Lucifer Yellow Injection Research Articles

Impaired Cognitive Function and Hippocampal Changes Following Chronic Diazepam Treatment in Middle-Aged Mice.

Background: Gamma-aminobutyric acid (GABA) type A receptors are positively allosterically modulated by benzodiazepine binding, leading to a potentiated response to GABA. Diazepam (DZP, a benzodiazepine) is widely prescribed for anxiety, epileptic discharge, and insomnia, and is also used as a muscle relaxant and anti-convulsant. However, some adverse effects – such as tolerance, dependence, withdrawal effects, and impairments in cognition and learning – are elicited by the long-term use of DZP. Clinical studies have reported that chronic DZP treatment increases the risk of dementia in older adults. Furthermore, several studies have reported that chronic DZP administration may affect neuronal activity in the hippocampus, dendritic spine structure, and cognitive performance. However, the effects of chronic DZP administration on cognitive function in aged mice is not yet completely understood.Methods: A behavioral test, immunohistochemical analysis of neurogenic and apoptotic markers, dendritic spine density analysis, and long-term potentiation (LTP) assay of the hippocampal CA1 and CA3 were performed in both young (8 weeks old) and middle-aged (12 months old) mice to investigate the effects of chronic DZP administration on cognitive function. The chronic intraperitoneal administration of DZP was performed by implanting an osmotic minipump. To assess spatial learning and memory ability, the Morris water maze test was performed. Dendritic spines were visualized using Lucifer yellow injection into the soma of hippocampal neurons, and spine density was analyzed. Moreover, the effects of exercise on DZP-induced changes in spine density and LTP in the hippocampus were assessed.Results: Learning performance was impaired by chronic DZP administration in middle-aged mice but not in young mice. LTP was attenuated by DZP administration in the CA1 of young mice and the CA3 of middle-aged mice. The spine density of hippocampal neurons was decreased by chronic DZP administration in the CA1 of both young and middle-aged mice as well as in the CA3 of middle-aged mice. Neither neurogenesis nor apoptosis in the hippocampus was affected by chronic DZP administration.Conclusion: The results of this study suggest that the effects of chronic DZP are different between young and middle-aged mice. The chronic DZP-induced memory retrieval performance impairment in middle-aged mice can likely be attributed to decreased LTP and dendritic spine density in hippocampal neurons in the CA3. Notably, prophylactic exercise suppressed the adverse effects of chronic DZP on LTP and spine maintenance in middle-aged mice.

Heterogeneity of tyrosine hydroxylase expressing neurons in the main olfactory bulb of the mouse

The structural features of dopamine (DA)-GABAergic neurons in the mouse main olfactory bulbs were examined, using both wild type and transgenic TH-GFP mice, with the combination of several methods; the immunocytochemistry, biotinylated dextran amine labeling, lucifer yellow injection in fixed slices, biocytin injection in live slice and the functional olfactory deprivation. DA-GABAergic neurons were clustered in the glomerular layer (GL) but they also scattered in other layers. DA-GABAergic juxtaglomerular neurons, were classified into 5 groups based on their structural features and named as follows: 1) Large periglomerular (LPG) cells with tuft-like glomerular dendritic branches and apparent axons extending to the distant glomeruli, which correspond to the " inhibitory juxtaglomerular association (IJGA) neurons" participating in the interglomerular association system. 2) Small periglomerular (SPG) cells including both axonic and anaxonic ones; the axonic SPG cells might correspond to the classical periglomerular cells. 3) Transglomerular cells extending dendritic processes spanning 2 or more glomeruli. 4) Incrusting cells extending their dendritic branches mainly in the periphery of the glomeruli. 5) Other various neurons not-yet classified. In the layers other than the GL various types of TH expressing neurons were scattered; some of them extended dendritic processes into the GL.

L-DOPA Oppositely Regulates Synaptic Strength and Spine Morphology in D1 and D2 Striatal Projection Neurons in Dyskinesia.

