bluuebunny/crossref_metadata_2025 huggingface dataset:
3-Hydroxyanthranilic acid oxygenase-containing astrocytic processes surround glutamate-containing axon terminals in the rat striatum
Authors: Roberts RC , McCarthy KE , Du F , Ottersen OP , Okuno E , Schwarcz R
Published: April 2018

Glutamate, the major transmitter of the corticostriatal pathway, is present in abundance in the striatum. 3-Hydroxyanthranilic acid oxygenase (3HAO) is the biosynthetic enzyme for quinolinic acid, an endogenous agonist of the NMDA glutamate receptor subtype and a potent neurotoxin. In order to explore the anatomical basis of possible functional interactions between glutamate and quinolinic acid in the rat striatum, pre- and postembedding immunocytochemical methods were used to localize 3HAO immunoreactivity (-i) and glutamate-i at the electron microscopic level. In accordance with previous light microscopic and biochemical studies, 3HAO-i was detected exclusively in astrocytes throughout the striatum. Notably, 3HAO-i was present in fine- caliber glial processes that often surrounded or abutted synaptic profiles, both asymmetric and symmetric. Glutamate-i was heavily deposited (3–13-fold higher gold particle density than tissue average) in axon terminals forming asymmetric synapses with spines and, occasionally, dendrites. In contrast, terminals forming symmetric synapses, dendrites, neuronal somata, and glial cells contained significantly less labeling than terminals forming asymmetric synapses. In double-labeled material, 3HAO-i was observed in glial processes that partially surrounded or were adjacent to glutamate-labeled terminals forming asymmetric synapses. 3HAO-labeled glial processes were also adjacent to unlabeled terminals forming symmetric synapses. Since quinolinic acid is known to enter the extracellular compartment readily, these results suggest that astrocytic quinolinic acid may participate in the regulation of glutamatergic neurotransmission in the rat striatum.
delta-Opioid receptor immunoreactivity: distribution in brainstem and spinal cord, and relationship to biogenic amines and enkephalin
Authors: Arvidsson U , Dado RJ , Riedl M , Lee JH , Law PY , Loh HH , Elde R , Wessendorf MW
Published: April 2018

We have recently developed antisera which recognize epitopes of the cloned delta-opioid receptor (DOR; Dado et al., 1993). In the present report we have further characterized these antisera, and raised additional antisera in rats. We used these antisera to determine the distribution of DOR-like immunoreactivity (-Ll) in rat spinal cord and brainstem in relation to serotoninergic, noradrenergic, and enkephalinergic neurons. We found DOR-Ll in fibers and varicosities distributed throughout the spinal cord gray matter, with highest densities in the superficial dorsal horn, in autonomic regions, around the central canal as well as in the ventral horn motor nuclei. In the brainstem a dense innervation of DOR-immunoreactive (-IR) fibers was found in several nuclei such as spinal trigeminal nuclei, midline raphe nuclei, parabrachial nuclei, periaqueductal gray matter (PAG), interpeduncular nucleus, ans substantia nigra. A group of DOR-positive cells was seen in the laterodorsal tegmental nucleus. In addition, a few DOR-IR cell bodies were demonstrated in the parabrachial nuclei, interpeduncular nucleus, PAG, and superior and inferior colliculi as well as around the central canal in the spinal cord. All DOR-positive cells showed a punctuate staining pattern within the cytoplasm of the cell body and in primary dendrites. No plasma membrane staining of cells or dendrites could be demonstrated using the DOR antisera. Double- labeling experiments for DOR and 5-hydroxytryptamine (5HT, serotonin) revealed that some 5HT-IR neurons in the raphe complex were surrounded by DOR-IR fibers. In the spinal cord a high degree of coexistence was found between DOR and 5HT in nerve fibers and varicosities in the neuropil around the motoneurons and in lamina V of the dorsal horn. In autonomic regions of the spinal cord, a low degree of colocalization was seen between DOR and 5HT; in the superficial dorsal horn no coexistence was found. Tyrosine hydroxylase (TH)-positive neurons in the brainstem (in the A5 area, locus coeruleus, and A7 area) were apposed by DOR-positive fibers. However, no coexistence could be seen between DOR and TH in any part of the spinal cord. A close relation, but no coexistence, was observed between DOR- and enkephalin (ENK)-IR fibers in the spinal cord ventral horn; in the intermediolateral nucleus a low degree of colocalization was observed. Thus, a delta- opioid receptor may affect the activity of descending serotoninergic and noradrenergic neurons by means of modulating the release of neurotransmitters from afferents to these neurons.(ABSTRACT TRUNCATED AT 400 WORDS)
Extracellular signal-regulated protein kinases (ERKs) and ERK kinase (MEK) in brain: regional distribution and regulation by chronic morphine
Authors: Ortiz J , Harris HW , Guitart X , Terwilliger RZ , Haycock JW , Nestler EJ
Published: April 2018

