorescence that corresponded to synaptic localization was present exclusively in the inner plexiform layer. Double-labeling experiments suggested that mGluR7 is expressed at the synaptic terminals of certain cone bipolar cells. Electron microscopy showed that mGluR7 was present both presynaptically, as an autoreceptor in cone bipolar cell ribbon synapses, and postsynaptically in amacrine cells. There are usually two postsynaptic processes at a bipolar cell ribbon synapse; however, the presynaptic aggregation of mGluR7 was restricted to one half of the active zone and therefore was opposed to only one of the postsynaptic processes. This selective localization of mGluR7 could differentially regulate the glutamate release from the ribbon synapse, thus leading to a differential activation of the postsynaptic neurons.
Use-Dependent Exaggeration of Neuronal Injury after Unilateral Sensorimotor Cortex Lesions
Authors: Kozlowski Dorothy A. , James Debra C. , Schallert Timothy
Published: April 2018

Unilateral injury to the forelimb representation area of the sensorimotor cortex (FL-SMC) in adult rats causes over-reliance on the unimpaired forelimb for postural-motor movements, as well as overgrowth of layer V pyramidal cell dendrites in the homotopic cortex of the noninjured hemisphere. The overgrowth appears to be use-dependent because it can be prevented by restricting movements of the unimpaired forelimb. Additionally, restricting the unimpaired forelimb in animals with FL-SMC damage results in significantly greater behavioral dysfunction when examined 2 d after cast removal (compared to that after impaired-limb immobilization, or no limb immobilization). In the present study, the long-term behavioral and anatomical effects of limb immobilization were examined. Animals with FL-SMC lesions were fitted with casts immediately after the lesion that immobilized the impaired forelimb, the unimpaired forelimb, or neither forelimb for 15 d. Immobilization of the nonimpaired forelimb resulted in chronic prevention of dendritic growth and severe and chronic behavioral deficits. In addition, immobilization of the nonimpaired forelimb resulted in a dramatic exaggeration of the neuronal injury, presumably attributable to forced overuse of the impaired limb. Immobilization of the impaired forelimb resulted in no detectable neural changes and in only slightly increased and longer-lasting behavioral asymmetries (compared to nonimmobilized, lesioned animals), presumably attributable to mild disuse of the impaired limb. Immobilization of a single forelimb in nonlesioned rats resulted in no significant behavioral or anatomical changes. Together, these results suggest that although behavioral experience can enhance neural growth after brain injury, the region surrounding the injury may be vulnerable to behavioral pressure during the early postlesion period.