Dopamine depletion in Parkinson's disease (PD) produces dendritic spine loss in striatal medium spiny neurons (MSNs) and increases their excitability. However, the synaptic changes that occur in MSNs in PD, in particular those induced by chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, are still poorly understood. We exposed BAC-transgenic D1-tomato and D2-eGFP mice to PD and dyskinesia model paradigms, enabling cell type-specific assessment of changes in synaptic physiology and morphology. The distinct fluorescence markers allowed us to identify D1 and D2 MSNs for analysis using intracellular sharp electrode recordings, electron microscopy, and 3D reconstructions with single-cell Lucifer Yellow injections. Dopamine depletion induced spine pruning in both types of MSNs, affecting mushroom and thin spines equally. Dopamine depletion also increased firing rate in both D1- and D2-MSNs, but reduced evoked-EPSP amplitude selectively in D2-MSNs. L-DOPA treatment that produced dyskinesia differentially affected synaptic properties in D1- and D2-MSNs. In D1-MSNs, spine density remained reduced but the remaining spines were enlarged, with bigger heads and larger postsynaptic densities. These morphological changes were accompanied by facilitation of action potential firing triggered by synaptic inputs. In contrast, although L-DOPA restored the number of spines in D2-MSNs, it resulted in shortened postsynaptic densities. These changes in D2-MSNs correlated with a decrease in synaptic transmission. Our findings indicate that L-DOPA-induced dyskinesia is associated with abnormal spine morphology, modified synaptic transmission, and altered EPSP-spike coupling, with distinct effects in D1- and D2-MSNs.

Calcium buffer proteins are specific markers of human retinal neurons.

Ca(2+)-buffer proteins (CaBPs) modulate the temporal and spatial characteristics of transient intracellular Ca(2+)-concentration changes in neurons in order to fine-tune the strength and duration of the output signal. CaBPs have been used as neurochemical markers to identify and trace neurons of several brain loci including the mammalian retina. The CaBP content of retinal neurons, however, varies between species and, thus, the results inferred from animal models cannot be utilised directly by clinical ophthalmologists. Moreover, the shortage of well-preserved human samples greatly impedes human retina studies at the cellular and network level. Our purpose has therefore been to examine the distribution of major CaBPs, including calretinin, calbindin-D28, parvalbumin and the recently discovered secretagogin in exceptionally well-preserved human retinal samples. Based on a combination of immunohistochemistry, Neurolucida tracing and Lucifer yellow injections, we have established a database in which the CaBP marker composition can be defined for morphologically identified cell types of the human retina. Hence, we describe the full CaBP make-up for a number of human retinal neurons, including HII horizontal cells, AII amacrine cells, type-1 tyrosine-hydroxylase-expressing amacrine cells and other lesser known neurons. We have also found a number of unidentified cells whose morphology remains to be characterised. We present several examples of the colocalisation of two or three CaBPs with slightly different subcellular distributions in the same cell strongly suggesting a compartment-specific division of labour of Ca(2+)-buffering by CaBPs. Our work thus provides a neurochemical framework for future ophthalmological studies and renders new information concerning the cellular and subcellular distribution of CaBPs for experimental neuroscience.

Structural plasticity in hippocampal cells related to the facilitative effect of intracranial self-stimulation on a spatial memory task.

Posttraining intracranial self-stimulation (SS) in the lateral hypothalamus facilitates the acquisition and retention of several implicit and explicit memory tasks. Here, intracellular injections of Lucifer yellow were used to assess morphological changes in hippocampal neurons that might be specifically related to the facilitative posttraining SS effect upon the acquisition and retention of a distributed spatial task in the Morris water maze. We examined the structure, size and branching complexity of cornus ammonis 1 (CA1) cells, and the spine density of CA1 pyramidal neurons and granular cells of the dentate gyrus (DG). Animals that received SS after each acquisition session performed faster and better than Sham ones--an improvement that was also evident in a probe trial 3 days after the last training session. The neuromorphological analysis revealed an increment in the size and branching complexity in apical CA1 dendritic arborization in SS-treated subjects as compared with Sham animals. Furthermore, increased spine density was observed in the CA1 field in SS animals, whereas no effects were observed in DG cells. Our results support the hypothesis that the facilitating effect of SS on the acquisition and retention of a spatial memory task could be related to structural plasticity in CA1 hippocampal cells.