Quantitative blot immunolabeling techniques were used to determine the concentrations of ERK1 (M(r) 44 kDa) and ERK2 (M(r) 42 kDa), the two major extracellular signal-regulated protein kinases, in different regions of rat brain. The aggregate ERK concentrations (ERK1 and ERK2) were relatively high in each of the brain regions studied, ranging from approximately 0.35 ng/microgram protein in cerebellum to approximately 1.2 ng/microgram protein in nucleus accumbens. However, differences in the regional distributions of ERK1 and ERK2 resulted in ratios of their relative abundance that differed by close to 10-fold among the regions studied. The ratios of ERK1 protein to ERK2 protein varied along a rostral-caudal gradient from a low of 0.16 in frontal cortex to a high of 1.5 in pons/medulla. In hypotonic homogenates from regions at either extreme of the gradient, ERK1 and ERK2 were both found to be predominantly (> 80%) soluble. In subcellular fractions prepared from sucrose homogenates of frontal cortex and pons/medulla, both ERK1 and ERK2 were enriched in the synaptosomal and cytosolic fractions, whereas ERK2 was also enriched in the microsomal fraction. By contrast, in subfractions containing purified nuclei, levels of ERK1 and ERK2 were about one-third of those seen in homogenates and, in subfractions enriched in mitochondria, both ERK1 and ERK2 were barely detectable. The catalytic activity of the ERKs paralleled their protein levels in all of the brain regions except the hippocampus, in which the activity and phosphotyrosine content were disproportionately high. As a possible explanation for this apparent disparity, the regional distribution of ERK kinase (MEK), which phosphorylates and activates the ERKs, was also investigated. The levels of immunoreactivity of the M(r) 45 kDa ERK kinase band differed by about threefold among the brain regions, with the highest levels being present in nucleus accumbens, hippocampus, substantia nigra, and caudate/putamen. Therefore, a higher concentration of ERK kinase immunoreactivity did not appear to account for the disproportionate levels of ERK activity and phosphotyrosine content in the hippocampus. Potential regulation of ERK and ERK kinase levels was also investigated in rats subjected to chronic morphine treatment. ERK1 and ERK2 levels were increased selectively in locus coeruleus and caudate/putamen after chronic morphine treatment, whereas ERK kinase immunoreactivity remained unchanged in all of the brain regions analyzed. In summary, the regional differences in ERK and ERK kinase expression and the region-specific regulation of ERK expression suggest that ERK-related signaling may play an important role in CNS function and its adaptive responses.
The CNS-specific hyaluronan-binding protein BEHAB is expressed in ventricular zones coincident with gliogenesis
Authors: Jaworski DM , Kelly GM , Hockfield S
Published: April 2018

Hyaluronan (HA) is a ubiquitous component of extracellular matrices, and in several systems it plays a central role in regulating cellular proliferation and differentiation. Cell, or tissue,-specific functions of HA are likely to be mediated by cell, or tissue,-specific HA-binding proteins. We previously hyaluronan-binding protein from rat and cat (Jaworski et al., 1994). In view of the potential role of HA in neural differentiation, we examined the expression of BEHAB during late embryonic and early postnatal development of the rat. BEHAB is expressed at very high levels in ventricular zones throughout the neuraxis. Expression is first detected at embryonic day 15 (E15) in the spinal cord, and is detected at progressively more rostral levels at later ages. BEHAB expression, like other features of neural development, follows both caudal-to-rostral and ventral-to-dorsal gradients. The timing of BEHAB expression parallels the timing of the generation of glial cells. In all areas of the CNS examined, BEHAB expression begins after the peak of neurogenesis and coincident with gliogenesis. The regulation of proliferation and differentiation by HA in other tissues, together with the expression of BEHAB in zones of mitotic activity coincident with the generation of glia, suggests that the extracellular matrix protein encoded by BEHAB could play a role in the generation or differentiation of CNS glia.
Optical signals from neurons with internally applied voltage-sensitive dyes
Authors: Antic S , Zecevic D
Published: April 2018

We carried out experiments to monitor optically the generation and spread of action potentials and subthreshold potentials in the processes of individual neurons in ganglia of the snail, Helix aspersa. The neurons were selectively stained by intracellular pressure injection of voltage-sensitive dyes. Optical signals were detected by a system for fast, multiple-site optical monitoring, utilizing a silicon photodiode array. After testing 30 voltage-sensitive dyes using absorption, we concluded that this mode was probably not sensitive enough to allow monitoring neuronal signals from distal processes. Satisfactory signals were obtained in fluorescence measurements using a newly synthesized styryl dye, JPW1114, specifically designed for intracellular application. There was an improvement in sensitivity (as defined by the signal-to-noise ratio) by a factor of about 50 over previously reported absorption and fluorescence signals from neuronal processes stained by either intra- or extracellular application of dyes. Recordings with good signal-to-noise ratio and adequate spatial and temporal resolution were obtained simultaneously from the cell body and long axonal branches. From this data, the site of action potential initiation was determined. Also, the propagation velocity of the action potential was calculated for different axonal segments; the results suggest that different regions have different velocities ranging from 0.53 m/sec to 0.07 m/sec. The present sensitivity was adequate to allow the recording of a 10 mV hyperpolarizing electrotonic response along axonal branches and to observe directly the decline of this passive response with distance from the site of stimulation. Relatively modest improvements in sensitivity will allow systematic analyses of the spread and summation of synaptic potentials in individual neurons.
Physiological subgroups of nonpyramidal cells with specific morphological characteristics in layer II/III of rat frontal cortex
Authors: Kawaguchi Y
Published: April 2018