The Effects of Cocaine Self-Administration on Dendritic Spine Density in the Rat Hippocampus Are Dependent on Genetic Background

Chronic exposure to cocaine induces modifications to neurons in the brain regions involved in addiction. Hence, we evaluated cocaine-induced changes in the hippocampal CA1 field in Fischer 344 (F344) and Lewis (LEW) rats, 2 strains that have been widely used to study genetic predisposition to drug addiction, by combining intracellular Lucifer yellow injection with confocal microscopy reconstruction of labeled neurons. Specifically, we examined the effects of cocaine self-administration on the structure, size, and branching complexity of the apical dendrites of CA1 pyramidal neurons. In addition, we quantified spine density in the collaterals of the apical dendritic arbors of these neurons. We found differences between these strains in several morphological parameters. For example, CA1 apical dendrites were more branched and complex in LEW than in F344 rats, while the spine density in the collateral dendrites of the apical dendritic arbors was greater in F344 rats. Interestingly, cocaine self-administration in LEW rats augmented the spine density, an effect that was not observed in the F344 strain. These results reveal significant structural differences in CA1 pyramidal cells between these strains and indicate that cocaine self-administration has a distinct effect on neuron morphology in the hippocampus of rats with different genetic backgrounds.

Neurons of the Dentate Molecular Layer in the Rabbit Hippocampus

The molecular layer of the dentate gyrus appears as the main entrance gate for information into the hippocampus, i.e., where the perforant path axons from the entorhinal cortex synapse onto the spines and dendrites of granule cells. A few dispersed neuronal somata appear intermingled in between and probably control the flow of information in this area. In rabbits, the number of neurons in the molecular layer increases in the first week of postnatal life and then stabilizes to appear permanent and heterogeneous over the individuals’ life span, including old animals. By means of Golgi impregnations, NADPH histochemistry, immunocytochemical stainings and intracellular labelings (lucifer yellow and biocytin injections), eight neuronal morphological types have been detected in the molecular layer of developing adult and old rabbits. Six of them appear as interneurons displaying smooth dendrites and GABA immunoreactivity: those here called as globoid, vertical, small horizontal, large horizontal, inverted pyramidal and polymorphic. Additionally there are two GABA negative types: the sarmentous and ectopic granular neurons. The distribution of the somata and dendritic trees of these neurons shows preferences for a definite sublayer of the molecular layer: small horizontal, sarmentous and inverted pyramidal neurons are preferably found in the outer third of the molecular layer; vertical, globoid and polymorph neurons locate the intermediate third, while large horizontal and ectopic granular neurons occupy the inner third or the juxtagranular molecular layer. Our results reveal substantial differences in the morphology and electrophysiological behaviour between each neuronal archetype in the dentate molecular layer, allowing us to propose a new classification for this neural population.

Retrograde Tracing with Fluorescent Microspheres Reveals Bifurcating Projections from Central Retina to Tectum and Thalamus in Chicks

The goal of this study is to demonstrate the dual-projection pattern of retinal ganglion cells (RGCs) projecting to the tectum and visual thalamus in chick using retrograde fluorescent tracers and also to define the morphological properties of these RGCs with dual projections by intracellular injection of Lucifer Yellow (LY) combined with immunohistochemistry. Thirty-two chicks received double injections of green and red fluorescent microspheres into their thalamus and tectum in the same side. In the central retina, most of the labelled RGCs were tec-RGCs (RGCs projecting to the tectum), a quarter was tha-RGCs (RGCs projecting to the thalamus), and almost all of the tha-RGCs were double-labelled RGCs. An intracellular injection of LY into the double-labelled RGCs showed all six groups of RGCs without specific populations in each group (J. Comp. Neurol., 2004, 469: 360). These dendritic patterns were mostly mono- and bistrata, which extended horizontally in the deeper part of the inner plexiform layer.