Physiological and morphological properties of nonpyramidal cells in layer II/III of frontal cortex of young rats were studied in vitro by whole-cell recording and intracellular staining with biocytin. Layer II/III nonpyramidal cells could be divided into four subgroups by their firing patterns in response to depolarizing current pulses and their patterns of dendritic and axonal arborizations. (1) Fast-spiking (FS) cells (35% of the total sample) showed an abrupt episode of nonadapting repetitive discharges of shorter-duration action potentials. FS cells had local and horizontal axonal arbors which did not enter layer I. This type of FS cell was immunoreactive for parvalbumin and included some basket cells. Three chandelier cells were identified as FS cells, although one chandelier cell was not identified as an FS cell. (2) Late- spiking (LS) cells (11%) exhibited slowly developing ramp depolarizations near threshold. LS cells were neurogliaform cells. (3) Low-threshold spike (LTS) cells (5%) had prominent low-threshold spikes when stimulated from hyperpolarizations. The main axons of LTS cells ascended, and the collaterals entered into layer I. (4) The remaining cells [regular-spiking nonpyramidal (RSNP) cells] (49%) could not be categorized into the above three subgroups. In some RSNP cells depolarizing pulses from hyperpolarizations induced fast depolarizing notches with small amplitude. RSNP cells had vertically elongated axonal fields, extending from layer I to V, sometimes to layer VI. This subgroup included double bouquet cells and bipolar cells. Each subgroup with a different firing mode may differentially contribute to neocortical laminar and columnar circuitry.
Cholinergic regulation of [Ca2+]i during cell division and differentiation in the mammalian retina
Authors: Wong RO
Published: April 2018

Transmitter-like molecules are thought to influence many aspects of neuronal development, often by regulating the levels of intracellular calcium. Using the Ca2+ sensitive dye, fura-2, this study demonstrates that in the rabbit retina, ACh analogs stimulate a rise in cytosolic free Ca2+ concentration ([Ca2+]i) in many cell types, and in cells at various stages of differentiation during embryonic and neonatal development. The elevation in [Ca2+]i in cells within the ventricular zone (VZ) resulted primarily from the activation of muscarinic receptors. By contrast, the cholinergic regulation of [Ca2+]i of ganglion cells and amacrine cells, cell types which migrate to their final destinations early in fetal life, was largely mediated by nicotinic receptors. The muscarinic response of the VZ cells was mediated by the M1, rather than the M2-type of muscarinic receptor. This response was abolished in the absence of extracellular Ca2+ and in the presence of NiCl2, but it was not affected by verapamil or omega- conotoxin, thus suggesting that while Ca2+ influx occurred, it did not involve L- and N-type voltage-gated Ca2+ channels. The muscarinic response in the VZ disappeared at the end of the period of cell division in the retina, just prior to eye opening. By contrast, nicotinic-induced changes in [Ca2+]i in ganglion cells and amacrine cells persisted throughout development. Since previous studies have implicated that the precursors of ganglion cells and amacrine cells also possess muscarinic receptors (Yamashita and Fukuda, 1993), the concomitant emergence of different functional cholinergic receptor (AChR) subtypes with differentiation in vivo suggests that ACh may play diverse and temporally regulated roles in the developing retina.
Synaptic NMDA receptor channels have a low open probability
Authors: Rosenmund C , Feltz A , Westbrook GL
Published: April 2018

Realistic estimates of channel-gating parameters of synaptic receptors are essential to an understanding of synaptic transmission and modulation. However, the gating of N-methyl-D-aspartate (NMDA) channels appears to differ, depending on recording conditions; thus, it remains unclear what measurements are most relevant to synaptic receptors. To further explore this discrepancy, we examined the open probability (Po) of NMDA channels in whole-cell and outside-out patch recording from cultured hippocampal neurons. Currents were evoked by rapid application of saturating concentrations of NMDA in the presence or absence of the “irreversible” open channel blocker, MK-801 ((+)-5-methyl-10,11-dihydro- 5H-dibenzo[a,d]cyclohepten-5,10-imine) . The reduction of the peak amplitude and the acceleration of the decay of the current in MK-801 were used to derive Po by fitting the current traces to a multistate kinetic model. The Po in whole cell was low (0.04), similar to that previously measured for synaptically activated NMDA channels. In contrast, ensemble average currents from outside-out patches were much more rapidly blocked in the presence of MK-801, indicative of a significantly higher Po. The Po also gradually increased with the duration of recording in both whole-cell and outside-out configurations, suggesting that channel gating is sensitive to mechanical alterations of the patch or that washout of cytoplasmic factors leads to an increase in channel open probability.
Differential effects of NGF and BDNF on voltage-gated calcium currents in embryonic basal forebrain neurons
Authors: Levine ES , Dreyfus CF , Black IB , Plummer MR
Published: April 2018