Expression of Asn-d-Trp-Phe-NH2 in the brain of the terrestrial slug Limax valentianus

The tripeptide Asn-d-Trp-Phe-NH(2) (NdWFamide) is a D-amino acid-containing cardioexcitatory peptide initially isolated from Aplysia. Previously we detected NdWFamide immunoreactivity in the visceral giant cells, the largest neurons in the brain of the terrestrial slug Limax located at the dorsal surface of the visceral ganglia. In the present study, we further analyzed the morphological features of these neurons by an intracellular injection of Lucifer yellow, and found that these neurons extend neurites out of the brain through at least 5 nerve bundles. We then isolated a gene and a cDNA clone potentially encoding a NdWFamide precursor, and investigated expression at the levels of mRNA and protein in Limax. The NdWFamide gene consists of 5 exons spanning at least 17 kb of the genome, and its open reading frame extends over 3 exons. The spatial expression pattern of NdWFamide mRNA was almost identical to that of the NdWFamide peptide, with some minor discrepancies in between. Although the most remarkable expression was evident in the visceral giant cells, we also found the expression of NdWFamide mRNA and peptide in the cerebral and pedal ganglia. These results suggest the involvement of NdWFamide in the regulation of a broad area of the slug's body.

Susceptibility to N-methyl-d-aspartate toxicity in morphological and functional types of cat retinal ganglion cells

To examine whether different types of retinal ganglion cells (RGCs) in the cat retina have different survival rates when exposed to N-methyl-D-aspartate (NMDA). NMDA injury was induced by intravitreal administration of NMDA at final concentrations of 0.2, 0.4, 0.6, and 0.8 mM. The total number of surviving RGCs and their distribution were counted by retrograde labeling with a fluorescent dye. Measurements of the proportions of the main RGC types (alpha, beta, and neither alpha nor beta cells) were obtained by using intracellular injections of Lucifer yellow. The mean percentage of surviving RGCs in the NMDA-injected retina was reduced to 59.4% (0.2 mM NMDA), 35.8% (0.4 mM), 10.8% (0.6 mM), and 14.1% (0.8 mM). At 0.2 mM, the survival rate of alpha cells was reduced to 56%, but that of beta cells remained at 81%. At 0.8 mM, the survival rate of alpha cells was 19%, while beta cells rapidly decreased to 9.9%. No difference was detected in NMDA vulnerability between ON- and OFF-center RGCs. Different RGC types display different susceptibilities to NMDA injury. A specific examination of the functions of different types of RGCs would be helpful in detecting retinal excitotoxicity such as in chronic retinal ischemia.

Olfactory ensheathing cell membrane properties are shaped by connectivity

Olfactory ensheathing cells (OECs) have been repeatedly implicated in mediating plasticity, particularly in situ in the olfactory nerve in which they support the extension of olfactory sensory neuron (OSN) axons from the olfactory epithelium to the olfactory bulb (OB). OECs are specialized glia whose processes surround OSN axon fascicles within the olfactory nerve and across the OB surface. Despite their purported importance in promoting axon extension, and following transplants, little is known about either morphology or biophysical properties of OECs in situ. In particular, cell-cell interactions that may influence OEC function are largely unexplored. Here, we studied OEC connectivity and morphology in slice preparations, preserving tissue structure and cell-cell interactions. Our analyses showed that OECs form a matrix of cellular projections surrounding axons, unique among glia, and express high levels of connexin-43. Lucifer Yellow injections revealed selective dye coupling among small subgroups of OECs. Two types of OECs were biophysically distinguished with whole-cell voltage-clamp recordings: (1) with low-input resistance (R(i)), linear current profiles, and frequently dye coupled; and (2) with high R(i), nonlinear current profiles, and infrequent dye coupling. Pharmacological blockade of gap junctions changed OEC membrane properties such that linear OECs became nonlinear. Double recordings indicated that the appearance of the nonlinear current profile was associated with the loss of electrical coupling between OECs. We conclude that the diversity of OEC current profiles can be explained by differences in gap-junction connectivity and discuss implications of this diversity for OEC influences on axon growth and excitability.

Abstract 1451: Hyperpolarization Induces Differentiation Of Human Cardiomyocyte Progenitor Cells

Background: Recently, we have isolated cardiomyocyte progenitor cells (hCMPCs) from human fetal and adult hearts. These cells differentiate into spontaneously beating cardiomyocytes when stimulated with 5-azacytidine. Subsequent stimulation by TGFβ enhances differentiation efficiency to nearly 100%. The underlying molecular mechanisms mediating this cardiomyogenic differentiation are not understood. In skeletal myoblasts, hyperpolarization-mediated activation of calcineurin signaling is crucial for myogenic differentiation. In hCMPCs, whole-cell patch clamp recordings showed a hyperpolarized membrane potential after stimulation with TGFβ or BMP. We hypothesized that hyperpolarization and calcineurin signaling regulate cardiomyogenic differentiation of hCMPCs after TGFβ stimulation. Methods & Results: To test whether hyperpolarization initiates cardiomyogenic differentiation, hyperpolarization was induced by 1) co-culture of hCMPCs with HEK 293 cells overexpressing a Kir2.1GFP fusion protein (KWGF cells) or 2) culture of hCMPCs overnight in medium containing low potassium concentrations. During co-culture, Lucifer Yellow dye injection in KWGF cells spread to neighboring hCMPCs, indicating cellular coupling. This resulted in stable hyperpolarization in hCMPCs, which could be blocked by addition of the gap junction inhibitor halothane. After two weeks, qPCR analysis revealed increased expression of cardiac sarcomeric genes in the hCMPCs in a dose-dependent manner. Induction of hyperpolarization by culturing hCMPCs with low potassium concentrations also resulted in increased expression of cardiac genes and the formation of spontaneously beating cells. Immunofluorescence staining revealed striated patterns of troponin I and α-actinin. Interestingly, hyperpolarization also increased intracellular calcium levels in hCMPCs, as measured by ratiometric imaging of indo-1 fluorescence, and, subsequently, a time-dependent increase in NFAT-Luciferase reporter activity, indicating activation of the calcineurin pathway. Conclusion: TGFβ and/or BMP-mediated hyperpolarization of hCMPCs induces calcineurin-mediated cardiomyogenic differentiation.

Satellite glial cells in sensory ganglia: Their possible contribution to inflammatory pain

Neurons in dorsal root ganglia (DRG) are surrounded by an envelope of satellite glial cells (SGCs). Little is known about SGC physiology and their interactions with neurons. In this work, we investigated changes in mouse DRG neurons and SGC following the induction of inflammation in the hind paw by the injection of complete Freund’s adjuvant (CFA). The electrophysiological properties of neurons were characterized by intracellular electrodes. Changes in coupling mediated by gap junctions between SGCs were monitored using intracellular injection of the fluorescent dye Lucifer yellow. Pain was assessed with von Frey hairs. We found that two weeks after CFA injection there was a 38% decrease in the threshold for firing an action potential in DRG neurons, consistent with neuronal hyperexcitability. Injection of Lucifer yellow into SGCs revealed that, compared with controls, coupling by gap junctions among SGCs surrounding adjacent neurons increased 2.7-, 3.2-, and 2.5-fold one week, two weeks, and one month, respectively, after CFA injection. In SGCs enveloping neurons that project into the inflamed paw this effect was more enhanced (5.4-fold). Interneuronal coupling was augmented by up to 7% after CFA injection. Pain threshold in the injected paw decreased by 13%, 16%, and 11% compared with controls at one week, two weeks, and one month, respectively, after CFA injection. Intraperitoneal injection of the gap junction blocker carbenoxolone prevented the inflammation-induced decrease in pain threshold. The results show that augmented glial coupling is one of the major events occurring in DRG following inflammation. The elevation in pain threshold after carbenoxolone administration provides indirect support for the idea that augmented intercellular coupling might contribute to chronic pain.