A number of studies have begun to describe the effects of nerve growth factor (NGF) and the closely related brain-derived neurotrophic factor (BDNF) on the function of basal forebrain neurons. Little is known, however, about the effects of neurotrophins on membrane calcium conductances, which may play a role in growth factor signal transduction as well as regulation of neuronal excitability. Using the whole-cell patch-clamp technique, we investigated the effects of both NGF and BDNF on voltage-gated Ca(2+)-channel currents in cultured embryonic basal forebrain neurons. Exposure for 4–6 d to NGF significantly increased both the L-type and N-type components of the whole-cell current. Conversely, similar exposure to BDNF had no effect on Ca(2+)-channel currents. Consequently, one of the important effects of NGF may be to enhance calcium entry via voltage-dependent channels.
GABAA receptor-mediated inhibition of rat substantia nigra dopaminergic neurons by pars reticulata projection neurons
Authors: Tepper JM , Martin LP , Anderson DR
Published: April 2018

Evidence from electrophysiological studies has suggested an inhibitory interaction between GABAergic neurons in substantia nigra pars reticulata and dopaminergic neurons in pars compacta. However, that this inhibitory interaction is due to a projection from pars reticulata to pars compacta has never been demonstrated directly, nor has the GABAergic neuron that mediates the interaction been identified either electrophysiologically or anatomically. To more closely examine interactions between substantia nigra pars reticulata GABA neurons and dopaminergic neurons, single unit extracellular recordings were obtained from antidromically identified nigrostriatal neurons and their response to antidromic activation of nigral GABAergic projection neurons observed. Stimulation of superior colliculus or thalamus produced a short latency inhibition of dopaminergic neurons. This inhibition was blocked by local application of bicuculline but not 2- hydroxysaclofen. Bicuculline caused most dopaminergic neurons to fire in a bursty mode, whereas saclofen caused most dopaminergic neurons to fire in a pacemaker-like mode. The thalamic-evoked inhibition was not affected by kainate lesions of the globus pallidus, but these lesions produced effects on firing pattern identical to those produced by saclofen. These data demonstrate a short latency inhibition of nigral dopaminergic neurons mediated by GABAA receptors that arises from the axon collaterals of pars reticulata projection neurons. We propose a model in which the firing pattern of nigral dopaminergic neurons in vivo is modulated differentially by disinhibition of GABAA inputs arising from pars reticulata projection neuron axon collaterals and disinhibition of pallidonigral GABAergic inputs mediated by GABAB receptors.
Selective increase in T-type calcium conductance of reticular thalamic neurons in a rat model of absence epilepsy
Authors: Tsakiridou E , Bertollini L , de Curtis M , Avanzini G , Pape HC
Published: April 2018

The properties of voltage-dependent calcium currents were compared in thalamic neurons acutely dissociated from a rat model of absence epilepsy, designated as Genetic Absence Epilepsy Rat from Strasbourg (GAERS), and from a Nonepileptic Control strain (NEC). Two populations of neurons were isolated: thalamocortical relay neurons of the nucleus ventrobasalis (VB) and neurons of the nucleus reticularis (RT) of the thalamus. Whole-cell patch-clamp analysis demonstrated an increase in the amplitude of the calcium (Ca2+) current with a low threshold of activation (IT) in RT neurons of GAERS in comparison to that of the seizure-free rat strain (-198 +/- 19 pA and -128 +/- 14 pA, respectively), whereas the sustained component (IL) was not significantly different. The kinetic properties, voltage dependence, and basic pharmacological sensitivity of the Ca2+ conductances were similar in the two populations of neurons. The amplitude of both IT and IL in RT neurons increased after birth, and differences in IT between GAERS and NEC attained significance after postnatal day 11. At corresponding ages, the Ca2+ currents in VB thalamocortical relay neurons were not altered in GAERS in comparison to those in NEC. We conclude that the selective increase in IT of RT neurons enhances the probability of recurrent intrathalamic burst activity, thereby strengthening the synchronizing mechanisms in thalamocortical systems, and, as such, represents a possible primary neuronal dysfunction that relates to the pathological increase in synchronization underlying the generation of bilateral and synchronous spike and wave discharges (SWDs) in an established genetic model of generalized epilepsy.
Survival of newly postmitotic motoneurons is transiently independent of exogenous trophic support
Authors: Mettling C , Gouin A , Robinson M , el M'Hamdi H , Camu W , Bloch-Gallego E , Buisson B , Tanaka H , Davies AM , Henderson CE
Published: April 2018

We compared the survival requirements of early- and late-born motoneurons from E5 chicken spinal cord. Density gradient centrifugation followed by immunopanning using SC1 antibody allowed us to purify two size classes of motoneuron. Large motoneurons retained by 6.8% metrizamide were shown by BrdU labeling in ovo to be born on average 1.5 d earlier than the small motoneurons recovered from the metrizamide pellet. Large motoneurons were both biochemically and functionally more mature: they expressed higher levels of choline acetyltransferase and low-affinity neurotrophin receptor, and had an acute requirement for trophic support from muscle-derived factors. After 24 hr in culture in basal medium, all early-born motoneurons died, whereas 60% of late-born motoneurons survived. Small motoneurons can develop into large motoneurons in ovo, suggesting that they represent a general transitional stage in motoneuron development. Our results suggest that a defined period elapses between birth of a motoneuron and its acquisition of trophic dependence, possibly corresponding to the time required for target innervation. This property may have important consequences for the timing and regulation of developmental motoneuron death.
Antinociception produced by an ascending spino-supraspinal pathway
Authors: Gear RW , Levine JD
Published: April 2018

Studies in mice and rats have shown that antinociception produced by intrathecal (i.t.) administration of opioids can be partially inhibited by intracerebroventricular (i.c.v.) administration of naloxone. In this study we tested the hypothesis that this inhibition by i.c.v. naloxone results from antagonism of supraspinal endogenous opioid-mediated antinociception produced by the action of i.t. opioids on an ascending antinociceptive pathway. In rats lightly anesthetized with urethane/alpha-chloralose, i.t. DAMGO, i.t. lidocaine, or spinal transection at T5-T6 all attenuated the trigeminal jaw opening reflex (JOR) (i.e., were antinociceptive), an effect that was antagonized in each case by i.c.v. naloxone. These findings support the suggestion that there exists a pathway that ascends from the spinal cord to a supraspinal site that tonically inhibits antinociception mediated by supraspinal opioids. When activity in this ascending pathway is suppressed (e.g., by i.t. opioids or local anesthetics or by spinal cord transection), antinociception mediated by supraspinal opioids is disinhibited. To determine the supraspinal site(s) at which endogenous opioid-dependent antinociception is evoked by i.t. opioids, we microinjected naloxone methiodide into several supraspinal sites. Microinjection of naloxone methiodide into nucleus accumbens but not into the rostral ventral medulla (RVM) or the periaqueductal gray matter (PAG) antagonized the suppression of the JOR produced by i.t. DAMGO or lidocaine. The possibility that this ascending pathway may represent a source of spinal input to mesolimbic circuitry involved in setting the state of sensorimotor reactivity to noxious stimuli is discussed.
Developing neurons use a putative pioneer's peripheral arbor to establish their terminal fields
Authors: Gan WB , Macagno ER
Published: April 2018

Pioneer neurons are known to guide later developing neurons during the initial phases of axonal outgrowth. To determine whether they are also important in the formation of terminal fields by the follower cells, we studied the role of a putative leech pioneer neuron, the pressure- sensitive (PD) neuron, in the establishment of other neurons' peripheral arbors. The PD neuron has a major axon that exits from its segmental ganglion to grow along the dorsal-posterior (DP) nerve to the dorsal body wall, where it arborizes extensively mainly in its own segment. It also has two minor axons that project to the two adjacent segments but branch to a lesser degree. We found that the peripheral projections of several later developing neurons, including the AP motor neuron and the TD sensory neuron, followed, with great precision, the major axon and peripheral arbor of the consegmental PD neuron, up to its fourth-order branches. When a PD neuron was ablated before it had grown to the body wall, the AP and TD axons grew normally toward and reached the target area, but then formed terminal arbors that were greatly reduced in size and abnormal in morphology. Further, if the ablation of a PD neuron was accompanied by the induction, in the same segment, of greater outgrowth of the minor axon of a PD neuron from the adjacent segment, the arbors of the same AP neurons grew along these novel PD neuron branches. These results demonstrate that the peripheral arbor of a PD neuron is a both necessary and sufficient template for the formation of normal terminal fields by certain later growing follower neurons.
Elements in the 5' flanking sequences of the mouse low-affinity NGF receptor gene direct appropriate CNS, but not PNS, expression in transgenic mice
Authors: Carroll SL , Schweitzer JB , Holtzman DM , Miller ML , Sclar GM , Milbrandt J
Published: April 2018

We have initiated a characterization of the cis-acting regulatory elements of the murine low-affinity NGF receptor (p75NGFR) gene. Despite studies in cultured cells that suggest the p75NGFR promoter is constitutive, a detailed analysis of this promoter in five lines of transgenic mice demonstrated a high degree of cell-type specificity: 8.4 kb of 5′ flanking sequence directs expression of a lacZ reporter to retinal and CNS neurons normally expressing p75NGFR. A transgene with 470 bp of 5′ flanking sequence is also expressed in the CNS, but its regulation is aberrant, with a loss of basal forebrain expression. In non-neural tissues, both transgenes were expressed only in the testis, kidney, anterior pituitary, and pancreatic islets; with the exception of the renal pattern of expression, transgene activity was confined to appropriate cells within these tissues. In contrast, although expression of both transgenes was prominent in adrenal medulla and gastrointestinal myenteric neurons, neither construct was active in several sensory or sympathetic ganglia that strongly express the endogenous p75NGFR gene, indicating that genetic elements necessary for expression in these neurons are not present in these promoter sequences. In addition, neither transgene was activated in Schwann cells during Wallerian degeneration of sciatic nerve. We conclude that regulation of the p75NGFR gene is complex, with the first 470 bp of 5′ flanking sequence sufficient for expression in enteric and CNS neurons and additional elements within the first 8.4 kb of 5′ flanking sequence required for restriction to appropriate CNS neurons. Further regulatory elements are possibly required for expression in at least some sensory and sympathetic neurons in the PNS and in Schwann cells. To identify potential regulatory elements in the 470 bp of 5′ flanking sequence from the smaller transgene, we compared the sequences of equivalent regions from the mouse, rat, and human p75NGFR genes. This “phylogenetic footprint” identified conserved motifs potentially important for the regulation of this gene in the CNS.
ATP modulation of synaptic transmission in the spinal substantia gelatinosa
Authors: Li J , Perl ER
Published: April 2018