Stress-Induced Alterations in Prefrontal Cortical Dendritic Morphology Predict Selective Impairments in Perceptual Attentional Set-Shifting

Stressful life events have been implicated clinically in the pathogenesis of mental illness, but the neural substrates that may account for this observation remain poorly understood. Attentional impairments symptomatic of these psychiatric conditions are associated with structural and functional abnormalities in a network of prefrontal cortical structures. Here, we examine whether chronic stress-induced dendritic alterations in the medial prefrontal cortex (mPFC) and orbital frontal cortex (OFC) underlie impairments in the behaviors that they subserve. After 21 d of repeated restraint stress, rats were tested on a perceptual attentional set-shifting task, which yields dissociable measures of reversal learning and attentional set-shifting, functions that are mediated by the OFC and mPFC, respectively. Intracellular iontophoretic injections of Lucifer yellow were performed in a subset of these rats to examine dendritic morphology in layer II/III pyramidal cells of the mPFC and lateral OFC. Chronic stress induced a selective impairment in attentional set-shifting and a corresponding retraction (20%) of apical dendritic arbors in the mPFC. In stressed rats, but not in controls, decreased dendritic arborization in the mPFC predicted impaired attentional set-shifting performance. In contrast, stress was not found to adversely affect reversal learning or dendritic morphology in the lateral OFC. Instead, apical dendritic arborization in the OFC was increased by 43%. This study provides the first direct evidence that dendritic remodeling in the prefrontal cortex may underlie the functional deficits in attentional control that are symptomatic of stress-related mental illnesses.

Role of Membrane Potential in Endothelium-Dependent Relaxation of Isolated Mouse Main Pulmonary Artery

The physiology of smooth muscle and endothelial cells of a particular vascular bed and from different species differs from each other. Acetylcholine causes an endothelium-dependent relaxation of preconstricted pulmonary arteries from the rat. This relaxation is mediated by nitric oxide (NO) plus a yet-unidentified endothelium-derived hyperpolarizing factor, which relaxes the smooth muscles by hyperpolarizing them. Our aim is to test whether these observations could be generalized to the smooth muscle cells from the mouse pulmonary artery. Smooth muscle or endothelial cell membrane potential of strips of murine pulmonary artery were measured simultaneously with the force developed by the strip. Acetylcholine hyperpolarized the endothelial cells. However, acetylcholine did not induce an endothelium-dependent hyperpolarization of the smooth muscle, while it relaxed the strip in an endothelium-dependent manner. This relaxation was abolished by an inhibitor of NO synthesis, nitro-L-arginine. Moreover, nitroglycerin relaxed the strips without changing the membrane potential of the smooth muscle cells. Injection of Lucifer yellow into the endothelial cells and the smooth muscle cells did not show heterocellular dye coupling. Furthermore, electron microscopy did not show gap junction plate at the myoendothelial junctions. We conclude that in the mouse main pulmonary artery, NO alone is responsible for the acetylcholine-induced endothelium-dependent vasodilatation, whereas the phenomenon called endothelium-derived hyperpolizing factor is not present. Therefore, caution should be taken when comparing different animal models to study pulmonary circulation and its reactivity.

Morphological properties of mouse retinal ganglion cells

The mouse retina offers an increasingly valuable model for vision research given the possibilities for genetic manipulation. Here we assess how the structural properties of mouse retinal ganglion cells relate to the stratification pattern of the dendrites of these neurons within the inner plexiform layer. For this purpose, we used 14 morphological measures to classify mouse retinal ganglion cells parametrically into different clusters. Retinal ganglion cells were labeled in one of three ways: Lucifer Yellow injection, ‘DiOlistics’ or transgenic expression of yellow fluorescent protein. The resulting analysis of 182 cells revealed 10 clusters of monostratified cells, with dendrites confined to either On or Off sublaminae of the inner plexiform layer, and four clusters of bistratified cells, dendrites spanning the On and Off sublaminae. We also sought to establish how these parametrically identified retinal ganglion cell clusters relate to cell types identified previously on the basis of immunocytochemical staining and the expression of yellow fluorescent protein. Cells labeled with an antibody against melanopsin were found to be located within a single cluster, while those labeled with the SMI-32 antibody were in four different clusters. Yellow fluorescent protein expressing cells were distributed within 13 of the 14 clusters identified here, which demonstrates that yellow fluorescent protein expression is a useful method for labeling virtually the entire population of mouse retinal ganglion cells. Collectively, these findings provide a valuable baseline for future studies dealing with the effects of genetic mutations on the morphological development of these neurons.