Actions of adenosine triphosphate (ATP) on neurons of the substantia gelatinosa (SG) were evaluated in spinal cord slices using tight-seal, whole-cell recordings. Bath-applied ATP activated a fast inward current and potentiated both glutamate-induced and synaptically evoked currents by acting through a purinergic receptor with the pharmacology of the P2 type. ATP also induced a delayed slow outward current and depressed synaptic currents that appeared to result from hydrolysis of ATP to adenosine. The inhibitory actions had features suggesting mediation by a P1-like purinergic receptor. Suramin, a putative P2 antagonist, inhibited ATP-induced fast inward currents but did not suppress synaptic currents evoked by dorsal root stimulation. It was concluded that in the SG, ATP released in synaptic regions acts as a synaptic modulator by augmenting excitatory amino acid actions and possibly by also producing a secondary adenosine inhibition.
Modular organization of the pontine nuclei: dendritic fields of identified pontine projection neurons in the rat respect the borders of cortical afferent fields
Authors: Schwarz C , Thier P
Published: April 2018

Cortical afferents transferring information destined for the cerebellum terminate in the pontine nuclei (PN) in a divergent and patchy fashion. We investigated whether the form of dendritic fields of pontine projection neurons which are postsynaptic to the cortical afferents are related to this patchy pattern. To this end we used a triple combination of (1) retrograde labeling (injection of Fluorogold into the brachium pontis), (2) anterograde labeling [injection of Dil into cortical areas A17 and Sml(forelimb)], and (3) subsequent intracellular fills of identified projection neurons (Lucifer yellow) in slightly fixed slices of pontine brainstem. In 64 projection neurons whose somata were located within 160 microns of the border defined by cortical afferent fields, most of the dendritic trees were found to respect the border. Strikingly, proximal dendrites which were oriented toward the border often bent in order to avoid the boundary. This observation was supported by a quantitative analysis. It revealed that overlap areas of dendritic fields with the neighboring compartment were significantly smaller than those of hypothetical, radially organized dendritic fields of the same size, indicating that the dendritic fields are indeed confined to single compartments. In a second series of experiments, double injections of the anterograde tracers Dil and DiAsp into adjacent sites within one cortical area (A17 or Sml) were made in order to test if the topology of the cortical map is preserved within individual pontine compartments. This, however, does not seem to be the case, since the terminal fields displayed a complex pattern of overlap and nonoverlap rather than a consistent shift of terminal fields expected in the case of preserved topology. The results of the present study are consistent with the view that pontine modules independently process information from different parts of individual cortical areas. We suggest that this characteristic property of the corticopontine projection system might be the morphological basis of the well established fact that somatotopically continuous sensory maps in the cortex are transformed into maps at the level of the cerebellar cortex, showing a fractured somatotopy.
Dopaminergic microtransplants into the substantia nigra of neonatal rats with bilateral 6-OHDA lesions. I. Evidence for anatomical reconstruction of the nigrostriatal pathway
Authors: Nikkah G , Cunningham MG , Cenci MA , McKay RD , Bjorklund A
Published: April 2018

Reconstruction of the nigrostriatal pathway by long axon growth derived from dopamine-rich ventral mesencephalic (VM) transplants grafted into the substantia nigra may enhance their functional integration as compared to VM grafts implanted ectopically into the striatum. Here we report on a novel approach by which fetal VM grafts are implanted unilaterally into the substantia nigra (SN) of 6-hydroxydopamine (6- OHDA)-lesioned neonatal pups at postnatal day 3 (P3) using a microtransplantation technique. The results demonstrate that homotopically placed dopaminergic neurons survive and integrate well into the previously 6-OHDA-lesioned neonatal SN region. Moreover, the tyrosine hydroxylase (TH)-positive neurons extended axons rostrally along the white matter tract of the internal capsule closely following the course of the original nigrostriatal pathway. The graft reestablished a TH-positive axon terminal network in the ipsilateral caudate-putamen, with the highest density in the medial and central parts. Retrograde labeling with Fluoro-Gold from the host striatum demonstrated that most of the transplant neurons giving rise to the graft-derived fiber outgrowth were TH-positive, but revealed also a small proportion of projecting neurons which were TH-negative. Amphetamine-induced striatal Fos expression was normalized in the caudate-putamen ipsilateral to the intranigral VM grafts, showing hyperexpression in some areas of the striatum, and the apomorphine- induced Fos expression seen in the 6-OHDA-lesioned animals was completely reversed on the grafted side. These findings indicate that the graft-derived dopaminergic reinnervation of the striatum is functional. The microtransplantation strategy may provide new avenues for the exploration of morphological and functional integration of fetal dopamine neurons in the nigrostriatal system and give new insights into the mechanisms controlling long-distance axon growth in the brain.
Visual topography in primate V2: multiple representation across functional stripes
Authors: Roe AW , Ts'o DY
Published: April 2018

The second visual cortical area (V2) of the primate is composed of repeating thin, pale, and thick cytochrome oxidase stripes containing primarily color-selective, broad-band oriented, and disparity-selective cells, respectively. We have now examined topography in V2 with respect to these functional subdivisions. Our data suggest that there are multiple, interleaved visual maps in V2, one for each of the color, orientation, and disparity domains. The same region of visual space is re-represented by each stripe within a stripe cycle, resulting in discontinuities or “jumps back” in representation at stripe borders. Adjacent stripe cycles represent adjacent regions of space such that the visual map is continuous from one stripe to the next like stripe. Receptive field size and scatter are significantly larger for thin stripes than for thick stripes. Unexpectedly, our data suggest two types of pale stripes within each stripe cycle, one with scatter similar to thin stripes and another to thick stripes. Some evidence also suggests the presence of multiple maps within individual stripes in V2. Consistent with functional clustering within single stripes (Ts'o et al., 1990b), we have recorded re-representations and topographic discontinuities coincident with functional borders within single stripes. These results suggest that multiple and interleaved mapping may be a common organizational strategy for representing multiple functional domains within a single cortical area.
Lissencephaly gene (LIS1) expression in the CNS suggests a role in neuronal migration
Authors: Reiner O , Albrecht U , Gordon M , Chianese KA , Wong C , Gal-Gerber O , Sapir T , Siracusa LD , Buchberg AM , Caskey CT
Published: April 2018

Miller-Dieker lissencephaly syndrome (MDS) is a human developmental brain malformation caused by neuronal migration defects resulting in abnormal layering of the cerebral cortex. LIS1, the gene defective in MDS, encodes a subunit of brain platelet-activating factor (PAF) acetylhydrolase which inactivates PAF, a neuroregulatory molecule. We have isolated murine cDNAs homologous to human LIS1 and mapped these to three different chromosomal loci (Lis1, Lis3, Lis4). The predicted sequences of murine Lis1 protein and its human homolog LIS1 are virtually identical. In the developing mouse and human, Lis1 and LIS1 genes were strongly expressed in the cortical plate. In the adult mouse Lis1 transcripts were abundant in cortex and hippocampus. The direct correlation between cortical defects in MDS patients and Lis1 expression in the murine cortex suggest that the mouse is a model system suitable to study the mechanistic basis of this intriguing genetic disease.
TRP, a protein essential for inositide-mediated Ca2+ influx is localized adjacent to the calcium stores in Drosophila photoreceptors
Authors: Pollock JA , Assaf A , Peretz A , Nichols CD , Mojet MH , Hardie RC , Minke B
Published: April 2018

The Drosophila transient receptor potential (trp) gene product (TRP) shows some structural similarity to vertebrate voltage-gated Ca2+ channels. It appears to function as a novel Ca2+ channel responsible for light stimulated, inositol trisphosphate (InsP3)-mediated Ca2+ entry in the fly retina. The subcellular localization of TRP protein was determined in this study using immunohistochemical staining with anti-TRP antibody (MAb83F6). TRP was localized to the base of the microvilli in a region adjacent to the presumed InsP3-sensitive Ca2+ stores. This specific localization was supported by measuring the magnitude of a TRP-dependent inward current that results from spontaneous activation of the light-sensitive channels during whole- cell recordings (the rundown current, RDC). We found that reduction of the microvilli area through genetic dissection with the opsin null mutant, ninaEora, was correlated with a pronounced enhancement of the TRP-dependent inward current relative to wild type, suggesting that the TRP-dependent current was not produced along the length of the microvilli. We suggest that the functional localization of the TRP protein is on the plasma membrane loop found along the base of the rhabdomeric microvillus. Thus, the TRP channel may function in concert with the InsP3-sensitive Ca2+ stores.
Endogenous opioids regulate long-term potentiation of synaptic inhibition in the dentate gyrus of rat hippocampus
Authors: Xie CW , Lewis DV
Published: April 2018

Long-term potentiation (LTP) of excitatory transmission in the hippocampus has been extensively studied as a synaptic model of learning and memory. Here we report a new form of LTP in which inhibitory synaptic signals are potentiated following tetanic stimulation of an opioid-containing excitatory pathway in the presence of opioid antagonists. The lateral perforant path (LPP) was stimulated at the dentate outer molecular layer of hippocampal slices. Evoked synaptic currents were recorded from dentate granule cells using whole- cell voltage-clamp techniques. A high-frequency stimulus train (100 Hz, 1 sec) delivered to the LPP in the presence of naloxone (1 microM) was found to induce a long-lasting potentiation (20 min to 2 hr) in the amplitude of gamma-aminobutyric acidA (GABAA) receptor-mediated inhibitory postsynaptic currents (IPSCs) of granule cells. Such a potentiation was not observed when tetanizing the LPP in control medium. Naloxone-revealed LTP of LPP-evoked IPSCs did not depend upon the presence of granule cell discharge, and was not accompanied by potentiation of mossy fiber-evoked IPSCs, indicating that feedforward, but not feedback, inhibitory circuits were involved. Induction of this LTP could be completely blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoic acid (D-APV). However, it was not significantly affected by hyperpolarization of granule cells. These results suggest that LTP may occur at the excitatory synapses between LPP terminals and GABAergic interneurons, rather than at the inhibitory synapses between interneurons and granule cells. Further examination using selective opioid antagonists demonstrated that blocking delta, but not mu and kappa, receptors is critical for inducing LTP of IPSCs in granule cells.
Differential spatiotemporal expression of K+ channel polypeptides in rat hippocampal neurons developing in situ and in vitro
Authors: Maletic-Savatic M , Lenn NJ , Trimmer JS
Published: April 2018