Recurrent spreading depression (SD) causes early opening of the blood-brain barrier (BBB)

SD is a depolarization wave propagating at a rate of about 3 mm/min in the cerebral cortex. It is characterized by an amplitude reduction of the electrocorticographic activity, a rise of the extracellular potassium to 60 mM and a reduction of the extracellular volume. Typical changes of cerebral blood flow (CBF) accompany SD, which consist of a short lasting spreading hyperemia followed by a long lasting spreading oligemia. SD is triggered by electrical, mechanical or toxic factors and is probably involved in the pathogenesis of migraine, focal ischemia, head trauma and cerebral hemorrhage. SD is assumed to be the pathophysiological correlate of the migraine aura. We have recently observed a patient with familial hemiplegic migraine (FHM) type II and a long-lasting migraine aura with fluctuating neurological symptoms. Quantitative analysis of early gadolinium-enhanced MR images revealed a left-hemispheric, cortical blood brain-barrier (BBB) disruption preceding delayed cortical edema in this ATP1A2 mutation carrier. As we know from the work by Strong et al. repetitive SDs can occur in the human brain tissue, which could explain the fluctuating symptoms in our FHM patient. Additionally, a recent publication by Gursoy-Ozdemir et al. showed a delayed enhancement of BBB permeability and subsequent cortical oedema following a single SD. Based on these observations, we studied here whether recurrent SD causes early BBB disruption in the rat cortex in vivo. We triggered SD with artificial cerebrospinal fluid (ACSF) containing a potassium concentration ([K+]ACSF) of 130 mM at an open parietal cranial window. SDs were monitored with an intracortical potassium sensitive microelectrode and laser-Doppler flowmetry. We also recorded the propagation of SDs to a distant closed frontal cranial window using an epidural Ag-AgCl electrode. BBB integrity was evaluated quantitatively by analyzing fluorescence in brain sections following the peripheral injection of Evan's Blue or Lucifer yellow. At the occipital window, [K+]ACSF evoked several SDs in all animals. In the first group (n = 10), one or more SDs propagated to the frontal window. In contrast, in the second group (n = 9), pretreatment with MK-801 (5 mg/kg BW i.v.), abolished SD propagation. Within two hours after the onset of the first SD, significantly enhanced fluorescence intensity at the frontal cortex was only measured in animals from group 1 with multiple propagated SDs. In conclusion, we provide evidence that repetitive SDs significantly increase blood-brain barrier permeability within two hours after the onset.

103 GAP JUNCTIONAL INTERCELLULAR COMMUNICATION IS DISPENSIBLE DURING REGULATION OF TIGHT JUNCTION MEMBRANE ASSEMBLY BY CELL CONTACT PATTERN AND PKC SIGNALING

Contact symmetries are involved in regulating cell lineage segregation during blastocyst biogenesis when tight junction (TJ) membrane assembly is restricted to the epithelial trophectoderm (TE). Manipulation of cell contact patterns by immunosurgical isolation of inner cell masses (ICMs) providing a contact-free cell surface serves as a switch to induce TE differentiation upon in vitro culture. In this model, protein kinase C (PKC)-mediated signaling up-regulates TJ membrane assembly. Whether signaling via gap junctional intercellular communication (GJIC) affects these processes is controversial. The current study investigates the interrelationship between changes in cell contact pattern, PKC signaling, and GJIC on TE differentiation and TJ assembly. Eight-cell embryos flushed from MF1 mice were cultured in T6/BSA to time development to early blastocyst stage (<2 h of cavitation). Laser confocal microscopy (BioRad MRC 600, BioRad Laboratories, Inc., Hertfordshire, UK) after immunostaining with antibodies against PKCδ, θ, λ, or ζ isoforms (Transduction Labs, Oxford, UK or Sigma) or junctional proteins (E-cadherin, ZO-2, Occludin, ZO-1α+, Desmoplakin) combined with ALEXA 488 conjugated secondary antibodies (Cambridge Bioscience, Oxford, UK) was used to determine the distribution of PKCs and junctional proteins in intact blastocysts and fully and partially isolated ICMs after immunosurgery and in vitro culture in DMEM + 10% FCS (Eckert et al. 2004 Reproduction 127, 653). While broad PKC activators (1 μM 12-O-tetradecanoylphorbol-13-acetate or Indolactam; Calbiochem, Nottingham, UK) accelerate membrane assembly of the TJ proteins ZO-2 and ZO-1α+ in fully isolated ICMs this up-regulation was suppressed in intact blastocysts (n = 32–47 per treatment and antibody) and in partially isolated ICMs (remnants of lysed TE remaining surrounding the ICM; n = 17–21 per treatment and antibody) for up to 24 h with no TJ protein detectable within the ICM, even after two consecutive rounds of TE lysis (n = 13–22 per treatment and antibody). When GJIC was inhibited during blastocyst formation in vitro and in cultured fully isolatd ICMs by 18 α-glycyrrhetinic acid (AGA, 65 μM; Sigma), cavitation rate and distribution of PKCs or junction assembly were not affected compared to controls (70–80% cavitated with characteristic distribution of junctional proteins and PKCs; P > 0.05, ANOVA; n = 15–20 per treatment, antibody, and cell contact pattern). When GJIC inhibition by AGA was confirmed by Lucifer yellow (Sigma) injection (no dye transfer in 82–100%, n = 14–17 per contact pattern), GJIC was also absent in 50% of fully isolated ICMs without AGA treatment, suggesting that cell contact modulation may affect GJIC. Taken together, our data suggest that cell contact pattern regulates TJ assembly via PKC signaling pathways and may also affect GJIC. GJIC appeared dispensable during cavitation, TJ assembly, and PKC signaling. A better understanding of the interrelationships between different signaling mechanisms may help to improve embryo culture methods and viability. Funding by the Wellcome Trust and MRC is gratefully acknowledged.

Characterization of displaced bipolar cells in the tiger salamander retina

In immunocytochemical studies of the tiger salamander retina, 17% of neurons in the outer nuclear layer did not label for recoverin, a photoreceptor marker. Lucifer yellow injection showed a population of cells in the ONL to be displaced bipolar cells, with axon terminals that stratified exclusively in the OFF sublamina of the inner plexiform layer (IPL), and predominately within the cone-dominated region of the OFF sublamina. Glutamate generated a dendritic cationic conductance increase in all displaced bipolar cells tested, and typical cone-dominated bipolar cell light responses were observed among displaced cells that stratified in the central IPL. We conclude that displaced bipolar cells in the tiger salamander retina are entirely OFF-center cells, and predominately cone-dominated cells.

Target-specific differences in somatodendritic morphology of layer V pyramidal neurons in rat motor cortex.

Dendritic geometry has been shown to be a critical determinant of information processing and neuronal computation. However, it is not known whether cortical projection neurons that target different subcortical nuclei have distinct dendritic morphologies. In this study, fast blue retrograde tracing in combination with intracellular Lucifer yellow injection and diaminobenzidine (DAB) photoconversion in fixed slices was used to study the morphological features of corticospinal, corticostriatal, and corticothalamic neurons in layer V of rat motor cortex. Marked differences in the distribution of soma, somal size, and dendritic profiles were found among the three groups of pyramidal neurons. Corticospinal neurons were large, were located in deep layer V, and had the most expansive dendritic fields. The apical dendrites of corticospinal pyramidal neurons were thick, spiny, and branched. In contrast, nearly all corticostriatal neurons were small cells located in superficial layer V. Their apical dendritic shafts were significantly more slender, though spiny like those of corticospinal neurons. Corticothalamic neurons, which were located in superficial layer V and in layer VI, had small or medium-sized soma, slender apical dendritic shafts, and dendrites that were largely spine free. This study indicates that, in layer V of rat motor cortex, each population of projection neurons has a unique somatodendritic morphology and suggests that distinct modes of cortical information processing are operative in corticospinal, corticostriatal, and corticothalamic neurons.