Hippocampal neurons are highly plastic in their excitable properties, both during development and in the adult brain. As voltage-sensitive K+ channels are major determinants of membrane excitability, one mechanism for generating plasticity is through regulation of K+ channel activity. To gain insights into the regulation of K+ channels in the hippocampus, we have analyzed the spatiotemporal expression patterns of five K+ channel polypeptides in rat hippocampal neurons developing in situ and in vitro. Delayed rectifier-type channels (Kv1.5, Kv2.1, and Kv2.2) are expressed on all neuronal somata and proximal dendrites, while A-type channels (Kv1.4 and Kv4.2) are present distally on distinct subpopulations of neurons. The development of these patterns in situ is monotonic; that is, while the time and spatial development varies among the channels, each K+ channel subtype initially appears in its adult pattern, suggesting that the mechanisms underlying spatial patterning operate through development. Immunoblots confirm the differential temporal expression of K+ channels in the developing hippocampus, and demonstrate developmentally regulated changes in the microheterogeneity of some K+ channel polypeptide species. Temporal expression patterns of all five K+ channels observed in situ are retained in vitro, while certain aspects of cellular and subcellular localization are altered for some of the K+ channel polypeptides studied. Similarities in K+ channel polypeptide expression in situ and in vitro indicate that the same regulatory mechanisms are controlling spatiotemporal patterning in both situations. However, differences between levels of expression for all subtypes studied except Kv2.1 indicate additional mechanisms operating in situ but absent in vitro that are important in determining polypeptide abundance.
Molecular, functional, and pharmacological characterization of the metabotropic glutamate receptor type 5 splice variants: comparison with mGluR1
Authors: Joly C , Gomeza J , Brabet I , Curry K , Bockaert J , Pin JP
Published: April 2018

The main excitatory neurotransmitter in the brain, glutamate (Glu), activates not only receptor-channels, but also receptors coupled to G- protein called metabotropic Glu receptors (mGluRs). Eight genes coding for mGluRs have been characterized to date giving rise to even more proteins due to alternative splicing phenomena. Here we characterized a splice variant of mGluR5, called mGluR5b which contains a 32 amino acid fragment inserted in the cytoplasmic tail, 50 residues after the 7th transmembrane domain. mGluR5b mRNAs are present in different regions of the adult rat brain and are expressed at a higher level than mGluR5a mRNA. Functional analysis of mGluR5a and mGluR5b revealed that they share all the properties of mGluR1a, but not those of mGluR1b or 1c. Like mGluR1a, both mGluR5a and mGluR5b activate a rapid and transient current in Xenopus oocytes. When expressed in LLC-PK1 cells, they show the same subcellular distribution as mGluR1a, and stimulate both inositol phosphate (IP) and cAMP production. Moreover, cells expressing mGluR5a or mGluR5b, like those expressing mGluR1a have a higher basal PLC activity that is not inhibited by glutamate-pyruvate transaminase (GPT), suggesting that these receptors have an intrinsic activity. Interestingly, the pharmacological profiles of mGluR5a and b are identical, but different from that of mGluR1a. Most agonists, except glutamate, are more potent on mGluR5a/b than on mGluR1a. Interestingly, the mGluR1a antagonists MCPG and 4CPG have no effect on mGluR5a/b; 4C3HPG which is a full antagonist at mGluR1a is a partial agonist at mGluR5a/b. These results indicate that the long C-terminal intracellular domain present only in mGluR1a and mGluR5a/b, although not well conserved, is likely to be involved in the specific functional properties of these receptors. Although the ligand recognition sites of mGluR5a/b and mGluR1a are highly conserved, these receptors have different pharmacology.
A dominant role of acid pH in inflammatory excitation and sensitization of nociceptors in rat skin, in vitro
Authors: Steen KH , Steen AE , Reeh PW
Published: April 2018

A major role of local acidosis in long lasting excitation and sensitization of cutaneous nociceptors has recently been demonstrated. In inflamed tissue, acid pH meets with a mixture of inflammatory mediators which, by themselves, stimulate nociceptors though being subject to profound tachyphylaxis. We have mimicked this condition in a rat skin-saphenous nerve preparation in vitro which allows direct application of chemicals to the isolated receptive fields at the corium side. Stimulant solutions used were CO2-saturated “synthetic interstitial fluid” (CO2-SIF, pH 6.1), and “inflammatory soup” (IS) in submaximal concentration containing bradykinin, 5-HT, histamine, prostaglandin E2 (all 10(-6) M in SIF at 38.5 degrees C and pH 7.0), and a combination made of CO2-saturated IS (CO2-IS, pH 6.1). Identified mechano-heat sensitive (“polymodal”) C-fiber terminals (n = 36) were treated with these solutions for 5 min at 10 min intervals or for 30 min of sustained stimulation: 20 units responded to CO2-SIF, 12 to IS, whereas 27 units (75%) were excited by CO2-IS. Thus, 6 out of 15 units insensitive to either of the two basic solutions were stimulated by their combination. This enhanced effect of CO2-IS was also expressed in shorter latencies (than with CO2-SIF) and in a significantly larger mean response magnitude of the fiber population: 152 spikes with the combination versus 45 spikes evoked by IS and 93 spikes by CO2-SIF (n = 25; p < 0.002 and < 0.02, respectively, Wilcoxon test).